The Reg1-interacting proteins, Bmh1, Bmh2, Ssb1, and Ssb2, have roles in maintaining glucose repression in Saccharomyces cerevisiae.

PubMed

Dombek, Kenneth M; Kacherovsky, Nataly; Young, Elton T

2004-09-10

In Saccharomyces cerevisiae, a type 1 protein phosphatase complex composed of the Glc7 catalytic subunit and the Reg1 regulatory subunit represses expression of many glucose-regulated genes. Here we show that the Reg1-interacting proteins Bmh1, Bmh2, Ssb1, and Ssb2 have roles in glucose repression. Deleting both BMH genes causes partially constitutive ADH2 expression without significantly increasing the level of Adr1 protein, the major activator of ADH2 expression. Adr1 and Bcy1, the regulatory subunit of cAMP-dependent protein kinase, are both required for this effect indicating that constitutive expression in Deltabmh1Deltabmh2 cells uses the same activation pathway that operates in Deltareg1 cells. Deletion of both BMH genes and REG1 causes a synergistic relief from repression, suggesting that Bmh proteins also act independently of Reg1 during glucose repression. A two-hybrid interaction with the Bmh proteins was mapped to amino acids 187-232, a region of Reg1 that is conserved in different classes of fungi. Deleting this region partially releases SUC2 from glucose repression. This indicates a role for the Reg1-Bmh interaction in glucose repression and also suggests a broad role for Bmh proteins in this process. An in vivo Reg1-Bmh interaction was confirmed by copurification of Bmh proteins with HA(3)-TAP-tagged Reg1. The nonconventional heat shock proteins Ssb1 and Ssb2 are also copurified with HA(3)-TAP-tagged Reg1. Deletion of both SSB genes modestly decreases repression of ADH2 expression in the presence of glucose, suggesting that Ssb proteins, perhaps through their interaction with Reg1, play a minor role in glucose repression.

Glucocorticoid and cytokine crosstalk: Feedback, feedforward, and co-regulatory interactions determine repression or resistance

PubMed Central

Shah, Suharsh; Altonsy, Mohammed O.; Gerber, Antony N.

2017-01-01

Inflammatory signals induce feedback and feedforward systems that provide temporal control. Although glucocorticoids can repress inflammatory gene expression, glucocorticoid receptor recruitment increases expression of negative feedback and feedforward regulators, including the phosphatase, DUSP1, the ubiquitin-modifying enzyme, TNFAIP3, or the mRNA-destabilizing protein, ZFP36. Moreover, glucocorticoid receptor cooperativity with factors, including nuclear factor-κB (NF-κB), may enhance regulator expression to promote repression. Conversely, MAPKs, which are inhibited by glucocorticoids, provide feedforward control to limit expression of the transcription factor IRF1, and the chemokine, CXCL10. We propose that modulation of feedback and feedforward control can determine repression or resistance of inflammatory gene expression toglucocorticoid. PMID:28283576

On the presence and role of human gene-body DNA methylation

PubMed Central

Jjingo, Daudi; Conley, Andrew B.; Yi, Soojin V.; Lunyak, Victoria V.; Jordan, I. King

2012-01-01

DNA methylation of promoter sequences is a repressive epigenetic mark that down-regulates gene expression. However, DNA methylation is more prevalent within gene-bodies than seen for promoters, and gene-body methylation has been observed to be positively correlated with gene expression levels. This paradox remains unexplained, and accordingly the role of DNA methylation in gene-bodies is poorly understood. We addressed the presence and role of human gene-body DNA methylation using a meta-analysis of human genome-wide methylation, expression and chromatin data sets. Methylation is associated with transcribed regions as genic sequences have higher levels of methylation than intergenic or promoter sequences. We also find that the relationship between gene-body DNA methylation and expression levels is non-monotonic and bell-shaped. Mid-level expressed genes have the highest levels of gene-body methylation, whereas the most lowly and highly expressed sets of genes both have low levels of methylation. While gene-body methylation can be seen to efficiently repress the initiation of intragenic transcription, the vast majority of methylated sites within genes are not associated with intragenic promoters. In fact, highly expressed genes initiate the most intragenic transcription, which is inconsistent with the previously held notion that gene-body methylation serves to repress spurious intragenic transcription to allow for efficient transcriptional elongation. These observations lead us to propose a model to explain the presence of human gene-body methylation. This model holds that the repression of intragenic transcription by gene-body methylation is largely epiphenomenal, and suggests that gene-body methylation levels are predominantly shaped via the accessibility of the DNA to methylating enzyme complexes. PMID:22577155

Msx1 Homeodomain Protein Represses the αGSU and GnRH Receptor Genes During Gonadotrope Development

PubMed Central

Xie, Huimin; Cherrington, Brian D.; Meadows, Jason D.; Witham, Emily A.

2013-01-01

Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LβT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at −114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program. PMID:23371388

Msx1 homeodomain protein represses the αGSU and GnRH receptor genes during gonadotrope development.

PubMed

Xie, Huimin; Cherrington, Brian D; Meadows, Jason D; Witham, Emily A; Mellon, Pamela L

2013-03-01

Multiple homeodomain transcription factors are crucial for pituitary organogenesis and cellular differentiation. A homeodomain repressor, Msx1, is expressed from the ventral aspect of the developing anterior pituitary and implicated in gonadotrope differentiation. Here, we find that Msx1 represses transcription of lineage-specific pituitary genes such as the common α-glycoprotein subunit (αGSU) and GnRH receptor (GnRHR) promoters in the mouse gonadotrope-derived cell lines, αT3-1 and LβT2. Repression of the mouse GnRHR promoter by Msx1 is mediated through a consensus-binding motif in the downstream activin regulatory element (DARE). Truncation and mutation analyses of the human αGSU promoter map Msx1 repression to a site at -114, located at the junctional regulatory element (JRE). Dlx activators are closely related to the Msx repressors, acting through the same elements, and Dlx3 and Dlx2 act as transcriptional activators for GnRHR and αGSU, respectively. Small interfering RNA knockdown of Msx1 in αT3-1 cells increases endogenous αGSU and GnRHR mRNA expression. Msx1 gene expression reaches its maximal expression at the rostral edge at e13.5. The subsequent decline in Msx1 expression specifically coincides with the onset of expression of both αGSU and GnRHR. The expression levels of both αGSU and GnRHR in Msx1-null mice at e18.5 are higher compared with wild type, further confirming a role for Msx1 in the repression of αGSU and GnRHR. In summary, Msx1 functions as a negative regulator early in pituitary development by repressing the gonadotrope-specific αGSU and GnRHR genes, but a temporal decline in Msx1 expression alleviates this repression allowing induction of GnRHR and αGSU, thus serving to time the onset of gonadotrope-specific gene program.

3'-UTR-located inverted Alu repeats facilitate mRNA translational repression and stress granule accumulation

PubMed Central

Fitzpatrick, Terry; Huang, Sui

2012-01-01

Alu repeats within human genes may potentially alter gene expression. Here, we show that 3′-UTR-located inverted Alu repeats significantly reduce expression of an AcGFP reporter gene. Mutational analysis demonstrates that the secondary structure, but not the primary nucleotide sequence, of the inverted Alu repeats is critical for repression. The expression levels and nucleocytoplasmic distribution of reporter mRNAs with or without 3′-UTR inverted Alu repeats are similar; suggesting that reporter gene repression is not due to changes in mRNA levels or mRNA nuclear sequestration. Instead, reporter gene mRNAs harboring 3′-UTR inverted Alu repeats accumulate in cytoplasmic stress granules. These findings may suggest a novel mechanism whereby 3′-UTR-located inverted Alu repeats regulate human gene expression through sequestration of mRNAs within stress granules. PMID:22688648

Expression and regulation of the penicillin G acylase gene from Proteus rettgeri cloned in Escherichia coli.

PubMed

Daumy, G O; Williams, J A; McColl, A S; Zuzel, T J; Danley, D

1986-10-01

The penicillin G acylase genes from the Proteus rettgeri wild type and from a hyperproducing mutant which is resistant to succinate repression were cloned in Escherichia coli K-12. Expression of both wild-type and mutant P. rettgeri acylase genes in E. coli K-12 was independent of orientation in the cloning vehicle and apparently resulted from recognition in E. coli of the P. rettgeri promoter sequences. The P. rettgeri acylase was secreted into the E. coli periplasmic space and was composed of subunits electrophoretically identical to those made in P. rettgeri. Expression of these genes in E. coli K-12 was not repressed by succinate as it is in P. rettgeri. Instead, expression of the enzymes was regulated by glucose catabolite repression.

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms.

PubMed

Alexander, Matthew R; Murgai, Meera; Moehle, Christopher W; Owens, Gary K

2012-04-02

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.

Glucocorticoid and cytokine crosstalk: Feedback, feedforward, and co-regulatory interactions determine repression or resistance.

PubMed

Newton, Robert; Shah, Suharsh; Altonsy, Mohammed O; Gerber, Antony N

2017-04-28

Inflammatory signals induce feedback and feedforward systems that provide temporal control. Although glucocorticoids can repress inflammatory gene expression, glucocorticoid receptor recruitment increases expression of negative feedback and feedforward regulators, including the phosphatase, DUSP1, the ubiquitin-modifying enzyme, TNFAIP3, or the mRNA-destabilizing protein, ZFP36. Moreover, glucocorticoid receptor cooperativity with factors, including nuclear factor-κB (NF-κB), may enhance regulator expression to promote repression. Conversely, MAPKs, which are inhibited by glucocorticoids, provide feedforward control to limit expression of the transcription factor IRF1, and the chemokine, CXCL10. We propose that modulation of feedback and feedforward control can determine repression or resistance of inflammatory gene expression toglucocorticoid. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

An upstream open reading frame represses expression of Lc, a member of the R/B family of maize transcriptional activators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Damiani, R.D. Jr.; Wessler, S.R.

1993-09-01

The R/B genes of maize encode a family of basic helix-loop-helix proteins that determine where and when the anthocyanin-pigment pathway will be expressed in the plant. Previous studies showed that allelic diversity among family members reflects differences in gene expression, specifically in transcription initiation. The authors present evidence that the R gene Lc is under translational control. They demonstrate that the 235-nt transcript leader of Lc represses expression 25- to 30-fold in an in vivo assay. Repression is mediated by the presence in cis of a 38-codon upstream open reading frame. Furthermore, the coding capacity of the upstream open readingmore » frame influences the magnitude of repression. It is proposed that translational control does not contribute to tissue specificity but prevents overexpression of the Lc protein. The diversity of promoter and 5' untranslated leader sequences among the R/B genes provides an opportunity to study the coevolution of transcriptional and translational mechanisms of gene regulation. 36 refs., 5 figs.« less

Pax6 Exerts Regional Control of Cortical Progenitor Proliferation via Direct Repression of Cdk6 and Hypophosphorylation of pRb

PubMed Central

Mi, Da; Carr, Catherine B.; Georgala, Petrina A.; Huang, Yu-Ting; Manuel, Martine N.; Jeanes, Emily; Niisato, Emi; Sansom, Stephen N.; Livesey, Frederick J.; Theil, Thomas; Hasenpusch-Theil, Kerstin; Simpson, T. Ian; Mason, John O.; Price, David J.

2013-01-01

Summary The mechanisms by which early spatiotemporal expression patterns of transcription factors such as Pax6 regulate cortical progenitors in a region-specific manner are poorly understood. Pax6 is expressed in a gradient across the developing cortex and is essential for normal corticogenesis. We found that constitutive or conditional loss of Pax6 increases cortical progenitor proliferation by amounts that vary regionally with normal Pax6 levels. We compared the gene expression profiles of equivalent Pax6-expressing progenitors isolated from Pax6+/+ and Pax6−/− cortices and identified many negatively regulated cell-cycle genes, including Cyclins and Cdks. Biochemical assays indicated that Pax6 directly represses Cdk6 expression. Cyclin/Cdk repression inhibits retinoblastoma protein (pRb) phosphorylation, thereby limiting the transcription of genes that directly promote the mechanics of the cell cycle, and we found that Pax6 inhibits pRb phosphorylation and represses genes involved in DNA replication. Our results indicate that Pax6’s modulation of cortical progenitor cell cycles is regional and direct. PMID:23622063

Expression of bvg-repressed genes in Bordetella pertussis is controlled by RisA through a novel c-di-GMP signaling pathway

USDA-ARS?s Scientific Manuscript database

The BvgAS two component system of Bordetella pertussis controls virulence factor expression. In addition, BvgAS controls expression of the bvg-repressed genes through the action of the repressor, BvgR. The transcription factor RisA is inhibited by BvgR, and when BvgR is not expressed RisA induces th...

NFE2 Induces miR-423-5p to Promote Gluconeogenesis and Hyperglycemia by Repressing the Hepatic FAM3A-ATP-Akt Pathway.

PubMed

Yang, Weili; Wang, Junpei; Chen, Zhenzhen; Chen, Ji; Meng, Yuhong; Chen, Liming; Chang, Yongsheng; Geng, Bin; Sun, Libo; Dou, Lin; Li, Jian; Guan, Youfei; Cui, Qinghua; Yang, Jichun

2017-07-01

Hepatic FAM3A expression is repressed under obese conditions, but the underlying mechanism remains unknown. This study determined the role and mechanism of miR-423-5p in hepatic glucose and lipid metabolism by repressing FAM3A expression. miR-423-5p expression was increased in the livers of obese diabetic mice and in patients with nonalcoholic fatty liver disease (NAFLD) with decreased FAM3A expression. miR-423-5p directly targeted FAM3A mRNA to repress its expression and the FAM3A-ATP-Akt pathway in cultured hepatocytes. Hepatic miR-423-5p inhibition suppressed gluconeogenesis and improved insulin resistance, hyperglycemia, and fatty liver in obese diabetic mice. In contrast, hepatic miR-423-5p overexpression promoted gluconeogenesis and hyperglycemia and increased lipid deposition in normal mice. miR-423-5p inhibition activated the FAM3A-ATP-Akt pathway and repressed gluconeogenic and lipogenic gene expression in diabetic mouse livers. The miR-423 precursor gene was further shown to be a target gene of NFE2, which induced miR-423-5p expression to repress the FAM3A-ATP-Akt pathway in cultured hepatocytes. Hepatic NFE2 overexpression upregulated miR-423-5p to repress the FAM3A-ATP-Akt pathway, promoting gluconeogenesis and lipid deposition and causing hyperglycemia in normal mice. In conclusion, under the obese condition, activation of the hepatic NFE2/miR-423-5p axis plays important roles in the progression of type 2 diabetes and NAFLD by repressing the FAM3A-ATP-Akt signaling pathway. © 2017 by the American Diabetes Association.

Dopamine Signaling Leads to Loss of Polycomb Repression and Aberrant Gene Activation in Experimental Parkinsonism

PubMed Central

Lerdrup, Mads; Gomes, Ana-Luisa; Kryh, Hanna; Spigolon, Giada; Caboche, Jocelyne; Fisone, Gilberto; Hansen, Klaus

2014-01-01

Polycomb group (PcG) proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation. The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP) experiments showed that increased H3K27me3S28p was accompanied by reduced PcG binding to regulatory regions of genes. An analysis of the genome wide distribution of L-DOPA-induced H3K27me3S28 phosphorylation by ChIP sequencing (ChIP-seq) in combination with expression analysis by RNA-sequencing (RNA-seq) showed that the induction of H3K27me3S28p correlated with increased expression of a subset of PcG repressed genes. We found that induction of H3K27me3S28p persisted during chronic L-DOPA administration to parkisonian mice and correlated with aberrant gene expression. We propose that dopaminergic transmission can activate PcG repressed genes in the adult brain and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson's disease. PMID:25254549

Mechanism of repression of the inhibin alpha-subunit gene by inducible 3',5'-cyclic adenosine monophosphate early repressor.

PubMed

Burkart, Anna D; Mukherjee, Abir; Mayo, Kelly E

2006-03-01

The rodent ovary is regulated throughout the reproductive cycle to maintain normal cyclicity. Ovarian follicular development is controlled by changes in gene expression in response to the gonadotropins FSH and LH. The inhibin alpha-subunit gene belongs to a group of genes that is positively regulated by FSH and negatively regulated by LH. Previous studies established an important role for inducible cAMP early repressor (ICER) in repression of alpha-inhibin. These current studies investigate the mechanisms of repression by ICER. It is not clear whether all four ICER isoforms expressed in the ovary can act as repressors of the inhibin alpha-subunit gene. EMSAs demonstrate binding of all isoforms to the inhibin alpha-subunit CRE (cAMP response element), and transfection studies demonstrate that all isoforms can repress the inhibin alpha-subunit gene. Repression by ICER is dependent on its binding to DNA as demonstrated by mutations to ICER's DNA-binding domain. These mutational studies also demonstrate that repression by ICER is not dependent on heterodimerization with CREB (CRE-binding protein). Competitive EMSAs show that ICER effectively competes with CREB for binding to the inhibin alpha CRE in vitro. Chromatin immunoprecipitation assays demonstrate a replacement of CREB dimers bound to the inhibin alpha CRE by ICER dimers in ovarian granulosa cells in response to LH signaling. Thus, there is a temporal association of transcription factors bound to the inhibin alpha-CRE controlling inhibin alpha-subunit gene expression.

Two Distinct Mechanisms Govern RpoS-Mediated Repression of Tick-Phase Genes during Mammalian Host Adaptation by Borrelia burgdorferi, the Lyme Disease Spirochete.

PubMed

Grove, Arianna P; Liveris, Dionysios; Iyer, Radha; Petzke, Mary; Rudman, Joseph; Caimano, Melissa J; Radolf, Justin D; Schwartz, Ira

2017-08-22

The alternative sigma factor RpoS plays a key role modulating gene expression in Borrelia burgdorferi , the Lyme disease spirochete, by transcribing mammalian host-phase genes and repressing σ 70 -dependent genes required within the arthropod vector. To identify cis regulatory elements involved in RpoS-dependent repression, we analyzed green fluorescent protein (GFP) transcriptional reporters containing portions of the upstream regions of the prototypical tick-phase genes ospAB , the glp operon, and bba74 As RpoS-mediated repression occurs only following mammalian host adaptation, strains containing the reporters were grown in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats. Wild-type spirochetes harboring ospAB - and glp-gfp constructs containing only the minimal (-35/-10) σ 70 promoter elements had significantly lower expression in DMCs relative to growth in vitro at 37°C; no reduction in expression occurred in a DMC-cultivated RpoS mutant harboring these constructs. In contrast, RpoS-mediated repression of bba74 required a stretch of DNA located between -165 and -82 relative to its transcriptional start site. Electrophoretic mobility shift assays employing extracts of DMC-cultivated B. burgdorferi produced a gel shift, whereas extracts from RpoS mutant spirochetes did not. Collectively, these data demonstrate that RpoS-mediated repression of tick-phase borrelial genes occurs by at least two distinct mechanisms. One (e.g., ospAB and the glp operon) involves primarily sequence elements near the core promoter, while the other (e.g., bba74 ) involves an RpoS-induced transacting repressor. Our results provide a genetic framework for further dissection of the essential "gatekeeper" role of RpoS throughout the B. burgdorferi enzootic cycle. IMPORTANCE Borrelia burgdorferi , the Lyme disease spirochete, modulates gene expression to adapt to the distinctive environments of its mammalian host and arthropod vector during its enzootic cycle. The alternative sigma factor RpoS has been referred to as a "gatekeeper" due to its central role in regulating the reciprocal expression of mammalian host- and tick-phase genes. While RpoS-dependent transcription has been studied extensively, little is known regarding the mechanism(s) of RpoS-mediated repression. We employed a combination of green fluorescent protein transcriptional reporters along with an in vivo model to define cis regulatory sequences responsible for RpoS-mediated repression of prototypical tick-phase genes. Repression of ospAB and the glp operon requires only sequences near their core promoters, whereas modulation of bba74 expression involves a putative RpoS-dependent repressor that binds upstream of the core promoter. Thus, Lyme disease spirochetes employ at least two different RpoS-dependent mechanisms to repress tick-phase genes within the mammal. Copyright © 2017 Grove et al.

Mutations Inactivating Herpes Simplex Virus 1 MicroRNA miR-H2 Do Not Detectably Increase ICP0 Gene Expression in Infected Cultured Cells or Mouse Trigeminal Ganglia.

PubMed

Pan, Dongli; Pesola, Jean M; Li, Gang; McCarron, Seamus; Coen, Donald M

2017-01-15

Herpes simplex virus 1 (HSV-1) latency entails the repression of productive ("lytic") gene expression. An attractive hypothesis to explain some of this repression involves inhibition of the expression of ICP0, a lytic gene activator, by a viral microRNA, miR-H2, which is completely complementary to ICP0 mRNA. To test this hypothesis, we engineered mutations that disrupt miR-H2 without affecting ICP0 in HSV-1. The mutant virus exhibited drastically reduced expression of miR-H2 but showed wild-type levels of infectious virus production and no increase in ICP0 expression in lytically infected cells, which is consistent with the weak expression of miR-H2 relative to the level of ICP0 mRNA in that setting. Following corneal inoculation of mice, the mutant was not significantly different from wild-type virus in terms of infectious virus production in the trigeminal ganglia during acute infection, mouse mortality, or the rate of reactivation from explanted latently infected ganglia. Critically, the mutant was indistinguishable from wild-type virus for the expression of ICP0 and other lytic genes in acutely and latently infected mouse trigeminal ganglia. The latter result may be related to miR-H2 being less effective in inhibiting ICP0 expression in transfection assays than a host microRNA, miR-138, which has previously been shown to inhibit lytic gene expression in infected ganglia by targeting ICP0 mRNA. Additionally, transfected miR-138 reduced lytic gene expression in infected cells more effectively than miR-H2. While this study provides little support for the hypothesis that miR-H2 promotes latency by inhibiting ICP0 expression, the possibility remains that miR-H2 might target other genes during latency. Herpes simplex virus 1 (HSV-1), which causes a variety of diseases, can establish lifelong latent infections from which virus can reactivate to cause recurrent disease. Latency is the most biologically interesting and clinically vexing feature of the virus. Ever since miR-H2's discovery as a viral microRNA bearing complete sequence complementarity to the mRNA for the important viral gene activator ICP0, inhibition of ICP0 expression by miR-H2 has been a major hypothesis to help explain the repression of lytic gene expression during latency. However, this hypothesis remained untested in latently infected animals. Using a miR-H2-deficient mutant virus, we found no evidence that miR-H2 represses the expression of ICP0 or other lytic genes in cells or mice infected with HSV-1. Although miR-H2 can repress ICP0 expression in transfection assays, such repression is weak. The results suggest that other mechanisms for miR-H2 activity and for the repression of lytic gene expression during latency deserve investigation. Copyright © 2017 American Society for Microbiology.

Bmi1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor

PubMed Central

Biehs, Brian; Hu, Jimmy Kuang-Hsien; Strauli, Nicolas B.; Sangiorgi, Eugenio; Jung, Heekyung; Heber, Ralf-Peter; Ho, Sunita; Goodwin, Alice F.; Dasen, Jeremy S.; Capecchi, Mario R.; Klein, Ophir D.

2013-01-01

The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs1, 2. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell cycle inhibitors p16ink4a and p19Arf3. However, deletion of Ink4a/Arf only partially rescues Bmi1 null phenotypes4, indicating that other important targets of BMI1 exist. Here, using the continuously-growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression, and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated1, 2, 5–7, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation. PMID:23728424

pH-Dependent DNA Distortion and Repression of Gene Expression by Pectobacterium atrosepticum PecS.

PubMed

Deochand, Dinesh K; Meariman, Jacob K; Grove, Anne

2016-07-15

Transcriptional activity is exquisitely sensitive to changes in promoter DNA topology. Transcription factors may therefore control gene activity by modulating the relative positioning of -10 and -35 promoter elements. The plant pathogen Pectobacterium atrosepticum, which causes soft rot in potatoes, must alter gene expression patterns to ensure growth in planta. In the related soft-rot enterobacterium Dickeya dadantii, PecS functions as a master regulator of virulence gene expression. Here, we report that P. atrosepticum PecS controls gene activity by altering promoter DNA topology in response to pH. While PecS binds the pecS promoter with high affinity regardless of pH, it induces significant DNA distortion only at neutral pH, the pH at which the pecS promoter is repressed in vivo. At pH ∼8, DNA distortions are attenuated, and PecS no longer represses the pecS promoter. A specific histidine (H142) located in a crevice between the dimerization- and DNA-binding regions is required for pH-dependent changes in DNA distortion and repression of gene activity, and mutation of this histidine renders the mutant protein incapable of repressing the pecS promoter. We propose that protonated PecS induces a DNA conformation at neutral pH in which -10 and -35 promoter elements are suboptimally positioned for RNA polymerase binding; on deprotonation of PecS, binding is no longer associated with significant changes in DNA conformation, allowing gene expression. We suggest that this mode of gene regulation leads to differential expression of the PecS regulon in response to alkalinization of the plant apoplast.

Light represses transcription of asparagine synthetase genes in photosynthetic and nonphotosynthetic organs of plants.

PubMed Central

Tsai, F Y; Coruzzi, G

1991-01-01

Asparagine synthetase (AS) mRNA in Pisum sativum accumulates preferentially in plants grown in the dark. Nuclear run-on experiments demonstrate that expression of both the AS1 and AS2 genes is negatively regulated by light at the level of transcription. A decrease in the transcriptional rate of the AS1 gene can be detected as early as 20 min after exposure to light. Time course experiments reveal that the levels of AS mRNA fluctuate dramatically during a "normal" light/dark cycle. This is due to a direct effect of light and not to changes associated with circadian rhythm. A novel finding is that the light-repressed expression of the AS1 gene is as dramatic in nonphotosynthetic organs such as roots as it is in leaves. Experiments demonstrate that the small amount of light which passes through the soil is sufficient to repress AS1 expression in roots, indicating that light has a direct effect on AS1 gene expression in roots. The negative regulation of AS gene expression by light was shown to be a general phenomenon in plants which also occurs in nonlegumes such as Nicotiana plumbaginifolia and Nicotiana tabacum. Thus, the AS genes can serve as a model with which to dissect the molecular basis for light-regulated transcriptional repression in plants. Images PMID:1681424

Hidden among the crowd: differential DNA methylation-expression correlations in cancer occur at important oncogenic pathways.

PubMed

Mosquera Orgueira, Adrián

2015-01-01

DNA methylation is a frequent epigenetic mechanism that participates in transcriptional repression. Variations in DNA methylation with respect to gene expression are constant, and, for unknown reasons, some genes with highly methylated promoters are sometimes overexpressed. In this study we have analyzed the expression and methylation patterns of thousands of genes in five groups of cancer and normal tissue samples in order to determine local and genome-wide differences. We observed significant changes in global methylation-expression correlation in all the neoplasms, which suggests that differential correlation events are frequent in cancer. A focused analysis in the breast cancer cohort identified 1662 genes whose correlation varies significantly between normal and cancerous breast, but whose DNA methylation and gene expression patterns do not change substantially. These genes were enriched in cancer-related pathways and repressive chromatin features across various model cell lines, such as PRC2 binding and H3K27me3 marks. Substantial changes in methylation-expression correlation indicate that these genes are subject to epigenetic remodeling, where the differential activity of other factors break the expected relationship between both variables. Our findings suggest a complex regulatory landscape where a redistribution of local and large-scale chromatin repressive domains at differentially correlated genes (DCGs) creates epigenetic hotspots that modulate cancer-specific gene expression.

Repression of Middle Sporulation Genes in Saccharomyces cerevisiae by the Sum1-Rfm1-Hst1 Complex Is Maintained by Set1 and H3K4 Methylation

PubMed Central

Jaiswal, Deepika; Jezek, Meagan; Quijote, Jeremiah; Lum, Joanna; Choi, Grace; Kulkarni, Rushmie; Park, DoHwan; Green, Erin M.

2017-01-01

The conserved yeast histone methyltransferase Set1 targets H3 lysine 4 (H3K4) for mono, di, and trimethylation and is linked to active transcription due to the euchromatic distribution of these methyl marks and the recruitment of Set1 during transcription. However, loss of Set1 results in increased expression of multiple classes of genes, including genes adjacent to telomeres and middle sporulation genes, which are repressed under normal growth conditions because they function in meiotic progression and spore formation. The mechanisms underlying Set1-mediated gene repression are varied, and still unclear in some cases, although repression has been linked to both direct and indirect action of Set1, associated with noncoding transcription, and is often dependent on the H3K4me2 mark. We show that Set1, and particularly the H3K4me2 mark, are implicated in repression of a subset of middle sporulation genes during vegetative growth. In the absence of Set1, there is loss of the DNA-binding transcriptional regulator Sum1 and the associated histone deacetylase Hst1 from chromatin in a locus-specific manner. This is linked to increased H4K5ac at these loci and aberrant middle gene expression. These data indicate that, in addition to DNA sequence, histone modification status also contributes to proper localization of Sum1. Our results also show that the role for Set1 in middle gene expression control diverges as cells receive signals to undergo meiosis. Overall, this work dissects an unexplored role for Set1 in gene-specific repression, and provides important insights into a new mechanism associated with the control of gene expression linked to meiotic differentiation. PMID:29066473

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

PubMed Central

Tan, Siyuan; Guschin, Dmitry; Davalos, Albert; Lee, Ya-Li; Snowden, Andrew W.; Jouvenot, Yann; Zhang, H. Steven; Howes, Katherine; McNamara, Andrew R.; Lai, Albert; Ullman, Chris; Reynolds, Lindsey; Moore, Michael; Isalan, Mark; Berg, Lutz-Peter; Campos, Bradley; Qi, Hong; Spratt, S. Kaye; Case, Casey C.; Pabo, Carl O.; Campisi, Judith; Gregory, Philip D.

2003-01-01

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics. PMID:14514889

Reduction of aflatoxin production by Aspergillus flavus and Aspergillus parasiticus in interaction with Streptomyces.

PubMed

Verheecke, C; Liboz, T; Anson, P; Diaz, R; Mathieu, F

2015-05-01

The aim of this study is to investigate aflatoxin gene expression during Streptomyces-Aspergillus interaction. Aflatoxins are carcinogenic compounds produced mainly by Aspergillus flavus and Aspergillus parasiticus. A previous study has shown that Streptomyces-A. flavus interaction can reduce aflatoxin content in vitro. Here, we first validated this same effect in the interaction with A. parasiticus. Moreover, we showed that growth reduction and aflatoxin content were correlated in A. parasiticus but not in A. flavus. Secondly, we investigated the mechanisms of action by reverse-transcriptase quantitative PCR. As microbial interaction can lead to variations in expression of household genes, the most stable [act1, βtub (and cox5 for A. parasiticus)] were chosen using geNorm software. To shed light on the mechanisms involved, we studied during the interaction the expression of five genes (aflD, aflM, aflP, aflR and aflS). Overall, the results of aflatoxin gene expression showed that Streptomyces repressed gene expression to a greater level in A. parasiticus than in A. flavus. Expression of aflR and aflS was generally repressed in both Aspergillus species. Expression of aflM was repressed and was correlated with aflatoxin B1 content. The results suggest that aflM expression could be a potential aflatoxin indicator in Streptomyces species interactions. Therefore, we demonstrate that Streptomyces can reduce aflatoxin production by both Aspergillus species and that this effect can be correlated with the repression of aflM expression. © 2015 The Authors.

Regulation of Nitrogen Metabolism by GATA Zinc Finger Transcription Factors in Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.

ABSTRACT Fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeastYarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism inY. lipolytica. Deletion of the GATA transcription factor genesgzf3andgzf2resulted in nitrogen source-specific growth defects and greater accumulation of lipids when the cells weremore » growing on a simple nitrogen source. Deletion ofgzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion ofgzf3results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, whilegzf2is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressormig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism. IMPORTANCENitrogen source is commonly used to control lipid production in industrial fungi. Here we identified regulators of nitrogen catabolite repression in the oleaginous yeastY. lipolyticato determine how the nitrogen source regulates lipid metabolism. We show that disruption of both activators and repressors of nitrogen catabolite repression leads to increased lipid accumulation via activation of carbon catabolite repression through an as yet uncharacterized method.« less

Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

PubMed Central

Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

2012-01-01

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression. PMID:22238674

Menin regulates Inhbb expression through an Akt/Ezh2-mediated H3K27 histone modification.

PubMed

Gherardi, Samuele; Ripoche, Doriane; Mikaelian, Ivan; Chanal, Marie; Teinturier, Romain; Goehrig, Delphine; Cordier-Bussat, Martine; Zhang, Chang X; Hennino, Ana; Bertolino, Philippe

2017-04-01

Although Men1 is a well-known tumour suppressor gene, little is known about the functions of Menin, the protein it encodes for. Since few years, numerous publications support a major role of Menin in the control of epigenetics gene regulation. While Menin interaction with MLL complex favours transcriptional activation of target genes through H3K4me3 marks, Menin also represses gene expression via mechanisms involving the Polycomb repressing complex (PRC). Interestingly, Ezh2, the PRC-methyltransferase that catalyses H3K27me3 repressive marks and Menin have been shown to co-occupy a large number of promoters. However, lack of binding between Menin and Ezh2 suggests that another member of the PRC complex is mediating this indirect interaction. Having found that ActivinB - a TGFβ superfamily member encoded by the Inhbb gene - is upregulated in insulinoma tumours caused by Men1 invalidation, we hypothesize that Menin could directly participate in the epigenetic-repression of Inhbb gene expression. Using Animal model and cell lines, we report that loss of Menin is directly associated with ActivinB-induced expression both in vivo and in vitro. Our work further reveals that ActivinB expression is mediated through a direct modulation of H3K27me3 marks on the Inhbb locus in Menin-KO cell lines. More importantly, we show that Menin binds on the promoter of Inhbb gene where it favours the recruitment of Ezh2 via an indirect mechanism involving Akt-phosphorylation. Our data suggests therefore that Menin could take an important part to the Ezh2-epigenetic repressive landscape in many cells and tissues through its capacity to modulate Akt phosphorylation. Copyright © 2017 Elsevier B.V. All rights reserved.

TCF7L1 recruits CtBP and HDAC1 to repress DICKKOPF4 gene expression in human colorectal cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eshelman, Melanie A.; Shah, Meera; Raup-Konsavage, Wesley M.

The T-cell factor/Lymphoid enhancer factor (TCF/LEF; hereafter TCF) family of transcription factors are critical regulators of colorectal cancer (CRC) cell growth. Of the four TCF family members, TCF7L1 functions predominantly as a repressor of gene expression. Few studies have addressed the role of TCF7L1 in CRC and only a handful of target genes regulated by this repressor are known. By silencing TCF7L1 expression in HCT116 cells, we show that it promotes cell proliferation and tumorigenesis in vivo by driving cell cycle progression. Microarray analysis of transcripts differentially expressed in control and TCF7L1-silenced CRC cells identified genes that control cell cycle kinetics andmore » cancer pathways. Among these, expression of the Wnt antagonist DICKKOPF4 (DKK4) was upregulated when TCF7L1 levels were reduced. We found that TCF7L1 recruits the C-terminal binding protein (CtBP) and histone deacetylase 1 (HDAC1) to the DKK4 promoter to repress DKK4 gene expression. In the absence of TCF7L1, TCF7L2 and β-catenin occupancy at the DKK4 promoter is stimulated and DKK4 expression is increased. These findings uncover a critical role for TCF7L1 in repressing DKK4 gene expression to promote the oncogenic potential of CRCs. - Highlights: • TCF7L1 promotes colorectal cancer cell proliferation and tumorigenesis. • DICKKOPF4 is directly regulated by TCF7L1. • TCF7L1 recruits CtBP and HDAC1 to repress DKK4 gene expression.« less

The Arabidopsis BRAHMA Chromatin-Remodeling ATPase Is Involved in Repression of Seed Maturation Genes in Leaves1[W][OA

PubMed Central

Tang, Xurong; Hou, Anfu; Babu, Mohan; Nguyen, Vi; Hurtado, Lidia; Lu, Qing; Reyes, Jose C.; Wang, Aiming; Keller, Wilfred A.; Harada, John J.; Tsang, Edward W.T.; Cui, Yuhai

2008-01-01

Synthesis and accumulation of seed storage proteins (SSPs) is an important aspect of the seed maturation program. Genes encoding SSPs are specifically and highly expressed in the seed during maturation. However, the mechanisms that repress the expression of these genes in leaf tissue are not well understood. To gain insight into the repression mechanisms, we performed a genetic screen for mutants that express SSPs in leaves. Here, we show that mutations affecting BRAHMA (BRM), a SNF2 chromatin-remodeling ATPase, cause ectopic expression of a subset of SSPs and other embryogenesis-related genes in leaf tissue. Consistent with the notion that such SNF2-like ATPases form protein complexes in vivo, we observed similar phenotypes for mutations of AtSWI3C, a BRM-interacting partner, and BSH, a SNF5 homolog and essential SWI/SNF subunit. Chromatin immunoprecipitation experiments show that BRM is recruited to the promoters of a number of embryogenesis genes in wild-type leaves, including the 2S genes, expressed in brm leaves. Consistent with its role in nucleosome remodeling, BRM appears to affect the chromatin structure of the At2S2 promoter. Thus, the BRM-containing chromatin-remodeling ATPase complex involved in many aspects of plant development mediates the repression of SSPs in leaf tissue. PMID:18508955

Developmentally linked human DNA hypermethylation is associated with down-modulation, repression, and upregulation of transcription

PubMed Central

Baribault, Carl; Ehrlich, Kenneth C.; Ponnaluri, V. K. Chaithanya; Pradhan, Sriharsa; Lacey, Michelle; Ehrlich, Melanie

2018-01-01

ABSTRACT DNA methylation can affect tissue-specific gene transcription in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). To elucidate the variety of associations between differentiation-related DNA hypermethylation and transcription, we used available epigenomic and transcriptomic profiles from 38 human cell/tissue types to focus on such relationships in 94 genes linked to hypermethylated DMRs in myoblasts (Mb). For 19 of the genes, promoter-region hypermethylation in Mb (and often a few heterologous cell types) was associated with gene repression but, importantly, DNA hypermethylation was absent in many other repressed samples. In another 24 genes, DNA hypermethylation overlapped cryptic enhancers or super-enhancers and correlated with down-modulated, but not silenced, gene expression. However, such methylation was absent, surprisingly, in both non-expressing samples and highly expressing samples. This suggests that some genes need DMR hypermethylation to help repress cryptic enhancer chromatin only when they are actively transcribed. For another 11 genes, we found an association between intergenic hypermethylated DMRs and positive expression of the gene in Mb. DNA hypermethylation/transcription correlations similar to those of Mb were evident sometimes in diverse tissues, such as aorta and brain. Our findings have implications for the possible involvement of methylated DNA in Duchenne's muscular dystrophy, congenital heart malformations, and cancer. This epigenomic analysis suggests that DNA methylation is not simply the inevitable consequence of changes in gene expression but, instead, is often an active agent for fine-tuning transcription in association with development. PMID:29498561

Differentially expressed genes in healthy and plum pox virus-infected Nicotiana benthamiana plants.

PubMed

Vozárová, Z; Žilová, M; Šubr, Z

2015-12-01

Viruses use both material and energy sources of their hosts and redirect the production of disposable compounds in order to make viral replication more efficient. Metabolism of infected organisms is modified by these enhanced requirements as well by their own defense response. Resulting complex story consists of many regulation events on various gene expression levels. Elucidating these processes may contribute to the knowledge on virus-host interactions and to evolving new antiviral strategies. In our work we applied a subtractive cloning technique to compare the transcriptomes of healthy and plum pox virus (PPV)-infected Nicotiana benthamiana plants. Several genes were found to be induced or repressed by the PPV infection. The induced genes were mainly related to general stress response or photosynthesis, several repressed genes could be connected with growth defects evoked by the infection. Interestingly, some genes usually up-regulated by fungal or bacterial infection were found repressed in PPV-infected plants. Potential involvement of particular differently expressed genes in the process of PPV infection is discussed.

Cloning and Characterizing Genes Involved in Monoterpene Induced Mammary Tumor Regression

DTIC Science & Technology

1998-05-01

Monoterpene -induced/repressed genes were identified in regressing rat mammary carcinomas treated with dietary limonene using a newly developed method...termed subtractive display. The subtractive display screen identified 42 monoterpene -induced genes comprising 9 known genes and 33 unidentified genes...as well as 58 monoterpene -repressed genes comprising 1 known gene and 57 unidentified genes. Several of the identified differentially expressed

Hidden among the crowd: differential DNA methylation-expression correlations in cancer occur at important oncogenic pathways

PubMed Central

Mosquera Orgueira, Adrián

2015-01-01

DNA methylation is a frequent epigenetic mechanism that participates in transcriptional repression. Variations in DNA methylation with respect to gene expression are constant, and, for unknown reasons, some genes with highly methylated promoters are sometimes overexpressed. In this study we have analyzed the expression and methylation patterns of thousands of genes in five groups of cancer and normal tissue samples in order to determine local and genome-wide differences. We observed significant changes in global methylation-expression correlation in all the neoplasms, which suggests that differential correlation events are frequent in cancer. A focused analysis in the breast cancer cohort identified 1662 genes whose correlation varies significantly between normal and cancerous breast, but whose DNA methylation and gene expression patterns do not change substantially. These genes were enriched in cancer-related pathways and repressive chromatin features across various model cell lines, such as PRC2 binding and H3K27me3 marks. Substantial changes in methylation-expression correlation indicate that these genes are subject to epigenetic remodeling, where the differential activity of other factors break the expected relationship between both variables. Our findings suggest a complex regulatory landscape where a redistribution of local and large-scale chromatin repressive domains at differentially correlated genes (DCGs) creates epigenetic hotspots that modulate cancer-specific gene expression. PMID:26029238

sae is essential for expression of the staphylococcal adhesins Eap and Emp.

PubMed

Harraghy, Niamh; Kormanec, Jan; Wolz, Christiane; Homerova, Dagmar; Goerke, Christiane; Ohlsen, Knut; Qazi, Saara; Hill, Philip; Herrmann, Mathias

2005-06-01

Eap and Emp are two Staphylococcus aureus adhesins initially described as extracellular matrix binding proteins. Eap has since emerged as being important in adherence to and invasion of eukaryotic cells, as well as being described as an immunomodulator and virulence factor in chronic infections. This paper describes the mapping of the transcription start point of the eap and emp promoters. Moreover, using reporter-gene assays and real-time PCR in defined regulatory mutants, environmental conditions and global regulators affecting expression of eap and emp were investigated. Marked differences were found in expression of eap and emp between strain Newman and the 8325 derivatives SH1000 and 8325-4. Moreover, both genes were repressed in the presence of glucose. Analysis of expression of both genes in various regulatory mutants revealed that sarA and agr were involved in their regulation, but the data suggested that there were additional regulators of both genes. In a sae mutant, expression of both genes was severely repressed. sae expression was also reduced in the presence of glucose, suggesting that repression of eap and emp in glucose-containing medium may, in part, be a consequence of a decrease in expression of sae.

Regulation of nitrogen metabolism by GATA zinc finger transcription factors in Yarrowia lipolytica

DOE PAGES

Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.; ...

2017-02-15

Here, fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast Yarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism in Y. lipolytica. Deletion of the GATA transcription factor genes gzf3 and gzf2 resulted in nitrogen source-specific growth defects and greatermore » accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion of gzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion of gzf3 results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, while gzf2 is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressor mig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism.« less

Regulation of nitrogen metabolism by GATA zinc finger transcription factors in Yarrowia lipolytica

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pomraning, Kyle R.; Bredeweg, Erin L.; Baker, Scott E.

Here, fungi accumulate lipids in a manner dependent on the quantity and quality of the nitrogen source on which they are growing. In the oleaginous yeast Yarrowia lipolytica, growth on a complex source of nitrogen enables rapid growth and limited accumulation of neutral lipids, while growth on a simple nitrogen source promotes lipid accumulation in large lipid droplets. Here we examined the roles of nitrogen catabolite repression and its regulation by GATA zinc finger transcription factors on lipid metabolism in Y. lipolytica. Deletion of the GATA transcription factor genes gzf3 and gzf2 resulted in nitrogen source-specific growth defects and greatermore » accumulation of lipids when the cells were growing on a simple nitrogen source. Deletion of gzf1, which is most similar to activators of genes repressed by nitrogen catabolite repression in filamentous ascomycetes, did not affect growth on the nitrogen sources tested. We examined gene expression of wild-type and GATA transcription factor mutants on simple and complex nitrogen sources and found that expression of enzymes involved in malate metabolism, beta-oxidation, and ammonia utilization are strongly upregulated on a simple nitrogen source. Deletion of gzf3 results in overexpression of genes with GATAA sites in their promoters, suggesting that it acts as a repressor, while gzf2 is required for expression of ammonia utilization genes but does not grossly affect the transcription level of genes predicted to be controlled by nitrogen catabolite repression. Both GATA transcription factor mutants exhibit decreased expression of genes controlled by carbon catabolite repression via the repressor mig1, including genes for beta-oxidation, highlighting the complex interplay between regulation of carbon, nitrogen, and lipid metabolism.« less

The Third Intron of the Interferon Regulatory Factor-8 Is an Initiator of Repressed Chromatin Restricting Its Expression in Non-Immune Cells

PubMed Central

Barnea-Yizhar, Ofer; Ram, Sigal; Kovalev, Ekaterina; Azriel, Aviva; Rand, Ulfert; Nakayama, Manabu; Hauser, Hansjörg; Gepstein, Lior; Levi, Ben-Zion

2016-01-01

Interferon Regulatory Factor-8 (IRF-8) serves as a key factor in the hierarchical differentiation towards monocyte/dendritic cell lineages. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF-8 expression in non-hematopoietic cells is still unknown. Here we show that the repression of IRF-8 expression in restrictive cells is mediated by its 3rd intron. Removal of this intron alleviates the repression of Bacterial Artificial Chromosome (BAC) IRF-8 reporter gene in these cells. Fine deletion analysis points to conserved regions within this intron mediating its restricted expression. Further, the intron alone selectively initiates gene silencing only in expression-restrictive cells. Characterization of this intron’s properties points to its role as an initiator of sustainable gene silencing inducing chromatin condensation with suppressive histone modifications. This intronic element cannot silence episomal transgene expression underlining a strict chromatin-dependent silencing mechanism. We validated this chromatin-state specificity of IRF-8 intron upon in-vitro differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes. Taken together, the IRF-8 3rd intron is sufficient and necessary to initiate gene silencing in non-hematopoietic cells, highlighting its role as a nucleation core for repressed chromatin during differentiation. PMID:27257682

Corticotropin-releasing hormone or dexamethasone regulates rat proopiomelanocortin transcription through Tpit/Pitx-responsive element in its promoter.

PubMed

Murakami, Itsuo; Takeuchi, Sakae; Kudo, Toshiyuki; Sutou, Shizuyo; Takahashi, Sumio

2007-05-01

Tpit/Pitx-responsive element (Tpit/PitxRE), which binds transcription factors Tpit and Pitx1, confers cell-type specific expression of proopiomelanocortin (POMC) gene in pituitary corticotrops where the gene expression is mainly regulated by corticotropin-releasing hormone (CRH) and glucocorticoids (Gcs). CRH stimulates POMC gene expression, which is mediated by the accumulation of intracellular cAMP and requires binding of Nur factors to Nur-responsive element (NurRE). Gcs antagonize NurRE-dependent POMC gene expression through direct interaction between glucocorticoid receptors and Nur factors. We examined whether Tpit/PitxRE and NurRE are involved in CRH/cAMP-induced activation and Gc-induced repression of POMC gene expression by reporter assay in AtT-20 corticotropic cells. Deletion and mutation of Tpit/PitxRE markedly reduced basal activity of the promoter, and those of NurRE decreased the levels of the CRH/cAMP-induced activation. Nifedipine, KN-62, and W-7, specific inhibitors of the L-type calcium channel, calmodulin-dependent protein kinase II, and calmodulin respectively, attenuated CRH/cAMP-induced activation of promoters with three copies of either Tpit/PitxRE or NurRE, indicating that both Tpit/PitxRE and NurRE mediate CRH-induced activation of POMC gene expression in a calcium-dependent manner. Deletion and mutation of Tpit/PitxRE abolished dexamethasone (DEX)-induced repression of POMC gene expression, while those of NurRE did not, indicating that Tpit/PitxRE predominantly mediates Gc-induced repression of POMC transcription. However, DEX treatment attenuated activities of promoters with three copies of either Tpit/PitxRE or NurRE, suggesting that Gcs act at NurRE as well as Tpit/PitxRE to repress POMC gene expression. We conclude that Tpit/PitxRE is an important element by which CRH and Gcs regulate the POMC gene expression.

Spot 42 Small RNA Regulates Arabinose-Inducible araBAD Promoter Activity by Repressing Synthesis of the High-Affinity Low-Capacity Arabinose Transporter

PubMed Central

Chen, Jiandong

2016-01-01

ABSTRACT The l-arabinose-inducible araBAD promoter (PBAD) enables tightly controlled and tunable expression of genes of interest in a broad range of bacterial species. It has been used successfully to study bacterial sRNA regulation, where PBAD drives expression of target mRNA translational fusions. Here we report that in Escherichia coli, Spot 42 sRNA regulates PBAD promoter activity by affecting arabinose uptake. We demonstrate that Spot 42 sRNA represses araF, a gene encoding the AraF subunit of the high-affinity low-capacity arabinose transporter AraFGH, through direct base-pairing interactions. We further show that endogenous Spot 42 sRNA is sufficient to repress araF expression under various growth conditions. Finally, we demonstrate this posttranscriptional repression has a biological consequence, decreasing the induction of PBAD at low levels of arabinose. This problem can be circumvented using strategies reported previously for avoiding all-or-none induction behavior, such as through constitutive expression of the low-affinity high-capacity arabinose transporter AraE or induction with a higher concentration of inducers. This work adds araF to the set of Spot 42-regulated genes, in agreement with previous studies suggesting that Spot 42, itself negatively regulated by the cyclic AMP (cAMP) receptor protein-cAMP complex, reinforces the catabolite repression network. IMPORTANCE The bacterial arabinose-inducible system is widely used for titratable control of gene expression. We demonstrate here that a posttranscriptional mechanism mediated by Spot 42 sRNA contributes to the functionality of the PBAD system at subsaturating inducer concentrations by affecting inducer uptake. Our finding extends the inputs into the known transcriptional control for the PBAD system and has implications for improving its usage for tunable gene expression. PMID:27849174

Opi1 mediates repression of phospholipid biosynthesis by phosphate limitation in the yeast Saccharomyces cerevisiae.

PubMed

Kliewe, Felix; Kumme, Jacqueline; Grigat, Mathias; Hintze, Stefan; Schüller, Hans-Joachim

2017-02-01

Structural genes of phospholipid biosynthesis in the yeast Saccharomyces cerevisiae are transcribed when precursor molecules inositol and choline (IC) are limiting. Gene expression is stimulated by the heterodimeric activator Ino2/Ino4, which binds to ICRE (inositol/choline-responsive element) promoter sequences. Activation is prevented by repressor Opi1, counteracting Ino2 when high concentrations of IC are available. Here we show that ICRE-dependent gene activation is repressed not only by an excess of IC but also under conditions of phosphate starvation. While PHO5 is activated by phosphate limitation, INO1 expression is repressed about 10-fold. Repression of ICRE-dependent genes by low phosphate is no longer observed in an opi1 mutant while repression is still effective in mutants of the PHO regulon (pho4, pho80, pho81 and pho85). In contrast, gene expression with high phosphate is reduced in the absence of pleiotropic sensor protein kinase Pho85. We could demonstrate that Pho85 binds to Opi1 in vitro and in vivo and that this interaction is increased in the presence of high concentrations of phosphate. Interestingly, Pho85 binds to two separate domains of Opi1 which have been previously shown to recruit pleiotropic corepressor Sin3 and activator Ino2, respectively. We postulate that Pho85 positively influences ICRE-dependent gene expression by phosphorylation-dependent weakening of Opi1 repressor, affecting its functional domains required for promoter recruitment and corepressor interaction. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Antisense transcriptional interference mediates condition-specific gene repression in budding yeast.

PubMed

Nevers, Alicia; Doyen, Antonia; Malabat, Christophe; Néron, Bertrand; Kergrohen, Thomas; Jacquier, Alain; Badis, Gwenael

2018-05-18

Pervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular antisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilized pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asRNAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chromatin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption.

Role of carbon source in the shift from oxidative to hydrolytic wood decomposition by Postia placenta.

PubMed

Zhang, Jiwei; Schilling, Jonathan S

2017-09-01

Brown rot fungi initiate wood decay using oxidative pretreatments to improve access for cellulolytic enzymes. These pretreatments are incompatible with enzymes, and we recently showed that Postia placenta overcomes this issue by delaying glycoside hydrolase (GH) gene upregulation briefly (<48h) until expression of oxidoreductases (ORs) is repressed. This implies an inducible cellulase system rather than a constitutive system, as often reported, and it remains unclear what cues this transition. To address this, we grew P. placenta along wood wafers and spatially mapped expression (via quantitative PCR) of twelve ORs and GHs targeted using functional genomics analyses. By layering expression patterns over solubilized sugar data (via HPLC) from wood, we observed solubilization of wood glucose, cellobiose, mannose, and xylose coincident with the OR-GH transition. We then tested effects of these soluble sugars, plus polymeric carbon sources (spruce powder, cellulose), on P. placenta gene expression in liquid cultures. Expression of ORs was strictly (aox1, cro5) or progressively repressed over time (qrd1, lcc1) by all soluble sugars, including cellobiose, but not by polymeric sources. Simple sugars repressed hemicellulase gene expression over time, but these sugars did not repress cellulases. Cellulase genes were upregulated, however, along with hemicellulases in the presence of soluble cellobiose and in the presence of polymeric carbon sources, relative to starvation (carbon-free). This verifies an inducible cellulase system in P. placenta that lacks carbon catabolite repression (CCR), and it suggests that brown rot fungi use soluble sugars, particularly cellobiose, to cue a critical oxidative-hydrolytic transition. Copyright © 2017 Elsevier Inc. All rights reserved.

Proto-oncogene FBI-1 (Pokemon/ZBTB7A) represses transcription of the tumor suppressor Rb gene via binding competition with Sp1 and recruitment of co-repressors.

PubMed

Jeon, Bu-Nam; Yoo, Jung-Yoon; Choi, Won-Il; Lee, Choong-Eun; Yoon, Ho-Geun; Hur, Man-Wook

2008-11-28

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp -308 to -188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp -65 to -56) and GC-box 2 (bp -18 to -9), the latter of which is also bound by FBI-1. We found that FRE3 (bp -244 to -236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression.

Integrated analysis of miRNA and mRNA expression data identifies multiple miRNAs regulatory networks for the tumorigenesis of colorectal cancer.

PubMed

Xu, Peng; Wang, Junhua; Sun, Bo; Xiao, Zhongdang

2018-06-15

Investigating the potential biological function of differential changed genes through integrating multiple omics data including miRNA and mRNA expression profiles, is always hot topic. However, how to evaluate the repression effect on target genes integrating miRNA and mRNA expression profiles are not fully solved. In this study, we provide an analyzing method by integrating both miRNAs and mRNAs expression data simultaneously. Difference analysis was adopted based on the repression score, then significantly repressed mRNAs were screened out by DEGseq. Pathway analysis for the significantly repressed mRNAs shows that multiple pathways such as MAPK signaling pathway, TGF-beta signaling pathway and so on, may correlated to the colorectal cancer(CRC). Focusing on the MAPK signaling pathway, a miRNA-mRNA network that centering the cell fate genes was constructed. Finally, the miRNA-mRNAs that potentially important in the CRC carcinogenesis were screened out and scored by impact index. Copyright © 2018 Elsevier B.V. All rights reserved.

The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during the first developmental cell lineage segregation.

PubMed

Huang, Daosheng; Guo, Guoji; Yuan, Ping; Ralston, Amy; Sun, Lingang; Huss, Mikael; Mistri, Tapan; Pinello, Luca; Ng, Huck Hui; Yuan, Guocheng; Ji, Junfeng; Rossant, Janet; Robson, Paul; Han, Xiaoping

2017-12-07

The first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and trophectoderm (TE). The transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4). However its downstream network in the developing embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify CDX2-regulated targets in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells. In addition, we examined the dynamics of gene expression changes using inducible CDX2 embryonic stem (ES) cells, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we identify putative novel regulatory elements that repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.

Nuclear factor I-A represses expression of the cell adhesion molecule L1

PubMed Central

2009-01-01

Background The neural cell adhesion molecule L1 plays a crucial role in development and plasticity of the nervous system. Neural cells thus require precise control of L1 expression. Results We identified a full binding site for nuclear factor I (NFI) transcription factors in the regulatory region of the mouse L1 gene. Electrophoretic mobility shift assay (EMSA) showed binding of nuclear factor I-A (NFI-A) to this site. Moreover, for a brain-specific isoform of NFI-A (NFI-A bs), we confirmed the interaction in vivo using chromatin immunoprecipitation (ChIP). Reporter gene assays showed that in neuroblastoma cells, overexpression of NFI-A bs repressed L1 expression threefold. Conclusion Our findings suggest that NFI-A, in particular its brain-specific isoform, represses L1 gene expression, and might act as a second silencer of L1 in addition to the neural restrictive silencer factor (NRSF). PMID:20003413

Mechanisms of epigenetic and cell-type specific regulation of Hey target genes in ES cells and cardiomyocytes.

PubMed

Weber, David; Heisig, Julia; Kneitz, Susanne; Wolf, Elmar; Eilers, Martin; Gessler, Manfred

2015-02-01

Hey bHLH transcription factors are critical effectors of Notch signaling. During mammalian heart development they are expressed in atrial and ventricular cardiomyocytes and in the developing endocardium. Hey knockout mice suffer from lethal cardiac defects, such as ventricular septum defects, valve defects and cardiomyopathy. Despite this functional relevance, little is known about the regulation of downstream targets in relevant cell types. The objective of this study was to elucidate the regulatory mechanisms by which Hey proteins affect gene expression in a cell type specific manner. We used an in vitro cardiomyocyte differentiation system with inducible Hey1 or Hey2 expression to study target gene regulation in cardiomyocytes (CM) generated from murine embryonic stem cells (ESC). The effects of Hey1 and Hey2 are largely redundant, but cell type specific. The number of regulated genes is comparable between ESC and CM, but the total number of binding sites is much higher, especially in ESC, targeting mainly genes involved in transcriptional regulation and developmental processes. Repression by Hey proteins generally correlates with the extent of Hey-binding to target promoters, Hdac recruitment and lower histone acetylation. Functionally, treatment with the Hdac inhibitor TSA abolished Hey target gene regulation. However, in CM the repressive effect of Hey-binding is lost for a subset of genes. These also lack Hey-dependent histone deacetylation in CM and are enriched for binding sites of cardiac specific activators like Srf, Nkx2-5, and Gata4. Ectopic Nkx2-5 overexpression in ESC blocks Hey-mediated repression of these genes. Thus, Hey proteins mechanistically repress target genes via Hdac recruitment and histone deacetylation. In CM Hey-repression is counteracted by cardiac activators, which recruit histone acetylases and prevent Hey mediated deacetylation and subsequent repression for a subset of genes. Copyright © 2014 Elsevier Ltd. All rights reserved.

RNAi mediates post-transcriptional repression of gene expression in fission yeast Schizosaccharomyces pombe

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smialowska, Agata, E-mail: smialowskaa@gmail.com; School of Life Sciences, Södertörn Högskola, Huddinge 141-89; Djupedal, Ingela

Highlights: • Protein coding genes accumulate anti-sense sRNAs in fission yeast S. pombe. • RNAi represses protein-coding genes in S. pombe. • RNAi-mediated gene repression is post-transcriptional. - Abstract: RNA interference (RNAi) is a gene silencing mechanism conserved from fungi to mammals. Small interfering RNAs are products and mediators of the RNAi pathway and act as specificity factors in recruiting effector complexes. The Schizosaccharomyces pombe genome encodes one of each of the core RNAi proteins, Dicer, Argonaute and RNA-dependent RNA polymerase (dcr1, ago1, rdp1). Even though the function of RNAi in heterochromatin assembly in S. pombe is established, its rolemore » in controlling gene expression is elusive. Here, we report the identification of small RNAs mapped anti-sense to protein coding genes in fission yeast. We demonstrate that these genes are up-regulated at the protein level in RNAi mutants, while their mRNA levels are not significantly changed. We show that the repression by RNAi is not a result of heterochromatin formation. Thus, we conclude that RNAi is involved in post-transcriptional gene silencing in S. pombe.« less

Genomic Analysis Reveals Contrasting PIFq Contribution to Diurnal Rhythmic Gene Expression in PIF-Induced and -Repressed Genes.

PubMed

Martin, Guiomar; Soy, Judit; Monte, Elena

2016-01-01

Members of the PIF quartet (PIFq; PIF1, PIF3, PIF4, and PIF5) collectively contribute to induce growth in Arabidopsis seedlings under short day (SD) conditions, specifically promoting elongation at dawn. Their action involves the direct regulation of growth-related and hormone-associated genes. However, a comprehensive definition of the PIFq-regulated transcriptome under SD is still lacking. We have recently shown that SD and free-running (LL) conditions correspond to "growth" and "no growth" conditions, respectively, correlating with greater abundance of PIF protein in SD. Here, we present a genomic analysis whereby we first define SD-regulated genes at dawn compared to LL in the wild type, followed by identification of those SD-regulated genes whose expression depends on the presence of PIFq. By using this sequential strategy, we have identified 349 PIF/SD-regulated genes, approximately 55% induced and 42% repressed by both SD and PIFq. Comparison with available databases indicates that PIF/SD-induced and PIF/SD-repressed sets are differently phased at dawn and mid-morning, respectively. In addition, we found that whereas rhythmicity of the PIF/SD-induced gene set is lost in LL, most PIF/SD-repressed genes keep their rhythmicity in LL, suggesting differential regulation of both gene sets by the circadian clock. Moreover, we also uncovered distinct overrepresented functions in the induced and repressed gene sets, in accord with previous studies in other examined PIF-regulated processes. Interestingly, promoter analyses showed that, whereas PIF/SD-induced genes are enriched in direct PIF targets, PIF/SD-repressed genes are mostly indirectly regulated by the PIFs and might be more enriched in ABA-regulated genes.

Primetime for Learning Genes.

PubMed

Keifer, Joyce

2017-02-11

Learning genes in mature neurons are uniquely suited to respond rapidly to specific environmental stimuli. Expression of individual learning genes, therefore, requires regulatory mechanisms that have the flexibility to respond with transcriptional activation or repression to select appropriate physiological and behavioral responses. Among the mechanisms that equip genes to respond adaptively are bivalent domains. These are specific histone modifications localized to gene promoters that are characteristic of both gene activation and repression, and have been studied primarily for developmental genes in embryonic stem cells. In this review, studies of the epigenetic regulation of learning genes in neurons, particularly the brain-derived neurotrophic factor gene ( BDNF ), by methylation/demethylation and chromatin modifications in the context of learning and memory will be highlighted. Because of the unique function of learning genes in the mature brain, it is proposed that bivalent domains are a characteristic feature of the chromatin landscape surrounding their promoters. This allows them to be "poised" for rapid response to activate or repress gene expression depending on environmental stimuli.

SnoN co-repressor binds and represses smad7 gene promoter.

PubMed

Briones-Orta, Marco A; Sosa-Garrocho, Marcela; Moreno-Alvarez, Paola; Fonseca-Sánchez, Miguel A; Macías-Silva, Marina

2006-03-17

SnoN and Ski oncoproteins are co-repressors for Smad proteins and repress TGF-beta-responsive gene expression. The smad7 gene is a TGF-beta target induced by Smad signaling, and its promoter contains the Smad-binding element (SBE) required for a positive regulation by the TGF-beta/Smad pathway. SnoN and Ski co-repressors also bind SBE but regulate negatively smad7 gene. Ski along with Smad4 binds and represses the smad7 promoter, whereas the repression mechanism by SnoN is not clear. Ski and SnoN overexpression inhibits smad7 reporter expression induced through TGF-beta signaling. Using chromatin immunoprecipitation assays, we found that SnoN binds smad7 promoter at the basal condition, whereas after a short TGF-beta treatment for 15-30 min SnoN is downregulated and no longer bound smad7 promoter. Interestingly, after a prolonged TGF-beta treatment SnoN is upregulated and returns to its position on the smad7 promoter, functioning probably as a negative feedback control. Thus, SnoN also seems to regulate negatively the TGF-beta-responsive smad7 gene by binding and repressing its promoter in a similar way to Ski.

Multiple Genes Repress Motility in Uropathogenic Escherichia coli Constitutively Expressing Type 1 Fimbriae▿ †

PubMed Central

Simms, Amy N.; Mobley, Harry L. T.

2008-01-01

Two surface organelles of uropathogenic Escherichia coli (UPEC), flagella and type 1 fimbriae, are critical for colonization of the urinary tract but mediate opposite actions. Flagella propel bacteria through urine and along mucus layers, while type 1 fimbriae allow bacteria to adhere to specific receptors present on uroepithelial cells. Constitutive expression of type 1 fimbriae leads to repression of motility and chemotaxis in UPEC strain CFT073, suggesting that UPEC may coordinately regulate motility and adherence. To identify genes involved in this regulation of motility by type 1 fimbriae, transposon mutagenesis was performed on a phase-locked type 1 fimbrial ON variant of strain CFT073 (CFT073 fim L-ON), followed by a screen for restoration of motility in soft agar. Functions of the genes identified included attachment, metabolism, transport, DNA mismatch repair, and transcriptional regulation, and a number of genes had hypothetical function. Isogenic deletion mutants of these genes were also constructed in CFT073 fim L-ON. Motility was partially restored in six of these mutants, including complementable mutations in four genes encoding known transcriptional regulators, lrhA, lrp, slyA, and papX; a mismatch repair gene, mutS; and one hypothetical gene, ydiV. Type 1 fimbrial expression in these mutants was unaltered, and the majority of these mutants expressed larger amounts of flagellin than the fim L-ON parental strain. Our results indicate that repression of motility in CFT073 fim L-ON is not solely due to the constitutive expression of type 1 fimbriae on the surfaces of the bacteria and that multiple genes may contribute to this repression. PMID:18359812

Orphan nuclear receptor TLX recruits histone deacetylases to repress transcription and regulate neural stem cell proliferation.

PubMed

Sun, Guoqiang; Yu, Ruth T; Evans, Ronald M; Shi, Yanhong

2007-09-25

TLX is a transcription factor that is essential for neural stem cell proliferation and self-renewal. However, the molecular mechanism of TLX-mediated neural stem cell proliferation and self-renewal is largely unknown. We show here that TLX recruits histone deacetylases (HDACs) to its downstream target genes to repress their transcription, which in turn regulates neural stem cell proliferation. TLX interacts with HDAC3 and HDAC5 in neural stem cells. The HDAC5-interaction domain was mapped to TLX residues 359-385, which contains a conserved nuclear receptor-coregulator interaction motif IXXLL. Both HDAC3 and HDAC5 have been shown to be recruited to the promoters of TLX target genes along with TLX in neural stem cells. Recruitment of HDACs led to transcriptional repression of TLX target genes, the cyclin-dependent kinase inhibitor, p21(CIP1/WAF1)(p21), and the tumor suppressor gene, pten. Either inhibition of HDAC activity or knockdown of HDAC expression led to marked induction of p21 and pten gene expression and dramatically reduced neural stem cell proliferation, suggesting that the TLX-interacting HDACs play an important role in neural stem cell proliferation. Moreover, expression of a TLX peptide containing the minimal HDAC5 interaction domain disrupted the TLX-HDAC5 interaction. Disruption of this interaction led to significant induction of p21 and pten gene expression and to dramatic inhibition of neural stem cell proliferation. Taken together, these findings demonstrate a mechanism for neural stem cell proliferation through transcriptional repression of p21 and pten gene expression by TLX-HDAC interactions.

Disruption of DNA methylation-dependent long gene repression in Rett syndrome

PubMed Central

Gabel, Harrison W.; Kinde, Benyam Z.; Stroud, Hume; Gilbert, Caitlin S.; Harmin, David A.; Kastan, Nathaniel R.; Hemberg, Martin; Ebert, Daniel H.; Greenberg, Michael E.

2015-01-01

Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism1. MECP2 encodes a methyl-DNA-binding protein2 that has been proposed to function as a transcriptional repressor, but despite numerous studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 regulates transcription3–9. Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains. We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits. In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain. These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain. PMID:25762136

Epigenetic Repression of Matrix Metalloproteinases in Myofibroblastic Hepatic Stellate Cells through Histone Deacetylases 4

PubMed Central

Qin, Lan; Han, Yuan-Ping

2010-01-01

Matrix metalloproteinases (MMPs), which are highly expressed in acute injury, are progressively repressed or silenced in fibrotic liver, favoring extracellular matrix accumulation, while the underlying mechanism is largely unknown. Similarly, normal/quiescent hepatic stellate cells (HSCs) express high levels of MMPs in response to injury signals, such as interleukin-1. After transdifferentiation, the myofibroblastic HSCs are incapable of expressing many MMPs; however, the major signaling pathways required for MMP expression are intact, indicating that repression is at the level of the chromatin. Indeed, both the MMP9 and MMP13 genes are inaccessible to transcription factors and RNA polymerase II, in association with impaired histone acetylation in their promoters. In accordance with impaired histone acetylation at the cellular level, histone deacetylase-4 is accumulated during HSC transdifferentiation. Furthermore, ectopic expression of histone deacetylase-4 in quiescent HSCs results in repression of MMP promoter activities as well as endogenous MMP9 protein expression. Thus, our findings suggest that a histone deacetylase-4-dependent mechanism underlies the epigenetic silencing of MMP genes during tissue fibrogenesis. PMID:20847282

The Sin3p PAH Domains Provide Separate Functions Repressing Meiotic Gene Transcription in Saccharomyces cerevisiae ▿

PubMed Central

Mallory, Michael J.; Law, Michael J.; Buckingham, Lela E.; Strich, Randy

2010-01-01

Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains. PMID:20971827

Proto-oncogene FBI-1 (Pokemon/ZBTB7A) Represses Transcription of the Tumor Suppressor Rb Gene via Binding Competition with Sp1 and Recruitment of Co-repressors*S⃞

PubMed Central

Jeon, Bu-Nam; Yoo, Jung-Yoon; Choi, Won-Il; Lee, Choong-Eun; Yoon, Ho-Geun; Hur, Man-Wook

2008-01-01

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp –308 to –188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp –65 to –56) and GC-box 2 (bp –18 to –9), the latter of which is also bound by FBI-1. We found that FRE3 (bp –244 to –236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression. PMID:18801742

Analysis of the SWI/SNF chromatin-remodeling complex during early heart development and BAF250a repression cardiac gene transcription during P19 cell differentiation

PubMed Central

Singh, Ajeet Pratap; Archer, Trevor K.

2014-01-01

The regulatory networks of differentiation programs and the molecular mechanisms of lineage-specific gene regulation in mammalian embryos remain only partially defined. We document differential expression and temporal switching of BRG1-associated factor (BAF) subunits, core pluripotency factors and cardiac-specific genes during post-implantation development and subsequent early organogenesis. Using affinity purification of BRG1 ATPase coupled to mass spectrometry, we characterized the cardiac-enriched remodeling complexes present in E8.5 mouse embryos. The relative abundance and combinatorial assembly of the BAF subunits provides functional specificity to Switch/Sucrose NonFermentable (SWI/SNF) complexes resulting in a unique gene expression profile in the developing heart. Remarkably, the specific depletion of the BAF250a subunit demonstrated differential effects on cardiac-specific gene expression and resulted in arrhythmic contracting cardiomyocytes in vitro. Indeed, the BAF250a physically interacts and functionally cooperates with Nucleosome Remodeling and Histone Deacetylase (NURD) complex subunits to repressively regulate chromatin structure of the cardiac genes by switching open and poised chromatin marks associated with active and repressed gene expression. Finally, BAF250a expression modulates BRG1 occupancy at the loci of cardiac genes regulatory regions in P19 cell differentiation. These findings reveal specialized and novel cardiac-enriched SWI/SNF chromatin-remodeling complexes, which are required for heart formation and critical for cardiac gene expression regulation at the early stages of heart development. PMID:24335282

Repression of anti-proliferative factor Tob1 in osteoarthritic cartilage

PubMed Central

Gebauer, Mathias; Saas, Joachim; Haag, Jochen; Dietz, Uwe; Takigawa, Masaharu; Bartnik, Eckart; Aigner, Thomas

2005-01-01

Osteoarthritis is the most common degenerative disorder of the modern world. However, many basic cellular features and molecular processes of the disease are poorly understood. In the present study we used oligonucleotide-based microarray analysis of genes of known or assumed relevance to the cellular phenotype to screen for relevant differences in gene expression between normal and osteoarthritic chondrocytes. Custom made oligonucleotide DNA arrays were used to screen for differentially expressed genes in normal (n = 9) and osteoarthritic (n = 10) cartilage samples. Real-time polymerase chain reaction (PCR) with gene-specific primers was used for quantification. Primary human adult articular chondrocytes and chondrosarcoma cell line HCS-2/8 were used to study changes in gene expression levels after stimulation with interleukin-1β and bone morphogenetic protein, as well as the dependence on cell differentiation. In situ hybridization with a gene-specific probe was applied to detect mRNA expression levels in fetal growth plate cartilage. Overall, more than 200 significantly regulated genes were detected between normal and osteoarthritic cartilage (P < 0.01). One of the significantly repressed genes, Tob1, encodes a protein belonging to a family involved in silencing cells in terms of proliferation and functional activity. The repression of Tob1 was confirmed by quantitative PCR and correlated to markers of chondrocyte activity and proliferation in vivo. Tob1 expression was also detected at a decreased level in isolated chondrocytes and in the chondrosarcoma cell line HCS-2/8. Again, in these cells it was negatively correlated with proliferative activity and positively with cellular differentiation. Altogether, the downregulation of the expression of Tob1 in osteoarthritic chondrocytes might be an important aspect of the cellular processes taking place during osteoarthritic cartilage degeneration. Activation, the reinitiation of proliferative activity and the loss of a stable phenotype are three major changes in osteoarthritic chondrocytes that are highly significantly correlated with the repression of Tob1 expression. PMID:15743474

Two cis elements collaborate to spatially repress transcription from a sea urchin promoter

NASA Technical Reports Server (NTRS)

Frudakis, T. N.; Wilt, F.

1995-01-01

The expression pattern of many territory-specific genes in metazoan embryos is maintained by an active process of negative spatial regulation. However, the mechanism of this strategy of gene regulation is not well understood in any system. Here we show that reporter constructs containing regulatory sequence for the SM30-alpha gene of Stronglyocentrotus purpuratus are expressed in a pattern congruent with that of the endogenous SM30 gene(s), largely as a result of active transcriptional repression in cell lineages in which the gene is not normally expressed. Chloramphenicol acetyl transferase assays of deletion constructs from the 2600-bp upstream region showed that repressive elements were present in the region from -1628 to -300. In situ hybridization analysis showed that the spatial fidelity of expression was severely compromised when the region from -1628 to -300 was deleted. Two highly repetitive sequence motifs, (G/A/C)CCCCT and (T/C)(T/A/C)CTTTT(T/A/C), are present in the -1628 to -300 region. Representatives of these elements were analyzed by gel mobility shift experiments and were found to interact specifically with protein in crude nuclear extracts. When oligonucleotides containing either sequence element were co-injected with a correctly regulated reporter as potential competitors, the reporter was expressed in inappropriate cells. When composite oligonucleotides, containing both sequence elements, were fused to a misregulated reporter, the expression of the reporter in inappropriate cells was suppressed. Comparison of composite oligonucleotides with oligonucleotides containing single constituent elements show that both sequence elements are required for effective spatial regulation. Thus, both individual elements are required, but only a composite element containing both elements is sufficient to function as a tissue-specific repressive element.

IBM1, a JmjC domain-containing histone demethylase, is involved in the regulation of RNA-directed DNA methylation through the epigenetic control of RDR2 and DCL3 expression in Arabidopsis

PubMed Central

Fan, Di; Dai, Yan; Wang, Xuncheng; Wang, Zhenjie; He, Hang; Yang, Hongchun; Cao, Ying; Deng, Xing Wang; Ma, Ligeng

2012-01-01

Small RNA-directed DNA methylation (RdDM) is an important epigenetic pathway in Arabidopsis that controls the expression of multiple genes and several developmental processes. RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3) are necessary factors in 24-nt small interfering RNA (siRNA) biogenesis, which is part of the RdDM pathway. Here, we found that Increase in BONSAI Methylation 1 (IBM1), a conserved JmjC family histone demethylase, is directly associated with RDR2 and DCL3 chromatin. The mutation of IBM1 induced the hypermethylation of H3K9 and DNA non-CG sites within RDR2 and DCL3, which repressed their expression. A genome-wide analysis suggested that the reduction in RDR2 and DCL3 expression affected siRNA biogenesis in a locus-specific manner and disrupted RdDM-directed gene repression. Together, our results suggest that IBM1 regulates gene expression through two distinct pathways: direct association to protect genes from silencing by preventing the coupling of histone and DNA methylation, and indirect silencing of gene expression through RdDM-directed repression. PMID:22772985

Genome-wide expression and methylation profiling in the aged rodent brain due to early-life Pb exposure and its relevance to aging.

PubMed

Dosunmu, Remi; Alashwal, Hany; Zawia, Nasser H

2012-06-01

In this study, we assessed global gene expression patterns in adolescent mice exposed to lead (Pb) as infants and their aged siblings to identify reprogrammed genes. Global expression on postnatal day 20 and 700 was analyzed and genes that were down- and up-regulated (≥2 fold) were identified, clustered and analyzed for their relationship to DNA methylation. About 150 genes were differentially expressed in old age. In normal aging, we observed an up-regulation of genes related to the immune response, metal-binding, metabolism and transcription/transduction coupling. Prior exposure to Pb revealed a repression in these genes suggesting that disturbances in developmental stages of the brain compromise the ability to defend against age-related stressors, thus promoting the neurodegenerative process. Overexpression and repression of genes corresponded with their DNA methylation profile. Published by Elsevier Ireland Ltd.

Combined SOM-portrayal of gene expression and DNA methylation landscapes disentangles modes of epigenetic regulation in glioblastoma.

PubMed

Hopp, Lydia; Löffler-Wirth, Henry; Galle, Jörg; Binder, Hans

2018-06-11

We present here a novel method that enables unraveling the interplay between gene expression and DNA methylation in complex diseases such as cancer. The method is based on self-organizing maps and allows for analysis of data landscapes from 'governed by methylation' to 'governed by expression'. We identified regulatory modules of coexpressed and comethylated genes in high-grade gliomas: two modes are governed by genes hypermethylated and underexpressed in IDH-mutated cases, while two other modes reflect immune and stromal signatures in the classical and mesenchymal subtypes. A fifth mode with proneural characteristics comprises genes of repressed and poised chromatin states active in healthy brain. Two additional modes enrich genes either in active or repressed chromatin states. The method disentangles the interplay between gene expression and methylation. It has the potential to integrate also mutation and copy number data and to apply to large sample cohorts.

Thyroid Hormone Receptor β Suppression of RUNX2 is Mediated by Brahma Related Gene 1 Dependent Chromatin Remodeling.

PubMed

Gillis, Noelle E; Taber, Thomas H; Bolf, Eric L; Beaudet, Caitlin M; Tomczak, Jennifer A; White, Jeffrey H; Stein, Janet L; Stein, Gary S; Lian, Jane B; Frietze, Seth; Carr, Frances E

2018-05-09

Thyroid hormone receptor beta (TRβ) suppresses tumor growth through regulation of gene expression, yet the associated TRβ-mediated changes in chromatin assembly are not known. The chromatin ATPase Brahma Related Gene 1 (BRG1, SMARCA4), a key component of chromatin remodeling complexes, is altered in many cancers, but its role in thyroid tumorigenesis and TRβ-mediated gene expression is unknown. We previously identified the oncogene runt-related transcription factor 2 (RUNX2) as a repressive target of TRβ. Here we report differential expression of BRG1 in non-malignant and malignant thyroid cells concordant with TRβ. BRG1 and TRβ have similar nuclear distribution patterns and significant co-localization. BRG1 interacts with TRβ and together are part of the regulatory complex at the RUNX2 promoter. Loss of BRG1 increases RUNX2 levels whereas re-introduction of TRβ and BRG1 synergistically decrease RUNX2 expression. RUNX2 promoter accessibility corresponded to RUNX2 expression levels. Inhibition of BRG1 activity ncreased accessibility of the RUNX2 promoter and corresponding expression. Our results reveal a novel mechanism of TRβ repression of oncogenic gene expression: TRβ recruitment of BRG1 to induce chromatin compaction and diminished RUNX2 expression. Therefore, BRG1-mediated chromatin remodeling may be obligatory for TRβ transcriptional repression and tumor suppressor function in thyroid tumorigenesis.

The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum

PubMed Central

Weber, Barbara; Lindell, Kristoffer; El Qaidi, Samir; Hjerde, Erik; Willassen, Nils-Peder

2011-01-01

Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ54 and the σ54-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment. PMID:21948044

The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum.

PubMed

Weber, Barbara; Lindell, Kristoffer; El Qaidi, Samir; Hjerde, Erik; Willassen, Nils-Peder; Milton, Debra L

2011-12-01

Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ(54) and the σ(54)-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilized vanT mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.

Orphan nuclear receptor TLX recruits histone deacetylases to repress transcription and regulate neural stem cell proliferation

PubMed Central

Sun, GuoQiang; Yu, Ruth T.; Evans, Ronald M.; Shi, Yanhong

2007-01-01

TLX is a transcription factor that is essential for neural stem cell proliferation and self-renewal. However, the molecular mechanism of TLX-mediated neural stem cell proliferation and self-renewal is largely unknown. We show here that TLX recruits histone deacetylases (HDACs) to its downstream target genes to repress their transcription, which in turn regulates neural stem cell proliferation. TLX interacts with HDAC3 and HDAC5 in neural stem cells. The HDAC5-interaction domain was mapped to TLX residues 359–385, which contains a conserved nuclear receptor–coregulator interaction motif IXXLL. Both HDAC3 and HDAC5 have been shown to be recruited to the promoters of TLX target genes along with TLX in neural stem cells. Recruitment of HDACs led to transcriptional repression of TLX target genes, the cyclin-dependent kinase inhibitor, p21CIP1/WAF1(p21), and the tumor suppressor gene, pten. Either inhibition of HDAC activity or knockdown of HDAC expression led to marked induction of p21 and pten gene expression and dramatically reduced neural stem cell proliferation, suggesting that the TLX-interacting HDACs play an important role in neural stem cell proliferation. Moreover, expression of a TLX peptide containing the minimal HDAC5 interaction domain disrupted the TLX–HDAC5 interaction. Disruption of this interaction led to significant induction of p21 and pten gene expression and to dramatic inhibition of neural stem cell proliferation. Taken together, these findings demonstrate a mechanism for neural stem cell proliferation through transcriptional repression of p21 and pten gene expression by TLX–HDAC interactions. PMID:17873065

Epigenetic involvement of Alien/ESET complex in thyroid hormone-mediated repression of E2F1 gene expression and cell proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Wei, E-mail: hongwei@tijmu.edu.cn; College of Basic Medicine, Tianjin Medical University, 300070 Tianjin; Li, Jinru

Highlights: Black-Right-Pointing-Pointer Corepressor Alien interacts with histone methyltransferase ESET in vivo. Black-Right-Pointing-Pointer Alien/ESET complex is recruited to nTRE of T3-responsive gene by liganded TR{beta}1. Black-Right-Pointing-Pointer ESET-mediated H3K9 methylation is required for liganded TR{beta}1-repressed transcription. Black-Right-Pointing-Pointer ESET is involved in T3-repressed G1/S phase transition and proliferation. -- Abstract: The ligand-bound thyroid hormone receptor (TR) is known to repress via a negative TRE (nTRE) the expression of E2F1, a key transcription factor that controls the G1/S phase transition. Alien has been identified as a novel interacting factor of E2F1 and acts as a corepressor of E2F1. The detailed molecular mechanism by whichmore » Alien inhibits E2F1 gene expression remains unclear. Here, we report that the histone H3 lysine 9 (H3K9) methyltransferase (HMT) ESET is an integral component of the corepressor Alien complex and the Alien/ESET complex is recruited to both sites, the E2F1 and the nTRE site of the E2F1 gene while the recruitment to the negative thyroid hormone response element (nTRE) is induced by the ligand-bound TR{beta}1 within the E2F1 gene promoter. We show that, overexpression of ESET promotes, whereas knockdown of ESET releases, the inhibition of TR{beta}1-regulated gene transcription upon T3 stimulation; and H3K9 methylation is required for TR{beta}1-repressed transcription. Furthermore, depletion of ESET impairs thyroid hormone-repressed proliferation as well as the G1/S transition of the cell cycle. Taken together, our data indicate that ESET is involved in TR{beta}1-mediated transcription repression and provide a molecular basis of thyroid hormone-induced repression of proliferation.« less

EBNA3C Directs Recruitment of RBPJ (CBF1) to Chromatin during the Process of Gene Repression in EBV Infected B Cells.

PubMed

Kalchschmidt, Jens S; Gillman, Adam C T; Paschos, Kostas; Bazot, Quentin; Kempkes, Bettina; Allday, Martin J

2016-01-01

It is well established that Epstein-Barr virus nuclear antigen 3C (EBNA3C) can act as a potent repressor of gene expression, but little is known about the sequence of events occurring during the repression process. To explore further the role of EBNA3C in gene repression-particularly in relation to histone modifications and cell factors involved-the three host genes previously reported as most robustly repressed by EBNA3C were investigated. COBLL1, a gene of unknown function, is regulated by EBNA3C alone and the two co-regulated disintegrin/metalloproteases, ADAM28 and ADAMDEC1 have been described previously as targets of both EBNA3A and EBNA3C. For the first time, EBNA3C was here shown to be the main regulator of all three genes early after infection of primary B cells. Using various EBV-recombinants, repression over orders of magnitude was seen only when EBNA3C was expressed. Unexpectedly, full repression was not achieved until 30 days after infection. This was accurately reproduced in established LCLs carrying EBV-recombinants conditional for EBNA3C function, demonstrating the utility of the conditional system to replicate events early after infection. Using this system, detailed chromatin immunoprecipitation analysis revealed that the initial repression was associated with loss of activation-associated histone modifications (H3K9ac, H3K27ac and H3K4me3) and was independent of recruitment of polycomb proteins and deposition of the repressive H3K27me3 modification, which were only observed later in repression. Most remarkable, and in contrast to current models of RBPJ in repression, was the observation that this DNA-binding factor accumulated at the EBNA3C-binding sites only when EBNA3C was functional. Transient reporter assays indicated that repression of these genes was dependent on the interaction between EBNA3C and RBPJ. This was confirmed with a novel EBV-recombinant encoding a mutant of EBNA3C unable to bind RBPJ, by showing this virus was incapable of repressing COBLL1 or ADAM28/ADAMDEC1 in newly infected primary B cells.

Regulation of metabolism in Escherichia coli during growth on mixtures of the non-glucose sugars: arabinose, lactose, and xylose.

PubMed

Ammar, Ehab M; Wang, Xiaoyi; Rao, Christopher V

2018-01-12

Catabolite repression refers to the process where the metabolism of one sugar represses the genes involved in metabolizing another sugar. While glucose provides the canonical example, many other sugars are also known to induce catabolite repression. However, less is known about the mechanism for catabolite repression by these non-glucose sugars. In this work, we investigated the mechanism of catabolite repression in the bacterium Escherichia coli during growth on lactose, L-arabinose, and D-xylose. The metabolism of these sugars is regulated in a hierarchical manner, where lactose is the preferred sugar, followed by L-arabinose, and then D-xylose. Previously, the preferential utilization of L-arabinose over D-xylose was found to result from transcriptional crosstalk. However, others have proposed that cAMP governs the hierarchical regulation of many non-glucose sugars. We investigated whether lactose-induced repression of L-arabinose and D-xylose gene expression is due to transcriptional crosstalk or cAMP. Our results demonstrate that it is due to cAMP and not transcriptional crosstalk. In addition, we found that repression is reciprocal, where both L-arabinose and D-xylose also repress the lactose gene expression, albeit to a lesser extent and also through a mechanism involving cAMP. Collectively, the results further our understanding of metabolism during growth on multiple sugars.

A novel dissociative steroid VBP15 reduces MUC5AC gene expression in airway epithelial cells but lacks the GRE mediated transcriptional properties of dexamethasone.

PubMed

Garvin, Lindsay M; Chen, Yajun; Damsker, Jesse M; Rose, Mary C

2016-06-01

Overproduction of secretory mucins contributes to morbidity/mortality in inflammatory lung diseases. Inflammatory mediators directly increase expression of mucin genes, but few drugs have been shown to directly repress mucin gene expression. IL-1β upregulates the MUC5AC mucin gene in part via the transcription factors NFκB while the glucocorticoid Dexamethasone (Dex) transcriptionally represses MUC5AC expression by Dex-activated GR binding to two GRE cis-sites in the MUC5AC promoter in lung epithelial cells. VBP compounds (ReveraGen BioPharma) maintain anti-inflammatory activity through inhibition of NFκB but exhibit reduced GRE-mediated transcriptional properties associated with adverse side-effects and thus have potential to minimize harmful side effects of long-term steroid therapy in inflammatory lung diseases. We investigated VBP15 efficacy as an anti-mucin agent in two types of airway epithelial cells and analyzed the transcription factor activity and promoter binding associated with VBP15-induced MUC5AC repression. VBP15 reduced MUC5AC mRNA abundance in a dose- and time-dependent manner similar to Dex in the presence or absence of IL-1β in A549 and differentiated human bronchial epithelial cells. Repression was abrogated in the presence of RU486, demonstrating a requirement for GR in the VBP15-induced repression of MUC5AC. Inhibition of NFκB activity resulted in reduced baseline expression of MUC5AC indicating that constitutive activity maintains MUC5AC production. Chromatin immunoprecipitation analysis demonstrated lack of GR and of p65 (NFκB) binding to composite GRE domains in the MUC5AC promoter following VBP15 exposure of cells, in contrast to Dex. These data demonstrate that VBP15 is a novel anti-mucin agent that mediates the reduction of MUC5AC gene expression differently than the classical glucocorticoid, Dex. Copyright © 2016 Elsevier Ltd. All rights reserved.

PHYTOCHROME INTERACTING FACTOR3 Associates with the Histone Deacetylase HDA15 in Repression of Chlorophyll Biosynthesis and Photosynthesis in Etiolated Arabidopsis Seedlings[W][OA

PubMed Central

Liu, Xuncheng; Chen, Chia-Yang; Wang, Ko-Ching; Luo, Ming; Tai, Ready; Yuan, Lianyu; Zhao, Minglei; Yang, Songguang; Tian, Gang; Cui, Yuhai; Hsieh, Hsu-Liang; Wu, Keqiang

2013-01-01

PHYTOCHROME INTERACTING FACTOR3 (PIF3) is a key basic helix-loop-helix transcription factor of Arabidopsis thaliana that negatively regulates light responses, repressing chlorophyll biosynthesis, photosynthesis, and photomorphogenesis in the dark. However, the mechanism for the PIF3-mediated transcription regulation remains largely unknown. In this study, we found that the REDUCED POTASSIUM DEPENDENCY3/HISTONE DEACETYLASE1-type histone deacetylase HDA15 directly interacted with PIF3 in vivo and in vitro. Genome-wide transcriptome analysis revealed that HDA15 acts mainly as a transcriptional repressor and negatively regulates chlorophyll biosynthesis and photosynthesis gene expression in etiolated seedlings. HDA15 and PIF3 cotarget to the genes involved in chlorophyll biosynthesis and photosynthesis in the dark and repress gene expression by decreasing the acetylation levels and RNA Polymerase II–associated transcription. The binding of HDA15 to the target genes depends on the presence of PIF3. In addition, PIF3 and HDA15 are dissociated from the target genes upon exposure to red light. Taken together, our results indicate that PIF3 associates with HDA15 to repress chlorophyll biosynthetic and photosynthetic genes in etiolated seedlings. PMID:23548744

L-rhamnose induction of Aspergillus nidulans α-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake.

PubMed

Tamayo-Ramos, Juan A; Flipphi, Michel; Pardo, Ester; Manzanares, Paloma; Orejas, Margarita

2012-02-21

Little is known about the structure and regulation of fungal α-L-rhamnosidase genes despite increasing interest in the biotechnological potential of the enzymes that they encode. Whilst the paradigmatic filamentous fungus Aspergillus nidulans growing on L-rhamnose produces an α-L-rhamnosidase suitable for oenological applications, at least eight genes encoding putative α-L-rhamnosidases have been found in its genome. In the current work we have identified the gene (rhaE) encoding the former activity, and characterization of its expression has revealed a novel regulatory mechanism. A shared pattern of expression has also been observed for a second α-L-rhamnosidase gene, (AN10277/rhaA). Amino acid sequence data for the oenological α-L-rhamnosidase were determined using MALDI-TOF mass spectrometry and correspond to the amino acid sequence deduced from AN7151 (rhaE). The cDNA of rhaE was expressed in Saccharomyces cerevisiae and yielded pNP-rhamnohydrolase activity. Phylogenetic analysis has revealed this eukaryotic α-L-rhamnosidase to be the first such enzyme found to be more closely related to bacterial rhamnosidases than other α-L-rhamnosidases of fungal origin. Northern analyses of diverse A. nidulans strains cultivated under different growth conditions indicate that rhaA and rhaE are induced by L-rhamnose and repressed by D-glucose as well as other carbon sources, some of which are considered to be non-repressive growth substrates. Interestingly, the transcriptional repression is independent of the wide domain carbon catabolite repressor CreA. Gene induction and glucose repression of these rha genes correlate with the uptake, or lack of it, of the inducing carbon source L-rhamnose, suggesting a prominent role for inducer exclusion in repression. The A. nidulans rhaE gene encodes an α-L-rhamnosidase phylogenetically distant to those described in filamentous fungi, and its expression is regulated by a novel CreA-independent mechanism. The identification of rhaE and the characterization of its regulation will facilitate the design of strategies to overproduce the encoded enzyme - or homologs from other fungi - for industrial applications. Moreover, A. nidulans α-L-rhamnosidase encoding genes could serve as prototypes for fungal genes coding for plant cell wall degrading enzymes regulated by a novel mechanism of CCR.

L-Rhamnose induction of Aspergillus nidulans α-L-rhamnosidase genes is glucose repressed via a CreA-independent mechanism acting at the level of inducer uptake

PubMed Central

2012-01-01

Background Little is known about the structure and regulation of fungal α-L-rhamnosidase genes despite increasing interest in the biotechnological potential of the enzymes that they encode. Whilst the paradigmatic filamentous fungus Aspergillus nidulans growing on L-rhamnose produces an α-L-rhamnosidase suitable for oenological applications, at least eight genes encoding putative α-L-rhamnosidases have been found in its genome. In the current work we have identified the gene (rhaE) encoding the former activity, and characterization of its expression has revealed a novel regulatory mechanism. A shared pattern of expression has also been observed for a second α-L-rhamnosidase gene, (AN10277/rhaA). Results Amino acid sequence data for the oenological α-L-rhamnosidase were determined using MALDI-TOF mass spectrometry and correspond to the amino acid sequence deduced from AN7151 (rhaE). The cDNA of rhaE was expressed in Saccharomyces cerevisiae and yielded pNP-rhamnohydrolase activity. Phylogenetic analysis has revealed this eukaryotic α-L-rhamnosidase to be the first such enzyme found to be more closely related to bacterial rhamnosidases than other α-L-rhamnosidases of fungal origin. Northern analyses of diverse A. nidulans strains cultivated under different growth conditions indicate that rhaA and rhaE are induced by L-rhamnose and repressed by D-glucose as well as other carbon sources, some of which are considered to be non-repressive growth substrates. Interestingly, the transcriptional repression is independent of the wide domain carbon catabolite repressor CreA. Gene induction and glucose repression of these rha genes correlate with the uptake, or lack of it, of the inducing carbon source L-rhamnose, suggesting a prominent role for inducer exclusion in repression. Conclusions The A. nidulans rhaE gene encodes an α-L-rhamnosidase phylogenetically distant to those described in filamentous fungi, and its expression is regulated by a novel CreA-independent mechanism. The identification of rhaE and the characterization of its regulation will facilitate the design of strategies to overproduce the encoded enzyme - or homologs from other fungi - for industrial applications. Moreover, A. nidulans α-L-rhamnosidase encoding genes could serve as prototypes for fungal genes coding for plant cell wall degrading enzymes regulated by a novel mechanism of CCR. PMID:22353731

Multiple regulatory elements for the glpA operon encoding anaerobic glycerol-3-phosphate dehydrogenase and the glpD operon encoding aerobic glycerol-3-phosphate dehydrogenase in Escherichia coli: further characterization of respiratory control.

PubMed

Iuchi, S; Cole, S T; Lin, E C

1990-01-01

In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the glpA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of glpA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpA1(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpA1(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function.

Repression of Virus-Induced Interferon A Promoters by Homeodomain Transcription Factor Ptx1

PubMed Central

Lopez, Sébastien; Island, Marie-Laure; Drouin, Jacques; Bandu, Marie-Thérese; Christeff, Nicolas; Darracq, Nicole; Barbey, Régine; Doly, Janine; Thomas, Dominique; Navarro, Sébastien

2000-01-01

Interferon A (IFN-A) genes are differentially expressed after virus induction. The differential expression of individual IFN-A genes is modulated by substitutions in the proximal positive virus responsive element A (VRE-A) of their promoters and by the presence or absence of a distal negative regulatory element (DNRE). The functional feature of the DNRE is to specifically act by repression of VRE-A activity. With the use of the yeast one-hybrid system, we describe here the identification of a specific DNRE-binding protein, the pituitary homeobox 1 (Ptx1 or Pitx1). Ptx1 is detectable in different cell types that differentially express IFN-A genes, and the endogenous Ptx1 protein binds specifically to the DNRE. Upon virus induction, Ptx1 negatively regulates the transcription of DNRE-containing IFN-A promoters, and the C-terminal region, as well as the homeodomain of the Ptx1 protein, is required for this repression. After virus induction, the expression of the Ptx1 antisense RNA leads to a significant increase of endogenous IFN-A gene transcription and is able to modify the pattern of differential expression of individual IFN-A genes. These studies suggest that Ptx1 contributes to the differential transcriptional strength of the promoters of different IFN-A genes and that these genes may provide new targets for transcriptional regulation by a homeodomain transcription factor. PMID:11003649

The N-CoR complex enables chromatin remodeler SNF2H to enhance repression by thyroid hormone receptor

PubMed Central

Alenghat, Theresa; Yu, Jiujiu; Lazar, Mitchell A

2006-01-01

Unliganded thyroid hormone receptor (TR) actively represses transcription via the nuclear receptor corepressor (N-CoR)/histone deacetylase 3 (HDAC3) complex. Although transcriptional activation by liganded receptors involves chromatin remodeling, the role of ATP-dependent remodeling in receptor-mediated repression is unknown. Here we report that SNF2H, the mammalian ISWI chromatin remodeling ATPase, is critical for repression of a genomically integrated, TR-regulated reporter gene. N-CoR and HDAC3 are both required for recruitment of SNF2H to the repressed gene. SNF2H does not interact directly with the N-CoR/HDAC3 complex, but binds to unacetylated histone H4 tails, suggesting that deacetylase activity of the corepressor complex is critical to SNF2H function. Indeed, HDAC3 as well as SNF2H are required for nucleosomal organization on the TR target gene. Consistent with these findings, reduction of SNF2H induces expression of an endogenous TR-regulated gene, dio1, in liver cells. Thus, although not apparent from studies of transiently transfected reporter genes, gene repression by TR involves the targeting of chromatin remodeling factors to repressed genes by the HDAC activity of nuclear receptor corepressors. PMID:16917504

Differential Responses of Soleus and Plantaris Muscle Fibers to Overloading

NASA Astrophysics Data System (ADS)

Kawano, Fuminori; Shibaguchi, Tsubasa; Ohira, Takashi; Nakai, Naoya; Ohira, Yoshinobu

2013-02-01

Responses of slow and fast fibers in soleus and plantaris muscles of adult rats to overloading by the tendon transection of synergists were studied. Overloading-related hypertrophy was noted in the slow fibers of plantaris and soleus, although the magnitude was greater in plantaris. Five genes with minor expression in slow soleus muscle were identified by microarray analysis. Base-line expressions of these genes in slow fibers of plantaris were also low. Further, repressive effects of overloading on these genes were seen in some fast fibers of plantaris, not in whole plantaris and soleus. The data suggested that the repression of particular genes might be related to the pronounced morphological response of fibers expressing type II, including I+II, myosin heavy chain (MyHC), although these genes with lower base-line expression in slow fibers did not respond to overloading.

Pod-1/Capsulin shows a sex- and stage-dependent expression pattern in the mouse gonad development and represses expression of Ad4BP/SF-1.

PubMed

Tamura, M; Kanno, Y; Chuma, S; Saito, T; Nakatsuji, N

2001-04-01

Mammalian sex-determination and differentiation are controlled by several genes, such as Sry, Sox-9, Dax-1 and Mullerian inhibiting substance (MIS), but their upstream and downstream genes are largely unknown. Ad4BP/SF-1, encoding a zinc finger transcription factor, plays important roles in gonadogenesis. Disruption of this gene caused disappearance of the urogenital system including the gonad. Ad4BP/SF-1, however, is also involved in the sex differentiation of the gonad at later stages, such as the regulation of steroid hormones and MIS. Pod-1/Capsulin, a member of basic helix-loop-helix transcription factors, is expressed in a pattern closely related but mostly complimentary to that of the Ad4BP/SF-1 expression in the developing gonad. In the co-transfection experiment using cultured cells, overexpression of Pod-1/Capsulin repressed expression of a reporter gene that carried the upstream regulatory region of the Ad4BP/SF-1 gene. Furthermore, forced expression of Pod-1/Capsulin repressed expression of Ad4BP/SF-1 in the Leydig cell-derived I-10 cells. These results suggest that Pod-1/Capsulin may play important roles in the development and sex differentiation of the mammalian gonad via transcriptional regulation of Ad4BP/SF-1.

The Ku Protein Complex Interacts with YY1, Is Up-Regulated in Human Heart Failure, and Represses α Myosin Heavy-Chain Gene Expression

PubMed Central

Sucharov, Carmen C.; Helmke, Steve M.; Langer, Stephen J.; Perryman, M. Benjamin; Bristow, Michael; Leinwand, Leslie

2004-01-01

Human heart failure is accompanied by repression of genes such as α myosin heavy chain (αMyHC) and SERCA2A and the induction of fetal genes such as βMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human αMyHC promoter. We have now identified a region of the αMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human αMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases αMyHC mRNA expression and increases skeletal α-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the αMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human αMyHC promoter during heart failure. PMID:15367688

Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection

PubMed Central

Ferrari, Roberto; Gou, Dawei; Jawdekar, Gauri; Johnson, Sarah A.; Nava, Miguel; Su, Trent; Yousef, Ahmed F.; Zemke, Nathan R.; Pellegrini, Matteo; Kurdistani, Siavash K.; Berk, Arnold J.

2015-01-01

SUMMARY Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGFβ-, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication. PMID:25525796

Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santhanam, U.; Ray, A.; Sehgal, P.B.

1991-09-01

The aberrant overexpression of interleukin 6 (IL-6) is implicated as an autocrine mechanism in the enhanced proliferation of the neoplastic cell elements in various B- and T-cell malignancies and in some carcinomas and sarcomas; many of these neoplasms have been shown to be associated with a mutated p53 gene. The possibility that wild-type (wt) p53, a nuclear tumor-suppressor protein, but not its transforming mutants might serve to repress IL-6 gene expression was investigated in HeLa cells. The authors transiently cotransfected these cells with constitutive cytomegalovirus (CMV) enhancer/promoter expression plasmids overproducing wt or mutant human or murine p53 and with appropriatemore » chloramphenicol acetyltransferase (CAT) reporter plasmids containing the promoter elements of human IL-6, c-fos, or {beta}-actin genes or of porcine major histocompatibility complex (MHC) class I gene in pN-38 to evaluate the effect of the various p53 species on these promoters. These observations identify transcriptional repression as a property of p53 and suggest that p53 and RB may be involved as transcriptional repressors in modulating IL-6 gene expression during cellular differentiation and oncogenesis.« less

The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes.

PubMed

Robbez-Masson, Luisa; Tie, Christopher H C; Conde, Lucia; Tunbak, Hale; Husovsky, Connor; Tchasovnikarova, Iva A; Timms, Richard T; Herrero, Javier; Lehner, Paul J; Rowe, Helen M

2018-05-04

Retrotransposons encompass half of the human genome and contribute to the formation of heterochromatin, which provides nuclear structure and regulates gene expression. Here, we asked if the human silencing hub (HUSH) complex is necessary to silence retrotransposons and whether it collaborates with TRIM28 and the chromatin remodeler ATRX at specific genomic loci. We show that the HUSH complex contributes to de novo repression and DNA methylation of a SVA retrotransposon reporter. By using naïve vs. primed mouse pluripotent stem cells, we reveal a critical role for the HUSH complex in naïve cells, implicating it in programming epigenetic marks in development. While the HUSH component FAM208A binds to endogenous retroviruses (ERVs) and long interspersed element-1s (LINE-1s or L1s), it is mainly required to repress evolutionarily young L1s (mouse-specific lineages less than 5 million years old). TRIM28, in contrast, is necessary to repress both ERVs and young L1s. Genes co-repressed by TRIM28 and FAM208A are evolutionarily young, or exhibit tissue-specific expression, are enriched in young L1s and display evidence for regulation through LTR promoters. Finally, we demonstrate that the HUSH complex is also required to repress L1 elements in human cells. Overall, these data indicate that the HUSH complex and TRIM28 co-repress young retrotransposons and new genes rewired by retrotransposon non-coding DNA. Published by Cold Spring Harbor Laboratory Press.

COACTIVATOR ACTIVATOR (CoAA) PREVENTS THE TRANSCRIPTIONAL ACTIVITY OF RUNT DOMAIN TRANSCRIPTION FACTORS

PubMed Central

Li, Xiaodong; Hoeppner, Luke H.; Jensen, Eric D.; Gopalakrishnan, Rajaram; Westendorf, Jennifer J.

2013-01-01

Runx proteins are essential for a number of developmental processes and are aberrantly expressed in many human cancers. Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation, hematopoiesis, and neuronal development. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. In this study, we identified CoAA as a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells. PMID:19585539

Transcriptional corepressor SMILE recruits SIRT1 to inhibit nuclear receptor estrogen receptor-related receptor gamma transactivation.

PubMed

Xie, Yuan-Bin; Park, Jeong-Hoh; Kim, Don-Kyu; Hwang, Jung Hwan; Oh, Sangmi; Park, Seung Bum; Shong, Minho; Lee, In-Kyu; Choi, Hueng-Sik

2009-10-16

SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a corepressor of the glucocorticoid receptor, constitutive androstane receptor, and hepatocyte nuclear factor 4alpha. Here we show that SMILE also represses estrogen receptor-related receptor gamma (ERRgamma) transactivation. Knockdown of SMILE gene expression increases ERRgamma activity. SMILE directly interacts with ERRgamma in vitro and in vivo. Domain mapping analysis showed that SMILE binds to the AF2 domain of ERRgamma. SMILE represses ERRgamma transactivation partially through competition with coactivators PGC-1alpha, PGC-1beta, and GRIP1. Interestingly, the repression of SMILE on ERRgamma is released by SIRT1 inhibitors, a catalytically inactive SIRT1 mutant, and SIRT1 small interfering RNA but not by histone protein deacetylase inhibitor. In vivo glutathione S-transferase pulldown and coimmunoprecipitation assays validated that SMILE physically interacts with SIRT1. Furthermore, the ERRgamma inverse agonist GSK5182 enhances the interaction of SMILE with ERRgamma and SMILE-mediated repression. Knockdown of SMILE or SIRT1 blocks the repressive effect of GSK5182. Moreover, chromatin immunoprecipitation assays revealed that GSK5182 augments the association of SMILE and SIRT1 on the promoter of the ERRgamma target PDK4. GSK5182 and adenoviral overexpression of SMILE cooperate to repress ERRgamma-induced PDK4 gene expression, and this repression is released by overexpression of a catalytically defective SIRT1 mutant. Finally, we demonstrated that ERRgamma regulates SMILE gene expression, which in turn inhibits ERRgamma. Overall, these findings implicate SMILE as a novel corepressor of ERRgamma and recruitment of SIRT1 as a novel repressive mechanism for SMILE and ERRgamma inverse agonist.

Genome-nuclear lamina interactions and gene regulation.

PubMed

Kind, Jop; van Steensel, Bas

2010-06-01

The nuclear lamina, a filamentous protein network that coats the inner nuclear membrane, has long been thought to interact with specific genomic loci and regulate their expression. Molecular mapping studies have now identified large genomic domains that are in contact with the lamina. Genes in these domains are typically repressed, and artificial tethering experiments indicate that the lamina can actively contribute to this repression. Furthermore, the lamina indirectly controls gene expression in the nuclear interior by sequestration of certain transcription factors. A variety of DNA-binding and chromatin proteins may anchor specific loci to the lamina, while histone-modifying enzymes partly mediate the local repressive effect of the lamina. Experimental tools are now available to begin to unravel the underlying molecular mechanisms. Copyright 2010 Elsevier Ltd. All rights reserved.

Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum.

PubMed

Xu, Tao; Li, Yongchao; He, Zhili; Van Nostrand, Joy D; Zhou, Jizhong

2017-01-01

Essential gene functions remain largely underexplored in bacteria. Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing; however, its genetic manipulation to reduce the formation of less-valuable acetate is technically challenging due to the essentiality of acetate-producing genes. Here we developed a Cas9 nickase-assisted chromosome-based RNA repression to stably manipulate essential genes in C. cellulolyticum . Our plasmid-based expression of antisense RNA (asRNA) molecules targeting the phosphotransacetylase ( pta ) gene successfully reduced the enzymatic activity by 35% in cellobiose-grown cells, metabolically decreased the acetate titer by 15 and 52% in wildtype transformants on cellulose and xylan, respectively. To control both acetate and lactate simultaneously, we transformed the repression plasmid into lactate production-deficient mutant and found the plasmid delivery reduced acetate titer by more than 33%, concomitant with negligible lactate formation. The strains with pta gene repression generally diverted more carbon into ethanol. However, further testing on chromosomal integrants that were created by double-crossover recombination exhibited only very weak repression because DNA integration dramatically lessened gene dosage. With the design of a tandem repetitive promoter-driven asRNA module and the use of a new Cas9 nickase genome editing tool, a chromosomal integrant (LM3P) was generated in a single step and successfully enhanced RNA repression, with a 27% decrease in acetate titer on cellulose in antibiotic-free medium. These results indicate the effectiveness of tandem promoter-driven RNA repression modules in promoting gene repression in chromosomal integrants. Our combinatorial method using a Cas9 nickase genome editing tool to integrate the gene repression module demonstrates easy-to-use and high-efficiency advantages, paving the way for stably manipulating genes, even essential ones, for functional characterization and microbial engineering.

Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum

PubMed Central

Xu, Tao; Li, Yongchao; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

2017-01-01

Essential gene functions remain largely underexplored in bacteria. Clostridium cellulolyticum is a promising candidate for consolidated bioprocessing; however, its genetic manipulation to reduce the formation of less-valuable acetate is technically challenging due to the essentiality of acetate-producing genes. Here we developed a Cas9 nickase-assisted chromosome-based RNA repression to stably manipulate essential genes in C. cellulolyticum. Our plasmid-based expression of antisense RNA (asRNA) molecules targeting the phosphotransacetylase (pta) gene successfully reduced the enzymatic activity by 35% in cellobiose-grown cells, metabolically decreased the acetate titer by 15 and 52% in wildtype transformants on cellulose and xylan, respectively. To control both acetate and lactate simultaneously, we transformed the repression plasmid into lactate production-deficient mutant and found the plasmid delivery reduced acetate titer by more than 33%, concomitant with negligible lactate formation. The strains with pta gene repression generally diverted more carbon into ethanol. However, further testing on chromosomal integrants that were created by double-crossover recombination exhibited only very weak repression because DNA integration dramatically lessened gene dosage. With the design of a tandem repetitive promoter-driven asRNA module and the use of a new Cas9 nickase genome editing tool, a chromosomal integrant (LM3P) was generated in a single step and successfully enhanced RNA repression, with a 27% decrease in acetate titer on cellulose in antibiotic-free medium. These results indicate the effectiveness of tandem promoter-driven RNA repression modules in promoting gene repression in chromosomal integrants. Our combinatorial method using a Cas9 nickase genome editing tool to integrate the gene repression module demonstrates easy-to-use and high-efficiency advantages, paving the way for stably manipulating genes, even essential ones, for functional characterization and microbial engineering. PMID:28936208

The miR172 target TOE3 represses AGAMOUS expression during Arabidopsis floral patterning.

PubMed

Jung, Jae-Hoon; Lee, Sangmin; Yun, Ju; Lee, Minyoung; Park, Chung-Mo

2014-02-01

microRNA172 (miR172) regulates phase transition and floral patterning in Arabidopsis by repressing targets that encode the APETALA2 (AP2) and AP2-like transcription factors. The miR172-mediated repression of the AP2 gene restricts AGAMOUS (AG) expression. In addition, most miR172 targets, including AP2, redundantly act as floral repressors, and the overexpression of the target genes causes delayed flowering. However, how miR172 targets other than AP2 regulate both of the developmental processes remains unclear. Here, we demonstrate that miR172-mediated repression of the TARGET OF EAT 3 (TOE3) gene is critical for floral patterning in Arabidopsis. Transgenic plants that overexpress a miR172-resistant TOE3 gene (rTOE3-ox) exhibit indeterminate flowers with numerous stamens and carpelloid organs, which is consistent with previous observations in transgenic plants that overexpress a miR172-resistant AP2 gene. TOE3 binds to the second intron of the AG gene. Accordingly, AG expression is significantly reduced in rTOE3-ox plants. TOE3 also interacts with AP2 in the nucleus. Given the major role of AP2 in floral patterning, miR172 likely regulates TOE3 in floral patterning, at least in part via AP2. In addition, a miR156 target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 directly activates TOE3 expression, revealing a novel signaling interaction between miR156 and miR172 in floral patterning. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis.

PubMed

Tran, Dinh Thi Minh; Phan, Trang Thi Phuong; Huynh, Thanh Kieu; Dang, Ngan Thi Kim; Huynh, Phuong Thi Kim; Nguyen, Tri Minh; Truong, Tuom Thi Tinh; Tran, Thuoc Linh; Schumann, Wolfgang; Nguyen, Hoang Duc

2017-07-25

Besides Escherichia coli, Bacillus subtilis is an important bacterial species for the production of recombinant proteins. Recombinant genes are inserted into shuttle expression vectors which replicate in both E. coli and in B. subtilis. The ligation products are first transformed into E. coli cells, analyzed for correct insertions, and the correct recombinant plasmids are then transformed into B. subtilis. A major problem using E. coli cells can be the strong basal level of expression of the recombinant protein which may interfere with the stability of the cells. To minimize this problem, we developed strong expression vectors being repressed in E. coli and inducer-free in B. subtilis. In general, induction of IPTG-inducible expression vectors is determined by the regulatory lacI gene encoding the LacI repressor in combination with the lacO operator on the promoter. To investigate the inducer-free properties of the vectors, we constructed inducer-free expression plasmids by removing the lacI gene and characterized their properties. First, we examined the ability to repress a reporter gene in E. coli, which is a prominent property facilitating the construction of the expression vectors carrying a target gene. The β-galactosidase (bgaB gene) basal levels expressed from Pgrac01-bgaB could be repressed at least twice in the E. coli cloning strain. Second, the inducer-free production of BgaB from four different plasmids with the Pgrac01 promoter in B. subtilis was investigated. As expected, BgaB expression levels of inducer-free constructs are at least 37 times higher than that of the inducible constructs in the absence of IPTG, and comparable to those in the presence of the inducer. Third, using efficient IPTG-inducible expression vectors containing the strong promoter Pgrac100, we could convert them into inducer-free expression plasmids. The BgaB production levels from the inducer-free plasmid in the absence of the inducer were at least 4.5 times higher than that of the inducible vector using the same promoter. Finally, we used gfp as a reporter gene in combination with the two promoters Pgrac01 and Pgrac100 to test the new vector types. The GFP expression levels could be repressed at least 1.5 times for the Pgrac01-gfp+ inducer-free construct in E. coli. The inducer-free constructs Pgrac01-gfp+ and Pgrac100-gfp+ allowed GFP expression at high levels from 23 × 10 4 to 32 × 10 4 RFU units and 9-13% of total intracellular proteins. We could reconfirm the two major advantages of the new inducer-free expression plasmids: (1) Strong repression of the target gene expression in the E. coli cloning strain, and (2) production of the target protein at high levels in B. subtilis in the absence of the inducer. We propose a general strategy to generate inducer-free expression vector by using IPTG-inducible vectors, and more specifically we developed inducer-free expression plasmids using IPTG-inducible promoters in the absence of the LacI repressor. These plasmids could be an excellent choice for high-level production of recombinant proteins in B. subtilis without the addition of inducer and at the same time maintaining a low basal level of the recombinant proteins in E. coli. The repression of the recombinant gene expression would facilitate cloning of genes that potentially inhibit the growth of E. coli cloning strains. The inducer-free expression plasmids will be extended versions of the current available IPTG-inducible expression vectors for B. subtilis, in which all these vectors use the same cognate promoters. These inducer-free and previously developed IPTG-inducible expression plasmids will be a useful cassette to study gene expression at a small scale up to a larger scale up for the production of recombinant proteins.

The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos.

PubMed

Wang, Zhe; Nakayama, Yukiko; Tsuda, Sachiko; Yamasu, Kyo

During vertebrate brain development, the gastrulation brain homeobox 2 gene (gbx2) is expressed in the forebrain, but its precise roles are still unknown. In this study, we addressed this issue in zebrafish (Danio rerio) first by carefully examining gbx2 expression in the developing forebrain. We showed that gbx2 was expressed in the telencephalon during late somitogenesis, from 18h post-fertilization (hpf) to 24 hpf, and in the thalamic primordium after 26 hpf. In contrast, another gbx gene, gbx1, was expressed in the anterior-most ventral telencephalon after 36 hpf. Thus, the expression patterns of these two gbx genes did not overlap, arguing against their redundant function in the forebrain. Two-color fluorescence in situ hybridization (FISH) showed close relationships between the telencephalic expression of gbx2 and other forebrain-forming genes, suggesting that their interactions contribute to the regionalization of the telencephalon. FISH further revealed that gbx2 is expressed in the ventricular region of the telencephalon. By using transgenic fish in which gbx2 can be induced by heat shock, we found that gbx2 induction at 16 hpf repressed the expression of emx3, dlx2a, and six3b in the ventral telencephalon. Among secreted factor genes, bmp2b and wnt1 were repressed in the vicinity of the gbx2 domain in the telencephalon. The expression of forebrain-forming genes was examined in mutant embryos lacking gbx2, showing emx3 and dlx2a to be upregulated in the subpallium at 24 hpf. Taken together, these findings indicate that gbx2 contributes to the development of the subpallium through its repressive activities against other telencephalon-forming genes. We further showed that inhibiting FGF signaling and activating Wnt signaling repressed gbx2 and affected the regionalization of the telencephalon, supporting a functional link between gbx2, intracellular signaling, and telencephalon development. Copyright © 2017 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

A concerted action of a paired-type homeobox gene, aristaless, and a homolog of Hox11/tlx homeobox gene, clawless, is essential for the distal tip development of the Drosophila leg.

PubMed

Kojima, Tetsuya; Tsuji, Takuya; Saigo, Kaoru

2005-03-15

The subdivision of the developing field by region-specific expression of genes encoding transcription factors is an essential step during appendage development in arthropod and vertebrates. In Drosophila leg development, the distal-most region (pretarsus) is specified by the expression of homeobox genes, aristaless and Lim1, and its immediate neighbor (distal tarsus) is specified by the expression of a pair of Bar homeobox genes. Here, we show that one additional gene, clawless, which is a homolog of vertebrate Hox11/tlx homeobox gene family and formerly known as C15, is specifically expressed in the pretarsus and cooperatively acts with aristaless to repress Bar and possibly to activate Lim1. Similar to aristaless, the maximal expression of clawless requires Lim1 and its co-factor, Chip. Bar attenuates aristaless and clawless expression through Lim1 repression. Aristaless and Clawless proteins form a complex capable of binding to specific DNA targets, which cannot be well recognized solely by Aristaless or Clawless.

Sugar regulation of SUGAR TRANSPORTER PROTEIN 1 (STP1) expression in Arabidopsis thaliana

PubMed Central

Cordoba, Elizabeth; Aceves-Zamudio, Denise Lizeth; Hernández-Bernal, Alma Fabiola; Ramos-Vega, Maricela; León, Patricia

2015-01-01

Sugars regulate the expression of many genes at the transcriptional level. In Arabidopsis thaliana, sugars induce or repress the expression of >1800 genes, including the STP1 (SUGAR TRANSPORTER PROTEIN 1) gene, which encodes an H+/monosaccharide cotransporter. STP1 transcript levels decrease more rapidly after the addition of low concentrations of sugars than the levels of other repressed genes, such as DIN6 (DARK-INDUCED 6). We found that this regulation is exerted at the transcriptional level and is initiated by phosphorylatable sugars. Interestingly, the sugar signal that modulates STP1 expression is transmitted through a HEXOKINASE 1-independent signalling pathway. Finally, analysis of the STP1 5′ regulatory region allowed us to delimit a region of 309bp that contains the cis elements implicated in the glucose regulation of STP1 expression. Putative cis-acting elements involved in this response were identified. PMID:25281700

Dynamic changes in gene expression during human trophoblast differentiation.

PubMed

Handwerger, Stuart; Aronow, Bruce

2003-01-01

The genetic program that directs human placental differentiation is poorly understood. In a recent study, we used DNA microarray analyses to determine genes that are dynamically regulated during human placental development in an in vitro model system in which highly purified cytotrophoblast cells aggregate spontaneously and fuse to form a multinucleated syncytium that expresses placental lactogen, human chorionic gonadotropin, and other proteins normally expressed by fully differentiated syncytiotrophoblast cells. Of the 6918 genes present on the Incyte Human GEM V microarray that we analyzed over a 9-day period, 141 were induced and 256 were downregulated by more than 2-fold. The dynamically regulated genes fell into nine distinct kinetic patterns of induction or repression, as detected by the K-means algorithm. Classifying the genes according to functional characteristics, the regulated genes could be divided into six overall categories: cell and tissue structural dynamics, cell cycle and apoptosis, intercellular communication, metabolism, regulation of gene expression, and expressed sequence tags and function unknown. Gene expression changes within key functional categories were tightly coupled to the morphological changes that occurred during trophoblast differentiation. Within several key gene categories (e.g., cell and tissue structure), many genes were strongly activated, while others with related function were strongly repressed. These findings suggest that trophoblast differentiation is augmented by "categorical reprogramming" in which the ability of induced genes to function is enhanced by diminished synthesis of other genes within the same category. We also observed categorical reprogramming in human decidual fibroblasts decidualized in vitro in response to progesterone, estradiol, and cyclic AMP. While there was little overlap between genes that are dynamically regulated during trophoblast differentiation versus decidualization, many of the categories in which genes were strongly activated also contained genes whose expression was strongly diminished. Taken together, these findings point to a fundamental role for simultaneous induction and repression of mRNAs that encode functionally related proteins during the differentiation process.

Repressors Nrg1 and Nrg2 Regulate a Set of Stress-Responsive Genes in Saccharomyces cerevisiae§

PubMed Central

Vyas, Valmik K.; Berkey, Cristin D.; Miyao, Takenori; Carlson, Marian

2005-01-01

The yeast Saccharomyces cerevisiae responds to environmental stress by rapidly altering the expression of large sets of genes. We report evidence that the transcriptional repressors Nrg1 and Nrg2 (Nrg1/Nrg2), which were previously implicated in glucose repression, regulate a set of stress-responsive genes. Genome-wide expression analysis identified 150 genes that were upregulated in nrg1Δ nrg2Δ double mutant cells, relative to wild-type cells, during growth in glucose. We found that many of these genes are regulated by glucose repression. Stress response elements (STREs) and STRE-like elements are overrepresented in the promoters of these genes, and a search of available expression data sets showed that many are regulated in response to a variety of environmental stress signals. In accord with these findings, mutation of NRG1 and NRG2 enhanced the resistance of cells to salt and oxidative stress and decreased tolerance to freezing. We present evidence that Nrg1/Nrg2 not only contribute to repression of target genes in the absence of stress but also limit induction in response to salt stress. We suggest that Nrg1/Nrg2 fine-tune the regulation of a set of stress-responsive genes. PMID:16278455

Specific repression of β-globin promoter activity by nuclear ferritin

PubMed Central

Broyles, Robert H.; Belegu, Visar; DeWitt, Christina R.; Shah, Sandeep N.; Stewart, Charles A.; Pye, Quentin N.; Floyd, Robert A.

2001-01-01

Developmental hemoglobin switching involves sequential globin gene activations and repressions that are incompletely understood. Earlier observations, described herein, led us to hypothesize that nuclear ferritin is a repressor of the adult β-globin gene in embryonic erythroid cells. Our data show that a ferritin-family protein in K562 cell nuclear extracts binds specifically to a highly conserved CAGTGC motif in the β-globin promoter at −153 to −148 bp from the cap site, and mutation of the CAGTGC motif reduces binding 20-fold in competition gel-shift assays. Purified human ferritin that is enriched in ferritin-H chains also binds the CAGTGC promoter segment. Expression clones of ferritin-H markedly repress β-globin promoter-driven reporter gene expression in cotransfected CV-1 cells in which the β-promoter has been stimulated with the transcription activator erythroid Krüppel-like factor (EKLF). We have constructed chloramphenicol acetyltransferase reporter plasmids containing either a wild-type or mutant β-globin promoter for the −150 CAGTGC motif and have compared the constructs for susceptibility to repression by ferritin-H in cotransfection assays. We find that stimulation by cotransfected EKLF is retained with the mutant promoter, whereas repression by ferritin-H is lost. Thus, mutation of the −150 CAGTGC motif not only markedly reduces in vitro binding of nuclear ferritin but also abrogates the ability of expressed ferritin-H to repress this promoter in our cell transfection assay, providing a strong link between DNA binding and function, and strong support for our proposal that nuclear ferritin-H is a repressor of the human β-globin gene. Such a repressor could be helpful in treating sickle cell and other genetic diseases. PMID:11481480

Inducible repression of multiple expansin genes leads to growth suppression during leaf development.

PubMed

Goh, Hoe-Han; Sloan, Jennifer; Dorca-Fornell, Carmen; Fleming, Andrew

2012-08-01

Expansins are cell wall proteins implicated in the control of plant growth via loosening of the extracellular matrix. They are encoded by a large gene family, and data linked to loss of single gene function to support a role of expansins in leaf growth remain limited. Here, we provide a quantitative growth analysis of transgenics containing an inducible artificial microRNA construct designed to down-regulate the expression of a number of expansin genes that an expression analysis indicated are expressed during the development of Arabidopsis (Arabidopsis thaliana) leaf 6. The results support the hypothesis that expansins are required for leaf growth and show that decreased expansin gene expression leads to a more marked repression of growth during the later stage of leaf development. In addition, a histological analysis of leaves in which expansin gene expression was suppressed indicates that, despite smaller leaves, mean cell size was increased. These data provide functional evidence for a role of expansins in leaf growth, indicate the importance of tissue/organ developmental context for the outcome of altered expansin gene expression, and highlight the separation of the outcome of expansin gene expression at the cellular and organ levels.

9-cis-retinoic acid represses estrogen-induced expression of the very low density apolipoprotein II gene.

PubMed

Schippers, I J; Kloppenburg, M; Snippe, L; Ab, G

1994-11-01

The chicken very low density apolipoprotein II (apoVLDLII) gene is estrogen-inducible and specifically expressed in liver. We examined the possible involvement of the retinoid X receptor (RXR) and its ligand 9-cis-retinoic acid (9-cis-RA) in the activation of the apoVLDLII promoter. We first concentrated on a potential RXR recognition site, which deviates at only one position from a perfect direct A/GGGTCA repeat spaced by one nucleotide (DR-1) and was earlier identified as a common HNF-4/COUP-TF recognition site. However, band shift analysis revealed that this imperfect DR-1 motif does not interact with RXR alpha-homodimers. In accordance with this observation we found that this regulatory element does not mediate transactivation through RXR alpha in the presence of 9-cis-RA. However, our experiments revealed another, unexpected, effect of 9-cis-RA. Instead of stimulating, 9-cis-RA attenuated estrogen-induced expression of transfected estrogen-responsive VLDL-CAT reporter plasmids. This repression appeared to take place through the main estrogen response element (ERE) of the gene. Importantly, 9-cis-RA also strongly repressed the estrogen-induced expression of the endogenous apoVLDLII gene in cultured chicken hepatoma cells.

Developmental control of transcriptional and proliferative potency during the evolutionary emergence of animals

PubMed Central

Arenas-Mena, Cesar; Coffman, James A.

2016-01-01

Summary It is proposed that the evolution of complex animals required repressive genetic mechanisms for controlling the transcriptional and proliferative potency of cells. Unicellular organisms are transcriptionally potent, able to express their full genetic complement as the need arises through their life cycle, whereas differentiated cells of multicellular organisms can only express a fraction of their genomic potential. Likewise, whereas cell proliferation in unicellular organisms is primarily limited by nutrient availability, cell proliferation in multicellular organisms is developmentally regulated. Repressive genetic controls limiting the potency of cells at the end of ontogeny would have stabilized the gene expression states of differentiated cells and prevented disruptive proliferation, allowing the emergence of diverse cell types and functional shapes. We propose that distal cis-regulatory elements represent the primary innovations that set the stage for the evolution of developmental gene regulatory networks and the repressive control of key multipotency and cell-cycle control genes. The testable prediction of this model is that the genomes of extant animals, unlike those of our unicellular relatives, encode gene regulatory circuits dedicated to the developmental control of transcriptional and proliferative potency. PMID:26173445

Adenovirus small E1A employs the lysine acetylases p300/CBP and tumor suppressor Rb to repress select host genes and promote productive virus infection.

PubMed

Ferrari, Roberto; Gou, Dawei; Jawdekar, Gauri; Johnson, Sarah A; Nava, Miguel; Su, Trent; Yousef, Ahmed F; Zemke, Nathan R; Pellegrini, Matteo; Kurdistani, Siavash K; Berk, Arnold J

2014-11-12

Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGF-β, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation

PubMed Central

Totsuka, Akane; Okamoto, Emi; Miyahara, Taira; Kouno, Takanobu; Cano, Emilio A.; Sasaki, Nobuhiro; Watanabe, Aiko; Tasaki, Keisuke; Nishihara, Masahiro; Ozeki, Yoshihiro

2018-01-01

In a previous study, two genes responsible for white flower phenotypes in carnation were identified. These genes encoded enzymes involved in anthocyanin synthesis, namely, flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR), and showed reduced expression in the white flower phenotypes. Here, we identify another candidate gene for white phenotype in carnation flowers using an RNA-seq analysis followed by RT-PCR. This candidate gene encodes a transcriptional regulatory factor of the basic helix-loop-helix (bHLH) type. In the cultivar examined here, both F3H and DFR genes produced active enzyme proteins; however, expression of DFR and of genes for enzymes involved in the downstream anthocyanin synthetic pathway from DFR was repressed in the absence of bHLH expression. Occasionally, flowers of the white flowered cultivar used here have red speckles and stripes on the white petals. We found that expression of bHLH occurred in these red petal segments and induced expression of DFR and the following downstream enzymes. Our results indicate that a member of the bHLH superfamily is another gene involved in anthocyanin synthesis in addition to structural genes encoding enzymes. PMID:29681756

Lithospermum erythrorhizon Root and its Naphthoquinones Repress SREBP1c and Activate PGC1α Through AMPKα.

PubMed

Velliquette, Rodney A; Rajgopal, Arun; Rebhun, John; Glynn, Kelly

2018-01-01

To examine specific molecular mechanisms involved in modulating hepatic lipogenesis and mitochondria biogenesis signals by Lithospermum erythrorhizon (gromwell) root extract. Stable cell lines with luciferase reporter constructs were generated to examine sterol regulatory element binding protein 1c (SREBP1c) and peroxisome proliferator-activated receptor gamma, coactivator 1 (PGC1) α promoter activity and estrogen-related receptor (ERR) α response element activity. Gene expression of SREBP1c, stearoyl coenzyme A desaturase 1, and PGC1α was measured by using reverse transcription polymerase chain reaction. Lipogenesis was measured in human hepatoma cells with Nile red staining and flow cytometry. Phosphorylation of AMP-activated protein kinase (AMPK) α was determined by using ELISA and Western blot. Gromwell root extract and its naphthoquinones dose-dependently repressed high glucose and liver X receptor α induction of SREBP1c promoter activity and gene expression. Hepatic lipogenesis was repressed, and PGC1α promoter and gene expression and ERRα response element activity were increased by gromwell root extract. Gromwell root extract, shikonin, and α-methyl-n-butyrylshikonin increased AMPKα phosphorylation, and inhibition of AMPK blunted the repression in SREBP1c promoter activity by gromwell root extract and its naphthoquinones. Data suggest that gromwell root extract and its naphthoquinones repress lipogenesis by increasing the phosphorylated state of AMPKα and stimulating mitochondrial biogenesis signals. © 2017 The Obesity Society.

Neural Progenitors Adopt Specific Identities by Directly Repressing All Alternative Progenitor Transcriptional Programs.

PubMed

Kutejova, Eva; Sasai, Noriaki; Shah, Ankita; Gouti, Mina; Briscoe, James

2016-03-21

In the vertebrate neural tube, a morphogen-induced transcriptional network produces multiple molecularly distinct progenitor domains, each generating different neuronal subtypes. Using an in vitro differentiation system, we defined gene expression signatures of distinct progenitor populations and identified direct gene-regulatory inputs corresponding to locations of specific transcription factor binding. Combined with targeted perturbations of the network, this revealed a mechanism in which a progenitor identity is installed by active repression of the entire transcriptional programs of other neural progenitor fates. In the ventral neural tube, sonic hedgehog (Shh) signaling, together with broadly expressed transcriptional activators, concurrently activates the gene expression programs of several domains. The specific outcome is selected by repressive input provided by Shh-induced transcription factors that act as the key nodes in the network, enabling progenitors to adopt a single definitive identity from several initially permitted options. Together, the data suggest design principles relevant to many developing tissues. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

H-NS represses transcription of the flagellin gene lafA of lateral flagella in Vibrio parahaemolyticus.

PubMed

Wang, Yan; Zhang, Yiquan; Yin, Zhe; Wang, Jie; Zhu, Yongzhe; Peng, Haoran; Zhou, Dongsheng; Qi, Zhongtian; Yang, Wenhui

2018-01-01

Swarming motility is ultimately mediated by the proton-powered lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf genes is tightly regulated by a number of environmental conditions and regulatory factors. The nucleoid-associated DNA-binding protein H-NS is a small and abundant protein that is widely distributed in bacteria, and H-NS-like protein-dependent expression of laf genes has been identified in Vibrio cholerae and V. parahaemolyticus. The data presented here show that H-NS acts as a repressor of the swarming motility in V. parahaemolyticus. A single σ 28 -dependent promoter was detected for lafA encoding the flagellin of the lateral flagella, and its activity was directly repressed by H-NS. Thus, H-NS represses swarming motility by directly acting on lafA. Briefly, this work revealed a novel function for H-NS as a repressor of the expression of lafA and swarming motility in V. parahaemolyticus.

BCL6 antagonizes NOTCH2 to maintain survival of human follicular lymphoma cells

PubMed Central

Valls, Ester; Lobry, Camille; Geng, Huimin; Wang, Ling; Cardenas, Mariano; Rivas, Martín; Cerchietti, Leandro; Oh, Philmo; Yang, Shao Ning; Oswald, Erin; Graham, Camille W.; Jiang, Yanwen; Hatzi, Katerina; Agirre, Xabier; Perkey, Eric; Li, Zhuoning; Tam, Wayne; Bhatt, Kamala; Leonard, John P.; Zweidler-McKay, Patrick A.; Maillard, Ivan; Elemento, Olivier; Ci, Weimin; Aifantis, Iannis; Melnick, Ari

2017-01-01

Summary Although the BCL6 transcriptional repressor is frequently expressed in human follicular lymphomas (FL), its biological role in this disease remains unknown. Herein we comprehensively identify the set of gene promoters directly targeted by BCL6 in primary human FLs. We noted that BCL6 binds and represses NOTCH2 and Notch pathway genes. Moreover, BCL6 and NOTCH2 pathway gene expression is inversely correlated in FL. Notably BCL6 up-regulation is associated with repression of Notch2 and its target genes in primary human and murine germinal center cells. Repression of Notch2 is an essential function of BCL6 in FL and GC B-cells since inducible expression of Notch2 abrogated GC formation in mice and kills FL cells. Indeed BCL6-targeting compounds or gene silencing leads to the induction of NOTCH2 activity and compromises survival of FL cells whereas NOTCH2 depletion or pathway antagonists rescue FL cells from such effects. Moreover, BCL6 inhibitors induced NOTCH2 expression and suppressed growth of human FL xenografts in vivo and primary human FL specimens ex vivo. These studies suggest that established FLs are thus dependent on BCL6 through its suppression of NOTCH2. PMID:28232365

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92

PubMed Central

Jin, Hyun Yong; Oda, Hiroyo; Chen, Pengda; Kang, Seung Goo; Valentine, Elizabeth; Liao, Lujian; Zhang, Yaoyang; Gonzalez-Martin, Alicia; Shepherd, Jovan; Head, Steven R.; Kim, Pyeung-Hyeun; Fu, Guo; Liu, Wen-Hsien; Han, Jiahuai

2017-01-01

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5’UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes. PMID:28241004

MCM-BP is required for repression of life-cycle specific genes transcribed by RNA polymerase I in the mammalian infectious form of Trypanosoma brucei.

PubMed

Kim, Hee-Sook; Park, Sung Hee; Günzl, Arthur; Cross, George A M

2013-01-01

Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes escape host immunity by differential expression of VSGs, we attempted to identify genes that contribute to VSG silencing, by performing a loss-of-silencing screen in T. brucei using a transposon-mediated random insertional mutagenesis. One identified gene, which we initially named LOS1, encodes a T. brucei MCM-Binding Protein (TbMCM-BP). Here we show that TbMCM-BP is essential for viability of infectious bloodstream-form (BF) trypanosome and is required for proper cell-cycle progression. Tandem affinity purification of TbMCM-BP followed by mass spectrometry identified four subunits (MCM4-MCM7) of the T. brucei MCM complex, a replicative helicase, and MCM8, a subunit that is uniquely co-purified with TbMCM-BP. TbMCM-BP is required not only for repression of subtelomeric VSGs but also for silencing of life-cycle specific, insect-stage genes, procyclin and procyclin-associated genes (PAGs), that are normally repressed in BF trypanosomes and are transcribed by RNA polymerase I. Our study uncovers a functional link between chromosome maintenance and RNA pol I-mediated gene silencing in T. brucei.

DNA methyltransferase DNMT3a contributes to neuropathic pain by repressing Kcna2 in primary afferent neurons

PubMed Central

Zhao, Jian-Yuan; Liang, Lingli; Gu, Xiyao; Li, Zhisong; Wu, Shaogen; Sun, Linlin; Atianjoh, Fidelis E.; Feng, Jian; Mo, Kai; Jia, Shushan; Lutz, Brianna Marie; Bekker, Alex; Nestler, Eric J.; Tao, Yuan-Xiang

2017-01-01

Nerve injury induces changes in gene transcription in dorsal root ganglion (DRG) neurons, which may contribute to nerve injury-induced neuropathic pain. DNA methylation represses gene expression. Here, we report that peripheral nerve injury increases expression of the DNA methyltransferase DNMT3a in the injured DRG neurons via the activation of the transcription factor octamer transcription factor 1. Blocking this increase prevents nerve injury-induced methylation of the voltage-dependent potassium (Kv) channel subunit Kcna2 promoter region and rescues Kcna2 expression in the injured DRG and attenuates neuropathic pain. Conversely, in the absence of nerve injury, mimicking this increase reduces the Kcna2 promoter activity, diminishes Kcna2 expression, decreases Kv current, increases excitability in DRG neurons and leads to spinal cord central sensitization and neuropathic pain symptoms. These findings suggest that DNMT3a may contribute to neuropathic pain by repressing Kcna2 expression in the DRG. PMID:28270689

A Novel Thyroid Hormone Mediated Regulation of HSV-1 Gene Expression and Replication is Specific to Neuronal Cells and Associated with Disruption of Chromatin Condensation

PubMed Central

Chen, Feng; Palem, Jay; Balish, Matthew; Figliozzi, Robert; Ajavon, Amakoe; Hsia, S Victor

2014-01-01

Previously we showed that thyroid hormone (T3) regulated the Herpes Simplex Virus Type -1 (HSV-1) gene expression and replication through its nuclear receptor TR via histone modification and chromatin remodeling in a neuroblastoma cell line neuro-2a cells (N2a). This observation suggested that T3 regulation may be neuron-specific and have implication in HSV-1 latency and reactivation. In this study, our in vitro latency/reactivation model demonstrated that removal of T3 can de-repress the HSV-1 replication and favor reactivation. Transfection studies and infection assays indicated that HSV-1 thymidine kinase (TK), a key viral gene during reactivation, was repressed by TR/T3 in cells with neuronal origin but not in non-neuronal cells. Additional studies showed that RCC1 (Regulator of Chromosome Condensation 1) was sequestered but efficiently detected upon viral infection in N2a cells. Western blot analyses indicated that addition of T3 repressed the RCC1 expression upon infection. It is likely that diminution of RCC1 upon infection in neuronal cells under the influence of TR/T3 may lead to repression of viral replication/gene expression thus promote latency. Together these results demonstrated that TR/T3 mediated regulation is specific to neuronal cells and differential chromosome condensation may play a critical role in this process. PMID:25346944

Msn2p and Msn4p Control a Large Number of Genes Induced at the Diauxic Transition Which Are Repressed by Cyclic AMP in Saccharomyces cerevisiae

PubMed Central

Boy-Marcotte, Emmanuelle; Perrot, Michel; Bussereau, Françoise; Boucherie, Hélian; Jacquet, Michel

1998-01-01

The multicopy suppressors of the snf1 defect, Msn2p and Msn4p transcription factors (Msn2/4p), activate genes through the stress-responsive cis element (CCCCT) in response to various stresses. This cis element is also the target for repression by the cyclic AMP (cAMP)-signaling pathway. We analyzed the two-dimensional gel electrophoresis pattern of protein synthesis of the msn2 msn4 double mutant and compared it with that of the wild-type strain during exponential growth phase and at the diauxic transition. Thirty-nine gene products (including those of ALD3, GDH3, GLK1, GPP2, HSP104, HXK1, PGM2, SOD2, SSA3, SSA4, TKL2, TPS1, and YBR149W) are dependent upon Msn2/4p for their induction at the diauxic transition. The expression of all these genes is repressed by cAMP. Thirty other genes identified during this study are still inducible in the mutant. A subset of these genes were found to be superinduced at the diauxic transition, and others were subject to cAMP repression (including ACH1, ADH2, ALD6, ATP2, GPD1, ICL1, and KGD2). We conclude from this analysis that Msn2/4p control a large number of genes induced at the diauxic transition but that other, as-yet-uncharacterized regulators, also contribute to this response. In addition, we show here that cAMP repression applies to both Msn2/4p-dependent and -independent control of gene expression at the diauxic shift. Furthermore, the fact that all the Msn2/4p gene targets are subject to cAMP repression suggests that these regulators could be targets for the cAMP-signaling pathway. PMID:9495741

Regulation of mRNA translation during mitosis.

PubMed

Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

2015-08-25

Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

MRG-1, an autosome-associated protein, silences X-linked genes and protects germline immortality in Caenorhabditis elegans

PubMed Central

Takasaki, Teruaki; Liu, Zheng; Habara, Yasuaki; Nishiwaki, Kiyoji; Nakayama, Jun-ichi; Inoue, Kunio; Sakamoto, Hiroshi; Strome, Susan

2008-01-01

MRG15, a mammalian protein related to the mortality factor MORF4, is required for cell proliferation and embryo survival. Our genetic analysis has revealed that the Caenorhabditis elegans ortholog MRG-1 serves similar roles. Maternal MRG-1 promotes embryo survival and is required for proliferation and immortality of the primordial germ cells (PGCs). As expected of a chromodomain protein, MRG-1 associates with chromatin. Unexpectedly, it is concentrated on the autosomes and not detectable on the X chromosomes. This association is not dependent on the autosome-enriched protein MES-4. Focusing on possible roles of MRG-1 in regulating gene expression, we determined that MRG-1 is required to maintain repression in the maternal germ line of transgenes on extrachromosomal arrays, and of several X-linked genes previously shown to depend on MES-4 for repression. MRG-1 is not required for PGCs to acquire transcriptional competence or for the turn-on of expression of several PGC-expressed genes (pgl-1, glh-1, glh-4 and nos-1). By contrast to this result in PGCs, MRG-1 is required for ectopic expression of those germline genes in somatic cells lacking the NuRD complex component MEP-1. We discuss how an autosome-enriched protein might repress genes on the X chromosome, promote PGC proliferation and survival, and influence the germ versus soma distinction. PMID:17215300

The Fur-Iron Complex Modulates Expression of the Quorum-Sensing Master Regulator, SmcR, To Control Expression of Virulence Factors in Vibrio vulnificus

PubMed Central

Kim, In Hwang; Wen, Yancheng; Son, Jee-Soo; Lee, Kyu-Ho

2013-01-01

The gene vvpE, encoding the virulence factor elastase, is a member of the quorum-sensing regulon in Vibrio vulnificus and displays enhanced expression at high cell density. We observed that this gene was repressed under iron-rich conditions and that the repression was due to a Fur (ferric uptake regulator)-dependent repression of smcR, a gene encoding a quorum-sensing master regulator with similarity to luxR in Vibrio harveyi. A gel mobility shift assay and a footprinting experiment demonstrated that the Fur-iron complex binds directly to two regions upstream of smcR (−82 to −36 and −2 to +27, with respect to the transcription start site) with differing affinities. However, binding of the Fur-iron complex is reversible enough to allow expression of smcR to be induced by quorum sensing at high cell density under iron-rich conditions. Under iron-limiting conditions, Fur fails to bind either region and the expression of smcR is regulated solely by quorum sensing. These results suggest that two biologically important environmental signals, iron and quorum sensing, converge to direct the expression of smcR, which then coordinates the expression of virulence factors. PMID:23716618

Mitochondrial functions mediate cellulase gene expression in Trichoderma reesei.

PubMed

Abrahão-Neto, J; Rossini, C H; el-Gogary, S; Henrique-Silva, F; Crivellaro, O; el-Dorry, H

1995-08-22

We examined the effects of inhibition of mitochondrial functions on the expression of two nuclear genes encoding the extracellular cellobiohydrolase I (cbh1) and endoglucanase I (egl1) of the cellulase system of the filamentous fungus Trichoderma reesei. The cbh1 and egl1 transcripts are repressed at a low oxygen tension, and by glucose at a concentration known to repress mitochondrial respiration. The transcripts are also down-regulated by chemical agents known to dissipate the proton electrochemical gradient of the inner mitochondrial membrane and blocking of the electron-transport chain, such as DNP and KCN, respectively. These results suggest that expression of those transcripts is influenced by the physiological state of the mitochondria. In addition, heterologous gene fusion shows that the sensitivity of the expression of those transcripts to the functional state of the mitochondria is transcriptionally controlled through the 5'-flanking DNA sequence of those genes.

Mapping the core of the Arabidopsis circadian clock defines the network structure of the oscillator.

PubMed

Huang, W; Pérez-García, P; Pokhilko, A; Millar, A J; Antoshechkin, I; Riechmann, J L; Mas, P

2012-04-06

In many organisms, the circadian clock is composed of functionally coupled morning and evening oscillators. In Arabidopsis, oscillator coupling relies on a core loop in which the evening oscillator component TIMING OF CAB EXPRESSION 1 (TOC1) was proposed to activate a subset of morning-expressed oscillator genes. Here, we show that TOC1 does not function as an activator but rather as a general repressor of oscillator gene expression. Repression occurs through TOC1 rhythmic association to the promoters of the oscillator genes. Hormone-dependent induction of TOC1 and analysis of RNA interference plants show that TOC1 prevents the activation of morning-expressed genes at night. Our study overturns the prevailing model of the Arabidopsis circadian clock, showing that the morning and evening oscillator loops are connected through the repressing activity of TOC1.

Cyclic di-GMP regulation of the bvg-repressed genes and the orphan response regulator RisA in Bordetella pertussis

USDA-ARS?s Scientific Manuscript database

Expression of Bordetella pertussis virulence factors is activated by the BvgAS two-component system. Under modulating growth conditions BvgAS indirectly represses another set of genes through the action of BvgR, a bvg-activated protein. BvgR blocks activation of the response regulator RisA which is ...

Transcriptional Repression of ATF4 Gene by CCAAT/Enhancer-binding Protein β (C/EBPβ) Differentially Regulates Integrated Stress Response*

PubMed Central

Dey, Souvik; Savant, Sudha; Teske, Brian F.; Hatzoglou, Maria; Calkhoven, Cornelis F.; Wek, Ronald C.

2012-01-01

Different environmental stresses induce the phosphorylation of eIF2 (eIF2∼P), repressing global protein synthesis coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of genes involved in metabolism and nutrient uptake, antioxidation, and regulation of apoptosis. Because ATF4 is a common downstream target that integrates signaling from different eIF2 kinases and their respective stress signals, the eIF2∼P/ATF4 pathway is collectively referred to as the integrated stress response. Although eIF2∼P elicits translational control in response to many different stresses, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2∼P. The rationale for this discordant induction of ATF4 expression and eIF2∼P in response to UV irradiation is that transcription of ATF4 is repressed, and therefore ATF4 mRNA is not available for preferential translation. In this study, we show that C/EBPβ is a transcriptional repressor of ATF4 during UV stress. C/EBPβ binds to critical elements in the ATF4 promoter, resulting in its transcriptional repression. Expression of C/EBPβ increases in response to UV stress, and the liver-enriched inhibitory protein (LIP) isoform of C/EBPβ, but not the liver-enriched activating protein (LAP) version, represses ATF4 transcription. Loss of the liver-enriched inhibitory protein isoform results in increased ATF4 mRNA levels in response to UV irradiation and subsequent recovery of ATF4 translation, leading to enhanced expression of its target genes. Together these results illustrate how eIF2∼P and translational control combined with transcription factors regulated by alternative signaling pathways can direct programs of gene expression that are specifically tailored to each environmental stress. PMID:22556424

Testis development requires the repression of Wnt4 by Fgf signaling

PubMed Central

Jameson, Samantha A.; Lin, Yi-Tzu; Capel, Blanche

2013-01-01

The bipotential gonad expresses genes associated with both the male and female pathways. Adoption of the male testicular fate is associated with the repression of many female genes including Wnt4. However, the importance of repression of Wnt4 to the establishment of male development was not previously determined. Deletion of either Fgf9 or Fgfr2 in an XY gonad resulted in up-regulation of Wnt4 and male-to-female sex reversal. We investigated whether the deletion if Wnt4 could rescue sex reversal in Fgf9 and Fgfr2 mutants. XY Fgf9/Wnt4 and Fgfr2/Wnt4 double mutants developed testes with male somatic and germ cells present, suggesting that the primary role of Fgf signaling is the repression of female-promoting genes. Thus, the decision to adopt the male fate is based not only on whether male genes, such as Sox9, are expressed, but also on the active repression of female genes, such as Wnt4. Because loss of Wnt4 results in the up-regulation of Fgf9, we also tested the possibility that derepression of Fgf9 was responsible for the aspects of male development observed in XX Wnt4 mutants. However, we found that the relationship between these two signaling factors is not symmetric: loss of Fgf9 in XX Wnt4−/− gonads does not rescue their partial female-to-male sex-reversal. PMID:22705479

Non-homeodomain regions of Hox proteins mediate activation versus repression of Six2 via a single enhancer site in vivo

PubMed Central

Yallowitz, Alisha R.; Gong, Ke-Qin; Swinehart, Ilea T.; Nelson, Lisa T.; Wellik, Deneen M.

2009-01-01

Summary Hox genes control many developmental events along the AP axis, but few target genes have been identified. Whether target genes are activated or repressed, what enhancer elements are required for regulation, and how different domains of the Hox proteins contribute to regulatory specificity is poorly understood. Six2 is genetically downstream of both the Hox11 paralogous genes in the developing mammalian kidney and Hoxa2 in branchial arch and facial mesenchyme. Loss-of-function of Hox11 leads to loss of Six2 expression and loss-of-function of Hoxa2 leads to expanded Six2 expression. Herein we demonstrate that a single enhancer site upstream of the Six2 coding sequence is responsible for both activation by Hox11 proteins in the kidney and repression by Hoxa2 in the branchial arch and facial mesenchyme in vivo. DNA binding activity is required for both activation and repression, but differential activity is not controlled by differences in the homeodomains. Rather, protein domains N- and C-terminal to the homeodomain confer activation versus repression activity. These data support a model in which the DNA binding specificity of Hox proteins in vivo may be similar, consistent with accumulated in vitro data, and that unique functions result mainly from differential interactions mediated by non-homeodomain regions of Hox proteins. PMID:19716816

Repression by Homeoprotein Pitx1 of Virus-Induced Interferon A Promoters Is Mediated by Physical Interaction and trans Repression of IRF3 and IRF7

PubMed Central

Island, Marie-Laure; Mesplede, Thibault; Darracq, Nicole; Bandu, Marie-Thérèse; Christeff, Nicolas; Djian, Philippe; Drouin, Jacques; Navarro, Sébastien

2002-01-01

Interferon A (IFN-A) genes are differentially expressed after virus induction. The differential expression of individual IFN-A genes is modulated by the specific transcription activators IFN regulatory factor 3 (IRF3) and IRF-7 and the homeoprotein transcription repressor Pitx1. We now show that repression by Pitx1 does not appear to be due to the recruitment of histone deacetylases. On the other hand, Pitx1 inhibits the IRF3 and IRF7 transcriptional activity of the IFN-A11 and IFN-A5 promoters and interacts physically with IRF3 and IRF7. Pitx1 trans-repression activity maps to specific C-terminal domains, and the Pitx1 homeodomain is involved in physical interaction with IRF3 or IRF7. IRF3 is able to bind to the antisilencer region of the IFN-A4 promoter, which overrides the repressive activity of Pitx1. These results indicate that interaction between the Pitx1 homeodomain and IRF3 or IRF7 and the ability of the Pitx1 C-terminal repressor domains to block IFN-A11 and IFN-A5 but not IFN-A4 promoter activities may contribute to our understanding of the complex differential transcriptional activation, repression, and antirepression of the IFN-A genes. PMID:12242290

Endogenous small RNAs and antibacterial immunity in plants.

PubMed

Jin, Hailing

2008-08-06

Small RNAs are non-coding regulatory RNA molecules that control gene expression by mediating mRNA degradation, translational inhibition, or chromatin modification. Virus-derived small RNAs induce silencing of viral RNAs and are essential for antiviral defense in both animal and plant systems. The role of host endogenous small RNAs on antibacterial immunity has only recently been recognized. Host disease resistance and defense responses are achieved by activation and repression of a large array of genes. Certain endogenous small RNAs in plants, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are induced or repressed in response to pathogen attack and subsequently regulate the expression of genes involved in disease resistance and defense responses by mediating transcriptional or post-transcriptional gene silencing. Thus, these small RNAs play an important role in gene expression reprogramming in plant disease resistance and defense responses. This review focuses on the recent findings of plant endogenous small RNAs in antibacterial immunity.

Molecular characterization and expression of microbial inulinase genes.

PubMed

Liu, Guang-Lei; Chi, Zhe; Chi, Zhen-Ming

2013-05-01

Many genes encoding exo- and endo-inulinases from bacteria, yeasts and filamentous fungi have been cloned and characterized. All the inulinases have several conserved motifs, such as WMND(E)PNGL, RDP, EC(V)P, SVEVF, Q and FS(T), which play an important role in inulinase catalysis and substrate binding. However, the exo-inulinases produced by yeasts has no conserved motif SVEVF and the yeasts do not produce any endo-inulinase. Exo- and endo-inulinases found in different microorganisms cluster separately at distant positions from each other. Most of the cloned inulinase genes have been expressed in Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia pastoris, Klyuveromyces lactis and Escherichia coli, respectively. The recombinant inulinases produced and the engineered hosts using the cloned inulinase genes have many potential applications. Expression of most of the inulinase genes is repressed by glucose and fructose and induced by inulin and sucrose. However, the detailed mechanisms of the repression and induction are still unknown.

miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila

PubMed Central

Xiong, Xiao-Peng; Chang, Kung-Yen; Ren, Xingjie; Ni, Jian-Quan; Rana, Tariq M.; Zhou, Rui

2016-01-01

microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity. PMID:27893816

miR-34 Modulates Innate Immunity and Ecdysone Signaling in Drosophila.

PubMed

Xiong, Xiao-Peng; Kurthkoti, Krishna; Chang, Kung-Yen; Li, Jian-Liang; Ren, Xingjie; Ni, Jian-Quan; Rana, Tariq M; Zhou, Rui

2016-11-01

microRNAs are endogenous small regulatory RNAs that modulate myriad biological processes by repressing target gene expression in a sequence-specific manner. Here we show that the conserved miRNA miR-34 regulates innate immunity and ecdysone signaling in Drosophila. miR-34 over-expression activates antibacterial innate immunity signaling both in cultured cells and in vivo, and flies over-expressing miR-34 display improved survival and pathogen clearance upon Gram-negative bacterial infection; whereas miR-34 knockout animals are defective in antibacterial defense. In particular, miR-34 achieves its immune-stimulatory function, at least in part, by repressing the two novel target genes Dlg1 and Eip75B. In addition, our study reveals a mutual repression between miR-34 expression and ecdysone signaling, and identifies miR-34 as a node in the intricate interplay between ecdysone signaling and innate immunity. Lastly, we identify cis-regulatory genomic elements and trans-acting transcription factors required for optimal ecdysone-mediated repression of miR-34. Taken together, our study enriches the repertoire of immune-modulating miRNAs in animals, and provides new insights into the interplay between steroid hormone signaling and innate immunity.

Analysis of hepatic gene transcription in mice expressing insulin-insensitive GSK3

PubMed Central

2005-01-01

GSK3 (glycogen synthase kinase-3) regulation is proposed to play a key role in the hormonal control of many cellular processes. Inhibition of GSK3 in animal models of diabetes leads to normalization of blood glucose levels, while high GSK3 activity has been reported in Type II diabetes. Insulin inhibits GSK3 by promoting phosphorylation of a serine residue (Ser-21 in GSK3α, Ser-9 in GSK3β), thereby relieving GSK3 inhibition of glycogen synthesis in muscle. GSK3 inhibition in liver reduces expression of the gluconeogenic genes PEPCK (phosphoenolpyruvate carboxykinase), G6Pase (glucose-6-phosphatase), as well as IGFBP1 (insulin-like growth factor binding protein-1). Overexpression of GSK3 in cells antagonizes insulin regulation of these genes. In the present study we demonstrate that regulation of these three genes by feeding is normal in mice that express insulin-insensitive GSK3. Therefore inactivation of GSK3 is not a prerequisite for insulin repression of these genes, despite the previous finding that GSK3 activity is absolutely required for maintaining their expression. Interestingly, insulin injection of wild-type mice, which activates PKB (protein kinase B) and inhibits GSK3 to a greater degree than feeding (50% versus 25%), does not repress these genes. We suggest for the first time that although pharmacological inhibition of GSK3 reduces hepatic glucose production even in insulin-resistant states, feeding can repress the gluconeogenic genes without inhibiting GSK3. PMID:16176184

An in vitro ES cell imprinting model shows that imprinted expression of the Igf2r gene arises from an allele-specific expression bias

PubMed Central

Latos, Paulina A.; Stricker, Stefan H.; Steenpass, Laura; Pauler, Florian M.; Huang, Ru; Senergin, Basak H.; Regha, Kakkad; Koerner, Martha V.; Warczok, Katarzyna E.; Unger, Christine; Barlow, Denise P.

2010-01-01

Genomic imprinting is an epigenetic process that results in parental-specific gene expression. Advances in understanding the mechanism that regulates imprinted gene expression in mammals have largely depended on generating targeted manipulations in embryonic stem (ES) cells that are analysed in vivo in mice. However, genomic imprinting consists of distinct developmental steps, some of which occur in post-implantation embryos, indicating that they could be studied in vitro in ES cells. The mouse Igf2r gene shows imprinted expression only in post-implantation stages, when repression of the paternal allele has been shown to require cis-expression of the Airn non-coding (nc) RNA and to correlate with gain of DNA methylation and repressive histone modifications. Here we follow the gain of imprinted expression of Igf2r during in vitro ES cell differentiation and show that it coincides with the onset of paternal-specific expression of the Airn ncRNA. Notably, although Airn ncRNA expression leads, as predicted, to gain of repressive epigenetic marks on the paternal Igf2r promoter, we unexpectedly find that the paternal Igf2r promoter is expressed at similar low levels throughout ES cell differentiation. Our results further show that the maternal and paternal Igf2r promoters are expressed equally in undifferentiated ES cells, but during differentiation expression of the maternal Igf2r promoter increases up to 10-fold, while expression from the paternal Igf2r promoter remains constant. This indicates, contrary to expectation, that the Airn ncRNA induces imprinted Igf2r expression not by silencing the paternal Igf2r promoter, but by generating an expression bias between the two parental alleles. PMID:19141673

tCRISPRi: tunable and reversible, one-step control of gene expression

NASA Astrophysics Data System (ADS)

Li, Xin-Tian; Jun, Yonggun; Erickstad, Michael J.; Brown, Steven D.; Parks, Adam; Court, Donald L.; Jun, Suckjoon

2016-12-01

The ability to control the level of gene expression is a major quest in biology. A widely used approach employs deletion of a nonessential gene of interest (knockout), or multi-step recombineering to move a gene of interest under a repressible promoter (knockdown). However, these genetic methods are laborious, and limited for quantitative study. Here, we report a tunable CRISPR-cas system, “tCRISPRi”, for precise and continuous titration of gene expression by more than 30-fold. Our tCRISPRi system employs various previous advancements into a single strain: (1) We constructed a new strain containing a tunable arabinose operon promoter PBAD to quantitatively control the expression of CRISPR-(d)Cas protein over two orders of magnitude in a plasmid-free system. (2) tCRISPRi is reversible, and gene expression is repressed under knockdown conditions. (3) tCRISPRi shows significantly less than 10% leaky expression. (4) Most important from a practical perspective, construction of tCRISPRi to target a new gene requires only one-step of oligo recombineering. Our results show that tCRISPRi, in combination with recombineering, provides a simple and easy-to-implement tool for gene expression control, and is ideally suited for construction of both individual strains and high-throughput tunable knockdown libraries.

A noncoding RNA transcribed from the AGAMOUS (AG) second intron binds to CURLY LEAF and represses AG expression in leaves.

PubMed

Wu, Hui-Wen; Deng, Shulin; Xu, Haiying; Mao, Hui-Zhu; Liu, Jun; Niu, Qi-Wen; Wang, Huan; Chua, Nam-Hai

2018-06-04

Dispersed H3K27 trimethylation (H3K27me3) of the AGAMOUS (AG) genomic locus is mediated by CURLY LEAF (CLF), a component of the Polycomb Repressive Complex (PRC) 2. Previous reports have shown that the AG second intron, which confers AG tissue-specific expression, harbors sequences targeted by several positive and negative regulators. Using RACE reverse transcription polymerase chain reaction, we found that the AG intron 2 encodes several noncoding RNAs. RNAi experiment showed that incRNA4 is needed for CLF repressive activity. AG-incRNA4RNAi lines showed increased leaf AG mRNA levels associated with a decrease of H3K27me3 levels; these plants displayed AG overexpression phenotypes. Genetic and biochemical analyses demonstrated that the AG-incRNA4 can associate with CLF to repress AG expression in leaf tissues through H3K27me3-mediated repression and to autoregulate its own expression level. The mechanism of AG-incRNA4-mediated repression may be relevant to investigations on tissue-specific expression of Arabidopsis MADS-box genes. © 2018 The Authors New Phytologist © 2018 New Phytologist Trust.

The effect of histone deacetylase inhibitors on AHSP expression

PubMed Central

Ziari, Katayoun; Ranjbaran, Reza; Nikouyan, Negin

2018-01-01

Alpha-hemoglobin stabilizing protein (AHSP) is a molecular chaperone that can reduce the damage caused by excess free α-globin to erythroid cells in patients with impaired β-globin chain synthesis. We assessed the effect of sodium phenylbutyrate and sodium valproate, two histone deacetylase inhibitors (HDIs) that are being studied for the treatment of hemoglobinopathies, on the expression of AHSP, BCL11A (all isoforms), γ-globin genes (HBG1/2), and some related transcription factors including GATA1, NFE2, EKLF, KLF4, and STAT3. For this purpose, the K562 cell line was cultured for 2, 4, and 6 days in the presence and absence of sodium phenylbutyrate and sodium valproate. Relative real-time qRT-PCR analysis of mRNA levels was performed to determine the effects of the two compounds on gene expression. Expression of all target mRNAs increased significantly (p < 0.05), except for the expression of BCL11A, which was down-regulated (p < 0.05) in the cells treated with both compounds relative to the levels measured for untreated cells. The findings indicated that sodium valproate had a more considerable effect than sodium phenylbutyrate (p < 0.0005) on BCL11A repression and the up-regulation of other studied genes. γ-Globin and AHSP gene expression continuously increased during the culture period in the treated cells, with the highest gene expression observed for 1 mM sodium valproate after 6 days. Both compounds repressed the expression of BCL11A (-XL, -L, -S) and up-regulated GATA1, NFE2, EKLF, KLF4, STAT3, AHSP, and γ-globin genes expression. Moreover, sodium valproate showed a stronger effect on repressing BCL11A and escalating the expression of other target genes. The findings of this in vitro experiment could be considered in selecting drugs for clinical use in patients with β-hemoglobinopathies. PMID:29389946

RNA-binding protein HuR sequesters microRNA-21 to prevent translation repression of proinflammatory tumor suppressor gene programmed cell death 4.

PubMed

Poria, D K; Guha, A; Nandi, I; Ray, P S

2016-03-31

Translation control of proinflammatory genes has a crucial role in regulating the inflammatory response and preventing chronic inflammation, including a transition to cancer. The proinflammatory tumor suppressor protein programmed cell death 4 (PDCD4) is important for maintaining the balance between inflammation and tumorigenesis. PDCD4 messenger RNA translation is inhibited by the oncogenic microRNA, miR-21. AU-rich element-binding protein HuR was found to interact with the PDCD4 3'-untranslated region (UTR) and prevent miR-21-mediated repression of PDCD4 translation. Cells stably expressing miR-21 showed higher proliferation and reduced apoptosis, which was reversed by HuR expression. Inflammatory stimulus caused nuclear-cytoplasmic relocalization of HuR, reversing the translation repression of PDCD4. Unprecedentedly, HuR was also found to bind to miR-21 directly, preventing its interaction with the PDCD4 3'-UTR, thereby preventing the translation repression of PDCD4. This suggests that HuR might act as a 'miRNA sponge' to regulate miRNA-mediated translation regulation under conditions of stress-induced nuclear-cytoplasmic translocation of HuR, which would allow fine-tuned gene expression in complex regulatory environments.

The multifaceted RisA regulon of Bordetella pertussis

PubMed Central

Coutte, Loïc; Huot, Ludovic; Antoine, Rudy; Slupek, Stephanie; Merkel, Tod J.; Chen, Qing; Stibitz, Scott; Hot, David; Locht, Camille

2016-01-01

The whooping cough agent Bordetella pertussis regulates the production of its virulence factors by the BvgA/S system. Phosphorylated BvgA activates the virulence-activated genes (vags) and represses the expression of the virulence-repressed genes (vrgs) via the activation of the bvgR gene. In modulating conditions, with MgSO4, the BvgA/S system is inactive, and the vrgs are expressed. Here, we show that the expression of almost all vrgs depends on RisA, another transcriptional regulator. We also show that some vags are surprisingly no longer modulated by MgSO4 in the risA− background. RisA also regulates the expression of other genes, including chemotaxis and flagellar operons, iron-regulated genes, and genes of unknown function, which may or may not be controlled by BvgA/S. We identified RisK as the likely cognate RisA kinase and found that it is important for expression of most, but not all RisA-regulated genes. This was confirmed using the phosphoablative RisAD60N and the phosphomimetic RisAD60E analogues. Thus the RisA regulon adds a new layer of complexity to B. pertussis virulence gene regulation. PMID:27620673

The multifaceted RisA regulon of Bordetella pertussis.

PubMed

Coutte, Loïc; Huot, Ludovic; Antoine, Rudy; Slupek, Stephanie; Merkel, Tod J; Chen, Qing; Stibitz, Scott; Hot, David; Locht, Camille

2016-09-13

The whooping cough agent Bordetella pertussis regulates the production of its virulence factors by the BvgA/S system. Phosphorylated BvgA activates the virulence-activated genes (vags) and represses the expression of the virulence-repressed genes (vrgs) via the activation of the bvgR gene. In modulating conditions, with MgSO4, the BvgA/S system is inactive, and the vrgs are expressed. Here, we show that the expression of almost all vrgs depends on RisA, another transcriptional regulator. We also show that some vags are surprisingly no longer modulated by MgSO4 in the risA(-) background. RisA also regulates the expression of other genes, including chemotaxis and flagellar operons, iron-regulated genes, and genes of unknown function, which may or may not be controlled by BvgA/S. We identified RisK as the likely cognate RisA kinase and found that it is important for expression of most, but not all RisA-regulated genes. This was confirmed using the phosphoablative RisAD(60)N and the phosphomimetic RisAD(60)E analogues. Thus the RisA regulon adds a new layer of complexity to B. pertussis virulence gene regulation.

Modulation of Estrogen Response Element-Driven Gene Expressions and Cellular Proliferation with Polar Directions by Designer Transcription Regulators

PubMed Central

Muyan, Mesut; Güpür, Gizem; Yaşar, Pelin; Ayaz, Gamze; User, Sırma Damla; Kazan, Hasan Hüseyin; Huang, Yanfang

2015-01-01

Estrogen receptor α (ERα), as a ligand-dependent transcription factor, mediates 17β-estradiol (E2) effects. ERα is a modular protein containing a DNA binding domain (DBD) and transcription activation domains (AD) located at the amino- and carboxyl-termini. The interaction of the E2-activated ERα dimer with estrogen response elements (EREs) of genes constitutes the initial step in the ERE-dependent signaling pathway necessary for alterations of cellular features. We previously constructed monomeric transcription activators, or monotransactivators, assembled from an engineered ERE-binding module (EBM) using the ERα-DBD and constitutively active ADs from other transcription factors. Monotransactivators modulated cell proliferation by activating and repressing ERE-driven gene expressions that simulate responses observed with E2-ERα. We reasoned here that integration of potent heterologous repression domains (RDs) into EBM could generate monotransrepressors that alter ERE-bearing gene expressions and cellular proliferation in directions opposite to those observed with E2-ERα or monotransactivators. Consistent with this, monotransrepressors suppressed reporter gene expressions that emulate the ERE-dependent signaling pathway. Moreover, a model monotransrepressor regulated DNA synthesis, cell cycle progression and proliferation of recombinant adenovirus infected ER-negative cells through decreasing as well as increasing gene expressions with polar directions compared with E2-ERα or monotransactivator. Our results indicate that an ‘activator’ or a ‘repressor’ possesses both transcription activating/enhancing and repressing/decreasing abilities within a chromatin context. Offering a protein engineering platform to alter signal pathway-specific gene expressions and cell growth, our approach could also be used for the development of tools for epigenetic modifications and for clinical interventions wherein multigenic de-regulations are an issue. PMID:26295471

MCM-BP Is Required for Repression of Life-Cycle Specific Genes Transcribed by RNA Polymerase I in the Mammalian Infectious Form of Trypanosoma brucei

PubMed Central

Kim, Hee-Sook; Park, Sung Hee; Günzl, Arthur; Cross, George A. M.

2013-01-01

Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes escape host immunity by differential expression of VSGs, we attempted to identify genes that contribute to VSG silencing, by performing a loss-of-silencing screen in T. brucei using a transposon-mediated random insertional mutagenesis. One identified gene, which we initially named LOS1, encodes a T. brucei MCM-Binding Protein (TbMCM-BP). Here we show that TbMCM-BP is essential for viability of infectious bloodstream-form (BF) trypanosome and is required for proper cell-cycle progression. Tandem affinity purification of TbMCM-BP followed by mass spectrometry identified four subunits (MCM4-MCM7) of the T. brucei MCM complex, a replicative helicase, and MCM8, a subunit that is uniquely co-purified with TbMCM-BP. TbMCM-BP is required not only for repression of subtelomeric VSGs but also for silencing of life-cycle specific, insect-stage genes, procyclin and procyclin-associated genes (PAGs), that are normally repressed in BF trypanosomes and are transcribed by RNA polymerase I. Our study uncovers a functional link between chromosome maintenance and RNA pol I-mediated gene silencing in T. brucei. PMID:23451133

An Arabidopsis mutant showing reduced feedback inhibition of photosynthesis.

PubMed

Van Oosten, J J; Gerbaud, A; Huijser, C; Dijkwel, P P; Chua, N H; Smeekens, S C

1997-11-01

Many plant genes are responsive to sugars but the mechanisms used by plants to sense sugars are unknown. A genetic approach has been used in Arabidopsis to identify genes involved in perception and transduction of sugar signals. For this purpose, an in vivo reporter system was established consisting of the light- and sugar-regulated plastocyanin promoter, fused to the luciferase coding sequence (PC-LUC construct). At the seedling stage, expression of the PC-LUC gene is repressed by sucrose, and a number of sucrose-uncoupled (sun) mutants were selected in which sucrose is unable to repress the activity of the PC promoter. Three mutants have been characterized in more detail. The sugar analog 2-deoxy-D-glucose (2DG) was used to repress whole plant photosynthesis, PC-LUC gene expression and total ribulose-1,5-bisphosphate activity. It was found that the sun6 mutation makes plants unresponsive to these 2DG-induced effects. Moreover, unlike wild-type plants, sun6 mutants are insensitive to elevated levels of glucose in the growth medium. These findings suggest that the SUN6 gene is active in a hexose-activated signal transduction pathway.

Herpes Simplex Virus Is Equipped with RNA- and Protein-Based Mechanisms To Repress Expression of ATRX, an Effector of Intrinsic Immunity

PubMed Central

Jurak, Igor; Silverstein, Leah B.; Sharma, Mayuri

2012-01-01

Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3′ untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection. PMID:22787211

Herpes simplex virus is equipped with RNA- and protein-based mechanisms to repress expression of ATRX, an effector of intrinsic immunity.

PubMed

Jurak, Igor; Silverstein, Leah B; Sharma, Mayuri; Coen, Donald M

2012-09-01

Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3' untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection.

Sall1 Maintains Nephron Progenitors and Nascent Nephrons by Acting as Both an Activator and a Repressor

PubMed Central

Kanda, Shoichiro; Tanigawa, Shunsuke; Ohmori, Tomoko; Taguchi, Atsuhiro; Kudo, Kuniko; Suzuki, Yutaka; Sato, Yuki; Hino, Shinjiro; Sander, Maike; Perantoni, Alan O.; Sugano, Sumio; Nakao, Mitsuyoshi

2014-01-01

The balanced self-renewal and differentiation of nephron progenitors are critical for kidney development and controlled, in part, by the transcription factor Six2, which antagonizes canonical Wnt signaling-mediated differentiation. A nuclear factor, Sall1, is expressed in Six2-positive progenitors as well as differentiating nascent nephrons, and it is essential for kidney formation. However, the molecular functions and targets of Sall1, especially the functions and targets in the nephron progenitors, remain unknown. Here, we report that Sall1 deletion in Six2-positive nephron progenitors results in severe progenitor depletion and apoptosis of the differentiating nephrons in mice. Analysis of mice with an inducible Sall1 deletion revealed that Sall1 activates genes expressed in progenitors while repressing genes expressed in differentiating nephrons. Sall1 and Six2 co-occupied many progenitor-related gene loci, and Sall1 bound to Six2 biochemically. In contrast, Sall1 did not bind to the Wnt4 locus suppressed by Six2. Sall1-mediated repression was also independent of its binding to DNA. Thus, Sall1 maintains nephron progenitors and their derivatives by a unique mechanism, which partly overlaps but is distinct from that of Six2: Sall1 activates progenitor-related genes in Six2-positive nephron progenitors and represses gene expression in Six2-negative differentiating nascent nephrons. PMID:24744442

Role of the BAHD1 Chromatin-Repressive Complex in Placental Development and Regulation of Steroid Metabolism

PubMed Central

Lakisic, Goran; Wendling, Olivia; Libertini, Emanuele; Radford, Elizabeth J.; Le Guillou, Morwenna; Champy, Marie-France; Wattenhofer-Donzé, Marie; Soubigou, Guillaume; Ait-Si-Ali, Slimane; Feunteun, Jean; Sorg, Tania; Coppée, Jean-Yves; Ferguson-Smith, Anne C.; Cossart, Pascale; Bierne, Hélène

2016-01-01

BAHD1 is a vertebrate protein that promotes heterochromatin formation and gene repression in association with several epigenetic regulators. However, its physiological roles remain unknown. Here, we demonstrate that ablation of the Bahd1 gene results in hypocholesterolemia, hypoglycemia and decreased body fat in mice. It also causes placental growth restriction with a drop of trophoblast glycogen cells, a reduction of fetal weight and a high neonatal mortality rate. By intersecting transcriptome data from murine Bahd1 knockout (KO) placentas at stages E16.5 and E18.5 of gestation, Bahd1-KO embryonic fibroblasts, and human cells stably expressing BAHD1, we also show that changes in BAHD1 levels alter expression of steroid/lipid metabolism genes. Biochemical analysis of the BAHD1-associated multiprotein complex identifies MIER proteins as novel partners of BAHD1 and suggests that BAHD1-MIER interaction forms a hub for histone deacetylases and methyltransferases, chromatin readers and transcription factors. We further show that overexpression of BAHD1 leads to an increase of MIER1 enrichment on the inactive X chromosome (Xi). In addition, BAHD1 and MIER1/3 repress expression of the steroid hormone receptor genes ESR1 and PGR, both playing important roles in placental development and energy metabolism. Moreover, modulation of BAHD1 expression in HEK293 cells triggers epigenetic changes at the ESR1 locus. Together, these results identify BAHD1 as a core component of a chromatin-repressive complex regulating placental morphogenesis and body fat storage and suggest that its dysfunction may contribute to several human diseases. PMID:26938916

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation.

PubMed

Zhyvoloup, Alexander; Melamed, Anat; Anderson, Ian; Planas, Delphine; Lee, Chen-Hsuin; Kriston-Vizi, Janos; Ketteler, Robin; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R M; Fassati, Ariberto

2017-07-01

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

Interlocked feedforward loops control cell-type-specific Rhodopsin expression in the Drosophila eye.

PubMed

Johnston, Robert J; Otake, Yoshiaki; Sood, Pranidhi; Vogt, Nina; Behnia, Rudy; Vasiliauskas, Daniel; McDonald, Elizabeth; Xie, Baotong; Koenig, Sebastian; Wolf, Reinhard; Cook, Tiffany; Gebelein, Brian; Kussell, Edo; Nakagoshi, Hideki; Desplan, Claude

2011-06-10

How complex networks of activators and repressors lead to exquisitely specific cell-type determination during development is poorly understood. In the Drosophila eye, expression patterns of Rhodopsins define at least eight functionally distinct though related subtypes of photoreceptors. Here, we describe a role for the transcription factor gene defective proventriculus (dve) as a critical node in the network regulating Rhodopsin expression. dve is a shared component of two opposing, interlocked feedforward loops (FFLs). Orthodenticle and Dve interact in an incoherent FFL to repress Rhodopsin expression throughout the eye. In R7 and R8 photoreceptors, a coherent FFL relieves repression by Dve while activating Rhodopsin expression. Therefore, this network uses repression to restrict and combinatorial activation to induce cell-type-specific expression. Furthermore, Dve levels are finely tuned to yield cell-type- and region-specific repression or activation outcomes. This interlocked FFL motif may be a general mechanism to control terminal cell-fate specification. Copyright © 2011 Elsevier Inc. All rights reserved.

Salt Sensitive Tet-Off-Like Systems to Knockdown Primordial Germ Cell Genes for Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus.

PubMed

Li, Hanbo; Su, Baofeng; Qin, Guyu; Ye, Zhi; Alsaqufi, Ahmed; Perera, Dayan A; Shang, Mei; Odin, Ramjie; Vo, Khoi; Drescher, David; Robinson, Dalton; Zhang, Dan; Abass, Nermeen; Dunham, Rex A

2017-05-31

Repressible knockdown approaches were investigated for transgenic sterilization in channel catfish, Ictalurus punctatus . Two primordial germ cell (PGC) marker genes, nanos and dead end , were targeted for knockdown, and an off-target gene, vasa , was monitored. Two potentially salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS) and zebrafish racemase (Rm), were each coupled with four knockdown strategies: ds-sh RNA targeting the 5' end (N1) or 3' end (N2) of channel catfish nanos , full-length cDNA sequence of channel catfish nanos for overexpression (cDNA) and ds-sh RNA targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with sodium chloride as the repressor compound. Spawning rates of full-sibling P₁ fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P₁ fish, most F₁ individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F₂ or F₃ are needed for evaluation.

Maternal Rest/Nrsf Regulates Zebrafish Behavior through snap25a/b

PubMed Central

Moravec, Cara E.; Samuel, John; Weng, Wei; Wood, Ian C.

2016-01-01

During embryonic development, regulation of gene expression is key to creating the many subtypes of cells that an organism needs throughout its lifetime. Recent work has shown that maternal genetics and environmental factors have lifelong consequences on diverse processes ranging from immune function to stress responses. The RE1-silencing transcription factor (Rest) is a transcriptional repressor that interacts with chromatin-modifying complexes to repress transcription of neural-specific genes during early development. Here we show that in zebrafish, maternally supplied rest regulates expression of target genes during larval development and has lifelong impacts on behavior. Larvae deprived of maternal rest are hyperactive and show atypical spatial preferences. Adult male fish deprived of maternal rest present with atypical spatial preferences in a novel environment assay. Transcriptome sequencing revealed 158 genes that are repressed by maternal rest in blastula stage embryos. Furthermore, we found that maternal rest is required for target gene repression until at least 6 dpf. Importantly, disruption of the RE1 sites in either snap25a or snap25b resulted in behaviors that recapitulate the hyperactivity phenotype caused by absence of maternal rest. Both maternal rest mutants and snap25a RE1 site mutants have altered primary motor neuron architecture that may account for the enhanced locomotor activity. These results demonstrate that maternal rest represses snap25a/b to modulate larval behavior and that early Rest activity has lifelong behavioral impacts. SIGNIFICANCE STATEMENT Maternal factors deposited in the oocyte have well-established roles during embryonic development. We show that, in zebrafish, maternal rest (RE1-silencing transcription factor) regulates expression of target genes during larval development and has lifelong impacts on behavior. The Rest transcriptional repressor interacts with chromatin-modifying complexes to limit transcription of neural genes. We identify several synaptic genes that are repressed by maternal Rest and demonstrate that snap25a/b are key targets of maternal rest that modulate larval locomotor activity. These results reveal that zygotic rest is unable to compensate for deficits in maternally supplied rest and uncovers novel temporal requirements for Rest activity, which has implications for the broad roles of Rest-mediated repression during neural development and in disease states. PMID:27605615

Population Level Purifying Selection and Gene Expression Shape Subgenome Evolution in Maize.

PubMed

Pophaly, Saurabh D; Tellier, Aurélien

2015-12-01

The maize ancestor experienced a recent whole-genome duplication (WGD) followed by gene erosion which generated two subgenomes, the dominant subgenome (maize1) experiencing fewer deletions than maize2. We take advantage of available extensive polymorphism and gene expression data in maize to study purifying selection and gene expression divergence between WGD retained paralog pairs. We first report a strong correlation in nucleotide diversity between duplicate pairs, except for upstream regions. We then show that maize1 genes are under stronger purifying selection than maize2. WGD retained genes have higher gene dosage and biased Gene Ontologies consistent with previous studies. The relative gene expression of paralogs across tissues demonstrates that 98% of duplicate pairs have either subfunctionalized in a tissuewise manner or have diverged consistently in their expression thereby preventing functional complementation. Tissuewise subfunctionalization seems to be a hallmark of transcription factors, whereas consistent repression occurs for macromolecular complexes. We show that dominant gene expression is a strong determinant of the strength of purifying selection, explaining the inferred stronger negative selection on maize1 genes. We propose a novel expression-based classification of duplicates which is more robust to explain observed polymorphism patterns than the subgenome location. Finally, upstream regions of repressed genes exhibit an enrichment in transposable elements which indicates a possible mechanism for expression divergence. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Prolactin Signaling through the Short Form of Its Receptor Represses Forkhead Transcription Factor FOXO3 and Its Target Gene Galt Causing a Severe Ovarian Defect

PubMed Central

Halperin, Julia; Devi, Sangeeta Y.; Elizur, Shai; Stocco, Carlos; Shehu, Aurora; Rebourcet, Diane; Unterman, Terry G.; Leslie, Nancy D.; Le, Jamie; Binart, Nadine; Gibori, Geula

2008-01-01

Prolactin (PRL) is a hormone with over 300 biological activities. Although the signaling pathway downstream of the long form of its receptor (RL) has been well characterized, little is known about PRL actions upon activation of the short form (RS). Here, we show that mice expressing only RS exhibit an ovarian phenotype of accelerated follicular recruitment followed by massive follicular death leading to premature ovarian failure. Consequently, RS-expressing ovaries of young adults are depleted of functional follicles and formed mostly by interstitium. We also show that activation of RS represses the expression of the transcription factor Forkhead box O3 (FOXO3) and that of the enzyme galactose-1-phosphate uridyltransferase (Galt), two proteins known to be essential for normal follicular development. Our finding that FOXO3 regulates the expression of Galt and enhances its transcriptional activity indicates that it is the repression of FOXO3 by PRL acting through RS that prevents Galt expression in the ovary and causes follicular death. Coexpression of RL with RS prevents PRL inhibition of Galt, and the ovarian defect is no longer seen in RS transgenic mice that coexpress RL, suggesting that RL prevents RS-induced ovarian impairment. In summary, we show that PRL signals through RS and causes, in the absence of RL, a severe ovarian pathology by repressing the expression of FOXO3 and that of its target gene Galt. We also provide evidence of a link between the premature ovarian failure seen in mice expressing RS and in mice with FOXO3 gene deletion as well as in human with Galt mutation. PMID:17975019

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system

PubMed Central

Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C.; Moon, Tae Seok

2016-01-01

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA–asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. PMID:26837577

Dynamic Repositioning of Dorsal to Two Different κB Motifs Controls Its Autoregulation during Immune Response in Drosophila

PubMed Central

Mrinal, Nirotpal; Nagaraju, Javaregowda

2010-01-01

Autoregulation is one of the mechanisms of imparting feedback control on gene expression. Positive autoregulatory feedback results in induction of a gene, and negative feedback leads to its suppression. Here, we report an interesting mechanism of autoregulation operating on Drosophila Rel gene dorsal that can activate as well as repress its expression. Using biochemical and genetic approaches, we show that upon immune challenge Dorsal regulates its activation as well as repression by dynamically binding to two different κB motifs, κBI (intronic κB) and κBP (promoter κB), present in the dorsal gene. Although the κBI motif functions as an enhancer, the κBP motif acts as a transcriptional repressor. Interestingly, Dorsal binding to these two motifs is dynamic; immediately upon immune challenge, Dorsal binds to the κBI leading to auto-activation, whereas at the terminal phase of the immune response, it is removed from the κBI and repositioned at the κBP, resulting in its repression. Furthermore, we show that repression of Dorsal as well as its binding to the κBP depends on the transcription factor AP1. Depletion of AP1 by RNA interference resulted in constitutive expression of Dorsal. In conclusion, this study suggests that during acute phase response dorsal is regulated by following two subcircuits: (i) Dl-κBI for activation and (ii) Dl-AP1-κBP for repression. These two subcircuits are temporally delineated and bring about overall regulation of dorsal during immune response. These results suggest the presence of a previously unknown mechanism of Dorsal autoregulation in immune-challenged Drosophila. PMID:20504768

Gene length as a biological timer to establish temporal transcriptional regulation

PubMed Central

Kirkconnell, Killeen S.; Magnuson, Brian; Paulsen, Michelle T.; Lu, Brian; Bedi, Karan; Ljungman, Mats

2017-01-01

ABSTRACT Transcriptional timing is inherently influenced by gene length, thus providing a mechanism for temporal regulation of gene expression. While gene size has been shown to be important for the expression timing of specific genes during early development, whether it plays a role in the timing of other global gene expression programs has not been extensively explored. Here, we investigate the role of gene length during the early transcriptional response of human fibroblasts to serum stimulation. Using the nascent sequencing techniques Bru-seq and BruUV-seq, we identified immediate genome-wide transcriptional changes following serum stimulation that were linked to rapid activation of enhancer elements. We identified 873 significantly induced and 209 significantly repressed genes. Variations in gene size allowed for a large group of genes to be simultaneously activated but produce full-length RNAs at different times. The median length of the group of serum-induced genes was significantly larger than the median length of all expressed genes, housekeeping genes, and serum-repressed genes. These gene length relationships were also observed in corresponding mouse orthologs, suggesting that relative gene size is evolutionarily conserved. The sizes of transcription factor and microRNA genes immediately induced after serum stimulation varied dramatically, setting up a cascade mechanism for temporal expression arising from a single activation event. The retention and expansion of large intronic sequences during evolution have likely played important roles in fine-tuning the temporal expression of target genes in various cellular response programs. PMID:28055303

Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants

DOE PAGES

Liang, Yan; Richardson, Sarah; Yan, Jingwei; ...

2017-01-17

Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repressmore » transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.« less

Endoribonuclease-Based Two-Component Repressor Systems for Tight Gene Expression Control in Plants

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Yan; Richardson, Sarah; Yan, Jingwei

Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits. In meeting these challenges we will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5'UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repressmore » transgene expression by >400-fold and to synchronize transgene repression. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. Here, we identified 54 orthologous systems from various bacteria, using a bioinformatics approach and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system.« less

MicroRNA MiR-17 retards tissue growth and represses fibronectin expression.

PubMed

Shan, Sze Wan; Lee, Daniel Y; Deng, Zhaoqun; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W; Zhang, Yaou; Xuan, Jim W; Yee, Siu-Pok; Siragam, Vinayakumar; Yang, Burton B

2009-08-01

MicroRNAs (miRNAs) are single-stranded regulatory RNAs, frequently expressed as clusters. Previous studies have demonstrated that the six-miRNA cluster miR-17~92 has important roles in tissue development and cancers. However, the precise role of each miRNA in the cluster is unknown. Here we show that overexpression of miR-17 results in decreased cell adhesion, migration and proliferation. Transgenic mice overexpressing miR-17 showed overall growth retardation, smaller organs and greatly reduced haematopoietic cell lineages. We found that fibronectin and the fibronectin type-III domain containing 3A (FNDC3A) are two targets that have their expression repressed by miR-17, both in vitro and in transgenic mice. Several lines of evidence support the notion that miR-17 causes cellular defects through its repression of fibronectin expression. Our single miRNA expression assay may be evolved to allow the manipulation of individual miRNA functions in vitro and in vivo. We anticipate that this could serve as a model for studying gene regulation by miRNAs in the development of gene therapy.

Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling

PubMed Central

Bourquin, Jean-Pierre; Subramanian, Aravind; Langebrake, Claudia; Reinhardt, Dirk; Bernard, Olivier; Ballerini, Paola; Baruchel, André; Cavé, Hélène; Dastugue, Nicole; Hasle, Henrik; Kaspers, Gertjan L.; Lessard, Michel; Michaux, Lucienne; Vyas, Paresh; van Wering, Elisabeth; Zwaan, Christian M.; Golub, Todd R.; Orkin, Stuart H.

2006-01-01

Individuals with Down syndrome (DS) are predisposed to develop acute megakaryoblastic leukemia (AMKL), characterized by expression of truncated GATA1 transcription factor protein (GATA1s) due to somatic mutation. The treatment outcome for DS-AMKL is more favorable than for AMKL in non-DS patients. To gain insight into gene expression differences in AMKL, we compared 24 DS and 39 non-DS AMKL samples. We found that non-DS-AMKL samples cluster in two groups, characterized by differences in expression of HOX/TALE family members. Both of these groups are distinct from DS-AMKL, independent of chromosome 21 gene expression. To explore alterations of the GATA1 transcriptome, we used cross-species comparison with genes regulated by GATA1 expression in murine erythroid precursors. Genes repressed after GATA1 induction in the murine system, most notably GATA-2, MYC, and KIT, show increased expression in DS-AMKL, suggesting that GATA1s fail to repress this class of genes. Only a subset of genes that are up-regulated upon GATA1 induction in the murine system show increased expression in DS-AMKL, including GATA1 and BACH1, a probable negative regulator of megakaryocytic differentiation located on chromosome 21. Surprisingly, expression of the chromosome 21 gene RUNX1, a known regulator of megakaryopoiesis, was not elevated in DS-AMKL. Our results identify relevant signatures for distinct AMKL entities and provide insight into gene expression changes associated with these related leukemias. PMID:16492768

Location of Promoter and Operator Sites in the Biotin Gene Cluster of Escherichia coli

PubMed Central

Cleary, Paul P.; Campbell, Allan; Chang, Robin

1972-01-01

Biotin independence in E. coli requires five closely linked genes, bioA, bioB, bioF, bioC, and bioD. The residual gene activity of deletion mutants has been studied by complementation and enzyme assays. Deletion of the left end of the bioA gene does not impair expression of the remaining genes, but deletions from the left extending into bioB abolish all gene expression. Nonsense mutations in bioB reduce expression of bioC, bioF, and bioD. Therefore, the four genes, bioB, bioF, bioC, and bioD, are transcribed as a unit from left to right, from a promotor located between bioA and bioB. Expression of the bio genes is repressible by added biotin. Deletions removing the left end of bioA do not affect repressibility of bioD. Therefore the operator, as well as the promoter, lie to the right of bioA. One deletion that removes bioA, bioB, and bioF renders the bioD gene constitutive, presumably by fusion to an unknown operon. Therefore, the operator lies to the left of bioC. PMID:4559599

Divergent role of the Hox gene Antennapedia in spiders is responsible for the convergent evolution of abdominal limb repression.

PubMed

Khadjeh, Sara; Turetzek, Natascha; Pechmann, Matthias; Schwager, Evelyn E; Wimmer, Ernst A; Damen, Wim G M; Prpic, Nikola-Michael

2012-03-27

Evolution often results in morphologically similar solutions in different organisms, a phenomenon known as convergence. However, there is little knowledge of the processes that lead to convergence at the genetic level. The genes of the Hox cluster control morphology in animals. They may also be central to the convergence of morphological traits, but whether morphological similarities also require similar changes in Hox gene function is disputed. In arthropods, body subdivision into a region with locomotory appendages ("thorax") and a region with reduced appendages ("abdomen") has evolved convergently in several groups, e.g., spiders and insects. In insects, legs develop in the expression domain of the Hox gene Antennapedia (Antp), whereas the Hox genes Ultrabithorax (Ubx) and abdominal-A mediate leg repression in the abdomen. Here, we show that, unlike Antp in insects, the Antp gene in the spider Achaearanea tepidariorum represses legs in the first segment of the abdomen (opisthosoma), and that Antp and Ubx are redundant in the following segment. The down-regulation of Antp in A. tepidariorum leads to a striking 10-legged phenotype. We present evidence from ectopic expression of the spider Antp gene in Drosophila embryos and imaginal tissue that this unique function of Antp is not due to changes in the Antp protein, but likely due to divergent evolution of cofactors, Hox collaborators or target genes in spiders and flies. Our results illustrate an interesting example of convergent evolution of abdominal leg repression in arthropods by altering the role of distinct Hox genes at different levels of their action.

Histone deacetylase inhibitor belinostat represses survivin expression through reactivation of transforming growth factor beta (TGFbeta) receptor II leading to cancer cell death.

PubMed

Chowdhury, Sanjib; Howell, Gillian M; Teggart, Carol A; Chowdhury, Aparajita; Person, Jonathan J; Bowers, Dawn M; Brattain, Michael G

2011-09-02

Survivin is a cancer-associated gene that functions to promote cell survival, cell division, and angiogenesis and is a marker of poor prognosis. Histone deacetylase inhibitors induce apoptosis and re-expression of epigenetically silenced tumor suppressor genes in cancer cells. In association with increased expression of the tumor suppressor gene transforming growth factor β receptor II (TGFβRII) induced by the histone deacetylase inhibitor belinostat, we observed repressed survivin expression. We investigated the molecular mechanisms involved in survivin down-regulation by belinostat downstream of reactivation of TGFβ signaling. We identified two mechanisms. At early time points, survivin protein half-life was decreased with its proteasomal degradation. We observed that belinostat activated protein kinase A at early time points in a TGFβ signaling-dependent mechanism. After longer times (48 h), survivin mRNA was also decreased by belinostat. We made the novel observation that belinostat mediated cell death through the TGFβ/protein kinase A signaling pathway. Induction of TGFβRII with concomitant survivin repression may represent a significant mechanism in the anticancer effects of this drug. Therefore, patient populations exhibiting high survivin expression with epigenetically silenced TGFβRII might potentially benefit from the use of this histone deacetylase inhibitor.

Regulation of mRNA translation during mitosis

PubMed Central

Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

2015-01-01

Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ∼200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function. DOI: http://dx.doi.org/10.7554/eLife.07957.001 PMID:26305499

Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression.

PubMed

Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Matagne, Valerie; Toro, Carlos A; Ramaswamy, Suresh; Plant, Tony M; Ojeda, Sergio R

2015-12-16

In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty.

Identification of a phorbol ester-repressible v-src-inducible gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simmons, D.L.; Levy, D.B.; Yannoni, Y.

1989-02-01

Chicken embryo fibroblasts (CEF) infected with a temperature-sensitive Rous sarcoma virus (RSV) mutant, tsNY72-4, express a set of pp60{sup v-src}-induced RNAs soon after shift to the permissive temperature. By subtractive and differential screening, the authors have cloned 12 of these sequences, 2 of which were c-fos and krox-24. Serum induced all the v-src-inducible genes tested, suggesting that these genes serve roles in normal cell division and are not specific to transformation per se. Significantly, however, v-src produced prolonged, and in some cases kinetically complex, patterns of induction compared to serum. For most of the clones, phorbol 12-tetradecanoate 13-acetate (TPA) inducedmore » mRNAs with kinetics similar to that of serum. However, one clone (CEF-4) was expressed in a biphasic manner. Another (CEF-10) was repressed by TPA at 1 hr, after which this mRNA was permanently induced. The pattern of repression-induction of CEF-10 mRNA is the inverse of protein kinase C (PKC) activity in the cell, suggesting that PKC actively represses this gene. In vivo expression of CEF-10 mRNA is restricted predominantly to the lung. A full-length CEF-10 cDNA encodes a 41-kDa protein that has an amino-terminal signal peptide for secretion, contains a markedly high number of cysteine residues, and shows no sequence similarity to known proteins.« less

Different Levels of Catabolite Repression Optimize Growth in Stable and Variable Environments

PubMed Central

New, Aaron M.; Cerulus, Bram; Govers, Sander K.; Perez-Samper, Gemma; Zhu, Bo; Boogmans, Sarah; Xavier, Joao B.; Verstrepen, Kevin J.

2014-01-01

Organisms respond to environmental changes by adapting the expression of key genes. However, such transcriptional reprogramming requires time and energy, and may also leave the organism ill-adapted when the original environment returns. Here, we study the dynamics of transcriptional reprogramming and fitness in the model eukaryote Saccharomyces cerevisiae in response to changing carbon environments. Population and single-cell analyses reveal that some wild yeast strains rapidly and uniformly adapt gene expression and growth to changing carbon sources, whereas other strains respond more slowly, resulting in long periods of slow growth (the so-called “lag phase”) and large differences between individual cells within the population. We exploit this natural heterogeneity to evolve a set of mutants that demonstrate how the frequency and duration of changes in carbon source can favor different carbon catabolite repression strategies. At one end of this spectrum are “specialist” strategies that display high rates of growth in stable environments, with more stringent catabolite repression and slower transcriptional reprogramming. The other mutants display less stringent catabolite repression, resulting in leaky expression of genes that are not required for growth in glucose. This “generalist” strategy reduces fitness in glucose, but allows faster transcriptional reprogramming and shorter lag phases when the cells need to shift to alternative carbon sources. Whole-genome sequencing of these mutants reveals that mutations in key regulatory genes such as HXK2 and STD1 adjust the regulation and transcriptional noise of metabolic genes, with some mutations leading to alternative gene regulatory strategies that allow “stochastic sensing” of the environment. Together, our study unmasks how variable and stable environments favor distinct strategies of transcriptional reprogramming and growth. PMID:24453942

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

PubMed Central

Butler, J S; Springer, M; Grunberg-Manago, M

1987-01-01

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo. PMID:2954162

AUU-to-AUG mutation in the initiator codon of the translation initiation factor IF3 abolishes translational autocontrol of its own gene (infC) in vivo.

PubMed

Butler, J S; Springer, M; Grunberg-Manago, M

1987-06-01

We previously showed that Escherichia coli translation initiation factor IF3 regulates the expression of its own gene infC at the translational level in vivo. Here we create two alterations in the infC gene and test their effects on translational autocontrol of infC expression in vivo by measuring beta-galactosidase activity expressed from infC-lacZ gene fusions under conditions of up to 4-fold derepression or 3-fold repression of infC expression. Replacement of the infC promoter with the trp promoter deletes 120 nucleotides of the infC mRNA 5' to the translation initiation site without affecting autogenous translational control. Mutation of the unusual AUU initiator codon of infC to the more common AUG initiator codon abolishes translation initiation factor IF3-dependent repression and derepression of infC expression in vivo. These results establish the AUU initiator codon of infC as an essential cis-acting element in autogenous translational control of translation initiation factor IF3 expression in vivo.

Repression of myoblast proliferation and fibroblast growth factor receptor 1 promoter activity by KLF10 protein.

PubMed

Parakati, Rajini; DiMario, Joseph X

2013-05-10

FGFR1 gene expression regulates myoblast proliferation and differentiation, and its expression is controlled by Krüppel-like transcription factors. KLF10 interacts with the FGFR1 promoter, repressing its activity and cell proliferation. KLF10 represses FGFR1 promoter activity and thereby myoblast proliferation. A model of transcriptional control of chicken FGFR1 gene regulation during myogenesis is presented. Skeletal muscle development is controlled by regulation of myoblast proliferation and differentiation into muscle fibers. Growth factors such as fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate cell proliferation and differentiation in numerous tissues, including skeletal muscle. Transcriptional regulation of FGFR1 gene expression is developmentally regulated by the Sp1 transcription factor, a member of the Krüppel-like factor (KLF) family of transcriptional regulators. Here, we show that another KLF transcription factor, KLF10, also regulates myoblast proliferation and FGFR1 promoter activity. Expression of KLF10 reduced myoblast proliferation by 86%. KLF10 expression also significantly reduced FGFR1 promoter activity in myoblasts and Sp1-mediated FGFR1 promoter activity in Drosophila SL2 cells. Southwestern blot, electromobility shift, and chromatin immunoprecipitation assays demonstrated that KLF10 bound to the proximal Sp factor binding site of the FGFR1 promoter and reduced Sp1 complex formation with the FGFR1 promoter at that site. These results indicate that KLF10 is an effective repressor of myoblast proliferation and represses FGFR1 promoter activity in these cells via an Sp1 binding site.

Telomeric repeat-containing RNA/G-quadruplex-forming sequences cause genome-wide alteration of gene expression in human cancer cells in vivo.

PubMed

Hirashima, Kyotaro; Seimiya, Hiroyuki

2015-02-27

Telomere erosion causes cell mortality, suggesting that longer telomeres enable more cell divisions. In telomerase-positive human cancer cells, however, telomeres are often kept shorter than those of surrounding normal tissues. Recently, we showed that cancer cell telomere elongation represses innate immune genes and promotes their differentiation in vivo. This implies that short telomeres contribute to cancer malignancy, but it is unclear how such genetic repression is caused by elongated telomeres. Here, we report that telomeric repeat-containing RNA (TERRA) induces a genome-wide alteration of gene expression in telomere-elongated cancer cells. Using three different cell lines, we found that telomere elongation up-regulates TERRA signal and down-regulates innate immune genes such as STAT1, ISG15 and OAS3 in vivo. Ectopic TERRA oligonucleotides repressed these genes even in cells with short telomeres under three-dimensional culture conditions. This appeared to occur from the action of G-quadruplexes (G4) in TERRA, because control oligonucleotides had no effect and a nontelomeric G4-forming oligonucleotide phenocopied the TERRA oligonucleotide. Telomere elongation and G4-forming oligonucleotides showed similar gene expression signatures. Most of the commonly suppressed genes were involved in the innate immune system and were up-regulated in various cancers. We propose that TERRA G4 counteracts cancer malignancy by suppressing innate immune genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

PPARγ partial agonist GQ-16 strongly represses a subset of genes in 3T3-L1 adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Milton, Flora Aparecida; Genomic Medicine, Houston Methodist Research Institute, Houston, TX; Cvoro, Aleksandra

Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists that improve insulin resistance but trigger side effects such as weight gain, edema, congestive heart failure and bone loss. GQ-16 is a PPARγ partial agonist that improves glucose tolerance and insulin sensitivity in mouse models of obesity and diabetes without inducing weight gain or edema. It is not clear whether GQ-16 acts as a partial agonist at all PPARγ target genes, or whether it displays gene-selective actions. To determine how GQ-16 influences PPARγ activity on a gene by gene basis, we compared effects of rosiglitazone (Rosi) and GQ-16 in mature 3T3-L1more » adipocytes using microarray and qRT-PCR. Rosi changed expression of 1156 genes in 3T3-L1, but GQ-16 only changed 89 genes. GQ-16 generally showed weak effects upon Rosi induced genes, consistent with partial agonist actions, but a subset of modestly Rosi induced and strongly repressed genes displayed disproportionately strong GQ-16 responses. PPARγ partial agonists MLR24 and SR1664 also exhibit disproportionately strong effects on transcriptional repression. We conclude that GQ-16 displays a continuum of weak partial agonist effects but efficiently represses some negatively regulated PPARγ responsive genes. Strong repressive effects could contribute to physiologic actions of GQ-16. - Highlights: • GQ-16 is an insulin sensitizing PPARγ ligand with reduced harmful side effects. • GQ-16 displays a continuum of weak partial agonist activities at PPARγ-induced genes. • GQ-16 exerts strong repressive effects at a subset of genes. • These inhibitor actions should be evaluated in models of adipose tissue inflammation.« less

Untangling the Web: The Diverse Functions of the PIWI/piRNA Pathway

PubMed Central

Mani, Sneha Ramesh; Juliano, Celina E.

2014-01-01

SUMMARY Small RNAs impact several cellular processes through gene regulation. Argonaute proteins bind small RNAs to form effector complexes that control transcriptional and post-transcriptional gene expression. PIWI proteins belong to the Argonaute protein family, and bind PIWI-interacting RNAs (piRNAs). They are highly abundant in the germline, but are also expressed in some somatic tissues. The PIWI/piRNA pathway has a role in transposon repression in Drosophila, which occurs both by epigenetic regulation and post-transcriptional degradation of transposon mRNAs. These functions are conserved, but clear differences in the extent and mechanism of transposon repression exist between species. Mutations in piwi genes lead to the upregulation of transposon mRNAs. It is hypothesized that this increased transposon mobilization leads to genomic instability and thus sterility, although no causal link has been established between transposon upregulation and genome instability. An alternative scenario could be that piwi mutations directly affect genomic instability, and thus lead to increased transposon expression. We propose that the PIWI/piRNA pathway controls genome stability in several ways: suppression of transposons, direct regulation of chromatin architecture and regulation of genes that control important biological processes related to genome stability. The PIWI/piRNA pathway also regulates at least some, if not many, protein-coding genes, which further lends support to the idea that piwi genes may have broader functions beyond transposon repression. An intriguing possibility is that the PIWI/piRNA pathway is using transposon sequences to coordinate the expression of large groups of genes to regulate cellular function. PMID:23712694

Acetylation of hMOF Modulates H4K16ac to Regulate DNA Repair Genes in Response to Oxidative Stress.

PubMed

Zhong, Jianing; Ji, Liying; Chen, Huiqian; Li, Xianfeng; Zhang, Jian'an; Wang, Xingxing; Wu, Weilin; Xu, Ying; Huang, Fei; Cai, Wanshi; Sun, Zhong Sheng

2017-01-01

Oxidative stress is considered to be a key risk state for a variety of human diseases. In response to oxidative stress, the regulation of transcriptional expression of DNA repair genes would be important to DNA repair and genomic stability. However, the overall pattern of transcriptional expression of DNA repair genes and the underlying molecular response mechanism to oxidative stress remain unclear. Here, by employing colorectal cancer cell lines following exposure to hydrogen peroxide, we generated expression profiles of DNA repair genes via RNA-seq and identified gene subsets that are induced or repressed following oxidative stress exposure. RRBS-seq analyses further indicated that transcriptional regulation of most of the DNA repair genes that were induced or repressed is independent of their DNA methylation status. Our analyses also indicate that hydrogen peroxide induces deacetylase SIRT1 which decreases chromatin affinity and the activity of histone acetyltransferase hMOF toward H4K16ac and results in decreased transcriptional expression of DNA repair genes. Taken together, our findings provide a potential mechanism by which oxidative stress suppresses DNA repair genes which is independent of the DNA methylation status of their promoters.

The HSV-1 Latency-Associated Transcript Functions to Repress Latent Phase Lytic Gene Expression and Suppress Virus Reactivation from Latently Infected Neurons

PubMed Central

Nicoll, Michael P.; Hann, William; Shivkumar, Maitreyi; Harman, Laura E. R.; Connor, Viv; Coleman, Heather M.; Proença, João T.; Efstathiou, Stacey

2016-01-01

Herpes simplex virus 1 (HSV-1) establishes life-long latent infection within sensory neurons, during which viral lytic gene expression is silenced. The only highly expressed viral gene product during latent infection is the latency-associated transcript (LAT), a non-protein coding RNA that has been strongly implicated in the epigenetic regulation of HSV-1 gene expression. We have investigated LAT-mediated control of latent gene expression using chromatin immunoprecipitation analyses and LAT-negative viruses engineered to express firefly luciferase or β-galactosidase from a heterologous lytic promoter. Whilst we were unable to determine a significant effect of LAT expression upon heterochromatin enrichment on latent HSV-1 genomes, we show that reporter gene expression from latent HSV-1 genomes occurs at a greater frequency in the absence of LAT. Furthermore, using luciferase reporter viruses we have observed that HSV-1 gene expression decreases during long-term latent infection, with a most marked effect during LAT-negative virus infection. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. Together, our data suggest that the HSV-1 LAT RNA represses HSV-1 gene expression in small populations of neurons within the mouse TG, a phenomenon that directly impacts upon the frequency of reactivation and the maintenance of the transcriptionally active latent reservoir. PMID:27055281

Comparative expression profiling of Nicotiana benthamiana leaves systemically infected with three fruit tree viruses.

PubMed

Dardick, Christopher

2007-08-01

Plant viruses cause a wide array of disease symptoms and cytopathic effects. Although some of these changes are virus specific, many appear to be common even among diverse viruses. Currently, little is known about the underlying molecular determinants. To identify gene expression changes that are concomitant with virus symptoms, we performed comparative expression profiling experiments on Nicotiana benthamiana leaves infected with one of three different fruit tree viruses that produce distinct symptoms: Plum pox potyvirus (PPV; leaf distortion and mosaic), Tomato ringspot nepovirus (ToRSV; tissue necrosis and general chlorosis), and Prunus necrotic ringspot ilarvirus (PNRSV; subtle chlorotic mottling). The numbers of statistically significant genes identified were consistent with the severity of the observed symptoms: 1,082 (ToRSV), 744 (PPV), and 89 (PNRSV). In all, 56% of the gene expression changes found in PPV-infected leaves also were altered by ToRSV, 87% of which changed in the same direction. Both PPV- and ToRSV-infected leaves showed widespread repression of genes associated with plastid functions. PPV uniquely induced the expression of large numbers of cytosolic ribosomal genes whereas ToRSV repressed the expression of plastidic ribosomal genes. How these and other observed expression changes might be associated with symptom development are discussed.

Blue light-mediated transcriptional activation and repression of gene expression in bacteria

PubMed Central

Jayaraman, Premkumar; Devarajan, Kavya; Chua, Tze Kwang; Zhang, Hanzhong; Gunawan, Erry; Poh, Chueh Loo

2016-01-01

Light-regulated modules offer unprecedented new ways to control cellular behavior in precise spatial and temporal resolution. The availability of such tools may dramatically accelerate the progression of synthetic biology applications. Nonetheless, current optogenetic toolbox of prokaryotes has potential issues such as lack of rapid and switchable control, less portable, low dynamic expression and limited parts. To address these shortcomings, we have engineered a novel bidirectional promoter system for Escherichia coli that can be induced or repressed rapidly and reversibly using the blue light dependent DNA-binding protein EL222. We demonstrated that by modulating the dosage of light pulses or intensity we could control the level of gene expression precisely. We show that both light-inducible and repressible system can function in parallel with high spatial precision in a single cell and can be switched stably between ON- and OFF-states by repetitive pulses of blue light. In addition, the light-inducible and repressible expression kinetics were quantitatively analysed using a mathematical model. We further apply the system, for the first time, to optogenetically synchronize two receiver cells performing different logic behaviors over time using blue light as a molecular clock signal. Overall, our modular approach layers a transformative platform for next-generation light-controllable synthetic biology systems in prokaryotes. PMID:27353329

Yeast carbon catabolite repression.

PubMed

Gancedo, J M

1998-06-01

Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources; some of these genes are also repressed by other sugars such as galactose, and the process is known as catabolite repression. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins, but there are different circuits of repression for different groups of genes. However, the protein kinase Snf1/Cat1 is shared by the various circuits and is therefore a central element in the regulatory process. Snf1 is not operative in the presence of glucose, and preliminary evidence suggests that Snf1 is in a dephosphorylated state under these conditions. However, the enzymes that phosphorylate and dephosphorylate Snf1 have not been identified, and it is not known how the presence of glucose may affect their activity. What has been established is that Snf1 remains active in mutants lacking either the proteins Grr1/Cat80 or Hxk2 or the Glc7 complex, which functions as a protein phosphatase. One of the main roles of Snf1 is to relieve repression by the Mig1 complex, but it is also required for the operation of transcription factors such as Adr1 and possibly other factors that are still unidentified. Although our knowledge of catabolite repression is still very incomplete, it is possible in certain cases to propose a partial model of the way in which the different elements involved in catabolite repression may be integrated.

Streptomyces-Aspergillus flavus interactions: impact on aflatoxin B accumulation.

PubMed

Verheecke, C; Liboz, T; Anson, P; Zhu, Y; Mathieu, F

2015-01-01

The aim of this work was to investigate the potential of Streptomyces sp. as biocontrol agents against aflatoxins in maize. As such, we assumed that Streptomyces sp. could provide a complementary approach to current biocontrol systems such as Afla-guard(®) and we focused on biocontrol that was able to have an antagonistic contact with A. flavus. A previous study showed that 27 (out of 38) Streptomyces sp. had mutual antagonism in contact with A. flavus. Among these, 16 Streptomyces sp. were able to reduce aflatoxin content to below 17% of the residual concentration. We selected six strains to understand the mechanisms involved in the prevention of aflatoxin accumulation. Thus, in interaction with A. flavus, we monitored by RT-qPCR the gene expression of aflD, aflM, aflP, aflR and aflS. All the Streptomyces sp. were able to reduce aflatoxin concentration (24.0-0.2% residual aflatoxin B1). They all impacted on gene expression, but only S35 and S38 were able to repress expression significantly. Indeed, S35 significantly repressed aflM expression and S38 significantly repressed aflR, aflM and aflP. S6 reduced aflatoxin concentrations (2.3% residual aflatoxin B1) and repressed aflS, aflM and enhanced aflR expression. In addition, the S6 strain (previously identified as the most reducing pure aflatoxin B1) was further tested to determine a potential adsorption mechanism. We did not observe any adsorption phenomenon. In conclusion, this study showed that Streptomyces sp. prevent the production of (aflatoxin gene expression) and decontamination of (aflatoxin B1 reduction) aflatoxins in vitro.

Chorion gene activation and repression is dependent on BmC/EBP expression and binding to cognate cis-elements.

PubMed

Papantonis, Argyris; Sourmeli, Sissy; Lecanidou, Rena

2008-05-09

From the different cis-elements clustered on silkmoth chorion gene promoters, C/EBP binding sites predominate. Their sequence composition and dispersal vary amongst promoters of diverse developmental specificity. Occupancy of these sites by BmC/EBP was examined through Southwestern and ChIP assays modified to suit ovarian follicular cells. For the genes studied, binding of BmC/EBP coincided with the respective stages of transcriptional activation. However, the factor was reloaded on promoter sequences long after individual gene repression. Furthermore, suppression of BmC/EBP transcription in developing follicles resulted in de-regulation of chorion gene expression. A biphasic function of BmC/EBP, according to which it may act as both an activator and a repressor during silkmoth choriogenesis, is considered under the light of the presented data.

ATF3 represses PPARγ expression and inhibits adipocyte differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jang, Min-Kyung; Jung, Myeong Ho, E-mail: jung0603@pusan.ac.kr

Highlights: • ATF3 decrease the expression of PPARγ and its target gene in 3T3-L1 adipocytes. • ATF3 represses the promoter activity of PPARγ2 gene. • ATF/CRE (−1537/−1530) is critical for ATF3-mediated downregulation of PPARγ. • ATF3 binds to the promoter region containing the ATF/CRE. • ER stress inhibits adipocyte differentiation through downregulation of PPARγ by ATF3. - Abstract: Activating transcription factor 3 (ATF3) is a stress-adaptive transcription factor that mediates cellular stress response signaling. We previously reported that ATF3 represses CCAAT/enhancer binding protein α (C/EBPα) expression and inhibits 3T3-L1 adipocyte differentiation. In this study, we explored potential role of ATF3more » in negatively regulating peroxisome proliferator activated receptor-γ (PPARγ). ATF3 decreased the expression of PPARγ and its target gene in 3T3-L1 adipocytes. ATF3 also repressed the activity of −2.6 Kb promoter of mouse PPARγ2. Overexpression of PPARγ significantly prevented the ATF3-mediated inhibition of 3T3-L1 differentiation. Transfection studies with 5′ deleted-reporters showed that ATF3 repressed the activity of −2037 bp promoter, whereas it did not affect the activity of −1458 bp promoter, suggesting that ATF3 responsive element is located between the −2037 and −1458. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 binds to ATF/CRE site (5′-TGACGTTT-3′) between −1537 and −1530. Mutation of the ATF/CRE site abrogated ATF3-mediated transrepression of the PPARγ2 promoter. Treatment with thapsigargin, endoplasmic reticulum (ER) stress inducer, increased ATF3 expression, whereas it decreased PPARγ expression. ATF3 knockdown significantly blocked the thapsigargin-mediated downregulation of PPARγ expression. Furthermore, overexpression of PPARγ prevented inhibition of 3T3-L1 differentiation by thapsigargin. Collectively, these results suggest that ATF3-mediated inhibition of PPARγ expression may contribute to inhibition of adipocyte differentiation during cellular stress including ER stress.« less

Wnt-Mediated Repression via Bipartite DNA Recognition by TCF in the Drosophila Hematopoietic System

PubMed Central

Zhang, Chen U.; Blauwkamp, Timothy A.; Burby, Peter E.; Cadigan, Ken M.

2014-01-01

The Wnt/β-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/β-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/β-catenin signaling and allosteric regulation of a transcription factor by DNA. PMID:25144371

Gene expression profiles in peripheral blood mononuclear cells of Chinese nickel refinery workers with high exposures to nickel and control subjects

PubMed Central

Arita, Adriana; Muñoz, Alexandra; Chervona, Yana; Niu, Jingping; Qu, Qingshan; Zhao, Najuan; Ruan, Ye; Kiok, Kathrin; Kluz, Thomas; Sun, Hong; Clancy, Hailey A.; Shamy, Magdy; Costa, Max

2012-01-01

Background Occupational exposure to nickel (Ni) is associated with an increased risk of lung and nasal cancers. Ni compounds exhibit weak mutagenic activity, alter the cell’s epigenetic homeostasis, and activate signaling pathways. However, changes in gene expression associated with Ni exposure have only been investigated in vitro. This study was conducted in a Chinese population to determine whether occupational exposure to Ni was associated with differential gene expression profiles in the peripheral blood mononuclear cells (PBMCs) of Ni-refinery workers when compared to referents. Methods Eight Ni-refinery workers and ten referents were selected. PBMC RNA was extracted and gene expression profiling was performed using Affymetrix exon arrays. Differentially expressed genes between both groups were identified in a global analysis. Results There were a total of 2756 differentially expressed genes (DEG) in the Ni-refinery workers relative to the control subjects (FDR adjusted p<0.05) with 770 up-regulated genes and 1986 down-regulated genes. DNA repair and epigenetic genes were significantly overrepresented (p< 0.0002) among the DEG. Of 31 DNA repair genes, 29 were repressed in the high exposure group and two were overexpressed. Of the 16 epigenetic genes 12 were repressed in the high exposure group and 4 were overexpressed. Conclusions The results of this study indicate that occupational exposure to Ni is associated with alterations in gene expression profiles in PBMCs of subjects. Impact Gene expression may be useful in identifying patterns of deregulation that precede clinical identification of Ni-induced cancers. PMID:23195993

Achieving large dynamic range control of gene expression with a compact RNA transcription–translation regulator

PubMed Central

2017-01-01

Abstract RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression leak, which impedes the construction of larger synthetic regulatory networks as leak propagation can interfere with desired network function. To address this, we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and ribosome binding site sequestration increases repression from 85% to 98%, or activation from 10-fold to over 900-fold, in response to cognate antisense RNAs. We also show that orthogonal repressive versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this mechanism, we use it to reduce network leak in an RNA-only cascade. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of more sophisticated regulatory networks. PMID:28387839

Functional analysis of the global repressor Tup1 for maltose metabolism in Saccharomyces cerevisiae: different roles of the functional domains.

PubMed

Lin, Xue; Yu, Ai-Qun; Zhang, Cui-Ying; Pi, Li; Bai, Xiao-Wen; Xiao, Dong-Guang

2017-11-09

Tup1 is a general transcriptional repressor of diverse gene families coordinately controlled by glucose repression, mating type, and other mechanisms in Saccharomyces cerevisiae. Several functional domains of Tup1 have been identified, each of which has differing effects on transcriptional repression. In this study, we aim to investigate the role of Tup1 and its domains in maltose metabolism of industrial baker's yeast. To this end, a battery of in-frame truncations in the TUP1 gene coding region were performed in the industrial baker's yeasts with different genetic background, and the maltose metabolism, leavening ability, MAL gene expression levels, and growth characteristics were investigated. The results suggest that the TUP1 gene is essential to maltose metabolism in industrial baker's yeast. Importantly, different domains of Tup1 play different roles in glucose repression and maltose metabolism of industrial baker's yeast cells. The Ssn6 interaction, N-terminal repression and C-terminal repression domains might play roles in the regulation of MAL transcription by Tup1 for maltose metabolism of baker's yeast. The WD region lacking the first repeat could influence the regulation of maltose metabolism directly, rather than indirectly through glucose repression. These findings lay a foundation for the optimization of industrial baker's yeast strains for accelerated maltose metabolism and facilitate future research on glucose repression in other sugar metabolism.

Gene repression via multiplex gRNA strategy in Y. lipolytica.

PubMed

Zhang, Jin-Lai; Peng, Yang-Zi; Liu, Duo; Liu, Hong; Cao, Ying-Xiu; Li, Bing-Zhi; Li, Chun; Yuan, Ying-Jin

2018-04-20

The oleaginous yeast Yarrowia lipolytica is a promising microbial cell factory due to their biochemical characteristics and native capacity to accumulate lipid-based chemicals. To create heterogenous biosynthesis pathway and manipulate metabolic flux in Y. lipolytica, numerous studies have been done for developing synthetic biology tools for gene regulation. CRISPR interference (CRISPRi), as an emerging technology, has been applied for specifically repressing genes of interest. In this study, we established CRISPRi systems in Y. lipolytica based on four different repressors, that was DNase-deactivated Cpf1 (dCpf1) from Francisella novicida, deactivated Cas9 (dCas9) from Streptococcus pyogenes, and two fusion proteins (dCpf1-KRAB and dCas9-KRAB). Ten gRNAs that bound to different regions of gfp gene were designed and the results indicated that there was no clear correlation between the repression efficiency and targeting sites no matter which repressor protein was used. In order to rapidly yield strong gene repression, a multiplex gRNAs strategy based on one-step Golden-brick assembly technology was developed. High repression efficiency 85% (dCpf1) and 92% (dCas9) were achieved in a short time by making three different gRNAs towards gfp gene simultaneously, which avoided the need of screening effective gRNA loci in advance. Moreover, two genes interference including gfp and vioE and three genes repression including vioA, vioB and vioE in protodeoxy-violaceinic acid pathway were also realized. Taken together, successful CRISPRi-mediated regulation of gene expression via four different repressors dCpf1, dCas9, dCpf1-KRAB and dCas9-KRAB in Y. lipolytica is achieved. And we demonstrate a multiplexed gRNA targeting strategy can efficiently achieve transcriptional simultaneous repression of several targeted genes and different sites of one gene using the one-step Golden-brick assembly. This timesaving method promised to be a potent transformative tool valuable for metabolic engineering, synthetic biology, and functional genomic studies of Y. lipolytica.

Salt Sensitive Tet-Off-Like Systems to Knockdown Primordial Germ Cell Genes for Repressible Transgenic Sterilization in Channel Catfish, Ictalurus punctatus

PubMed Central

Li, Hanbo; Su, Baofeng; Qin, Guyu; Ye, Zhi; Alsaqufi, Ahmed; Perera, Dayan A.; Shang, Mei; Odin, Ramjie; Vo, Khoi; Drescher, David; Robinson, Dalton; Zhang, Dan; Abass, Nermeen; Dunham, Rex A.

2017-01-01

Repressible knockdown approaches were investigated for transgenic sterilization in channel catfish, Ictalurus punctatus. Two primordial germ cell (PGC) marker genes, nanos and dead end, were targeted for knockdown, and an off-target gene, vasa, was monitored. Two potentially salt sensitive repressible promoters, zebrafish adenylosuccinate synthase 2 (ADSS) and zebrafish racemase (Rm), were each coupled with four knockdown strategies: ds-sh RNA targeting the 5′ end (N1) or 3′ end (N2) of channel catfish nanos, full-length cDNA sequence of channel catfish nanos for overexpression (cDNA) and ds-sh RNA targeting channel catfish dead end (DND). Each construct had an untreated group and treated group with sodium chloride as the repressor compound. Spawning rates of full-sibling P1 fish exposed or not exposed to the constructs as treated and untreated embryos were 93% and 59%, respectively, indicating potential sterilization of fish and repression of the constructs. Although the mRNA expression data of PGC marker genes were inconsistent in P1 fish, most F1 individuals were able to downregulate the target genes in untreated groups and repress the knockdown process in treated groups. The results indicate that repressible transgenic sterilization is feasible for reproductive control of fish, but more data from F2 or F3 are needed for evaluation. PMID:28561774

Digoxin reveals a functional connection between HIV-1 integration preference and T-cell activation

PubMed Central

Planas, Delphine; Merritt, Andy; Routy, Jean-Pierre; Ancuta, Petronela; Bangham, Charles R. M.

2017-01-01

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation. PMID:28727807

The Yersinia pestis gcvB gene encodes two small regulatory RNA molecules

PubMed Central

McArthur, Sarah D; Pulvermacher, Sarah C; Stauffer, George V

2006-01-01

Background In recent years it has become clear that small non-coding RNAs function as regulatory elements in bacterial virulence and bacterial stress responses. We tested for the presence of the small non-coding GcvB RNAs in Y. pestis as possible regulators of gene expression in this organism. Results In this study, we report that the Yersinia pestis KIM6 gcvB gene encodes two small RNAs. Transcription of gcvB is activated by the GcvA protein and repressed by the GcvR protein. The gcvB-encoded RNAs are required for repression of the Y. pestis dppA gene, encoding the periplasmic-binding protein component of the dipeptide transport system, showing that the GcvB RNAs have regulatory activity. A deletion of the gcvB gene from the Y. pestis KIM6 chromosome results in a decrease in the generation time of the organism as well as a change in colony morphology. Conclusion The results of this study indicate that the Y. pestis gcvB gene encodes two small non-coding regulatory RNAs that repress dppA expression. A gcvB deletion is pleiotropic, suggesting that the sRNAs are likely involved in controlling genes in addition to dppA. PMID:16768793

The phytochemical 3,3'-diindolylmethane decreases expression of AR-controlled DNA damage repair genes through repressive chromatin modifications and is associated with DNA damage in prostate cancer cells.

PubMed

Palomera-Sanchez, Zoraya; Watson, Gregory W; Wong, Carmen P; Beaver, Laura M; Williams, David E; Dashwood, Roderick H; Ho, Emily

2017-09-01

Androgen receptor (AR) is a transcription factor involved in normal prostate physiology and prostate cancer (PCa) development. 3,3'-Diindolylmethane (DIM) is a promising phytochemical agent against PCa that affects AR activity and epigenetic regulators in PCa cells. However, whether DIM suppresses PCa via epigenetic regulation of AR target genes is unknown. We assessed epigenetic regulation of AR target genes in LNCaP PCa cells and showed that DIM treatment led to epigenetic suppression of AR target genes involved in DNA repair (PARP1, MRE11, DNA-PK). Decreased expression of these genes was accompanied by an increase in repressive chromatin marks, loss of AR occupancy and EZH2 recruitment to their regulatory regions. Decreased DNA repair gene expression was associated with an increase in DNA damage (γH2Ax) and up-regulation of genomic repeat elements LINE1 and α-satellite. Our results suggest that DIM suppresses AR-dependent gene transcription through epigenetic modulation, leading to DNA damage and genome instability in PCa cells. Published by Elsevier Inc.

Regulation of sugar transport and metabolism by the Candida albicans Rgt1 transcriptional repressor.

PubMed

Sexton, Jessica A; Brown, Victoria; Johnston, Mark

2007-10-01

The ability of the fungal pathogen Candida albicans to cause systemic infections depends in part on the function of Hgt4, a cell surface sugar sensor. The orthologues of Hgt4 in Saccharomyces cerevisiae, Snf3 and Rgt2, initiate a signalling cascade that inactivates Rgt1, a transcriptional repressor of genes encoding hexose transporters. To determine whether Hgt4 functions similarly through the C. albicans orthologue of Rgt1, we analysed Cargt1 deletion mutants. We found that Cargt1 mutants are sensitive to the glucose analogue 2-deoxyglucose, a phenotype probably due to uncontrolled expression of genes encoding glucose transporters. Indeed, transcriptional profiling revealed that expression of about two dozen genes, including multiple HGT genes encoding hexose transporters, is increased in the Cargt1 mutant in the absence of sugars, suggesting that CaRgt1 represses expression of several HGT genes under this condition. Some of the HGT genes (probably encoding high-affinity transporters) are also repressed by high levels of glucose, and we show that this repression is mediated by CaMig1, the orthologue of the major glucose-activated repressor in S. cerevisiae, but not by its paralogue CaMig2. Therefore, CaRgt1 and CaMig1 collaborate to control expression of C. albicans hexose transporters in response to different levels of sugars. We were surprised to find that CaRgt1 also regulates expression of GAL1, suggesting that regulation of galactose metabolism in C. albicans is unconventional. Finally, Cargt1 mutations cause cells to hyperfilament, and suppress the hypofilamented phenotype of an hgt4 mutant, indicating that the Hgt4 glucose sensor may affect filamentation by modulating sugar import and metabolism via CaRgt1. Copyright 2007 John Wiley & Sons, Ltd.

Chromatin Redistribution of the DEK Oncoprotein Represses hTERT Transcription in Leukemias12

PubMed Central

Karam, Maroun; Thenoz, Morgan; Capraro, Valérie; Robin, Jean-Philippe; Pinatel, Christiane; Lancon, Agnès; Galia, Perrine; Sibon, David; Thomas, Xavier; Ducastelle-Lepretre, Sophie; Nicolini, Franck; El-Hamri, Mohamed; Chelghoun, Youcef; Wattel, Eric; Mortreux, Franck

2014-01-01

Although numerous factors have been found to modulate hTERT transcription, the mechanism of its repression in certain leukemias remains unknown. We show here that DEK represses hTERT transcription through its enrichment on the hTERT promoter in cells from chronic and acute myeloid leukemias, chronic lymphocytic leukemia, but not acute lymphocytic leukemias where hTERT is overexpressed. We isolated DEK from the hTERT promoter incubated with nuclear extracts derived from fresh acute myelogenous leukemia (AML) cells and from cells expressing Tax, an hTERT repressor encoded by the human T cell leukemia virus type 1. In addition to the recruitment of DEK, the displacement of two potent known hTERT transactivators from the hTERT promoter characterized both AML cells and Tax-expressing cells. Reporter and chromatin immunoprecipitation assays permitted to map the region that supports the repressive effect of DEK on hTERT transcription, which was proportionate to the level of DEK-promoter association but not with the level of DEK expression. Besides hTERT repression, this context of chromatin redistribution of DEK was found to govern about 40% of overall transcriptional modifications, including those of cancer-prone genes. In conclusion, DEK emerges as an hTERT repressor shared by various leukemia subtypes and seems involved in the deregulation of numerous genes associated with leukemogenesis. PMID:24563617

O-GlcNAc cycling: Emerging Roles in Development and Epigenetics

PubMed Central

Love, Dona C.; Krause, Michael W.; Hanover, John A.

2010-01-01

The nutrient-sensing hexosamine signaling pathway modulates the levels of O-linked N-acetylglucosamine (O-GlcNAc) on key targets impacting cellular signaling, protein turnover and gene expression. O-GlcNAc cycling may be deregulated in neurodegenerative disease, cancer, and diabetes. Studies in model organisms demonstrate that the O-GlcNAc transferase (OGT/Sxc) is essential for Polycomb group (PcG) repression of the homeotic genes, clusters of genes responsible for the adult body plan. Surprisingly, from flies to man, the O-GlcNAcase (OGA, MGEA5) gene is embedded within the NK cluster, the most evolutionarily ancient of three homeobox gene clusters regulated by PcG repression. PcG repression also plays a key role in maintaining stem cell identity, recruiting the DNA methyltransferase machinery for imprinting, and in X-chromosome inactivation. Intriguingly, the Ogt gene resides near the Xist locus in vertebrates and is subject to regulation by PcG-dependent X-inactivation. OGT is also an enzymatic component of the human dosage compensation complex. These ‘evo-devo’ relationships linking O-GlcNAc cycling to higher order chromatin structure provide insights into how nutrient availability may influence the epigenetic regulation of gene expression. O-GlcNAc cycling at promoters and PcG repression represent concrete mechanisms by which nutritional information may be transmitted across generations in the intra-uterine environment. Thus, the nutrient-sensing hexosamine signaling pathway may be a key contributor to the metabolic deregulation resulting from prenatal exposure to famine, or the ‘vicious cycle’ observed in children of mothers with type-2 diabetes and metabolic disease. PMID:20488252

The CsrR/CsrS two-component system of group A Streptococcus responds to environmental Mg2+.

PubMed

Gryllos, Ioannis; Levin, James C; Wessels, Michael R

2003-04-01

Group A streptococci control expression of key virulence determinants via the two-component sensorregulator system CsrRCsrS. The membrane-bound sensor CsrS is thought to respond to previously unknown environmental signal(s) by controlling phosphorylation of its cognate regulator component CsrR. Phosphorylation of CsrR increases its affinity for binding to the promoter regions of Csr-regulated genes to repress transcription. Here we show that environmental Mg(2+) concentration is a potent and specific stimulus for CsrRCsrS-mediated regulation. We studied the effect of divalent cations on expression of the Csr-regulated hyaluronic acid capsule genes (hasABC) by measuring chloramphenicol acetyltransferase (CAT) activity in a reporter strain of group A Streptococcus carrying a has operon promoter-cat fusion. Addition of Mg(2+), but not of Ca(2+), Mn(2+), or Zn(2+), repressed capsule gene expression by up to 80% in a dose-dependent fashion. The decrease in capsule gene transcription was associated with a marked reduction in cell-associated capsular polysaccharide. RNA hybridization analysis demonstrated reduced expression of the Csr-regulated hasABC operon, streptokinase (ska), and streptolysin S (sagA) during growth in the presence of 15 mM Mg(2+) for the wild-type strain 003CAT but not for an isogenic csrS mutant. We propose that Mg(2+) binds to CsrS to induce phosphorylation of CsrR and subsequent repression of virulence gene expression. The low concentration of Mg(2+) in extracellular body fluids predicts that the CsrRCsrS system is maintained in the inactive state during infection, thereby allowing maximal expression of critical virulence determinants in the human host.

Alteration of BRCA1 expression affects alcohol-induced transcription of RNA Pol III-dependent genes.

PubMed

Zhong, Qian; Shi, Ganggang; Zhang, Yanmei; Lu, Lei; Levy, Daniel; Zhong, Shuping

2015-02-01

Emerging evidence has indicated that alcohol consumption is an established risk factor for breast cancer. Deregulation of RNA polymerase III (Pol III) transcription enhances cellular Pol III gene production, leading to an increase in translational capacity to promote cell transformation and tumor formation. We have reported that alcohol intake increases Pol III gene transcription to promote cell transformation and tumor formation in vitro and in vivo. Studies revealed that tumor suppressors, pRb, p53, PTEN and Maf1 repress the transcription of Pol III genes. BRCA1 is a tumor suppressor and its mutation is tightly related to breast cancer development. However, it is not clear whether BRCA1 expression affects alcohol-induced transcription of Pol III genes. At the present studies, we report that restoring BRCA1 in HCC 1937 cells, which is a BRCA1 deficient cell line, represses Pol III gene transcription. Expressing mutant or truncated BRCA1 in these cells does not affect the ability of repression on Pol III genes. Our analysis has demonstrated that alcohol induces Pol III gene transcription. More importantly, overexpression of BRCA1 in estrogen receptor positive (ER+) breast cancer cells (MCF-7) decreases the induction of tRNA(Leu) and 5S rRNA genes by alcohol, whereas reduction of BRCA1 by its siRNA slightly increases the transcription of the class of genes. This suggests that BRCA1 is associated with alcohol-induced deregulation of Pol III genes. These studies for the first time demonstrate the role of BRCA1 in induction of Pol III genes by alcohol and uncover a novel mechanism of alcohol-associated breast cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Steady-state and dynamic gene expression programs in Saccharomyces cerevisiae in response to variation in environmental nitrogen

PubMed Central

Airoldi, Edoardo M.; Miller, Darach; Athanasiadou, Rodoniki; Brandt, Nathan; Abdul-Rahman, Farah; Neymotin, Benjamin; Hashimoto, Tatsu; Bahmani, Tayebeh; Gresham, David

2016-01-01

Cell growth rate is regulated in response to the abundance and molecular form of essential nutrients. In Saccharomyces cerevisiae (budding yeast), the molecular form of environmental nitrogen is a major determinant of cell growth rate, supporting growth rates that vary at least threefold. Transcriptional control of nitrogen use is mediated in large part by nitrogen catabolite repression (NCR), which results in the repression of specific transcripts in the presence of a preferred nitrogen source that supports a fast growth rate, such as glutamine, that are otherwise expressed in the presence of a nonpreferred nitrogen source, such as proline, which supports a slower growth rate. Differential expression of the NCR regulon and additional nitrogen-responsive genes results in >500 transcripts that are differentially expressed in cells growing in the presence of different nitrogen sources in batch cultures. Here we find that in growth rate–controlled cultures using nitrogen-limited chemostats, gene expression programs are strikingly similar regardless of nitrogen source. NCR expression is derepressed in all nitrogen-limiting chemostat conditions regardless of nitrogen source, and in these conditions, only 34 transcripts exhibit nitrogen source–specific differential gene expression. Addition of either the preferred nitrogen source, glutamine, or the nonpreferred nitrogen source, proline, to cells growing in nitrogen-limited chemostats results in rapid, dose-dependent repression of the NCR regulon. Using a novel means of computational normalization to compare global gene expression programs in steady-state and dynamic conditions, we find evidence that the addition of nitrogen to nitrogen-limited cells results in the transient overproduction of transcripts required for protein translation. Simultaneously, we find that that accelerated mRNA degradation underlies the rapid clearing of a subset of transcripts, which is most pronounced for the highly expressed NCR-regulated permease genes GAP1, MEP2, DAL5, PUT4, and DIP5. Our results reveal novel aspects of nitrogen-regulated gene expression and highlight the need for a quantitative approach to study how the cell coordinates protein translation and nitrogen assimilation to optimize cell growth in different environments. PMID:26941329

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

PubMed

Lee, Young Je; Hoynes-O'Connor, Allison; Leong, Matthew C; Moon, Tae Seok

2016-03-18

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

PubMed Central

Salusjärvi, Laura; Kankainen, Matti; Soliymani, Rabah; Pitkänen, Juha-Pekka; Penttilä, Merja; Ruohonen, Laura

2008-01-01

Background Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of Saccharomyces cerevisiae for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising S. cerevisiae grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose. Results Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators HAP4, CAT8 and SIP1-2 and 4. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor RGT1 and its target genes HXT2-3, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells. Conclusion The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant S. cerevisiae strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings. PMID:18533012

Global Expression Profiling in Atopic Eczema Reveals Reciprocal Expression of Inflammatory and Lipid Genes

PubMed Central

Sääf, Annika M.; Tengvall-Linder, Maria; Chang, Howard Y.; Adler, Adam S.; Wahlgren, Carl-Fredrik; Scheynius, Annika; Nordenskjöld, Magnus; Bradley, Maria

2008-01-01

Background Atopic eczema (AE) is a common chronic inflammatory skin disorder. In order to dissect the genetic background several linkage and genetic association studies have been performed. Yet very little is known about specific genes involved in this complex skin disease, and the underlying molecular mechanisms are not fully understood. Methodology/Findings We used human DNA microarrays to identify a molecular picture of the programmed responses of the human genome to AE. The transcriptional program was analyzed in skin biopsy samples from lesional and patch-tested skin from AE patients sensitized to Malassezia sympodialis (M. sympodialis), and corresponding biopsies from healthy individuals. The most notable feature of the global gene-expression pattern observed in AE skin was a reciprocal expression of induced inflammatory genes and repressed lipid metabolism genes. The overall transcriptional response in M. sympodialis patch-tested AE skin was similar to the gene-expression signature identified in lesional AE skin. In the constellation of genes differentially expressed in AE skin compared to healthy control skin, we have identified several potential susceptibility genes that may play a critical role in the pathological condition of AE. Many of these genes, including genes with a role in immune responses, lipid homeostasis, and epidermal differentiation, are localized on chromosomal regions previously linked to AE. Conclusions/Significance Through genome-wide expression profiling, we were able to discover a distinct reciprocal expression pattern of induced inflammatory genes and repressed lipid metabolism genes in skin from AE patients. We found a significant enrichment of differentially expressed genes in AE with cytobands associated to the disease, and furthermore new chromosomal regions were found that could potentially guide future region-specific linkage mapping in AE. The full data set is available at http://microarray-pubs.stanford.edu/eczema. PMID:19107207

DNA-Demethylase Regulated Genes Show Methylation-Independent Spatiotemporal Expression Patterns

PubMed Central

Schumann, Ulrike; Lee, Joanne; Kazan, Kemal; Ayliffe, Michael; Wang, Ming-Bo

2017-01-01

Recent research has indicated that a subset of defense-related genes is downregulated in the Arabidopsis DNA demethylase triple mutant rdd (ros1 dml2 dml3) resulting in increased susceptibility to the fungal pathogen Fusarium oxysporum. In rdd plants these downregulated genes contain hypermethylated transposable element sequences (TE) in their promoters, suggesting that this methylation represses gene expression in the mutant and that these sequences are actively demethylated in wild-type plants to maintain gene expression. In this study, the tissue-specific and pathogen-inducible expression patterns of rdd-downregulated genes were investigated and the individual role of ROS1, DML2, and DML3 demethylases in these spatiotemporal regulation patterns was determined. Large differences in defense gene expression were observed between pathogen-infected and uninfected tissues and between root and shoot tissues in both WT and rdd plants, however, only subtle changes in promoter TE methylation patterns occurred. Therefore, while TE hypermethylation caused decreased gene expression in rdd plants it did not dramatically effect spatiotemporal gene regulation, suggesting that this latter regulation is largely methylation independent. Analysis of ros1-3, dml2-1, and dml3-1 single gene mutant lines showed that promoter TE hypermethylation and defense-related gene repression was predominantly, but not exclusively, due to loss of ROS1 activity. These data demonstrate that DNA demethylation of TE sequences, largely by ROS1, promotes defense-related gene expression but does not control spatiotemporal expression in Arabidopsis. Summary: Ros1-mediated DNA demethylation of promoter transposable elements is essential for activation of defense-related gene expression in response to fungal infection in Arabidopsis thaliana. PMID:28894455

KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands

PubMed Central

Farcas, Anca M; Blackledge, Neil P; Sudbery, Ian; Long, Hannah K; McGouran, Joanna F; Rose, Nathan R; Lee, Sheena; Sims, David; Cerase, Andrea; Sheahan, Thomas W; Koseki, Haruhiko; Brockdorff, Neil; Ponting, Chris P; Kessler, Benedikt M; Klose, Robert J

2012-01-01

CpG islands (CGIs) are associated with most mammalian gene promoters. A subset of CGIs act as polycomb response elements (PREs) and are recognized by the polycomb silencing systems to regulate expression of genes involved in early development. How CGIs function mechanistically as nucleation sites for polycomb repressive complexes remains unknown. Here we discover that KDM2B (FBXL10) specifically recognizes non-methylated DNA in CGIs and recruits the polycomb repressive complex 1 (PRC1). This contributes to histone H2A lysine 119 ubiquitylation (H2AK119ub1) and gene repression. Unexpectedly, we also find that CGIs are occupied by low levels of PRC1 throughout the genome, suggesting that the KDM2B-PRC1 complex may sample CGI-associated genes for susceptibility to polycomb-mediated silencing. These observations demonstrate an unexpected and direct link between recognition of CGIs by KDM2B and targeting of the polycomb repressive system. This provides the basis for a new model describing the functionality of CGIs as mammalian PREs. DOI: http://dx.doi.org/10.7554/eLife.00205.001 PMID:23256043

Molecular cloning of a catalase cDNA from Nicotiana glutinosa L. and its repression by tobacco mosaic virus infection.

PubMed

Yi, S Y; Yu, S H; Choi, D

1999-06-30

Recent reports revealed that catalase has a role in the plant defense mechanism against a broad range of pathogens through being inhibited by salicylic acid (SA). During an effort to clone disease resistance-responsive genes, a cDNA encoding catalase (Ngcat1; Nicotiana glutinosa cat1) was isolated from a tobacco cDNA library. In N. glutinosa, catalase is encoded by a small gene family. The deduced amino acid sequence of the Ngcat1 cDNA has 98% homology with the cat1 gene of N. plumbaginifolia. The Ngcat1 expression is controlled by the circadian clock, and its mRNA level is the most abundant in leaves. Both the expression of Ngcat1 mRNA and its enzyme activity in the tobacco plant undergoing a hypersensitive response (HR) to TMV infection were repressed. The repression of the mRNA level was also observed following treatment with SA. These results imply that SA may act as an inhibitor of catalase transcription during the HR of tobacco. Cloning and expression of the Ngcat1 in tobacco following pathogen infection and SA treatment are presented.

Transcriptional repression of epithelial cell adhesion molecule (EpCAM) contributes to p53 control of breast cancer invasion

PubMed Central

Sankpal, NV; Willman, MW; Fleming, TP; Mayfield, J; Gillanders, WE

2014-01-01

p53 is a tumor suppressor gene with well-characterized roles in cell cycle regulation, apoptosis and the maintenance of genome stability. Recent evidence suggests that p53 may also contribute to the regulation of migration and invasion. Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein that is overexpressed in the majority of human epithelial carcinomas, including breast and colorectal carcinomas. We demonstrate by chromatin immunoprecipitation assays that p53 interacts with a candidate p53 binding site within the EpCAM gene. p53-mediated transcriptional repression of EpCAM was confirmed in gain-of-function, and loss-of-function experimental systems. Induction of wildtype p53 was associated with a significant dose-dependent decrease in EpCAM expression; conversely, specific ablation of p53 was associated with a significant increase in EpCAM expression. At the functional level, specific ablation of p53 expression is associated with increased breast cancer invasion, and this effect is abrogated by concomitant specific ablation of EpCAM expression. Taken together, these biochemical and functional data are the first demonstration that (1) wildtype p53 protein binds to a response element within the EpCAM gene and negatively regulates EpCAM expression, and (2) transcriptional repression of EpCAM contributes to p53 control of breast cancer invasion. PMID:19141643

Yeast Carbon Catabolite Repression†

PubMed Central

Gancedo, Juana M.

1998-01-01

Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources; some of these genes are also repressed by other sugars such as galactose, and the process is known as catabolite repression. The different sugars produce signals which modify the conformation of certain proteins that, in turn, directly or through a regulatory cascade affect the expression of the genes subject to catabolite repression. These genes are not all controlled by a single set of regulatory proteins, but there are different circuits of repression for different groups of genes. However, the protein kinase Snf1/Cat1 is shared by the various circuits and is therefore a central element in the regulatory process. Snf1 is not operative in the presence of glucose, and preliminary evidence suggests that Snf1 is in a dephosphorylated state under these conditions. However, the enzymes that phosphorylate and dephosphorylate Snf1 have not been identified, and it is not known how the presence of glucose may affect their activity. What has been established is that Snf1 remains active in mutants lacking either the proteins Grr1/Cat80 or Hxk2 or the Glc7 complex, which functions as a protein phosphatase. One of the main roles of Snf1 is to relieve repression by the Mig1 complex, but it is also required for the operation of transcription factors such as Adr1 and possibly other factors that are still unidentified. Although our knowledge of catabolite repression is still very incomplete, it is possible in certain cases to propose a partial model of the way in which the different elements involved in catabolite repression may be integrated. PMID:9618445

Aberrant expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice.

PubMed

Hwang, Soo Seok; Kim, Kiwan; Lee, Wonyong; Lee, Gap Ryol

2012-08-03

The Th2 locus control region (LCR) has been shown to be a crucial cis-acting element for Th2 cytokine expression and Th2 cell differentiation. To study the role of Th2 LCR in ifng locus regulation, we examined the expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice. We found IFN-γ to be aberrantly up-regulated. In addition, histone 3(H3)-acetylation and histone 3 lysine 4 (H3-K4)-methylation greatly increased at the ifng locus of the Th2 cells. GATA-3 and STAT6 bound to the ifng promoter in Th2 cells from the wild type but not from the Th2 LCR-deficient mice, and they directly repressed ifng expression in transient reporter assay. Moreover, ectopic expression of GATA-3 and STAT6-VT repressed the aberrant expression of the ifng gene and restored repressive chromatin state at the ifng locus in Th2 cells from Th2 LCR-deficient mice. These results suggest that expression of the ifng gene and chromatin remodeling of the ifng locus are under the control of a Th2 LCR-mediated Th2 differentiation program. Copyright © 2012 Elsevier Inc. All rights reserved.

Rice NAD+-dependent histone deacetylase OsSRT1 represses glycolysis and regulates the moonlighting function of GAPDH as a transcriptional activator of glycolytic genes.

PubMed

Zhang, Hua; Zhao, Yu; Zhou, Dao-Xiu

2017-12-01

Sirtuins, a family of proteins with homology to the yeast silent information regulator 2 (Sir2), are NAD+-dependent histone deacetylases and play crucial roles in energy sensing and regulation in yeast and animal cells. Plants are autotrophic organisms and display distinct features of carbon and energy metabolism. It remains largely unexplored whether and how plant cells sense energy/redox status to control carbon metabolic flux under various growth conditions. In this work, we show that the rice nuclear sirtuin OsSRT1 not only functions as an epigenetic regulator to repress glycolytic genes expression and glycolysis in seedlings, but also inhibits transcriptional activity of glyceraldehyde-3-phosphatedehydrogenase (GAPDH) that is enriched on glycolytic genes promoters and stimulates their expression. We show that OsSRT1 reduces GAPDH lysine acetylation and nuclear accumulation that are enhanced by oxidative stress. Mass spectrometry identified six acetylated lysines regulated by OsSRT1. OsSRT1-dependent lysine deacetylation of OsGAPDH1 represses transcriptional activity of the protein. The results indicate that OsSRT1 represses glycolysis by both regulating epigenetic modification of histone and inhibiting the moonlighting function of GAPDH as a transcriptional activator of glycolytic genes in rice. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome expression analysis in yeast reveals novel transcriptional regulation by inositol and choline and new regulatory functions for Opi1p, Ino2p, and Ino4p.

PubMed

Santiago, Teresa C; Mamoun, Choukri Ben

2003-10-03

In Saccharomyces cerevisiae, genes encoding phospholipid-synthesizing enzymes are regulated by inositol and choline (IC). The current model suggests that when these precursors become limiting, the transcriptional complex Ino2p-Ino4p activates the expression of these genes, whereas repression requires Opi1p and occurs when IC are available. In this study, microarray-based expression analysis was performed to assess the global transcriptional response to IC in a wild-type strain and in the opi1delta, ino2delta, and ino4delta null mutant strains. Fifty genes were either activated or repressed by IC in the wild-type strain, including three already known IC-repressed genes. We demonstrated that the IC response was not limited to genes involved in membrane biogenesis, but encompassed various metabolic pathways such as biotin synthesis, one-carbon compound metabolism, nitrogen-containing compound transport and degradation, cell wall organization and biogenesis, and acetyl-CoA metabolism. The expression of a large number of IC-regulated genes did not change in the opi1delta, ino2delta, and ino4delta strains, thus implicating new regulatory elements in the IC response. Our studies revealed that Opi1p, Ino2p, and Ino4p have dual regulatory activities, acting in both positive and negative transcriptional regulation of a large number of genes, most of which are not regulated by IC and only a subset of which is involved in membrane biogenesis. These data provide the first global response profile of yeast to IC and reveal novel regulatory mechanisms by these precursors.

MYC association with cancer risk and a new model of MYC-mediated repression.

PubMed

Cole, Michael D

2014-07-01

MYC is one of the most frequently mutated and overexpressed genes in human cancer but the regulation of MYC expression and the ability of MYC protein to repress cellular genes (including itself) have remained mysterious. Recent genome-wide association studies show that many genetic polymorphisms associated with disease risk map to distal regulatory elements that regulate the MYC promoter through large chromatin loops. Cancer risk-associated single-nucleotide polymorphisms (SNPs) contain more potent enhancer activity, promoting higher MYC levels and a greater risk of disease. The MYC promoter is also subject to complex regulatory circuits and limits its own expression by a feedback loop. A model for MYC autoregulation is discussed which involves a signaling pathway between the PTEN (phosphatase and tensin homolog) tumor suppressor and repressive histone modifications laid down by the EZH2 methyltransferase. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Roles of the first and second round of DNA replication in the regulation of zygotic gene activation in mice.

PubMed

Sonehara, Hiroki; Nagata, Masao; Aoki, Fugaku

2008-10-01

In the mouse embryo, expression of zygotic genes starts in the S/G2 phase of the 1-cell stage and greatly increases during the 2-cell stage. Although the timing of zygotic gene activation (ZGA) is thus established, the mechanism regulating ZGA is poorly understood. Previous studies using reporter genes have suggested that a transcriptionally repressive state is established during the 2-cell stage and that the first and second rounds of DNA replication are involved in this process. To further elucidate the respective roles of the two rounds of DNA replication in ZGA, we analyzed the expression of four ZGA genes (hsp70.1, eif-1a, muerv and zscan4d) in embryos whose DNA replication was inhibited by treatment with aphidicolin, an inhibitor of DNA polymerase. Inhibiting the first round increased the expression levels of hsp70.1, eif-1a and zscan4d but decreased that of muerv, while inhibiting the second round increased the expression levels of all four genes. These results suggest that the transcriptionally repressive state seems to be established after the second round of DNA replication.

Stage-specific control of early B cell development by the transcription factor Ikaros

PubMed Central

Gültekin, Sinan; Dakic, Aleksandar; Axelsson, Elin; Minnich, Martina; Ebert, Anja; Werner, Barbara; Roth, Mareike; Cimmino, Luisa; Dickins, Ross A.; Zuber, Johannes; Jaritz, Markus; Busslinger, Meinrad

2018-01-01

Ikaros is an essential regulator of lymphopoiesis. Here, we studied the B-cell-specific function of Ikaros by conditional Ikzf1 inactivation in pro-B cells. B-cell development was arrested at an aberrant ‘pro-B’ cell stage characterized by increased cell adhesion and loss of pre-B cell receptor signaling. Ikaros was found to activate genes coding for pre-BCR signal transducers and to repress genes involved in the downregulation of pre-BCR signaling and upregulation of the integrin signaling pathway. Unexpectedly, derepression of Aiolos expression could not compensate for the loss of Ikaros in pro-B cells. Ikaros induced or suppressed active chromatin at regulatory elements of activated or repressed target genes. Notably, Ikaros binding and target gene expression was dynamically regulated at distinct stages of early B-lymphopoiesis. PMID:24509509

Cooperative gene regulation by microRNA pairs and their identification using a computational workflow

PubMed Central

Schmitz, Ulf; Lai, Xin; Winter, Felix; Wolkenhauer, Olaf; Vera, Julio; Gupta, Shailendra K.

2014-01-01

MicroRNAs (miRNAs) are an integral part of gene regulation at the post-transcriptional level. Recently, it has been shown that pairs of miRNAs can repress the translation of a target mRNA in a cooperative manner, which leads to an enhanced effectiveness and specificity in target repression. However, it remains unclear which miRNA pairs can synergize and which genes are target of cooperative miRNA regulation. In this paper, we present a computational workflow for the prediction and analysis of cooperating miRNAs and their mutual target genes, which we refer to as RNA triplexes. The workflow integrates methods of miRNA target prediction; triplex structure analysis; molecular dynamics simulations and mathematical modeling for a reliable prediction of functional RNA triplexes and target repression efficiency. In a case study we analyzed the human genome and identified several thousand targets of cooperative gene regulation. Our results suggest that miRNA cooperativity is a frequent mechanism for an enhanced target repression by pairs of miRNAs facilitating distinctive and fine-tuned target gene expression patterns. Human RNA triplexes predicted and characterized in this study are organized in a web resource at www.sbi.uni-rostock.de/triplexrna/. PMID:24875477

Separate necdin domains bind ARNT2 and HIF1{alpha} and repress transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedman, Eitan R.; Fan Chenming

2007-11-09

PWS is caused by the loss of expression of a set of maternally imprinted genes including NECDIN (NDN). NDN is expressed in post-mitotic neurons and plays an essential role in PWS as mouse models lacking only the Ndn gene mimic aspects of this disease. Patients haploid for SIM1 develop a PW-like syndrome. Here, we report that NDN directly interacts with ARNT2, a bHLH-PAS protein and dimer partner for SIM1. We also found that NDN can interact with HIF1{alpha}. We showed that NDN can repress transcriptional activation mediated by ARNT2:SIM1 as well as ARNT2:HIF1{alpha}. The N-terminal 115 residues of NDN aremore » sufficient for interaction with the bHLH domains of ARNT2 or HIF1{alpha} but not for transcriptional repression. Using GAL4-NDN fusion proteins, we determined that NDN possesses multiple repression domains. We thus propose that NDN regulates neuronal function and hypoxic response by regulating the activities of the ARNT2:SIM1 and ARNT2:HIF1{alpha} dimers, respectively.« less

Epigenetic regulation of puberty via Zinc finger protein-mediated transcriptional repression

PubMed Central

Lomniczi, Alejandro; Wright, Hollis; Castellano, Juan Manuel; Matagne, Valerie; Toro, Carlos A.; Ramaswamy, Suresh; Plant, Tony M.; Ojeda, Sergio R.

2015-01-01

In primates, puberty is unleashed by increased GnRH release from the hypothalamus following an interval of juvenile quiescence. GWAS implicates Zinc finger (ZNF) genes in timing human puberty. Here we show that hypothalamic expression of several ZNFs decreased in agonadal male monkeys in association with the pubertal reactivation of gonadotropin secretion. Expression of two of these ZNFs, GATAD1 and ZNF573, also decreases in peripubertal female monkeys. However, only GATAD1 abundance increases when gonadotropin secretion is suppressed during late infancy. Targeted delivery of GATAD1 or ZNF573 to the rat hypothalamus delays puberty by impairing the transition of a transcriptional network from an immature repressive epigenetic configuration to one of activation. GATAD1 represses transcription of two key puberty-related genes, KISS1 and TAC3, directly, and reduces the activating histone mark H3K4me2 at each promoter via recruitment of histone demethylase KDM1A. We conclude that GATAD1 epitomizes a subset of ZNFs involved in epigenetic repression of primate puberty. PMID:26671628

Transcriptional responses to glucose at different glycolytic rates in Saccharomyces cerevisiae.

PubMed

Elbing, Karin; Ståhlberg, Anders; Hohmann, Stefan; Gustafsson, Lena

2004-12-01

The addition of glucose to Saccharomyces cerevisiae cells causes reprogramming of gene expression. Glucose is sensed by membrane receptors as well as (so far elusive) intracellular sensing mechanisms. The availability of four yeast strains that display different hexose uptake capacities allowed us to study glucose-induced effects at different glycolytic rates. Rapid glucose responses were observed in all strains able to take up glucose, consistent with intracellular sensing. The degree of long-term responses, however, clearly correlated with the glycolytic rate: glucose-stimulated expression of genes encoding enzymes of the lower part of glycolysis showed an almost linear correlation with the glycolytic rate, while expression levels of genes encoding gluconeogenic enzymes and invertase (SUC2) showed an inverse correlation. Glucose control of SUC2 expression is mediated by the Snf1-Mig1 pathway. Mig1 dephosphorylation upon glucose addition is known to lead to repression of target genes. Mig1 was initially dephosphorylated upon glucose addition in all strains able to take up glucose, but remained dephosphorylated only at high glycolytic rates. Remarkably, transient Mig1-dephosphorylation was accompanied by the repression of SUC2 expression at high glycolytic rates, but stimulated SUC2 expression at low glycolytic rates. This suggests that Mig1-mediated repression can be overruled by factors mediating induction via a low glucose signal. At low and moderate glycolytic rates, Mig1 was partly dephosphorylated both in the presence of phosphorylated, active Snf1, and unphosphorylated, inactive Snf1, indicating that Mig1 was actively phosphorylated and dephosphorylated simultaneously, suggesting independent control of both processes. Taken together, it appears that glucose addition affects the expression of SUC2 as well as Mig1 activity by both Snf1-dependent and -independent mechanisms that can now be dissected and resolved as early and late/sustained responses.

MTA3 regulates CGB5 and Snail genes in trophoblast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ying; Miyazaki, Jun; Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake

Highlights: •Impaired MTA3, raised CGB5 and Snail expression are associated with preeclampsia. •Knock-down of MTA3 causes up-regulation of CGB5 and Snail genes in BeWo cells. •MTA3 occupies CGB5 and Snail gene promoters in BeWo cells. -- Abstract: Secreted by the placental trophoblast, human chorionic gonadotropin (hCG) is an important hormone during pregnancy and is required for the maintenance of pregnancy. Previous studies have shown that dys-regulation of hCG expression is associated with preeclampsia. However, the exact relationship between altered hCG levels and development of preeclampsia is unknown. Metastasis associated protein 3 (MTA3), a chromatin remodeling protein, is abundantly expressed inmore » the placental trophoblasts, but its function is unknown. In breast cancer, MTA3 has been shown to repress the expression of Snail and cell migration. However, whether MTA3 acts similarly in the trophoblast has not been investigated. In the present study, we examined the role of MTA3 in regulating the hCG β-subunit gene (gene name: CGB5) and Snail expression in the trophoblast cell line, BeWo, as well as its relevance to the high hCG expression levels seen in preeclampsia. First, we investigated MTA3 expression in preeclamptic placenta as compared to normal control placenta via gene expression microarray and qRT-PCR and found that MTA3 was significantly down-regulated, whereas both CGB5 and Snail were up-regulated in preeclamptic placenta. Secondly, we knocked down MTA3 gene in trophoblast cell line BeWo and found Snail and hCG were both up-regulated, suggesting that MTA3 represses Snail and hCG gene expression in trophoblasts. Next, we cloned the CGB5 and Snail promoters into the pGL3-basic vector individually and found that silencing of MTA3 by siRNA resulted in an increase of both CGB5 and Snail promoter activities. To confirm that this MTA3 inhibition is a direct effect, we performed a chromatin immune-precipitation (ChIP) assay and found that MTA3 occupied the proximal promoter regions of both Snail and hCG within BeWo cells. Furthermore, we examined MTA3 expression in placental trophoblast by immunohistochemistry and found that MTA3 expression was higher in villous cytotrophoblasts versus syncytiotrophoblasts, which supports an inverse association of MTA3 with hCG expression. Lastly, using the well-characterized trophoblast fusion model, we examined MTA3 and hCG levels in forskolin-treated BeWo cells and found that MTA3 down-regulation was accompanied by an up-regulation of hCG. These data further suggest that MTA3 is repressing placental hCG expression. In summary, MTA3 plays a critical role in repressing hCG and Snail in placenta trophoblast and its deregulation is associated with preeclampsia.« less

batman Interacts with polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor.

PubMed

Faucheux, M; Roignant, J-Y; Netter, S; Charollais, J; Antoniewski, C; Théodore, L

2003-02-01

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.

batman Interacts with Polycomb and trithorax Group Genes and Encodes a BTB/POZ Protein That Is Included in a Complex Containing GAGA Factor

PubMed Central

Faucheux, M.; Roignant, J.-Y.; Netter, S.; Charollais, J.; Antoniewski, C.; Théodore, L.

2003-01-01

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes. PMID:12556479

The BTB and CNC homology 1 (BACH1) target genes are involved in the oxidative stress response and in control of the cell cycle.

PubMed

Warnatz, Hans-Jörg; Schmidt, Dominic; Manke, Thomas; Piccini, Ilaria; Sultan, Marc; Borodina, Tatiana; Balzereit, Daniela; Wruck, Wasco; Soldatov, Alexey; Vingron, Martin; Lehrach, Hans; Yaspo, Marie-Laure

2011-07-01

The regulation of gene expression in response to environmental signals and metabolic imbalances is a key step in maintaining cellular homeostasis. BTB and CNC homology 1 (BACH1) is a heme-binding transcription factor repressing the transcription from a subset of MAF recognition elements at low intracellular heme levels. Upon heme binding, BACH1 is released from the MAF recognition elements, resulting in increased expression of antioxidant response genes. To systematically address the gene regulatory networks involving BACH1, we combined chromatin immunoprecipitation sequencing analysis of BACH1 target genes in HEK 293 cells with knockdown of BACH1 using three independent types of small interfering RNAs followed by transcriptome profiling using microarrays. The 59 BACH1 target genes identified by chromatin immunoprecipitation sequencing were found highly enriched in genes showing expression changes after BACH1 knockdown, demonstrating the impact of BACH1 repression on transcription. In addition to known and new BACH1 targets involved in heme degradation (HMOX1, FTL, FTH1, ME1, and SLC48A1) and redox regulation (GCLC, GCLM, and SLC7A11), we also discovered BACH1 target genes affecting cell cycle and apoptosis pathways (ITPR2, CALM1, SQSTM1, TFE3, EWSR1, CDK6, BCL2L11, and MAFG) as well as subcellular transport processes (CLSTN1, PSAP, MAPT, and vault RNA). The newly identified impact of BACH1 on genes involved in neurodegenerative processes and proliferation provides an interesting basis for future dissection of BACH1-mediated gene repression in neurodegeneration and virus-induced cancerogenesis.

Sulfate-Dependent Repression of Genes That Function in Organosulfur Metabolism in Bacillus subtilis Requires Spx

PubMed Central

Erwin, Kyle N.; Nakano, Shunji; Zuber, Peter

2005-01-01

Oxidative stress in Bacillus subtilis results in the accumulation of Spx protein, which exerts both positive and negative transcriptional control over a genome-wide scale through its interaction with the RNA polymerase α subunit. Previous microarray transcriptome studies uncovered a unique class of genes that are controlled by Spx-RNA polymerase interaction under normal growth conditions that do not promote Spx overproduction. These genes were repressed by Spx when sulfate was present as a sole sulfur source. The genes include those of the ytmI, yxeI, and ssu operons, which encode products resembling proteins that function in the uptake and desulfurization of organic sulfur compounds. Primer extension and analysis of operon-lacZ fusion expression revealed that the operons are repressed by sulfate and cysteine; however, Spx functioned only in sulfate-dependent repression. Both the ytmI operon and the divergently transcribed ytlI, encoding a LysR-type regulator that positively controls ytmI operon transcription, are repressed by Spx in sulfate-containing media. The CXXC motif of Spx, which is necessary for redox sensitive control of Spx activity in response to oxidative stress, is not required for sulfate-dependent repression. The yxeL-lacZ and ssu-lacZ fusions were also repressed in an Spx-dependent manner in media containing sulfate as the sole sulfur source. This work uncovers a new role for Spx in the control of sulfur metabolism in a gram-positive bacterium under nonstressful growth conditions. PMID:15937167

VEGF and VEGFB Play Balancing Roles in Adipose Differentiation, Gene Expression, and Function.

PubMed

Jin, Honghong; Li, Dan; Wang, Xutong; Jia, Jia; Chen, Yang; Yao, Yapeng; Zhao, Chunlan; Lu, Xiaodan; Zhang, Shujie; Togo, Jacques; Ji, Yan; Zhang, Luqing; Feng, Xuechao; Zheng, Yaowu

2018-05-01

Obesity is the result of abnormal adipose development and energy metabolism. Using vascular endothelial growth factor (VEGF) B-knockout and inducible VEGF downregulation mouse models, we have shown that VEGFB inactivation caused expansion of white adipose, whitening of brown adipose, an increase in fat accumulation, and a reduction in energy consumption. At the same time, expression of the white adipose-associated genes was increased and brown adipose-associated genes decreased. VEGF repression, in contrast, induced brown adipose expansion and brown adipocyte development in white adipose, increased energy expenditure, upregulated brown adipose-associated genes, and downregulated white adipose-associated genes. When VEGFB-knockout and VEGF-repressed mice are crossed together, VEGF and VEGFB can counteractively regulate large numbers of genes and efficiently reverse each other's roles. These genes, under counteractive VEGF and VEGFB regulations, include transcription factors, adhesion molecules, and metabolic enzymes. This balancing role is confirmed by morphologic and functional changes. This study reports that VEGF and VEGFB counteractively regulate adipose development and function in energy metabolism.

Molecular characterization of SMILE as a novel corepressor of nuclear receptors.

PubMed

Xie, Yuan-Bin; Nedumaran, Balachandar; Choi, Hueng-Sik

2009-07-01

SMILE (small heterodimer partner interacting leucine zipper protein) has been identified as a coregulator in ER signaling. In this study, we have examined the effects of SMILE on other NRs (nuclear receptors). SMILE inhibits GR, CAR and HNF4 alpha-mediated transactivation. Knockdown of SMILE gene expression increases the transactivation of the NRs. SMILE interacts with GR, CAR and HNF4 alpha in vitro and in vivo. SMILE and these NRs colocalize in the nucleus. SMILE binds to the ligand-binding domain or AF2 domain of the NRs. Competitions between SMILE and the coactivators GRIP1 or PGC-1 alpha have been demonstrated in vitro and in vivo. Furthermore, an intrinsic repressive activity of SMILE is observed in Gal4-fusion system, and the intrinsic repressive domain is mapped to the C-terminus of SMILE, spanning residues 203-354. Moreover, SMILE interacts with specific HDACs (histone deacetylases) and SMILE-mediated repression is released by HDAC inhibitor trichostatin A, in a NR-specific manner. Finally, ChIP (chromatin immunoprecipitation) assays reveal that SMILE associates with the NRs on the target gene promoters. Adenoviral overexpression of SMILE represses GR-, CAR- and HNF4 alpha-mediated target gene expression. Overall, these results suggest that SMILE functions as a novel corepressor of NRs via competition with coactivators and the recruitment of HDACs.

Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting.

PubMed

Masuda, Junko; Kawamoto, Hiroshi; Strober, Warren; Takayama, Eiji; Mizutani, Akifumi; Murakami, Hiroshi; Ikawa, Tomokatsu; Kitani, Atsushi; Maeno, Narumi; Shigehiro, Tsukasa; Satoh, Ayano; Seno, Akimasa; Arun, Vaidyanath; Kasai, Tomonari; Fuss, Ivan J; Katsura, Yoshimoto; Seno, Masaharu

2016-12-01

Transplantation of hematopoietic stem and progenitor cells (HSCs) i.e., self-renewing cells that retain multipotentiality, is now a widely performed therapy for many hematopoietic diseases. However, these cells are present in low number and are subject to replicative senescence after extraction; thus, the acquisition of sufficient numbers of cells for transplantation requires donors able to provide repetitive blood samples and/or methods of expanding cell numbers without disturbing cell multipotentiality. Previous studies have shown that HSCs maintain their multipotentiality and self-renewal activity if TCF3 transcription function is blocked under B cell differentiating conditions. Taking advantage of this finding to devise a new approach to HSC expansion in vitro, we constructed an episomal expression vector that specifically targets and transiently represses the TCF3 gene. This consisted of a vector encoding a transcription activator-like effector (TALE) fused to a Krüppel-associated box (KRAB) repressor. We showed that this TALE-KRAB vector repressed expression of an exogenous reporter gene in HEK293 and COS-7 cell lines and, more importantly, efficiently repressed endogenous TCF3 in a human B lymphoma cell line. These findings suggest that this vector can be used to maintain multipotentiality in HSC being subjected to a long-term expansion regimen prior to transplantation.

SUMOylation of the KRAB zinc-finger transcription factor PARIS/ZNF746 regulates its transcriptional activity.

PubMed

Nishida, Tamotsu; Yamada, Yoshiji

2016-05-13

Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS in a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. Copyright © 2016 Elsevier Inc. All rights reserved.

Regulation of the plasma cell transcription factor Blimp-1 gene by Bach2 and Bcl6.

PubMed

Ochiai, Kyoko; Muto, Akihiko; Tanaka, Hiromu; Takahashi, Shinichiro; Igarashi, Kazuhiko

2008-03-01

B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differentiation. Prior to the terminal differentiation into plasma cells, Blimp-1 expression is suppressed in B cells by transcription repressors BTB and CNC homology 2 (Bach2) and B cell lymphoma 6 (Bcl6). Bach2 binds to the Maf recognition element (MARE) of the promoter upstream region of the Blimp-1 gene (Prdm1) by forming a heterodimer with MafK. Bach2 and Bcl6 were found to interact with each other in B cells. While both Bach2 and Bcl6 possess the BTB domain which mediates protein-protein interactions, they interacted in a BTB-independent manner. Bcl6 is known to repress Prdm1 through a Bcl6 recognition element 1 in the intron 5, in which a putative, evolutionarily conserved MARE was identified. Both repressed the expression of a reporter gene containing the intron 5 region depending on the presence of the respective binding sites in 18-81 pre-B cells. Co-expression of Bach2 and Bcl6 resulted in further repression of the reporter plasmid. Chromatin immunoprecipitation assays showed MafK to bind to the intron MARE in various B cell lines, thus suggesting that it binds as a heterodimer with Bach2. Therefore, the interaction between Bach2 and Bcl6 might be crucial for the proper repression of Prdm1 in B cells.

Mutual regulatory interactions of the trunk gap genes during blastoderm patterning in the hemipteran Oncopeltus fasciatus.

PubMed

Ben-David, Jonathan; Chipman, Ariel D

2010-10-01

The early embryo of the milkweed bug, Oncopeltus fasciatus, appears as a single cell layer - the embryonic blastoderm - covering the entire egg. It is at this blastoderm stage that morphological domains are first determined, long before the appearance of overt segmentation. Central to the process of patterning the blastoderm into distinct domains are a group of transcription factors known as gap genes. In Drosophila melanogaster these genes form a network of interactions, and maintain sharp expression boundaries through strong mutual repression. Their restricted expression domains define specific areas along the entire body. We have studied the expression domains of the four trunk gap gene homologues in O. fasciatus and have determined their interactions through dsRNA gene knockdown experiments, followed by expression analyses. While the blastoderm in O. fasciatus includes only the first six segments of the embryo, the expression domains of the gap genes within these segments are broadly similar to those in Drosophila where the blastoderm includes all 15 segments. However, the interactions between the gap genes are surprisingly different from those in Drosophila, and mutual repression between the genes seems to play a much less significant role. This suggests that the well-studied interaction pattern in Drosophila is evolutionarily derived, and has evolved from a less strongly interacting network. Copyright © 2010 Elsevier Inc. All rights reserved.

Chromosome-wide mechanisms to decouple gene expression from gene dose during sex-chromosome evolution

PubMed Central

Wheeler, Bayly S; Anderson, Erika; Frøkjær-Jensen, Christian; Bian, Qian; Jorgensen, Erik; Meyer, Barbara J

2016-01-01

Changes in chromosome number impair fitness by disrupting the balance of gene expression. Here we analyze mechanisms to compensate for changes in gene dose that accompanied the evolution of sex chromosomes from autosomes. Using single-copy transgenes integrated throughout the Caenorhabditis elegans genome, we show that expression of all X-linked transgenes is balanced between XX hermaphrodites and XO males. However, proximity of a dosage compensation complex (DCC) binding site (rex site) is neither necessary to repress X-linked transgenes nor sufficient to repress transgenes on autosomes. Thus, X is broadly permissive for dosage compensation, and the DCC acts via a chromosome-wide mechanism to balance transcription between sexes. In contrast, no analogous X-chromosome-wide mechanism balances transcription between X and autosomes: expression of compensated hermaphrodite X-linked transgenes is half that of autosomal transgenes. Furthermore, our results argue against an X-chromosome dosage compensation model contingent upon rex-directed positioning of X relative to the nuclear periphery. DOI: http://dx.doi.org/10.7554/eLife.17365.001 PMID:27572259

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

PubMed Central

Schreiner, Sabrina; Bürck, Carolin; Glass, Mandy; Groitl, Peter; Wimmer, Peter; Kinkley, Sarah; Mund, Andreas; Everett, Roger D.; Dobner, Thomas

2013-01-01

Death domain–associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein–protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling. PMID:23396441

JAZF1 promotes proliferation of C2C12 cells, but retards their myogenic differentiation through transcriptional repression of MEF2C and MRF4—Implications for the role of Jazf1 variants in oncogenesis and type 2 diabetes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuasa, Katsutoshi; Aoki, Natsumi; Hijikata, Takao, E-mail: hijikata@musashino-u.ac.jp

Single-nucleotide polymorphisms associated with type 2 diabetes (T2D) have been identified in Jazf1, which is also involved in the oncogenesis of endometrial stromal tumors. To understand how Jazf1 variants confer a risk of tumorigenesis and T2D, we explored the functional roles of JAZF1 and searched for JAZF1 target genes in myogenic C2C12 cells. Consistent with an increase of Jazf1 transcripts during myoblast proliferation and their decrease during myogenic differentiation in regenerating skeletal muscle, JAZF1 overexpression promoted cell proliferation, whereas it retarded myogenic differentiation. Examination of myogenic genes revealed that JAZF1 overexpression transcriptionally repressed MEF2C and MRF4 and their downstream genes.more » AMP deaminase1 (AMPD1) was identified as a candidate for JAZF1 target by gene array analysis. However, promoter assays of Ampd1 demonstrated that mutation of the putative binding site for the TR4/JAZF1 complex did not alleviate the repressive effects of JAZF1 on promoter activity. Instead, JAZF1-mediated repression of Ampd1 occurred through the MEF2-binding site and E-box within the Ampd1 proximal regulatory elements. Consistently, MEF2C and MRF4 expression enhanced Ampd1 promoter activity. AMPD1 overexpression and JAZF1 downregulation impaired AMPK phosphorylation, while JAZF1 overexpression also reduced it. Collectively, these results suggest that aberrant JAZF1 expression contributes to the oncogenesis and T2D pathogenesis. - Highlights: • JAZF1 promotes cell cycle progression and proliferation of myoblasts. • JAZF1 retards myogenic differentiation and hypertrophy of myotubes. • JAZF1 transcriptionally represses Mef2C and Mrf4 expression. • JAZF1 has an impact on the phosphorylation of AMPK.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishida, Tamotsu, E-mail: nishida@gene.mie-u.ac.jp; Yamada, Yoshiji

Parkin-interacting substrate (PARIS), a member of the family of Krüppel-associated box (KRAB)-containing zinc-finger transcription factors, is a substrate of the ubiquitin E3 ligase parkin. PARIS represses the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), although the underlying mechanisms remain largely unknown. In the present study, we demonstrate that PARIS can be SUMOylated, and its SUMOylation plays a role in the repression of PGC-1a promoter activity. Protein inhibitor of activated STAT y (PIASy) was identified as an interacting protein of PARIS and shown to enhance its SUMOylation. PIASy repressed PGC-1a promoter activity, and this effect was attenuated by PARIS inmore » a manner dependent on its SUMOylation status. Co-expression of SUMO-1 with PIASy completely repressed PGC-1a promoter activity independently of PARIS expression. PARIS-mediated PGC-1a promoter repression depended on the activity of histone deacetylases (HDAC), whereas PIASy repressed the PGC-1a promoter in an HDAC-independent manner. Taken together, these results suggest that PARIS and PIASy modulate PGC-1a gene transcription through distinct molecular mechanisms. -- Highlights: •PARIS can be SUMOylated in vivo and in vitro. •SUMOylation of PARIS functions in the repression of PGC-1a promoter activity. •PIASy interacts with PARIS and enhances its SUMOylation. •PIASy influences PARIS-mediated repression of PGC-1a promoter activity.« less

Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins

PubMed Central

Frederix, Marijke; Edwards, Anne; Swiderska, Anna; Stanger, Andrew; Karunakaran, Ramakrishnan; Williams, Alan; Abbruscato, Pamela; Sanchez-Contreras, Maria; Poole, Philip S; Downie, J Allan

2014-01-01

In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots. PMID:24942546

The rice YABBY1 gene is involved in the feedback regulation of gibberellin metabolism.

PubMed

Dai, Mingqiu; Zhao, Yu; Ma, Qian; Hu, Yongfeng; Hedden, Peter; Zhang, Qifa; Zhou, Dao-Xiu

2007-05-01

Gibberellin (GA) biosynthesis is regulated by feedback control providing a mechanism for GA homeostasis in plants. However, regulatory elements involved in the feedback control are not known. In this report, we show that a rice (Oryza sativa) YABBY1 (YAB1) gene had a similar expression pattern as key rice GA biosynthetic genes GA3ox2 and GA20ox2. Overexpression of YAB1 in transgenic rice resulted in a semidwarf phenotype that could be fully rescued by applied GA. Quantification of the endogenous GA content revealed increases of GA(20) and decreases of GA(1) levels in the overexpression plants, in which the transcripts of the biosynthetic gene GA3ox2 were decreased. Cosuppression of YAB1 in transgenic plants induced expression of GA3ox2. The repression of GA3ox2 could be obtained upon treatment by dexamethasone of transgenic plants expressing a YAB1-glucocorticoid receptor fusion. Importantly, we show that YAB1 bound to a GA-responsive element within the GA3ox2 promoter. In addition, the expression of YAB1 was deregulated in GA biosynthesis and signaling mutants and could be either transiently induced by GA or repressed by a GA inhibitor. Finally, either overexpression or cosuppression of YAB1 impaired GA-mediated repression of GA3ox2. These data together suggest that YAB1 is involved in the feedback regulation of GA biosynthesis in rice.

Regulatory regions in the promoters of the Saccharomyces cerevisiae PYC1 and PYC2 genes encoding isoenzymes of pyruvate carboxylase.

PubMed

Menéndez, J; Gancedo, C

1998-07-15

We have identified regions in the promoters of the PYC1 and PYC2 genes from Saccharomyces cerevisiae involved in their regulation in different culture conditions. In the case of PYC1, a UAS in the region between -330/-297 and three repressing sequences with the common central core CCGCC at positions -457, -432 and -399 were identified. Specific binding of nuclear proteins to the -330/-214 DNA fragment was abolished in rtg mutants suggesting a role for the RTG genes in the control of PYC1 expression. In the case of the PYC2 promoter, elimination of a fragment from -417 to -291 brings about a two-fold decrease in the expression in repressed conditions and a similar increase in derepression.

Trichostatin A enhances estrogen receptor-alpha repression in MCF-7 breast cancer cells under hypoxia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noh, Hyunggyun; Park, Joonwoo; Shim, Myeongguk

Estrogen receptor (ER) is a crucial determinant of resistance to endocrine therapy, which may change during the progression of breast cancer. We previously showed that hypoxia induces ESR1 gene repression and ERα protein degradation via proteasome-mediated pathway in breast cancer cells. HDAC plays important roles in the regulation of histone and non-histone protein post-translational modification. HDAC inhibitors can induce epigenetic changes and have therapeutic potential for targeting various cancers. Trichostatin A exerts potent antitumor activities against breast cancer cells in vitro and in vivo. In this report, we show that TSA augments ESR1 gene repression at the transcriptional level and downregulates ERαmore » protein expression under hypoxic conditions through a proteasome-mediated pathway. TSA-induced estrogen response element-driven reporter activity in the absence of estrogen was synergistically enhanced under hypoxia; however, TSA inhibited cell proliferation under both normoxia and hypoxia. Our data show that the hypoxia-induced repression of ESR1 and degradation of ERα are enhanced by concomitant treatment with TSA. These findings expand our understanding of hormone responsiveness in the tumor microenvironment; however, additional in-depth studies are required to elucidate the detailed mechanisms of TSA-induced ERα regulation under hypoxia. - Highlights: • TSA augments ESR1 gene repression at the transcriptional level under hypoxia. • TSA downregulates ERα protein expression under hypoxia. • TSA-induced ERα regulation under hypoxia is essential for understanding the behavior and progression of breast cancer.« less

A transcription activator-like effector (TALE) induction system mediated by proteolysis.

PubMed

Copeland, Matthew F; Politz, Mark C; Johnson, Charles B; Markley, Andrew L; Pfleger, Brian F

2016-04-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications owing to their customizable DNA-binding specificity. In this work we expanded the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded after induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator-agnostic.

A transcription activator-like effector induction system mediated by proteolysis

PubMed Central

Copeland, Matthew F.; Politz, Mark C.; Johnson, Charles B.; Markley, Andrew L.; Pfleger, Brian F.

2016-01-01

Simple and predictable trans-acting regulatory tools are needed in the fields of synthetic biology and metabolic engineering to build complex genetic circuits and optimize the levels of native and heterologous gene products. Transcription activator-like effectors (TALEs) are bacterial virulence factors that have recently gained traction in biotechnology applications due to their customizable DNA binding specificity. In this work we expand the versatility of these transcription factors to create an inducible TALE system by inserting tobacco-etch virus (TEV) protease recognition sites into the TALE backbone. The resulting engineered TALEs maintain transcriptional repression of their target genes in Escherichia coli, but are degraded following the induction of the TEV protease, thereby promoting expression of the previously repressed target gene of interest. This TALE-TEV technology enables both repression and induction of plasmid or chromosomal target genes in a manner analogous to traditional repressor proteins but with the added flexibility of being operator agnostic. PMID:26854666

GRHL3/GET1 and Trithorax Group Members Collaborate to Activate the Epidermal Progenitor Differentiation Program

PubMed Central

Hopkin, Amelia Soto; Gordon, William; Klein, Rachel Herndon; Espitia, Francisco; Daily, Kenneth; Zeller, Michael; Baldi, Pierre; Andersen, Bogi

2012-01-01

The antagonistic actions of Polycomb and Trithorax are responsible for proper cell fate determination in mammalian tissues. In the epidermis, a self-renewing epithelium, previous work has shown that release from Polycomb repression only partially explains differentiation gene activation. We now show that Trithorax is also a key regulator of epidermal differentiation, not only through activation of genes repressed by Polycomb in progenitor cells, but also through activation of genes independent of regulation by Polycomb. The differentiation associated transcription factor GRHL3/GET1 recruits the ubiquitously expressed Trithorax complex to a subset of differentiation genes. PMID:22829784

Gravity Plays an Important Role in Muscle Development and the Differentiation of Contractile Protein Phenotype

NASA Technical Reports Server (NTRS)

Adams, Gregory A.; Haddad, Fadia; Baldwin, Kenneth M.

2003-01-01

Several muscles in the body exist mainly to work against gravity. Whether gravity is important in the development of these muscles is not known. By examining the basic proteins that compose muscle, questions about the role of gravity in muscle development can be answered. Myosin heavy chains (MHCs) are a family of proteins critically important for muscle contraction. Several types of MHCs exist (e.g., neonatal, slow, fast), and each type is produced by a particular gene. Neonatal MHCs are produced early in life. Slow MHCs are important in antigravity muscles, and fast MHCs are found in fast-twitch power muscles. The gene that is turned on or expressed will determine which MHC is produced. Early in development, antigravity skeletal muscles (muscles that work against gravity) normally produce a combination of the neonatal/embryonic MHCs. The expression of these primitive MHCs is repressed early in development; and the adult slow and fast MHC genes become fully expressed. We tested the hypothesis that weightbearing activity is critical for inducing the normal expression of the slow MHC gene typically expressed in adult antigravity muscles. Also, we hypothesized that thyroid hormone, but not opposition to gravity, is necessary for expressing the adult fast IIb MHC gene essential for high-intensity muscle performance. Groups of normal thyroid and thyroid-deficient neonatal rats were studied after their return from the 16-day Neurolab mission and compared to matched controls. The results suggest: (1) Weightlessness impaired body and limb skeletal muscle growth in both normal and thyroid-deficient animals. Antigravity muscles were impaired more than those used primarily for locomotion andor nonweightbearing activity. (2) Systemic and muscle expression of insulin-like growth factor-I (IGF-I), an important body and tissue growth factor, was depressed in flight animals. (3) Normal slow, type I MHC gene expression was markedly repressed in the normal thyroid flight group. (4) Fast IIb MHC gene expression was enhanced in fast-twitch muscles of normal thyroid animals exposed to spaceflight; however, thyroid deficiency markedly repressed expression of this gene independently of spaceflight. In summary, the absence of gravity, when imposed at critical stages of development, impaired body and skeletal muscle growth, as well as expression of the MHC gene family of motor proteins. This suggests that normal weightbearing activity is essential for establishing body and muscle growth in neonatal animals, and for expressing the motor gene essential for supporting antigravity functions.

Metabolic Adaptation to Nutrients Involves Coregulation of Gene Expression by the RNA Helicase Dbp2 and the Cyc8 Corepressor in Saccharomyces cerevisiae.

PubMed

Wang, Siwen; Xing, Zheng; Pascuzzi, Pete E; Tran, Elizabeth J

2017-07-05

Cells fine-tune their metabolic programs according to nutrient availability in order to maintain homeostasis. This is achieved largely through integrating signaling pathways and the gene expression program, allowing cells to adapt to nutritional change. Dbp2, a member of the DEAD-box RNA helicase family in Saccharomyces cerevisiae , has been proposed to integrate gene expression with cellular metabolism. Prior work from our laboratory has reported the necessity of DBP2 in proper gene expression, particularly for genes involved in glucose-dependent regulation. Here, by comparing differentially expressed genes in dbp2 ∆ to those of 700 other deletion strains from other studies, we find that CYC8 and TUP1 , which form a complex and inhibit transcription of numerous genes, corepress a common set of genes with DBP2 Gene ontology (GO) annotations reveal that these corepressed genes are related to cellular metabolism, including respiration, gluconeogenesis, and alternative carbon-source utilization genes. Consistent with a direct role in metabolic gene regulation, loss of either DBP2 or CYC8 results in increased cellular respiration rates. Furthermore, we find that corepressed genes have a propensity to be associated with overlapping long noncoding RNAs and that upregulation of these genes in the absence of DBP2 correlates with decreased binding of Cyc8 to these gene promoters. Taken together, this suggests that Dbp2 integrates nutrient availability with energy homeostasis by maintaining repression of glucose-repressed, Cyc8-targeted genes across the genome. Copyright © 2017 Wang et al.

[Construction of screening system for mutation of negative regulatory genes in Streptomyces].

PubMed

Zhu, Yu; Feng, Chi; Tan, Huarong; Tian, Yuqing

2013-10-04

We aimed to create a novel report system for screening the mutation of the negative regulatory genes, especially for those repressing the expression of cryptic antibiotics clusters. We used marker-free gene disruption strategy, which combines with the "REDIRECT (Rapid Efficient Directed Recombination Time Saving)" technology and in vivo site-specific recombination by Streptomyces phage phiBT1 integrase, to construct a scbR2/inoA double mutant strain of S. coelicolor M145. This strain was used as the host of the report system. For the construction of the reporter plasmid, the ScbR2 repressed promoter of cpkO from CPK (cryptic polyketide) cluster was used to drive the expression of a promoterless conserved gene inoA of S. coelicolor. Then the reporter plasmid was introduced into the host strain described above to test the availability of inoA as a reporter gene in this system. The scbR2/inoA double mutant strain gave rise to a bald pheno type on MM medium in the absence of inositol, and produced yellow pigmented secondary metabolite by the disruption of scbR2 to release the repression of cpkO, a pathway specific activator gene situated in CPK cluster. After introducing the reporter plasmid into this test stain, the resulting strain recovered the phenotype as wild-type strain, indicating that the promoter of cpkO can drive the expression of inoA in scbR2 mutant and consequently restore the biosynthesis of inositol. Our results indicated that inoA can be used as a novel reporter gene for Streptomyces, especially for detecting the activation of the "silent" promoter. This report system might be available for screening the mutation of the negative regulatory genes for the cryptic secondary metabolic gene clusters.

SUV39H1 interacts with HTLV-1 Tax and abrogates Tax transactivation of HTLV-1 LTR

PubMed Central

Kamoi, Koju; Yamamoto, Keiyu; Misawa, Aya; Miyake, Ariko; Ishida, Takaomi; Tanaka, Yuetsu; Mochizuki, Manabu; Watanabe, Toshiki

2006-01-01

Background Tax is the oncoprotein of HTLV-1 which deregulates signal transduction pathways, transcription of genes and cell cycle regulation of host cells. Transacting function of Tax is mainly mediated by its protein-protein interactions with host cellular factors. As to Tax-mediated regulation of gene expression of HTLV-1 and cellular genes, Tax was shown to regulate histone acetylation through its physical interaction with histone acetylases and deacetylases. However, functional interaction of Tax with histone methyltransferases (HMTase) has not been studied. Here we examined the ability of Tax to interact with a histone methyltransferase SUV39H1 that methylates histone H3 lysine 9 (H3K9) and represses transcription of genes, and studied the functional effects of the interaction on HTLV-1 gene expression. Results Tax was shown to interact with SUV39H1 in vitro, and the interaction is largely dependent on the C-terminal half of SUV39H1 containing the SET domain. Tax does not affect the methyltransferase activity of SUV39H1 but tethers SUV39H1 to a Tax containing complex in the nuclei. In reporter gene assays, co-expression of SUV39H1 represses Tax transactivation of HTLV-1 LTR promoter activity, which was dependent on the methyltransferase activity of SUV39H1. Furthermore, SUV39H1 expression is induced along with Tax in JPX9 cells. Chromatin immunoprecipitation (ChIP) analysis shows localization of SUV39H1 on the LTR after Tax induction, but not in the absence of Tax induction, in JPX9 transformants retaining HTLV-1-Luc plasmid. Immunoblotting shows higher levels of SUV39H1 expression in HTLV-1 transformed and latently infected cell lines. Conclusion Our study revealed for the first time the interaction between Tax and SUV39H1 and apparent tethering of SUV39H1 by Tax to the HTLV-1 LTR. It is speculated that Tax-mediated tethering of SUV39H1 to the LTR and induction of the repressive histone modification on the chromatin through H3 K9 methylation may be the basis for the dose-dependent repression of Tax transactivation of LTR by SUV39H1. Tax-induced SUV39H1 expression, Tax-SUV39H1 interaction and tethering to the LTR may provide a support for an idea that the above sequence of events may form a negative feedback loop that self-limits HTLV-1 viral gene expression in infected cells. PMID:16409643

Arabidopsis Polycomb Repressive Complex 2 binding sites contain putative GAGA factor binding motifs within coding regions of genes

PubMed Central

2013-01-01

Background Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes. Results We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs). Conclusions Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3. PMID:24001316

Enterovirus Control of Translation and RNA Granule Stress Responses.

PubMed

Lloyd, Richard E

2016-03-30

Enteroviruses such as poliovirus (PV) and coxsackievirus B3 (CVB3) have evolved several parallel strategies to regulate cellular gene expression and stress responses to ensure efficient expression of the viral genome. Enteroviruses utilize their encoded proteinases to take over the cellular translation apparatus and direct ribosomes to viral mRNAs. In addition, viral proteinases are used to control and repress the two main types of cytoplasmic RNA granules, stress granules (SGs) and processing bodies (P-bodies, PBs), which are stress-responsive dynamic structures involved in repression of gene expression. This review discusses these processes and the current understanding of the underlying mechanisms with respect to enterovirus infections. In addition, the review discusses accumulating data suggesting linkage exists between RNA granule formation and innate immune sensing and activation.

NRSF-dependent epigenetic mechanisms contribute to programming of stress-sensitive neurons by neonatal experience, promoting resilience.

PubMed

Singh-Taylor, A; Molet, J; Jiang, S; Korosi, A; Bolton, J L; Noam, Y; Simeone, K; Cope, J; Chen, Y; Mortazavi, A; Baram, T Z

2018-03-01

Resilience to stress-related emotional disorders is governed in part by early-life experiences. Here we demonstrate experience-dependent re-programming of stress-sensitive hypothalamic neurons, which takes place through modification of neuronal gene expression via epigenetic mechanisms. Specifically, we found that augmented maternal care reduced glutamatergic synapses onto stress-sensitive hypothalamic neurons and repressed expression of the stress-responsive gene, Crh. In hypothalamus in vitro, reduced glutamatergic neurotransmission recapitulated the repressive effects of augmented maternal care on Crh, and this required recruitment of the transcriptional repressor repressor element-1 silencing transcription factor/neuron restrictive silencing factor (NRSF). Increased NRSF binding to chromatin was accompanied by sequential repressive epigenetic changes which outlasted NRSF binding. chromatin immunoprecipitation-seq analyses of NRSF targets identified gene networks that, in addition to Crh, likely contributed to the augmented care-induced phenotype, including diminished depression-like and anxiety-like behaviors. Together, we believe these findings provide the first causal link between enriched neonatal experience, synaptic refinement and induction of epigenetic processes within specific neurons. They uncover a novel mechanistic pathway from neonatal environment to emotional resilience.

Mycotoxigenic Fusarium and Deoxynivalenol Production Repress Chitinase Gene Expression in the Biocontrol Agent Trichoderma atroviride P1

PubMed Central

Lutz, Matthias P.; Feichtinger, Georg; Défago, Geneviève; Duffy, Brion

2003-01-01

Mycotoxin contamination associated with head blight of wheat and other grains caused by Fusarium culmorum and F. graminearum is a chronic threat to crop, human, and animal health throughout the world. One of the most important toxins in terms of human exposure is deoxynivalenol (DON) (formerly called vomitoxin), an inhibitor of protein synthesis with a broad spectrum of toxigenicity against animals. Certain Fusarium toxins have additional antimicrobial activity, and the phytotoxin fusaric acid has recently been shown to modulate fungus-bacterium interactions that affect plant health (Duffy and Défago, Phytopathology 87:1250-1257, 1997). The potential impact of DON on Fusarium competition with other microorganisms has not been described previously. Any competitive advantage conferred by DON would complicate efforts to control Fusarium during its saprophytic growth on crop residues that are left after harvest and constitute the primary inoculum reservoir for outbreaks in subsequent plantings. We examined the effect of the DON mycotoxin on ecological interactions between pathogenic Fusarium and Trichoderma atroviride strain P1, a competitor fungus with biocontrol activity against a wide range of plant diseases. Expression of the Trichoderma chitinase genes, ech42 and nag1, which contribute to biocontrol activity, was monitored in vitro and on crop residues of two maize cultivars by using goxA reporter gene fusions. We found that DON-producing F. culmorum and F. graminearum strains repressed expression of nag1-gox. DON-negative wild-type Fusarium strains and a DON-negative mutant with an insertional disruption in the tricothecene biosynthetic gene, tri5, had no effect on antagonist gene expression. The role of DON as the principal repressor above other pathogen factors was confirmed. Exposure of Trichoderma to synthetic DON or to a non-DON-producing Fusarium mutant resulted in the same level of nag1-gox repression as the level observed with DON-producing Fusarium. DON repression was specific for nag1-gox and had no effect, either positive or negative, on expression of another key chitinase gene, ech42. This is the first demonstration that a target pathogen down-regulates genes in a fungal biocontrol agent, and our results provide evidence that mycotoxins have a novel ecological function as factors in Fusarium competitiveness. PMID:12788701

Global gene expression analysis of the heat shock response in the phytopathogen Xylella fastidiosa.

PubMed

Koide, Tie; Vêncio, Ricardo Z N; Gomes, Suely L

2006-08-01

Xylella fastidiosa is a phytopathogenic bacterium that is responsible for diseases in many economically important crops. Although different strains have been studied, little is known about X. fastidiosa stress responses. One of the better characterized stress responses in bacteria is the heat shock response, which induces the expression of specific genes to prevent protein misfolding and aggregation and to promote degradation of the irreversibly denatured polypeptides. To investigate X. fastidiosa genes involved in the heat shock response, we performed a whole-genome microarray analysis in a time course experiment. Globally, 261 genes were induced (9.7%) and 222 genes were repressed (8.3%). The expression profiles of the differentially expressed genes were grouped, and their expression patterns were validated by quantitative reverse transcription-PCR experiments. We determined the transcription start sites of six heat shock-inducible genes and analyzed their promoter regions, which allowed us to propose a putative consensus for sigma(32) promoters in Xylella and to suggest additional genes as putative members of this regulon. Besides the induction of classical heat shock protein genes, we observed the up-regulation of virulence-associated genes such as vapD and of genes for hemagglutinins, hemolysin, and xylan-degrading enzymes, which may indicate the importance of heat stress to bacterial pathogenesis. In addition, we observed the repression of genes related to fimbriae, aerobic respiration, and protein biosynthesis and the induction of genes related to the extracytoplasmic stress response and some phage-related genes, revealing the complex network of genes that work together in response to heat shock.

Sumoylation of the net inhibitory domain (NID) is stimulated by PIAS1 and has a negative effect on the transcriptional activity of Net.

PubMed

Wasylyk, Christine; Criqui-Filipe, Paola; Wasylyk, Bohdan

2005-01-27

Net (Elk-3, Sap-2, Erp) and the related ternary complex factors Elk-1 and Sap-1 are effectors of multiple signalling pathways at the transcriptional level and play a key role in the dynamic regulation of gene expression. Net is distinct from Elk-1 and Sap-1, in that it is a strong repressor of transcription that is converted to an activator by the Ras/Erk signalling pathway. Two autonomous repression domains of Net, the NID and the CID, mediate repression. We have previously shown that the co-repressor CtBP is implicated in repression by the CID. In this report we show that repression by the NID involves a different pathway, sumoylation by Ubc9 and PIAS1. PIAS1 interacts with the NID in the two-hybrid assay and in vitro. Ubc9 and PIAS1 stimulate sumoylation in vivo of lysine 162 in the NID. Sumoylation of lysine 162 increases repression by Net and decreases the positive activity of Net. These results increase our understanding of how one of the ternary complex factors regulates transcription, and contribute to the understanding of how different domains of a transcription factor participate in the complexity of regulation of gene expression.

Gene expression changes in uterine myomas in response to ulipristal acetate treatment.

PubMed

Courtoy, Guillaume E; Donnez, Jacques; Ambroise, Jérôme; Arriagada, Pablo; Luyckx, Mathieu; Marbaix, Etienne; Dolmans, Marie-Madeleine

2018-05-07

Does ulipristal acetate (UPA) modify the expression of genes related to apoptosis or the extracellular matrix in uterine myomas and are any modifications associated with a clinical response? Targeted analysis of 176 apoptosis- or extracellular-matrix-related genes was conducted using polymerase chain reaction (PCR) arrays. Relevant results were validated by quantitative PCR. Four groups were established: responsive short-term (one course, n = 9), responsive long-term (two to four courses, n = 9), non-responsive (n = 9), and the control group who was not given any hormone therapy (n = 9). The clinical response was monitored by medical imagery and considered significant when volume reduction was greater than 25%. Compared with untreated myomas, significant changes in expression of four genes were found in UPA-treated myomas. Gene expression of integrin subunit beta 4 was repressed by UPA treatment (fold change [FC] = -12.50, P < 0.001, q < 0.001), tenascin-C expression was downregulated in UPA-responsive patients (FC = -2.50, P = 0.010, q = 0.090), survivin was repressed in short-term UPA-responsive tumours (FC = -7.69, P < 0.001, q = 0.010), and catenin delta 2 gene expression was upregulated in non-responsive myomas (FC = +7.36, P < 0.001, q = 0.010). This characterization provides the first molecular distinction between myomas responsive or non-responsive to UPA treatment. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

SSTY proteins colocalize with the postmeiotic sex chromatin and interact with regulators of its expression

PubMed Central

Comptour, Aurélie; Moretti, Charlotte; Serrentino, Maria-Elisabetta; Auer, Jana; Ialy-Radio, Côme; Ward, Monika A.; Touré, Aminata; Vaiman, Daniel; Cocquet, Julie

2014-01-01

In mammals, X- and Y-encoded genes are transcriptionally shut down during male meiosis, but the expression of many of them is (re)activated, after meiosis, in spermatids. Postmeiotic XY gene expression is timely regulated by active epigenetic marks, which are de novo incorporated in the sex chromatin of spermatids, and by repressive epigenetic marks inherited from meiosis; alteration in this process leads to male infertility. In the mouse, postmeiotic XY gene expression is known to depend on genetic information carried by the male specific region of the Y chromosome long arm (MSYq). The MSYq gene Sly has been shown to be a key regulator of postmeiotic sex chromosome gene expression and necessary for the maintenance/recruitment of repressive epigenetic marks on the sex chromatin, but studies suggest that another MSYq gene may be required. The best candidate to date is Ssty, an MSYq multicopy gene of unknown function. Here, we show that SSTY proteins are specifically expressed in round and elongating spermatids and colocalize with the postmeiotic sex chromatin. Moreover, SSTY proteins interact with SLY protein and its X-linked homolog SLX/SLXL1, and may be required for the localization of SLX/SLY proteins in the spermatid nucleus and sex chromatin. Our data suggest that SSTY is a second MSYq factor involved in the control of XY gene expression during sperm differentiation. As Slx/Slxl1 and Sly genes have been shown to be involved in the XY intragenomic conflict which affects the offspring sex-ratio, Ssty might constitute another actor of this conflict. PMID:24456183

Localized epigenetic silencing of a damage-activated WNT enhancer limits regeneration in mature Drosophila imaginal discs

PubMed Central

Harris, Robin E; Setiawan, Linda; Saul, Josh; Hariharan, Iswar K

2016-01-01

Many organisms lose the capacity to regenerate damaged tissues as they mature. Damaged Drosophila imaginal discs regenerate efficiently early in the third larval instar (L3) but progressively lose this ability. This correlates with reduced damage-responsive expression of multiple genes, including the WNT genes wingless (wg) and Wnt6. We demonstrate that damage-responsive expression of both genes requires a bipartite enhancer whose activity declines during L3. Within this enhancer, a damage-responsive module stays active throughout L3, while an adjacent silencing element nucleates increasing levels of epigenetic silencing restricted to this enhancer. Cas9-mediated deletion of the silencing element alleviates WNT repression, but is, in itself, insufficient to promote regeneration. However, directing Myc expression to the blastema overcomes repression of multiple genes, including wg, and restores cellular responses necessary for regeneration. Localized epigenetic silencing of damage-responsive enhancers can therefore restrict regenerative capacity in maturing organisms without compromising gene functions regulated by developmental signals. DOI: http://dx.doi.org/10.7554/eLife.11588.001 PMID:26840050

A dynamic interplay of nucleosome and Msn2 binding regulates kinetics of gene activation and repression following stress

PubMed Central

Elfving, Nils; Chereji, Răzvan V.; Bharatula, Vasudha; Björklund, Stefan; Morozov, Alexandre V.; Broach, James R.

2014-01-01

The transcription factor Msn2 mediates a significant proportion of the environmental stress response, in which a common cohort of genes changes expression in a stereotypic fashion upon exposure to any of a wide variety of stresses. We have applied genome-wide chromatin immunoprecipitation and nucleosome profiling to determine where Msn2 binds under stressful conditions and how that binding affects, and is affected by, nucleosome positioning. We concurrently determined the effect of Msn2 activity on gene expression following stress and demonstrated that Msn2 stimulates both activation and repression. We found that some genes responded to both intermittent and continuous Msn2 nuclear occupancy while others responded only to continuous occupancy. Finally, these studies document a dynamic interplay between nucleosomes and Msn2 such that nucleosomes can restrict access of Msn2 to its canonical binding sites while Msn2 can promote reposition, expulsion and recruitment of nucleosomes to alter gene expression. This interplay may allow the cell to discriminate between different types of stress signaling. PMID:24598258

Transcriptome alteration in Phytophthora infestans in response to phenazine-1-carboxylic acid production by Pseudomonas fluorescens strain LBUM223.

PubMed

Roquigny, Roxane; Novinscak, Amy; Arseneault, Tanya; Joly, David L; Filion, Martin

2018-06-19

Phytophthora infestans is responsible for late blight, one of the most important potato diseases. Phenazine-1-carboxylic acid (PCA)-producing Pseudomonas fluorescens strain LBUM223 isolated in our laboratory shows biocontrol potential against various plant pathogens. To characterize the effect of LBUM223 on the transcriptome of P. infestans, we conducted an in vitro time-course study. Confrontational assay was performed using P. infestans inoculated alone (control) or with LBUM223, its phzC- isogenic mutant (not producing PCA), or exogenically applied PCA. Destructive sampling was performed at 6, 9 and 12 days and the transcriptome of P. infestans was analysed using RNA-Seq. The expression of a subset of differentially expressed genes was validated by RT-qPCR. Both LBUM223 and exogenically applied PCA significantly repressed P. infestans' growth at all times. Compared to the control treatment, transcriptomic analyses showed that the percentages of all P. infestans' genes significantly altered by LBUM223 and exogenically applied PCA increased as time progressed, from 50 to 61% and from to 32 to 46%, respectively. When applying an absolute cut-off value of 3 fold change or more for all three harvesting times, 207 genes were found significantly differentially expressed by PCA, either produced by LBUM223 or exogenically applied. Gene ontology analysis revealed that both treatments altered the expression of key functional genes involved in major functions like phosphorylation mechanisms, transmembrane transport and oxidoreduction activities. Interestingly, even though no host plant tissue was present in the in vitro system, PCA also led to the overexpression of several genes encoding effectors. The mutant only slightly repressed P. infestans' growth and barely altered its transcriptome. Our study suggests that PCA is involved in P. infestans' growth repression and led to important transcriptomic changes by both up- and down-regulating gene expression in P. infestans over time. Different metabolic functions were altered and many effectors were found to be upregulated, suggesting their implication in biocontrol.

Heat shock instructs hESCs to exit from the self-renewal program through negative regulation of OCT4 by SAPK/JNK and HSF1 pathway.

PubMed

Byun, Kyunghee; Kim, Taek-Kyun; Oh, Jeehyun; Bayarsaikhan, Enkhjargal; Kim, Daesik; Lee, Min Young; Pack, Chan-Gi; Hwang, Daehee; Lee, Bonghee

2013-11-01

Environmental factors affect self-renewal of stem cells by modulating the components of self-renewal networks. Heat shock, an environmental factor, induces heat shock factors (HSFs), which up-regulate stress response-related genes. However, the link of heat shock to self-renewal of stem cells has not been elucidated yet. Here, we present the direct link of heat shock to a core stem cell regulator, OCT4, in the self-renewal network through SAPK/JNK and HSF1 pathway. We first showed that heat shock initiated differentiation of human embryonic stem cells (hESCs). Gene expression analysis revealed that heat shock increased the expression of many genes involved in cellular processes related to differentiation of stem cells. We then examined the effects of HSFs induced by heat shock on core self-renewal factors. Among HSFs, heat shock induced mainly HSF1 in hESCs. The HSF1 repressed the expression of OCT4, leading to the differentiation of hESCs and the above differentiation-related gene expression change. We further examined the effects of the upstream MAP (mitogen-activated protein) kinases of HSF1 on the repression of OCT4 expression by HSF1. Among the MAP kinases, SAPK/JNK controlled predominantly the repression of the OCT4 expression by HSF1. The direct link of heat shock to the core self-renewal regulator through SAPK/JNK and HSF1 provides a fundamental basis for understanding the effect of heat and other stresses involving activation of HSF1 on the self-renewal program and further controlling differentiation of hESCs in a broad spectrum of stem cell applications using these stresses. © 2013.

The RNA helicase DDX17 controls the transcriptional activity of REST and the expression of proneural microRNAs in neuronal differentiation.

PubMed

Lambert, Marie-Pierre; Terrone, Sophie; Giraud, Guillaume; Benoit-Pilven, Clara; Cluet, David; Combaret, Valérie; Mortreux, Franck; Auboeuf, Didier; Bourgeois, Cyril F

2018-06-21

The Repressor Element 1-silencing transcription factor (REST) represses a number of neuronal genes in non-neuronal cells or in undifferentiated neural progenitors. Here, we report that the DEAD box RNA helicase DDX17 controls important REST-related processes that are critical during the early phases of neuronal differentiation. First, DDX17 associates with REST, promotes its binding to the promoter of a subset of REST-targeted genes and co-regulates REST transcriptional repression activity. During neuronal differentiation, we observed a downregulation of DDX17 along with that of the REST complex that contributes to the activation of neuronal genes. Second, DDX17 and its paralog DDX5 regulate the expression of several proneural microRNAs that are known to target the REST complex during neurogenesis, including miR-26a/b that are also direct regulators of DDX17 expression. In this context, we propose a new mechanism by which RNA helicases can control the biogenesis of intronic miRNAs. We show that the processing of the miR-26a2 precursor is dependent on RNA helicases, owing to an intronic regulatory region that negatively impacts on both miRNA processing and splicing of its host intron. Our work places DDX17 in the heart of a pathway involving REST and miRNAs that allows neuronal gene repression.

Use of transcriptomic data for extending a model of the AppA/PpsR system in Rhodobacter sphaeroides.

PubMed

Pandey, Rakesh; Armitage, Judith P; Wadhams, George H

2017-12-28

Photosynthetic (PS) gene expression in Rhodobacter sphaeroides is regulated in response to changes in light and redox conditions mainly by PrrB/A, FnrL and AppA/PpsR systems. The PrrB/A and FnrL systems activate the expression of them under anaerobic conditions while the AppA/PpsR system represses them under aerobic conditions. Recently, two mathematical models have been developed for the AppA/PpsR system and demonstrated how the interaction between AppA and PpsR could lead to a phenotype in which PS genes are repressed under semi-aerobic conditions. These models have also predicted that the transition from aerobic to anaerobic growth mode could occur via a bistable regime. However, they lack experimentally quantifiable inputs and outputs. Here, we extend one of them to include such quantities and combine all relevant micro-array data publically available for a PS gene of this bacterium and use that to parameterise the model. In addition, we hypothesise that the AppA/PpsR system alone might account for the observed trend of PS gene expression under semi-aerobic conditions. Our extended model of the AppA/PpsR system includes the biological input of atmospheric oxygen concentration and an output of photosynthetic gene expression. Following our hypothesis that the AppA/PpsR system alone is sufficient to describe the overall trend of PS gene expression we parameterise the model and suggest that the rate of AppA reduction in vivo should be faster than its oxidation. Also, we show that despite both the reduced and oxidised forms of PpsR binding to the PS gene promoters in vitro, binding of the oxidised form as a repressor alone is sufficient to reproduce the observed PS gene expression pattern. Finally, the combination of model parameters which fit the biological data well are broadly consistent with those which were previously determined to be required for the system to show (i) the repression of PS genes under semi-aerobic conditions, and (ii) bistability. We found that despite at least three pathways being involved in the regulation of photosynthetic genes, the AppA/PpsR system alone is capable of accounting for the observed trends in photosynthetic gene expression seen at different oxygen levels.

Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

PubMed

Amabile, Angelo; Migliara, Alessandro; Capasso, Paola; Biffi, Mauro; Cittaro, Davide; Naldini, Luigi; Lombardo, Angelo

2016-09-22

Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Emerin and histone deacetylase 3 (HDAC3) cooperatively regulate expression and nuclear positions of MyoD, Myf5, and Pax7 genes during myogenesis

PubMed Central

Demmerle, Justin; Koch, Adam J.; Holaska, James M.

2016-01-01

The spatial organization of chromatin is critical in establishing cell-type dependent gene expression programs. The inner nuclear membrane protein emerin has been implicated in regulating global chromatin architecture. We show emerin associates with genomic loci of muscle differentiation promoting factors in murine myogenic progenitors, including Myf5 and MyoD. Prior to their transcriptional activation Myf5 and MyoD loci localized to the nuclear lamina in proliferating progenitors and moved to the nucleoplasm upon transcriptional activation during differentiation. The Pax7 locus, which is transcribed in proliferating progenitors, localized to the nucleoplasm and Pax7 moved to the nuclear lamina upon repression during differentiation. Localization of Myf5, MyoD, and Pax7 to the nuclear lamina and proper temporal expression of these genes required emerin and HDAC3. Interestingly, activation of HDAC3 catalytic activity rescued both Myf5 localization to the nuclear lamina and its expression. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear lamina by activating the catalytic activity of HDAC3 to regulate the coordinated spatiotemporal expression of myogenic differentiation genes. PMID:24062260

Divergent Expression Patterns and Function Implications of Four nanos Genes in a Hermaphroditic Fish, Epinephelus coioides.

PubMed

Sun, Zhi-Hui; Wang, Yang; Lu, Wei-Jia; Li, Zhi; Liu, Xiao-Chun; Li, Shui-Sheng; Zhou, Li; Gui, Jian-Fang

2017-03-23

Multiple nanos genes have been characterized in several fishes, but the functional implications of their various expression patterns remain unclear. In this study, we identified and characterized four nanos genes from a hermaphroditic fish orange-spotted grouper, Epinephelus coioides . Ecnanos1a and Ecnanos1b show divergent expression patterns, and the dynamic expression change of Ecnanos1a in pituitaries during sex change is associated with testis differentiation and spermatogenesis. Ecnanos2 and Ecnanos3 might be germline stem cells (GSCs) and primordial germ cells (PGCs)-specific markers, respectively. Significantly, Ecnanos3 3'-untranslated region (UTR) is necessary for PGC specific expression, where a non-canonical "GCACGTTT" sequence is required for miR-430-mediated repression of Ecnanos3 RNA. Furthermore, grouper Dead end (Dnd) can relieve miR-430 repression in PGCs by associating with a 23 bp U-rich region (URR) in Ecnanos3 3'-UTR. The current study revealed the functional association of multiple nanos genes with PGC formation and germ cell development in orange-spotted grouper, and opened up new possibilities for developing biotechnologies through utilizing the associations between Ecnanos3 and PGCs or between Ecnanos2 and GSCs in the hermaphroditic fish.

Active and Repressive Chromatin Are Interspersed without Spreading in an Imprinted Gene Cluster in the Mammalian Genome

PubMed Central

Regha, Kakkad; Sloane, Mathew A.; Huang, Ru; Pauler, Florian M.; Warczok, Katarzyna E.; Melikant, Balázs; Radolf, Martin; Martens, Joost H.A.; Schotta, Gunnar; Jenuwein, Thomas; Barlow, Denise P.

2010-01-01

SUMMARY The Igf2r imprinted cluster is an epigenetic silencing model in which expression of a ncRNA silences multiple genes in cis. Here, we map a 250 kb region in mouse embryonic fibroblast cells to show that histone modifications associated with expressed and silent genes are mutually exclusive and localized to discrete regions. Expressed genes were modified at promoter regions by H3K4me3 + H3K4me2 + H3K9Ac and on putative regulatory elements flanking active promoters by H3K4me2 + H3K9Ac. Silent genes showed two types of nonoverlapping profile. One type spread over large domains of tissue-specific silent genes and contained H3K27me3 alone. A second type formed localized foci on silent imprinted gene promoters and a nonexpressed pseudogene and contained H3K9me3 + H4K20me3 ± HP1. Thus, mammalian chromosome arms contain active chromatin interspersed with repressive chromatin resembling the type of heterochromatin previously considered a feature of centromeres, telomeres, and the inactive X chromosome. PMID:17679087

Roles of polypyrimidine tract binding proteins in major immediate-early gene expression and viral replication of human cytomegalovirus.

PubMed

Cosme, Ruth S Cruz; Yamamura, Yasuhiro; Tang, Qiyi

2009-04-01

Human cytomegalovirus (HCMV), a member of the beta subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns.

Roles of Polypyrimidine Tract Binding Proteins in Major Immediate-Early Gene Expression and Viral Replication of Human Cytomegalovirus▿

PubMed Central

Cosme, Ruth S. Cruz; Yamamura, Yasuhiro; Tang, Qiyi

2009-01-01

Human cytomegalovirus (HCMV), a member of the β subgroup of the family Herpesviridae, causes serious health problems worldwide. HCMV gene expression in host cells is a well-defined sequential process: immediate-early (IE) gene expression, early-gene expression, DNA replication, and late-gene expression. The most abundant IE gene, major IE (MIE) gene pre-mRNA, needs to be spliced before being exported to the cytoplasm for translation. In this study, the regulation of MIE gene splicing was investigated; in so doing, we found that polypyrimidine tract binding proteins (PTBs) strongly repressed MIE gene production in cotransfection assays. In addition, we discovered that the repressive effects of PTB could be rescued by splicing factor U2AF. Taken together, the results suggest that PTBs inhibit MIE gene splicing by competing with U2AF65 for binding to the polypyrimidine tract in pre-mRNA. In intron deletion mutation assays and RNA detection experiments (reverse transcription [RT]-PCR and real-time RT-PCR), we further observed that PTBs target all the introns of the MIE gene, especially intron 2, and affect gene splicing, which was reflected in the variation in the ratio of pre-mRNA to mRNA. Using transfection assays, we demonstrated that PTB knockdown cells induce a higher degree of MIE gene splicing/expression. Consistently, HCMV can produce more viral proteins and viral particles in PTB knockdown cells after infection. We conclude that PTB inhibits HCMV replication by interfering with MIE gene splicing through competition with U2AF for binding to the polypyrimidine tract in MIE gene introns. PMID:19144709

PcG and trxG in plants - friends or foes.

PubMed

Pu, Li; Sung, Zinmay Renee

2015-05-01

The highly-conserved Polycomb group (PcG) and trithorax group (trxG) proteins play major roles in regulating gene expression and maintaining developmental states in many organisms. However, neither the recruitment of Polycomb repressive complexes (PRC) nor the mechanisms of PcG and trxG-mediated gene silencing and activation are well understood. Recent progress in Arabidopsis research challenges the dominant model of PRC2-dependent recruitment of PRC1 to target genes. Moreover, evidence indicates that diverse forms of PRC1, with shared components, are a common theme in plants and mammals. Although trxG is known to antagonize PcG, emerging data reveal that trxG can also repress gene expression, acting cooperatively with PcG. We discuss these recent findings and highlight the employment of diverse epigenetic mechanisms during development in plants and animals. Copyright © 2015 Elsevier Ltd. All rights reserved.

The transcription repressor, ZEB1, cooperates with CtBP2 and HDAC1 to suppress IL-2 gene activation in T cells.

PubMed

Wang, Jun; Lee, Seungsoo; Teh, Charis En-Yi; Bunting, Karen; Ma, Lina; Shannon, M Frances

2009-03-01

Activation of T cells leads to the induction of many cytokine genes that are required for appropriate immune responses, including IL-2, a key cytokine for T cell proliferation and homeostasis. The activating transcription factors such as nuclear factor of activated T cells, nuclear factor kappaB/Rel and activated protein-1 family members that regulate inducible IL-2 gene expression have been well documented. However, negative regulation of the IL-2 gene is less studied. Here we examine the role of zinc finger E-box-binding protein (ZEB) 1, a homeodomain/Zn finger transcription factor, as a repressor of IL-2 gene transcription. We show here that ZEB1 is expressed in non-stimulated and stimulated T cells and using chromatin immunoprecipitation assays we show that ZEB1 binds to the IL-2 promoter. Over-expression of ZEB1 can repress IL-2 promoter activity, as well as endogenous IL-2 mRNA production in EL-4 T cells, and this repression is dependent on the ZEB-binding site at -100. ZEB1 cooperates with the co-repressor C-terminal-binding protein (CtBP) 2 and with histone deacetylase 1 to repress the IL-2 promoter and this cooperation depends on the ZEB-binding site in the promoter as well as the Pro-X-Asp-Leu-Ser protein-protein interaction domain in CtBP2. Thus, ZEB1 may function to recruit a repressor complex to the IL-2 promoter.

Genome-wide identification and characterization of TCP genes involved in ovule development of Phalaenopsis equestris

PubMed Central

Lin, Yu-Fu; Chen, You-Yi; Hsiao, Yu-Yun; Shen, Ching-Yu; Hsu, Jui-Ling; Yeh, Chuan-Ming; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Liu, Zhong-Jian; Tsai, Wen-Chieh

2016-01-01

TEOSINTE-BRANCHED/CYCLOIDEA/PCF (TCP) proteins are plant-specific transcription factors known to have a role in multiple aspects of plant growth and development at the cellular, organ and tissue levels. However, there has been no related study of TCPs in orchids. Here we identified 23 TCP genes from the genome sequence of Phalaenopsis equestris. Phylogenetic analysis distinguished two homology classes of PeTCP transcription factor families: classes I and II. Class II was further divided into two subclasses, CIN and CYC/TB1. Spatial and temporal expression analysis showed that PePCF10 was predominantly expressed in ovules at early developmental stages and PeCIN8 had high expression at late developmental stages in ovules, with overlapping expression at day 16 after pollination. Subcellular localization and protein–protein interaction analyses revealed that PePCF10 and PeCIN8 could form homodimers and localize in the nucleus. However, PePCF10 and PeCIN8 could not form heterodimers. In transgenic Arabidopsis thaliana plants (overexpression and SRDX, a super repression motif derived from the EAR-motif of the repression domain of tobacco ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR 3 and SUPERMAN, dominantly repressed), the two genes helped regulate cell proliferation. Together, these results suggest that PePCF10 and PeCIN8 play important roles in orchid ovule development by modulating cell division. PMID:27543606

Repression of the Low Affinity Iron Transporter Gene FET4

PubMed Central

Caetano, Soraia M.; Menezes, Regina; Amaral, Catarina; Rodrigues-Pousada, Claudina; Pimentel, Catarina

2015-01-01

Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, causing several cellular damages and eventually leading to death. In the yeast Saccharomyces cerevisiae, the transcription factor Yap1 plays a key role in the regulation of several genes involved in metal stress response. We have previously shown that Yap1 represses the expression of FET4, a gene encoding a low affinity iron transporter able to transport metals other than iron. Here, we have studied the relevance of this repression in cell tolerance to cadmium. Our results indicate that genomic deletion of Yap1 increases FET4 transcript and protein levels. In addition, the cadmium toxicity exhibited by this strain is completely reversed by co-deletion of FET4 gene. These data correlate well with the increased intracellular levels of cadmium observed in the mutant yap1. Rox1, a well known aerobic repressor of hypoxic genes, conveys the Yap1-mediated repression of FET4. We further show that, in a scenario where the activity of Yap1 or Rox1 is compromised, cells activate post-transcriptional mechanisms, involving the exoribonuclease Xrn1, to compensate the derepression of FET4. Our data thus reveal a novel protection mechanism against cadmium toxicity mediated by Yap1 that relies on the aerobic repression of FET4 and results in the impairment of cadmium uptake. PMID:26063801

Eos Negatively Regulates Human γ-globin Gene Transcription during Erythroid Differentiation

PubMed Central

Yu, Hai-Chuan; Zhao, Hua-Lu; Wu, Zhi-Kui; Zhang, Jun-Wu

2011-01-01

Background Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4), a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. Methodology/Principal Findings Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs). DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3) of the β-globin locus control region (LCR), the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C) assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. Conclusions/Significance Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation. PMID:21829552

Bacterial Competition Reveals Differential Regulation of the pks Genes by Bacillus subtilis

PubMed Central

Vargas-Bautista, Carol; Rahlwes, Kathryn

2014-01-01

Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305–310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis. PMID:24187085

Bacterial competition reveals differential regulation of the pks genes by Bacillus subtilis.

PubMed

Vargas-Bautista, Carol; Rahlwes, Kathryn; Straight, Paul

2014-02-01

Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305-310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for B. subtilis.

Crosstalk between virulence loci: regulation of Salmonella enterica pathogenicity island 1 (SPI-1) by products of the std fimbrial operon.

PubMed

López-Garrido, Javier; Casadesús, Josep

2012-01-01

Invasion of intestinal epithelial cells is a critical step in Salmonella infection and requires the expression of genes located in Salmonella pathogenicity island 1 (SPI-1). A key factor for SPI-1 expression is DNA adenine (Dam) methylation, which activates synthesis of the SPI-1 transcriptional activator HilD. Dam-dependent regulation of hilD is postranscriptional (and therefore indirect), indicating the involvement of unknown cell functions under Dam methylation control. A genetic screen has identified the std fimbrial operon as the missing link between Dam methylation and SPI-1. We show that all genes in the std operon are part of a single transcriptional unit, and describe three previously uncharacterized ORFs (renamed stdD, stdE, and stdF). We present evidence that two such loci (stdE and stdF) are involved in Dam-dependent control of Salmonella SPI-1: in a Dam(-) background, deletion of stdE or stdF suppresses SPI-1 repression; in a Dam(+) background, constitutive expression of StdE and/or StdF represses SPI-1. Repression of SPI-1 by products of std operon explains the invasion defect of Salmonella Dam(-) mutants, which constitutively express the std operon. Dam-dependent repression of std in the ileum may be required to permit invasion, as indicated by two observations: constitutive expression of StdE and StdF reduces invasion of epithelial cells in vitro (1,000 fold) and attenuates Salmonella virulence in the mouse model (>60 fold). In turn, crosstalk between std and SPI-1 may play a role in intestinal infections by preventing expression of SPI-1 in the caecum, an intestinal compartment in which the std operon is known to be expressed.

Phosphate assimilation in Rhizobium (Sinorhizobium) meliloti: identification of a pit-like gene.

PubMed

Bardin, S D; Voegele, R T; Finan, T M

1998-08-01

Rhizobium meliloti mutants defective in the phoCDET-encoded phosphate transport system form root nodules on alfalfa plants that fail to fix nitrogen (Fix-). We have previously reported that two classes of second-site mutations can suppress the Fix- phenotype of phoCDET mutants to Fix+. Here we show that one of these suppressor loci (sfx1) contains two genes, orfA and pit, which appear to form an operon transcribed in the order orfA-pit. The Pit protein is homologous to various phosphate transporters, and we present evidence that three suppressor mutations arose from a single thymidine deletion in a hepta-thymidine sequence centered 54 nucleotides upstream of the orfA transcription start site. This mutation increased the level of orfA-pit transcription. These data, together with previous biochemical evidence, show that the orfA-pit genes encode a Pi transport system that is expressed in wild-type cells grown with excess Pi but repressed in cells under conditions of Pi limitation. In phoCDET mutant cells, orfA-pit expression is repressed, but this repression is alleviated by the second-site suppressor mutations. Suppression increases orfA-pit expression compensating for the deficiencies in phosphate assimilation and symbiosis of the phoCDET mutants.

p53 targets chromatin structure alteration to repress alpha-fetoprotein gene expression.

PubMed

Ogden, S K; Lee, K C; Wernke-Dollries, K; Stratton, S A; Aronow, B; Barton, M C

2001-11-09

Many of the functions ascribed to p53 tumor suppressor protein are mediated through transcription regulation. We have shown that p53 represses hepatic-specific alpha-fetoprotein (AFP) gene expression by direct interaction with a composite HNF-3/p53 DNA binding element. Using solid-phase, chromatin-assembled AFP DNA templates and analysis of chromatin structure and transcription in vitro, we find that p53 binds DNA and alters chromatin structure at the AFP core promoter to regulate transcription. Chromatin assembled in the presence of hepatoma extracts is activated for AFP transcription with an open, accessible core promoter structure. Distal (-850) binding of p53 during chromatin assembly, but not post-assembly, reverses transcription activation concomitant with promoter inaccessibility to restriction enzyme digestion. Inhibition of histone deacetylase activity by trichostatin-A (TSA) addition, prior to and during chromatin assembly, activated chromatin transcription in parallel with increased core promoter accessibility. Chromatin immunoprecipitation analyses showed increased H3 and H4 acetylated histones at the core promoter in the presence of TSA, while histone acetylation remained unchanged at the site of distal p53 binding. Our data reveal that p53 targets chromatin structure alteration at the core promoter, independently of effects on histone acetylation, to establish repressed AFP gene expression.

Mi2β Is Required for γ-Globin Gene Silencing: Temporal Assembly of a GATA-1-FOG-1-Mi2 Repressor Complex in β-YAC Transgenic Mice

PubMed Central

Costa, Flávia C.; Fedosyuk, Halyna; Chazelle, Allen M.; Neades, Renee Y.; Peterson, Kenneth R.

2012-01-01

Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the −566 GATA motif of the Aγ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of Aγ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the −566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1–mediated repressor complex was disrupted by the −566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting −566 Aγ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis. PMID:23284307

Mi2β is required for γ-globin gene silencing: temporal assembly of a GATA-1-FOG-1-Mi2 repressor complex in β-YAC transgenic mice.

PubMed

Costa, Flávia C; Fedosyuk, Halyna; Chazelle, Allen M; Neades, Renee Y; Peterson, Kenneth R

2012-01-01

Activation of γ-globin gene expression in adults is known to be therapeutic for sickle cell disease. Thus, it follows that the converse, alleviation of repression, would be equally effective, since the net result would be the same: an increase in fetal hemoglobin. A GATA-1-FOG-1-Mi2 repressor complex was recently demonstrated to be recruited to the -566 GATA motif of the (A)γ-globin gene. We show that Mi2β is essential for γ-globin gene silencing using Mi2β conditional knockout β-YAC transgenic mice. In addition, increased expression of (A)γ-globin was detected in adult blood from β-YAC transgenic mice containing a T>G HPFH point mutation at the -566 GATA silencer site. ChIP experiments demonstrated that GATA-1 is recruited to this silencer at day E16, followed by recruitment of FOG-1 and Mi2 at day E17 in wild-type β-YAC transgenic mice. Recruitment of the GATA-1-mediated repressor complex was disrupted by the -566 HPFH mutation at developmental stages when it normally binds. Our data suggest that a temporal repression mechanism is operative in the silencing of γ-globin gene expression and that either a trans-acting Mi2β knockout deletion mutation or the cis-acting -566 (A)γ-globin HPFH point mutation disrupts establishment of repression, resulting in continued γ-globin gene transcription during adult definitive erythropoiesis.

Human native lipoprotein-induced de novo DNA methylation is associated with repression of inflammatory genes in THP-1 macrophages.

PubMed

Rangel-Salazar, Rubén; Wickström-Lindholm, Marie; Aguilar-Salinas, Carlos A; Alvarado-Caudillo, Yolanda; Døssing, Kristina B V; Esteller, Manel; Labourier, Emmanuel; Lund, Gertrud; Nielsen, Finn C; Rodríguez-Ríos, Dalia; Solís-Martínez, Martha O; Wrobel, Katarzyna; Wrobel, Kazimierz; Zaina, Silvio

2011-11-25

We previously showed that a VLDL- and LDL-rich mix of human native lipoproteins induces a set of repressive epigenetic marks, i.e. de novo DNA methylation, histone 4 hypoacetylation and histone 4 lysine 20 (H4K20) hypermethylation in THP-1 macrophages. Here, we: 1) ask what gene expression changes accompany these epigenetic responses; 2) test the involvement of candidate factors mediating the latter. We exploited genome expression arrays to identify target genes for lipoprotein-induced silencing, in addition to RNAi and expression studies to test the involvement of candidate mediating factors. The study was conducted in human THP-1 macrophages. Native lipoprotein-induced de novo DNA methylation was associated with a general repression of various critical genes for macrophage function, including pro-inflammatory genes. Lipoproteins showed differential effects on epigenetic marks, as de novo DNA methylation was induced by VLDL and to a lesser extent by LDL, but not by HDL, and VLDL induced H4K20 hypermethylation, while HDL caused H4 deacetylation. The analysis of candidate factors mediating VLDL-induced DNA hypermethylation revealed that this response was: 1) surprisingly, mediated exclusively by the canonical maintenance DNA methyltransferase DNMT1, and 2) independent of the Dicer/micro-RNA pathway. Our work provides novel insights into epigenetic gene regulation by native lipoproteins. Furthermore, we provide an example of DNMT1 acting as a de novo DNA methyltransferase independently of canonical de novo enzymes, and show proof of principle that de novo DNA methylation can occur independently of a functional Dicer/micro-RNA pathway in mammals.

SRSF3 represses the expression of PDCD4 protein by coordinated regulation of alternative splicing, export and translation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Seung Kuk; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

2016-02-05

Gene expression is regulated at multiple steps, such as transcription, splicing, export, degradation and translation. Considering diverse roles of SR proteins, we determined whether the tumor-related splicing factor SRSF3 regulates the expression of the tumor-suppressor protein, PDCD4, at multiple steps. As we have reported previously, knockdown of SRSF3 increased the PDCD4 protein level in SW480 colon cancer cells. More interestingly, here we showed that the alternative splicing and the nuclear export of minor isoforms of pdcd4 mRNA were repressed by SRSF3, but the translation step was unaffected. In contrast, only the translation step of the major isoform of pdcd4 mRNAmore » was repressed by SRSF3. Therefore, overexpression of SRSF3 might be relevant to the repression of all isoforms of PDCD4 protein levels in most types of cancer cell. We propose that SRSF3 could act as a coordinator of the expression of PDCD4 protein via two mechanisms on two alternatively spliced mRNA isoforms.« less

Paradoxical Role of DNA Methylation in Activation of FoxA2 Gene Expression during Endoderm Development*

PubMed Central

Bahar Halpern, Keren; Vana, Tal; Walker, Michael D.

2014-01-01

The transcription factor FoxA2 is a master regulator of endoderm development and pancreatic beta cell gene expression. To elucidate the mechanisms underlying the activation of the FoxA2 gene during differentiation, we have compared the epigenetic status of undifferentiated human embryonic stem cells (hESCs), hESC-derived early endoderm stage cells (CXCR4+ cells), and pancreatic islet cells. Unexpectedly, a CpG island in the promoter region of the FoxA2 gene displayed paradoxically high levels of DNA methylation in expressing tissues (CXCR4+, islets) and low levels in nonexpressing tissues. This CpG island region was found to repress reporter gene expression and bind the Polycomb group protein SUZ12 and the DNA methyltransferase (DNMT)3b preferentially in undifferentiated hESCs as compared with CXCR4+ or islets cells. Consistent with this, activation of FoxA2 gene expression, but not CXCR4 or SOX17, was strongly inhibited by 5-aza-2′-deoxycytidine and by knockdown of DNMT3b. We hypothesize that in nonexpressing tissues, the lack of DNA methylation allows the binding of DNA methyltransferases and repressing proteins, such as Polycomb group proteins; upon differentiation, DNMT activation leads to CpG island methylation, causing loss of repressor protein binding. These results suggest a novel and unexpected role for DNA methylation in the activation of FoxA2 gene expression during differentiation. PMID:25016019

Cuf2 Is a Novel Meiosis-Specific Regulatory Factor of Meiosis Maturation

PubMed Central

Ioannoni, Raphael; Beaudoin, Jude; Lopez-Maury, Luis; Codlin, Sandra; Bahler, Jurg; Labbe, Simon

2012-01-01

Background Meiosis is the specialized form of the cell cycle by which diploid cells produce the haploid gametes required for sexual reproduction. Initiation and progression through meiosis requires that the expression of the meiotic genes is precisely controlled so as to provide the correct gene products at the correct times. During meiosis, four temporal gene clusters are either induced or repressed by a cascade of transcription factors. Principal Findings In this report a novel copper-fist-type regulator, Cuf2, is shown to be expressed exclusively during meiosis. The expression profile of the cuf2+ mRNA revealed that it was induced during middle-phase meiosis. Both cuf2+ mRNA and protein levels are unregulated by copper addition or starvation. The transcription of cuf2+ required the presence of a functional mei4+ gene encoding a key transcription factor that activates the expression of numerous middle meiotic genes. Microscopic analyses of cells expressing a functional Cuf2-GFP protein revealed that Cuf2 co-localized with both homologous chromosomes and sister chromatids during the meiotic divisions. Cells lacking Cuf2 showed an elevated and sustained expression of several of the middle meiotic genes that persisted even during late meiosis. Moreover, cells carrying disrupted cuf2Δ/cuf2Δ alleles displayed an abnormal morphology of the forespore membranes and a dramatic reduction of spore viability. Significance Collectively, the results revealed that Cuf2 functions in the timely repression of the middle-phase genes during meiotic differentiation. PMID:22558440

The DREAM complex: Master coordinator of cell cycle dependent gene expression

PubMed Central

Sadasivam, Subhashini; DeCaprio, James A.

2014-01-01

Preface The dimerization partner (DP), retinoblastoma (RB)-like, E2F and MuvB (DREAM) complex provides a previously unsuspected unifying role in the cell cycle by directly linking p130, p107, E2F, BMYB and FOXM1. DREAM mediates gene repression during G0 and coordinates periodic gene expression with peaks during G1/S and G2/M. Perturbations in DREAM regulation shift the balance from quiescence towards proliferation and contribute to increased mitotic gene expression levels frequently observed in cancers with poor prognosis. PMID:23842645

Researchers use Modified CRISPR Systems to Modulate Gene Expression on a Genomic Scale

Cancer.gov

Cancer Target Discovery and Development Network (CTD2) researchers at the University of California, San Francisco, developed a CRISPR system that can regulate both gene repression and activation with fewer off-target effects.

Regulation of the expression of plant resistance gene SNC1 by a protein with a conserved BAT2 domain.

PubMed

Li, Yingzhong; Tessaro, Mark J; Li, Xin; Zhang, Yuelin

2010-07-01

Plant Resistance (R) genes encode immune receptors that recognize pathogens and activate defense responses. Because of fitness costs associated with maintaining R protein-mediated resistance, expression levels of R genes have to be tightly regulated. However, mechanisms on how R-gene expression is regulated are poorly understood. Here we show that MODIFIER OF snc1, 1 (MOS1) regulates the expression of SUPPRESSOR OF npr1-1, CONSTITUTIVE1 (SNC1), which encodes a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat type of R protein in Arabidopsis (Arabidopsis thaliana). In the mos1 loss-of-function mutant plants, snc1 expression is repressed and constitutive resistance responses mediated by snc1 are lost. The repression of snc1 expression in mos1 is released by knocking out DECREASE IN DNA METHYLATION1. In mos1 mutants, DNA methylation in a region upstream of SNC1 is altered. Furthermore, expression of snc1 transgenes using the native promoter does not require MOS1, indicating that regulation of SNC1 expression by MOS1 is at the chromatin level. Map-based cloning of MOS1 revealed that it encodes a novel protein with a HLA-B ASSOCIATED TRANSCRIPT2 (BAT2) domain that is conserved in plants and animals. Our study on MOS1 suggests that BAT2 domain-containing proteins may function in regulation of gene expression at chromatin level.

Edible blue-green algae reduce the production of pro-inflammatory cytokines by inhibiting NF-κB pathway in macrophages and splenocytes

PubMed Central

Ku, Chai Siah; Pham, Tho X.; Park, Youngki; Kim, Bohkyung; Shin, Min; Kang, Insoo; Lee, Jiyoung

2013-01-01

Background Chronic inflammation contributes to the development of pathological disorders including insulin resistance and atherosclerosis. Identification of anti-inflammatory natural products can prevent the inflammatory diseases. Methods Anti-inflammatory effects of blue-green algae (BGA), i.e., Nostoc commune var. Sphaeroides Kützing (NO) and Spirulina Platensis (SP), were compared in RAW 264.7 and mouse bone marrow-derived macrophages (BMM) as well as splenocytes from apolipoprotein E knockout (apoE−/−) mice fed BGA. Results When macrophages pretreated with 100 μg/ml NO lipid extract (NOE) or SP lipid extract (SPE) were activated by lipopolysaccharide (LPS), expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), and IL-6, were significantly repressed. NOE and SPE also significantly repressed the expression of TNFα and IL-1β in BMM. LPS-induced secretion of IL-6 was lower in splenocytes from apoE−/− fed an atherogenic diet containing 5% NO or SP for 12 weeks. In RAW 264.7 macrophages, NOE and SPE markedly decreased nuclear translocation of NF-κB. The degree of repression of pro-inflammatory gene expression by algal extracts was much stronger than that of SN50, an inhibitor of NF-κB nuclear translocation. Trichostatin A, a pan histone deacetylase inhibitor, increased basal expression of IL-1β and attenuated the repression of the gene expression by SPE. SPE significantly down-regulated mRNA abundance of 11 HDAC isoforms, consequently increasing acetylated histone 3 levels. Conclusion NOE and SPE repress pro-inflammatory cytokine expression and secretion in macrophages and splenocytes via inhibition of NF-κB pathway. Histone acetylation state is likely involved in the inhibition. General significance This study underscores natural products can exert anti-inflammatory effects by epigenetic modifications such as histone acetylation. PMID:23357040

The yeast ADH7 promoter enables gene expression under pronounced translation repression caused by the combined stress of vanillin, furfural, and 5-hydroxymethylfurfural.

PubMed

Ishida, Yoko; Nguyen, Trinh Thi My; Izawa, Shingo

2017-06-20

Lignocellulosic biomass conversion inhibitors such as vanillin, furfural, and 5-hydroxymethylfurfural (HMF) inhibit the growth of and fermentation by Saccharomyces cerevisiae. A high concentration of each fermentation inhibitor represses translation and increases non-translated mRNAs. We previously reported that the mRNAs of ADH7 and BDH2, which encode putative NADPH- and NADH-dependent alcohol dehydrogenases, respectively, were efficiently translated even with translation repression in response to severe vanillin stress. However, the combined effects of these fermentation inhibitors on the expression of ADH7 and BDH2 remain unclear. We herein demonstrated that exposure to a combined stress of vanillin, furfural, and HMF repressed translation. The protein synthesis of Adh7, but not Bdh2 was significantly induced under combined stress conditions, even though the mRNA levels of ADH7 and BDH2 were up-regulated. Additionally, adh7Δ cells were more sensitive to the combined stress than wild-type and bdh2Δ cells. These results suggest that induction of the ADH7 expression plays a role in the tolerance to the combined stress of vanillin, furfural, and HMF. Furthermore, we succeeded in improving yeast tolerance to the combined stress by controlling the expression of ALD6 with the ADH7 promoter. Our results demonstrate that the ADH7 promoter can overcome the pronounced translation repression caused by the combined stress of vanillin, furfural, and HMF, and also suggest a new gene engineering strategy to breed robust and optimized yeasts for bioethanol production from a lignocellulosic biomass. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of a Truncated ATHB17 Protein in Maize Increases Ear Weight at Silking

PubMed Central

Creelman, Robert A.; Griffith, Cara; Ahrens, Jeffrey E.; Taylor, J. Philip; Murphy, Lesley R.; Manjunath, Siva; Thompson, Rebecca L.; Lingard, Matthew J.; Back, Stephanie L.; Larue, Huachun; Brayton, Bonnie R.; Burek, Amanda J.; Tiwari, Shiv; Adam, Luc; Morrell, James A.; Caldo, Rico A.; Huai, Qing; Kouadio, Jean-Louis K.; Kuehn, Rosemarie; Sant, Anagha M.; Wingbermuehle, William J.; Sala, Rodrigo; Foster, Matt; Kinser, Josh D.; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E.; Huang, Mingya G.; Kuriakose, Saritha V.; Skottke, Kyle; Repetti, Peter P.; Reuber, T. Lynne; Ruff, Thomas G.; Petracek, Marie E.; Loida, Paul J.

2014-01-01

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize. PMID:24736658

Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

PubMed

Rice, Elena A; Khandelwal, Abha; Creelman, Robert A; Griffith, Cara; Ahrens, Jeffrey E; Taylor, J Philip; Murphy, Lesley R; Manjunath, Siva; Thompson, Rebecca L; Lingard, Matthew J; Back, Stephanie L; Larue, Huachun; Brayton, Bonnie R; Burek, Amanda J; Tiwari, Shiv; Adam, Luc; Morrell, James A; Caldo, Rico A; Huai, Qing; Kouadio, Jean-Louis K; Kuehn, Rosemarie; Sant, Anagha M; Wingbermuehle, William J; Sala, Rodrigo; Foster, Matt; Kinser, Josh D; Mohanty, Radha; Jiang, Dongming; Ziegler, Todd E; Huang, Mingya G; Kuriakose, Saritha V; Skottke, Kyle; Repetti, Peter P; Reuber, T Lynne; Ruff, Thomas G; Petracek, Marie E; Loida, Paul J

2014-01-01

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12.

PubMed

Maurer, Lisa M; Yohannes, Elizabeth; Bondurant, Sandra S; Radmacher, Michael; Slonczewski, Joan L

2005-01-01

Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with an alpha level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins.

Grape hexokinases are involved in the expression regulation of sucrose synthase- and cell wall invertase-encoding genes by glucose and ABA.

PubMed

Wang, Xiu-Qin; Zheng, Li-Li; Lin, Hao; Yu, Fei; Sun, Li-Hui; Li, Li-Mei

2017-05-01

Hexokinase (HXK, EC 2.7.1.1) is a multifunctional protein that both is involved in catalyzing the first step of glycolysis and plays an important role in sugar signaling. However, the supporting genetic evidence on hexokinases (CsHXKs) from grape (Vitis vinifera L. cv. Cabernet Sauvignon) berries has been lacking. Here, to investigate the role of CsHXK isoforms as glucose (Glc) and abscisic acid (ABA) sensors, we cloned two hexokinase isozymes, CsHXK1 and CsHXK2 with highly conserved genomic structure of nine exons and eight introns. We also found adenosine phosphate binding, substrate recognition and connection sites in their putative proteins. During grape berry development, the expression profiles of two CsHXK isoforms, sucrose synthases (SuSys) and cell wall invertase (CWINV) genes increased concomitantly with high levels of endogenous Glc and ABA. Furthermore, we showed that in wild type grape berry calli (WT), glucose repressed the expression levels of sucrose synthase (SuSy) and cell wall invertase (CWINV) genes, while ABA increased their expression levels. ABA could not only effectively improve the expression levels of SuSy and CWINV, but also block the repression induced by glucose on the expression of both genes. However, after silencing CsHXK1 or CsHXK2 in grape calli, SuSy and CWINV expression were enhanced, and the expressions of the two genes are insensitive in response to Glc treatment. Interestingly, exogenous ABA alone could not or less increase SuSy and CWINV expression in silencing CsHXK1 or CsHXK2 grape calli compared to WT. Meantime, ABA could not block the repression induced by glucose on the expression of SuSy and CWINV in CsHXK1 or CsHXK2 mutants. Therefore, Glc signal transduction depends on the regulation of CsHXK1 or CsHXK2. ABA signal was also disturbed by CsHXK1 or CsHXK2 silencing. The present results provide new insights into the regulatory role of Glc and ABA on the enzymes related to sugar metabolism in grape berry.

MYC/MIZ1-dependent gene repression inversely coordinates the circadian clock with cell cycle and proliferation.

PubMed

Shostak, Anton; Ruppert, Bianca; Ha, Nati; Bruns, Philipp; Toprak, Umut H; Eils, Roland; Schlesner, Matthias; Diernfellner, Axel; Brunner, Michael

2016-06-24

The circadian clock and the cell cycle are major cellular systems that organize global physiology in temporal fashion. It seems conceivable that the potentially conflicting programs are coordinated. We show here that overexpression of MYC in U2OS cells attenuates the clock and conversely promotes cell proliferation while downregulation of MYC strengthens the clock and reduces proliferation. Inhibition of the circadian clock is crucially dependent on the formation of repressive complexes of MYC with MIZ1 and subsequent downregulation of the core clock genes BMAL1 (ARNTL), CLOCK and NPAS2. We show furthermore that BMAL1 expression levels correlate inversely with MYC levels in 102 human lymphomas. Our data suggest that MYC acts as a master coordinator that inversely modulates the impact of cell cycle and circadian clock on gene expression.

Epigenetic and SP1-mediated regulation is involved in the repression of galactokinase 1 gene in the liver of neonatal piglets born to betaine-supplemented sows.

PubMed

Cai, Demin; Yuan, Mengjie; Liu, Haoyu; Han, Zhengqiang; Pan, Shifeng; Yang, Yang; Zhao, Ruqian

2017-08-01

In this study, we sought to investigate the effects of maternal betaine supplementation on the expression and regulation of GALK1 gene in the liver of neonatal piglets. Sixteen sows of two groups were fed control or betaine-supplemented diets (3 g/kg), respectively, throughout the pregnancy. Newborn piglets were individually weighed immediately after birth, and one male piglet close to mean body weight from the same litter was selected and killed before suckling. Serum samples of newborn piglets were analyzed for biochemical indexes, hormone and amino acid levels. Liver samples were analyzed for GALK1 expression by real-time PCR and western blotting, while GALK1 regulational mechanism was analyzed by methylated DNA immunoprecipitation, chromatin immunoprecipitation and microRNAs expression. Betaine-exposed neonatal piglets had lower serum concentration of galactose, which was associated with significantly down-regulated hepatic GALK1 expression. The repression of GALK1 mRNA expression was associated with DNA hypermethylation and more enriched repression histone mark H3K27me3 on its promoter. Binding sites of SP1, GR and STAT3 were predicted on GALK1 promoter, and decreased SP1 protein content and lower SP1 binding to GALK1 promoter were detected in the liver of betaine-exposed piglets. Furthermore, the expression of miRNA-149 targeting GALK1 was up-regulated in the liver of betaine-exposed piglets, along with elevated miRNAs-processing enzymes Dicer and Ago2. Our results suggest that maternal dietary betaine supplementation during gestation suppresses GALK1 expression in the liver of neonatal piglets, which involves complex gene regulation mechanisms including DNA methylation, histone modification, miRNAs expression and SP1-mediated transcriptional modulation.

A real-time control system of gene expression using ligand-bound nucleic acid aptamer for metabolic engineering.

PubMed

Wang, Jing; Cui, Xun; Yang, Le; Zhang, Zhe; Lv, Liping; Wang, Haoyuan; Zhao, Zhenmin; Guan, Ningzi; Dong, Lichun; Chen, Rachel

2017-07-01

Artificial control of bio-functions through regulating gene expression is one of the most important and attractive technologies to build novel living systems that are useful in the areas of chemical synthesis, nanotechnology, pharmacology, cell biology. Here, we present a novel real-time control system of gene regulation that includes an enhancement element by introducing duplex DNA aptamers upstream promoter and a repression element by introducing a RNA aptamer upstream ribosome binding site. With the presence of ligands corresponding to the DNA aptamers, the expression of the target gene can be potentially enhanced at the transcriptional level by strengthening the recognition capability of RNAP to the recognition region and speeding up the separation efficiency of the unwinding region due to the induced DNA bubble around the thrombin-bound aptamers; while with the presence of RNA aptamer ligand, the gene expression can be repressed at the translational level by weakening the recognition capability of ribosome to RBS due to the shielding of RBS by the formed aptamer-ligand complex upstream RBS. The effectiveness and potential utility of the developed gene regulation system were demonstrated by regulating the expression of ecaA gene in the cell-free systems. The realistic metabolic engineering application of the system has also tested by regulating the expression of mgtC gene and thrombin cDNA in Escherichia coli JD1021 for controlling metabolic flux and improving thrombin production, verifying that the real-time control system of gene regulation is able to realize the dynamic regulation of gene expression with potential applications in bacterial physiology studies and metabolic engineering. Copyright © 2017. Published by Elsevier Inc.

Balancing gene expression without library construction via a reusable sRNA pool.

PubMed

Ghodasara, Amar; Voigt, Christopher A

2017-07-27

Balancing protein expression is critical when optimizing genetic systems. Typically, this requires library construction to vary the genetic parts controlling each gene, which can be expensive and time-consuming. Here, we develop sRNAs corresponding to 15nt 'target' sequences that can be inserted upstream of a gene. The targeted gene can be repressed from 1.6- to 87-fold by controlling sRNA expression using promoters of different strength. A pool is built where six sRNAs are placed under the control of 16 promoters that span a ∼103-fold range of strengths, yielding ∼107 combinations. This pool can simultaneously optimize up to six genes in a system. This requires building only a single system-specific construct by placing a target sequence upstream of each gene and transforming it with the pre-built sRNA pool. The resulting library is screened and the top clone is sequenced to determine the promoter controlling each sRNA, from which the fold-repression of the genes can be inferred. The system is then rebuilt by rationally selecting parts that implement the optimal expression of each gene. We demonstrate the versatility of this approach by using the same pool to optimize a metabolic pathway (β-carotene) and genetic circuit (XNOR logic gate). © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Convergence of hepcidin deficiency, systemic iron overloading, heme accumulation, and REV-ERBα/β activation in aryl hydrocarbon receptor-elicited hepatotoxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fader, Kelly A.; Nault, Rance

Persistent aryl hydrocarbon receptor (AhR) agonists elicit dose-dependent hepatic lipid accumulation, oxidative stress, inflammation, and fibrosis in mice. Iron (Fe) promotes AhR-mediated oxidative stress by catalyzing reactive oxygen species (ROS) production. To further characterize the role of Fe in AhR-mediated hepatotoxicity, male C57BL/6 mice were orally gavaged with sesame oil vehicle or 0.01–30 μg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every 4 days for 28 days. Duodenal epithelial and hepatic RNA-Seq data were integrated with hepatic AhR ChIP-Seq, capillary electrophoresis protein measurements, and clinical chemistry analyses. TCDD dose-dependently repressed hepatic expression of hepcidin (Hamp and Hamp2), the master regulator of systemic Fe homeostasis, resultingmore » in a 2.6-fold increase in serum Fe with accumulating Fe spilling into urine. Total hepatic Fe levels were negligibly increased while transferrin saturation remained unchanged. Furthermore, TCDD elicited dose-dependent gene expression changes in heme biosynthesis including the induction of aminolevulinic acid synthase 1 (Alas1) and repression of uroporphyrinogen decarboxylase (Urod), leading to a 50% increase in hepatic hemin and a 13.2-fold increase in total urinary porphyrins. Consistent with this heme accumulation, differential gene expression suggests that heme activated BACH1 and REV-ERBα/β, causing induction of heme oxygenase 1 (Hmox1) and repression of fatty acid biosynthesis, respectively. Collectively, these results suggest that Hamp repression, Fe accumulation, and increased heme levels converge to promote oxidative stress and the progression of TCDD-elicited hepatotoxicity. - Highlights: • TCDD represses hepatic hepcidin expression, leading to systemic iron overloading. • Dysregulation of heme biosynthesis is consistent with heme and porphyrin accumulation. • Heme-activated REV-ERBα/β repress circadian-regulated hepatic lipid metabolism. • Disruption of iron homeostasis promotes TCDD-elicited steatohepatitis with fibrosis.« less

Identification of polycomb and trithorax group responsive elements in the regulatory region of the Drosophila homeotic gene Sex combs reduced

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gindhart, J.G. Jr.; Kaufman, T.C.

1995-02-01

The Drosophilia homeotic gene Sex combs reduced (Scr) is necessary for the establishment and maintenance of the morphological identity of the labial and prothoracic segments. In the early embryo, its expression pattern is established through the activity of several gap and segmentation gene products, as well as other transcription factors. Once established, the Polycomb group (Pc-G) and trithorax group (trx-G) gene products maintain the spatial pattern of Scr expression for the remainder of development. We report the identification of DNA fragments in the Scr regulatory region that may be important for its regulation by Polycomb and trithorax group gene products.more » When DNA fragments containing these regulatory sequences are subcloned into P-element vectors containing a white minigene, transformants containing these constructs exhibit mosaic patterns of pigmentation in the adult eye, indicating that white minigene expression is repressed in a clonally heritable manner. The size of pigmented and nonpigmented clones in the adult eye suggests that the event determining whether a cell in the eye anlagen will express white occurs at least as early as the first larval instar. The amount of white minigene repression is reduced in some Polycomb group mutants, whereas repression is enhanced in flies mutant for a subset of trithorax group loci. The repressor activity of one fragment, normally located in Scr Intron 2, is increased when it is able to homologously pair, a property consistent with genetic data suggesting that Scr exhibits transvection. Another Scr regulatory fragment, normally located 40 kb upstream of the Scr promoter, silences ectopic expression of an Scr-lacZ fusion gene in the embryo and does so in a Polycomb-dependent manner. We propose that the regulatory sequences located within these DNA fragments may normally mediate the regulation of Scr by proteins encoded by members of Polycomb and trithorax group loci. 98 refs., 6 figs., 4 tabs.« less

Hypoxia and PGE2 Regulate MiTF-CX During Cervical Ripening

PubMed Central

Hari Kishore, Annavarapu; Li, Xiang-Hong

2012-01-01

The mechanisms by which the cervix remains closed during the massive uterine expansion of pregnancy are unknown. IL-8 is important for recruitment of immune cells into the cervical stroma, matrix remodeling, and dilation of the cervix during labor. Previously, we have shown that several cytokine genes transcriptionally repressed in the cervix during gestation are activated during cervical ripening and dilation. IL-8 gene expression is repressed in cervical stromal cells during pregnancy by the transcription factor microphthalmia-associated transcription factor (MiTF-CX). Here, we tested the hypothesis that hypoxia and the transcription factor hypoxia inducible factor-1α (HIF-1α) may regulate MiTF-CX and cervical ripening. Using tissues from women during pregnancy before and after cervical ripening, we show that, during cervical ripening, HIF-1α was stabilized and relocalized to the nucleus. Further, we found that hypoxia and two hypoxia mimetics that stabilize HIF-1α activated the transcriptional repressor differentiated embryo chondrocyte-expressed gene 1, which bound to sites in the MiTF-CX promoter crucial for its positive autoregulation. Ectopic overexpression of MiTF-CX abrogated hypoxia-induced up-regulation of IL-8 gene expression. We also show that activation of HIF-1α induced cyclooxygenase-2 and that prostaglandin E2 repressed MiTF-CX. We conclude that hypoxia and stabilization of the transcription factor HIF-1α result in up-regulation of differentiated embryo chondrocyte-expressed gene 1, loss of MiTF, and absence of MiTF binding to the IL-8 promoter, which in turn leads to up-regulation of IL-8 gene expression. Hypoxia also up-regulated cyclooxygenase-2, leading to prostaglandin E2-mediated loss of MiTF in cervical stromal cells. The results support a pivotal role for hypoxia and HIF-1α in the cervical ripening process during pregnancy. PMID:23144021

A mutation affecting carbon catabolite repression suppresses growth defects in pyruvate carboxylase mutants from Saccharomyces cerevisiae.

PubMed

Blázquez, M A; Gamo, F J; Gancedo, C

1995-12-18

Yeasts with disruptions in the genes PYC1 and PYC2 encoding the isoenzymes of pyruvate carboxylase cannot grow in a glucose-ammonium medium (Stucka et al. (1991) Mol. Gen. Genet. 229, 307-315). We have isolated a dominant mutation, BPC1-1, that allows growth in this medium of yeasts with interrupted PYC1 and PYC2 genes. The BPC1-1 mutation abolishes catabolite repression of a series of genes and allows expression of the enzymes of the glyoxylate cycle during growth in glucose. A functional glyoxylate cycle is necessary for suppression as a disruption of gene ICL1 encoding isocitrate lyase abolished the phenotypic effect of BPC1-1 on growth in glucose-ammonium. Concurrent expression from constitutive promoters of genes ICL1 and MLS1 (encoding malate synthase) also suppressed the growth phenotype of pyc1 pyc2 mutants. The mutation BPC1-1 is either allelic or closely linked to the mutation DGT1-1.

Vitamin D receptor-mediated control of Soggy, Wise, and Hairless gene expression in keratinocytes.

PubMed

Hsieh, Jui-Cheng; Estess, Rudolf C; Kaneko, Ichiro; Whitfield, G Kerr; Jurutka, Peter W; Haussler, Mark R

2014-02-01

The vitamin D receptor (VDR), but not its hormonal ligand, 1,25-dihydroxyvitamin D3 (1,25D), is required for the progression of the mammalian hair cycle. We studied three genes relevant to hair cycle signaling, DKKL1 (Soggy), SOSTDC1 (Wise), and HR (Hairless), to determine whether their expression is regulated by VDR and/or its 1,25D ligand. DKKL1 mRNA was repressed 49-72% by 1,25D in primary human and CCD-1106 KERTr keratinocytes; a functional vitamin D responsive element (VDRE) was identified at -9590 bp in murine Soggy. Similarly, SOSTDC1 mRNA was repressed 41-59% by 1,25D in KERTr and primary human keratinocytes; a functional VDRE was located at -6215 bp in human Wise. In contrast, HR mRNA was upregulated 1.56- to 2.77-fold by 1,25D in primary human and KERTr keratinocytes; a VDRE (TGGTGAgtgAGGACA) consisting of an imperfect direct repeat separated by three nucleotides (DR3) was identified at -7269 bp in the human Hairless gene that mediated dramatic induction, even in the absence of 1,25D ligand. In parallel, a DR4 thyroid hormone responsive element, TGGTGAggccAGGACA, was identified at +1304 bp in the human HR gene that conferred tri-iodothyronine (T3)-independent transcriptional activation. Because the thyroid hormone receptor controls HR expression in the CNS, whereas VDR functions in concert with the HR corepressor specifically in skin, a model is proposed wherein unliganded VDR upregulates the expression of HR, the gene product of which acts as a downstream comodulator to feedback-repress DKKL1 and SOSTDC1, resulting in integration of bone morphogenic protein and Wnt signaling to drive the mammalian hair cycle and/or influencing epidermal function.

Polycomb repressive complex 2 epigenomic signature defines age-associated hypermethylation and gene expression changes

PubMed Central

Dozmorov, Mikhail G

2015-01-01

Although age-associated gene expression and methylation changes have been reported throughout the literature, the unifying epigenomic principles of aging remain poorly understood. Recent explosion in availability and resolution of functional/regulatory genome annotation data (epigenomic data), such as that provided by the ENCODE and Roadmap Epigenomics projects, provides an opportunity for the identification of epigenomic mechanisms potentially altered by age-associated differentially methylated regions (aDMRs) and regulatory signatures in the promoters of age-associated genes (aGENs). In this study we found that aDMRs and aGENs identified in multiple independent studies share a common Polycomb Repressive Complex 2 signature marked by EZH2, SUZ12, CTCF binding sites, repressive H3K27me3, and activating H3K4me1 histone modification marks, and a “poised promoter” chromatin state. This signature is depleted in RNA Polymerase II-associated transcription factor binding sites, activating H3K79me2, H3K36me3, H3K27ac marks, and an “active promoter” chromatin state. The PRC2 signature was shown to be generally stable across cell types. When considering the directionality of methylation changes, we found the PRC2 signature to be associated with aDMRs hypermethylated with age, while hypomethylated aDMRs were associated with enhancers. In contrast, aGENs were associated with the PRC2 signature independently of the directionality of gene expression changes. In this study we demonstrate that the PRC2 signature is the common epigenomic context of genomic regions associated with hypermethylation and gene expression changes in aging. PMID:25880792

Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

PubMed

Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

2013-12-06

Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

Presenilins Regulate Neurotrypsin Gene Expression and Neurotrypsin-dependent Agrin Cleavage via Cyclic AMP Response Element-binding Protein (CREB) Modulation*

PubMed Central

Almenar-Queralt, Angels; Kim, Sonia N.; Benner, Christopher; Herrera, Cheryl M.; Kang, David E.; Garcia-Bassets, Ivan; Goldstein, Lawrence S. B.

2013-01-01

Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment. PMID:24145027

A negative regulator encoded by a rice WRKY gene represses both abscisic acid and gibberellins signaling in aleurone cells.

PubMed

Zhang, Zhong-Lin; Shin, Margaret; Zou, Xiaolu; Huang, Jianzhi; Ho, Tun-hua David; Shen, Qingxi J

2009-05-01

Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in aleurone cells represses the expression of two reporter constructs: the beta-glucuronidase gene driven by the GA-inducible Amy32b alpha-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and -71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and -71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling.

Genome engineering and gene expression control for bacterial strain development.

PubMed

Song, Chan Woo; Lee, Joungmin; Lee, Sang Yup

2015-01-01

In recent years, a number of techniques and tools have been developed for genome engineering and gene expression control to achieve desired phenotypes of various bacteria. Here we review and discuss the recent advances in bacterial genome manipulation and gene expression control techniques, and their actual uses with accompanying examples. Genome engineering has been commonly performed based on homologous recombination. During such genome manipulation, the counterselection systems employing SacB or nucleases have mainly been used for the efficient selection of desired engineered strains. The recombineering technology enables simple and more rapid manipulation of the bacterial genome. The group II intron-mediated genome engineering technology is another option for some bacteria that are difficult to be engineered by homologous recombination. Due to the increasing demands on high-throughput screening of bacterial strains having the desired phenotypes, several multiplex genome engineering techniques have recently been developed and validated in some bacteria. Another approach to achieve desired bacterial phenotypes is the repression of target gene expression without the modification of genome sequences. This can be performed by expressing antisense RNA, small regulatory RNA, or CRISPR RNA to repress target gene expression at the transcriptional or translational level. All of these techniques allow efficient and rapid development and screening of bacterial strains having desired phenotypes, and more advanced techniques are expected to be seen. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

CRISPRi-sRNA: Transcriptional-Translational Regulation of Extracellular Electron Transfer in Shewanella oneidensis.

PubMed

Cao, Yingxiu; Li, Xiaofei; Li, Feng; Song, Hao

2017-09-15

Extracellular electron transfer (EET) in Shewanella oneidensis MR-1, which is one of the most well-studied exoelectrogens, underlies many microbial electrocatalysis processes, including microbial fuel cells, microbial electrolysis cells, and microbial electrosynthesis. However, regulating the efficiency of EET remains challenging due to the lack of efficient genome regulation tools that regulate gene expression levels in S. oneidensis. Here, we systematically established a transcriptional regulation technology, i.e., clustered regularly interspaced short palindromic repeats interference (CRISPRi), in S. oneidensis MR-1 using green fluorescent protein (GFP) as a reporter. We used this CRISPRi technology to repress the expression levels of target genes, individually and in combination, in the EET pathways (e.g., the MtrCAB pathway and genes affecting the formation of electroactive biofilms in S. oneidensis), which in turn enabled the efficient regulation of EET efficiency. We then established a translational regulation technology, i.e., Hfq-dependent small regulatory RNA (sRNA), in S. oneidensis by repressing the GFP reporter and mtrA, which is a critical gene in the EET pathways in S. oneidensis. To achieve coordinated transcriptional and translational regulation at the genomic level, the CRISPRi and Hfq-dependent sRNA systems were incorporated into a single plasmid harbored in a recombinant S. oneidensis strain, which enabled an even higher efficiency of mtrA gene repression in the EET pathways than that achieved by the CRISPRi and Hfq-dependent sRNA system alone, as exhibited by the reduced electricity output. Overall, we developed a combined CRISPRi-sRNA method that enabled the synergistic transcriptional and translational regulation of target genes in S. oneidensis. This technology involving CRISPRi-sRNA transcriptional-translational regulation of gene expression at the genomic level could be applied to other microorganisms.

Escherichia coli mutant with altered respiratory control of the frd operon.

PubMed Central

Iuchi, S; Kuritzkes, D R; Lin, E C

1985-01-01

In wild-type Escherichia coli, fumarate reductase encoded by the frd operon is inducible by its substrate in the absence of molecular oxygen and nitrate. Synthesis of this enzyme under permissive conditions requires the fnr+ gene product, which is believed to be a pleiotropic regulatory protein that activates transcription. A spontaneous mutant was isolated in which the expression of the frd operon no longer depended on the presence of fumarate or the fnr+ gene product. Aerobic repression of the operon was abolished, but nitrate repression remained intact. Transductional analysis showed that the mutation was closely linked to the frd locus. The mutant phenotype strongly suggests that repression by molecular oxygen and nitrate is mediated by different mechanisms. PMID:3882660

Allele-specific DNA methylation and its interplay with repressive histone marks at promoter-mutant TERT genes

PubMed Central

Stern, Josh Lewis; Paucek, Richard D.; Huang, Franklin W.; Ghandi, Mahmoud; Nwumeh, Ronald; Costello, James C.; Cech, Thomas R.

2017-01-01

SUMMARY A mutation in the promoter of the Telomerase Reverse Transcriptase (TERT) gene is the most frequent noncoding mutation in cancer. The mutation drives unusual monoallelic expression of TERT, allowing immortalization. Here we find that DNA methylation of the TERT CpG Island (CGI) is also allele-specific in multiple cancers. The expressed allele is hypomethylated, which is opposite to cancers without TERT promoter mutations. The continued presence of Polycomb repressive complex 2 (PRC2) on the inactive allele suggests that histone marks of repressed chromatin may be causally linked to high DNA methylation. Consistent with this hypothesis, TERT promoter DNA containing 5-methyl-CpG has much increased affinity for PRC2 in vitro. Thus, CpG methylation and histone marks appear to collaborate to maintain the two TERT alleles in different epigenetic states in TERT promoter-mutant cancers. Finally, in several cancers DNA methylation levels at the TERT CGI correlate with altered patient survival. PMID:29281820

Mutation of praR in Rhizobium leguminosarum enhances root biofilms, improving nodulation competitiveness by increased expression of attachment proteins.

PubMed

Frederix, Marijke; Edwards, Anne; Swiderska, Anna; Stanger, Andrew; Karunakaran, Ramakrishnan; Williams, Alan; Abbruscato, Pamela; Sanchez-Contreras, Maria; Poole, Philip S; Downie, J Allan

2014-08-01

In Rhizobium leguminosarum bv. viciae, quorum-sensing is regulated by CinR, which induces the cinIS operon. CinI synthesizes an AHL, whereas CinS inactivates PraR, a repressor. Mutation of praR enhanced biofilms in vitro. We developed a light (lux)-dependent assay of rhizobial attachment to roots and demonstrated that mutation of praR increased biofilms on pea roots. The praR mutant out-competed wild-type for infection of pea nodules in mixed inoculations. Analysis of gene expression by microarrays and promoter fusions revealed that PraR represses its own transcription and mutation of praR increased expression of several genes including those encoding secreted proteins (the adhesins RapA2, RapB and RapC, two cadherins and the glycanase PlyB), the polysaccharide regulator RosR, and another protein similar to PraR. PraR bound to the promoters of several of these genes indicating direct repression. Mutations in rapA2, rapB, rapC, plyB, the cadherins or rosR did not affect the enhanced root attachment or nodule competitiveness of the praR mutant. However combinations of mutations in rapA, rapB and rapC abolished the enhanced attachment and nodule competitiveness. We conclude that relief of PraR-mediated repression determines a lifestyle switch allowing the expression of genes that are important for biofilm formation on roots and the subsequent initiation of infection of legume roots. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Coordinated Gene Regulation in the Initial Phase of Salt Stress Adaptation*

PubMed Central

Vanacloig-Pedros, Elena; Bets-Plasencia, Carolina; Pascual-Ahuir, Amparo; Proft, Markus

2015-01-01

Stress triggers complex transcriptional responses, which include both gene activation and repression. We used time-resolved reporter assays in living yeast cells to gain insights into the coordination of positive and negative control of gene expression upon salt stress. We found that the repression of “housekeeping” genes coincides with the transient activation of defense genes and that the timing of this expression pattern depends on the severity of the stress. Moreover, we identified mutants that caused an alteration in the kinetics of this transcriptional control. Loss of function of the vacuolar H+-ATPase (vma1) or a defect in the biosynthesis of the osmolyte glycerol (gpd1) caused a prolonged repression of housekeeping genes and a delay in gene activation at inducible loci. Both mutants have a defect in the relocation of RNA polymerase II complexes at stress defense genes. Accordingly salt-activated transcription is delayed and less efficient upon partially respiratory growth conditions in which glycerol production is significantly reduced. Furthermore, the loss of Hog1 MAP kinase function aggravates the loss of RNA polymerase II from housekeeping loci, which apparently do not accumulate at inducible genes. Additionally the Def1 RNA polymerase II degradation factor, but not a high pool of nuclear polymerase II complexes, is needed for efficient stress-induced gene activation. The data presented here indicate that the finely tuned transcriptional control upon salt stress is dependent on physiological functions of the cell, such as the intracellular ion balance, the protective accumulation of osmolyte molecules, and the RNA polymerase II turnover. PMID:25745106

AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs

PubMed Central

El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

2015-01-01

Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs. PMID:26162096

AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs.

PubMed

El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

2015-01-01

Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

Regulation of MET by FOXP2, genes implicated in higher cognitive dysfunction and autism risk.

PubMed

Mukamel, Zohar; Konopka, Genevieve; Wexler, Eric; Osborn, Gregory E; Dong, Hongmei; Bergman, Mica Y; Levitt, Pat; Geschwind, Daniel H

2011-08-10

Autism spectrum disorder (ASD) is a highly heritable, behaviorally defined, heterogeneous disorder of unknown pathogenesis. Several genetic risk genes have been identified, including the gene encoding the receptor tyrosine kinase MET, which regulates neuronal differentiation and growth. An ASD-associated polymorphism disrupts MET gene transcription, and there are reduced levels of MET protein expression in the mature temporal cortex of subjects with ASD. To address the possible neurodevelopmental contribution of MET to ASD pathogenesis, we examined the expression and transcriptional regulation of MET by a transcription factor, FOXP2, which is implicated in regulation of cognition and language, two functions altered in ASD. MET mRNA expression in the midgestation human fetal cerebral cortex is strikingly restricted, localized to portions of the temporal and occipital lobes. Within the cortical plate of the temporal lobe, the pattern of MET expression is highly complementary to the expression pattern of FOXP2, suggesting the latter may play a role in repression of gene expression. Consistent with this, MET and FOXP2 also are reciprocally expressed by differentiating normal human neuronal progenitor cells (NHNPs) in vitro, leading us to assess whether FOXP2 transcriptionally regulates MET. Indeed, FOXP2 binds directly to the 5' regulatory region of MET, and overexpression of FOXP2 results in transcriptional repression of MET. The expression of MET in restricted human neocortical regions, and its regulation in part by FOXP2, is consistent with genetic evidence for MET contributing to ASD risk.

Epigenetic control of effector gene expression in the plant pathogenic fungus Leptosphaeria maculans.

PubMed

Soyer, Jessica L; El Ghalid, Mennat; Glaser, Nicolas; Ollivier, Bénédicte; Linglin, Juliette; Grandaubert, Jonathan; Balesdent, Marie-Hélène; Connolly, Lanelle R; Freitag, Michael; Rouxel, Thierry; Fudal, Isabelle

2014-03-01

Plant pathogens secrete an arsenal of small secreted proteins (SSPs) acting as effectors that modulate host immunity to facilitate infection. SSP-encoding genes are often located in particular genomic environments and show waves of concerted expression at diverse stages of plant infection. To date, little is known about the regulation of their expression. The genome of the Ascomycete Leptosphaeria maculans comprises alternating gene-rich GC-isochores and gene-poor AT-isochores. The AT-isochores harbor mosaics of transposable elements, encompassing one-third of the genome, and are enriched in putative effector genes that present similar expression patterns, namely no expression or low-level expression during axenic cultures compared to strong induction of expression during primary infection of oilseed rape (Brassica napus). Here, we investigated the involvement of one specific histone modification, histone H3 lysine 9 methylation (H3K9me3), in epigenetic regulation of concerted effector gene expression in L. maculans. For this purpose, we silenced the expression of two key players in heterochromatin assembly and maintenance, HP1 and DIM-5 by RNAi. By using HP1-GFP as a heterochromatin marker, we observed that almost no chromatin condensation is visible in strains in which LmDIM5 was silenced by RNAi. By whole genome oligoarrays we observed overexpression of 369 or 390 genes, respectively, in the silenced-LmHP1 and -LmDIM5 transformants during growth in axenic culture, clearly favouring expression of SSP-encoding genes within AT-isochores. The ectopic integration of four effector genes in GC-isochores led to their overexpression during growth in axenic culture. These data strongly suggest that epigenetic control, mediated by HP1 and DIM-5, represses the expression of at least part of the effector genes located in AT-isochores during growth in axenic culture. Our hypothesis is that changes of lifestyle and a switch toward pathogenesis lift chromatin-mediated repression, allowing a rapid response to new environmental conditions.

Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum

PubMed Central

Reyes-Dominguez, Yazmid; Boedi, Stefan; Sulyok, Michael; Wiesenberger, Gerlinde; Stoppacher, Norbert; Krska, Rudolf; Strauss, Joseph

2012-01-01

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin. PMID:22100541

Transcriptional Networks Controlled by NKX2-1 in the Development of Forebrain GABAergic Neurons

DOE PAGES

Sandberg, Magnus; Flandin, Pierre; Silberberg, Shanni; ...

2016-09-21

The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissivemore » chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach gives a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants.« less

A ternary AppA-PpsR-DNA complex mediates light regulation of photosynthesis-related gene expression.

PubMed

Winkler, Andreas; Heintz, Udo; Lindner, Robert; Reinstein, Jochen; Shoeman, Robert L; Schlichting, Ilme

2013-07-01

The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources, depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the bacterium for using light energy. Illumination, however, enhances repression under semiaerobic conditions. Here, we describe molecular details of two proteins mediating oxygen and light control of photosynthesis-gene expression: the light-sensing antirepressor AppA and the transcriptional repressor PpsR. Our crystal structures of both proteins and their complex and hydrogen/deuterium-exchange data show that light activation of AppA-PpsR2 affects the PpsR effector region within the complex. DNA binding studies demonstrate the formation of a light-sensitive ternary AppA-PpsR-DNA complex. We discuss implications of these results for regulation by light and oxygen, highlighting new insights into blue light-mediated signal transduction.

A Regulatory Circuit Composed of a Transcription Factor, IscR, and a Regulatory RNA, RyhB, Controls Fe-S Cluster Delivery.

PubMed

Mandin, Pierre; Chareyre, Sylvia; Barras, Frédéric

2016-09-20

Fe-S clusters are cofactors conserved through all domains of life. Once assembled by dedicated ISC and/or SUF scaffolds, Fe-S clusters are conveyed to their apo-targets via A-type carrier proteins (ATCs). Escherichia coli possesses four such ATCs. ErpA is the only ATC essential under aerobiosis. Recent studies reported a possible regulation of the erpA mRNA by the small RNA (sRNA) RyhB, which controls the expression of many genes under iron starvation. Surprisingly, erpA has not been identified in recent transcriptomic analysis of the iron starvation response, thus bringing into question the actual physiological significance of the putative regulation of erpA by RyhB. Using an sRNA library, we show that among 26 sRNAs, only RyhB represses the expression of an erpA-lacZ translational fusion. We further demonstrate that this repression occurs during iron starvation. Using mutational analysis, we show that RyhB base pairs to the erpA mRNA, inducing its disappearance. In addition, IscR, the master regulator of Fe-S homeostasis, represses expression of erpA at the transcriptional level when iron is abundant, but depleting iron from the medium alleviates this repression. The conjunction of transcriptional derepression by IscR and posttranscriptional repression by RyhB under Fe-limiting conditions is best described as an incoherent regulatory circuit. This double regulation allows full expression of erpA at iron concentrations for which Fe-S biogenesis switches from the ISC to the SUF system. We further provide evidence that this regulatory circuit coordinates ATC usage to iron availability. Regulatory small RNAs (sRNAs) have emerged as major actors in the control of gene expression in the last few decades. Relatively little is known about how these regulators interact with classical transcription factors to coordinate genetic responses. We show here how an sRNA, RyhB, and a transcription factor, IscR, regulate expression of an essential gene, erpA, in the bacterium E. coli ErpA is involved in the biogenesis of Fe-S clusters, an important class of cofactors involved in a plethora of cellular reactions. Interestingly, we show that RyhB and IscR repress expression of erpA under opposite conditions in regard to iron concentration, forming a regulatory circuit called an "incoherent network." This incoherent network serves to maximize expression of erpA at iron concentrations where it is most needed. Altogether, our study paves the way for a better understanding of mixed regulatory networks composed of RNAs and transcription factors. Copyright © 2016 Mandin et al.

Non-additive interactions involving two distinct elements mediate sloppy-paired regulation by pair-rule transcription factors

PubMed Central

Prazak, Lisa; Fujioka, Miki; Gergen, J. Peter

2010-01-01

The relatively simple combinatorial rules responsible for establishing the initial metameric expression of sloppy-paired-1 (slp1) in the Drosophila blastoderm embryo make this system an attractive model for investigating the mechanism of regulation by pair rule transcription factors. This investigation of slp1 cis-regulatory architecture identifies two distinct elements, a proximal early stripe element (PESE) and a distal early stripe element (DESE) located from −3.1 kb to −2.5 kb and from −8.1 kb to −7.1 kb upstream of the slp1 promoter, respectively, that mediate this early regulation. The proximal element expresses only even-numbered stripes and mediates repression by Even-skipped (Eve) as well as by the combination of Runt and Fushi-tarazu (Ftz). A 272 basepair sub-element of PESE retains Eve-dependent repression, but is expressed throughout the even-numbered parasegments due to the loss of repression by Runt and Ftz. In contrast, the distal element expresses both odd and even-numbered stripes and also drives inappropriate expression in the anterior half of the odd-numbered parasegments due to an inability to respond to repression by Eve. Importantly, a composite reporter gene containing both early stripe elements recapitulates pair-rule gene-dependent regulation in a manner beyond what is expected from combining their individual patterns. These results indicate interactions involving distinct cis-elements contribute to the proper integration of pair-rule regulatory information. A model fully accounting for these results proposes that metameric slp1 expression is achieved through the Runt-dependent regulation of interactions between these two pair-rule response elements and the slp1 promoter. PMID:20435028

CIP, a cardiac Isl1-interacting protein, represses cardiomyocyte hypertrophy.

PubMed

Huang, Zhan-Peng; Young Seok, Hee; Zhou, Bin; Chen, Jinghai; Chen, Jian-Fu; Tao, Yazhong; Pu, William T; Wang, Da-Zhi

2012-03-16

Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic growth) of cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well-established, the molecular events that inhibit or repress cardiac hypertrophy are less known. To identify and investigate novel regulators that modulate cardiac hypertrophy. Here, we report the identification, characterization, and functional examination of a novel cardiac Isl1-interacting protein (CIP). CIP was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization, and reporter gene tracing demonstrated that CIP is highly expressed in cardiomyocytes of developing and adult hearts. Yeast two-hybrid screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification of cardiac progenitor cells in the second heart field, as a cofactor of CIP. CIP directly interacted with Isl1, and we mapped the domains of these two proteins, which mediate their interaction. We show that CIP represses the transcriptional activity of Isl1 in the activation of the myocyte enhancer factor 2C. The expression of CIP was dramatically reduced in hypertrophic cardiomyocytes. Most importantly, overexpression of CIP repressed agonist-induced cardiomyocyte hypertrophy. Our studies therefore identify CIP as a novel regulator of cardiac hypertrophy.

Zhx2 and Zbtb20: Novel regulators of postnatal alpha-fetoprotein repression and their potential role in gene reactivation during liver cancer

PubMed Central

Peterson, Martha L.; Ma, Chunhong; Spear, Brett T.

2012-01-01

The mouse alpha-fetoprotein (AFP) gene is abundantly expressed in the fetal liver, normally silent in the adult liver but is frequently reactivated in hepatocellular carcinoma. The basis for AFP expression in the fetal liver has been studied extensively. However, the basis for AFP reactivation during hepatocarcinogenesis is not well understood. Two novel factors that control postnatal AFP repression, Zhx2 and Zbtb20, were recently identified. Here, we review the transcription factors that regulate AFP in the fetal liver, as well as Zhx2 and Zbtb20, and raise the possibility that the loss of these postnatal repressors may be involved in AFP reactivation in liver cancer. PMID:21216289

Regulation of FOXO1-mediated transcription and cell proliferation by PARP-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakamaki, Jun-ichi; Daitoku, Hiroaki; Yoshimochi, Kenji

2009-05-08

Forkhead box O (FOXO) transcription factors play an important role in a wide range of biological processes, including cell cycle control, apoptosis, detoxification of reactive oxygen species, and gluconeogenesis through regulation of gene expression. In this study, we demonstrated that PARP-1 functions as a negative regulator of FOXO1. We showed that PARP-1 directly binds to and poly(ADP-ribosyl)ates FOXO1 protein. PARP-1 represses FOXO1-mediated expression of cell cycle inhibitor p27{sup Kip1} gene. Notably, poly(ADP-ribosyl)ation activity was not required for the repressive effect of PARP-1 on FOXO1 function. Furthermore, knockdown of PARP-1 led to a decrease in cell proliferation in a manner dependentmore » on FOXO1 function. Chromatin immunoprecipitation experiments confirmed that PARP-1 is recruited to the p27{sup Kip1} gene promoter through a binding to FOXO1. These results suggest that PARP-1 acts as a corepressor for FOXO1, which could play an important role in proper cell proliferation by regulating p27{sup Kip1} gene expression.« less

Structure and Function of the Macrolide Biosensor Protein, MphR(A), with and without Erythromycin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Jianting; Sagar, Vatsala; Smolinsky, Adam

2009-09-02

The regulatory protein MphR(A) has recently seen extensive use in synthetic biological applications, such as metabolite sensing and exogenous control of gene expression. This protein negatively regulates the expression of a macrolide 2{prime}-phosphotransferase I resistance gene (mphA) via binding to a 35-bp DNA operator upstream of the start codon and is de-repressed by the presence of erythromycin. Here, we present the refined crystal structure of the MphR(A) protein free of erythromycin and that of the MphR(A) protein with bound erythromycin at 2.00- and 1.76-{angstrom} resolutions, respectively. We also studied the DNA binding properties of the protein and identified mutants ofmore » MphR(A) that are defective in gene repression and ligand binding in a cell-based reporter assay. The combination of these two structures illustrates the molecular basis of erythromycin-induced gene expression and provides a framework for additional applied uses of this protein in the isolation and engineered biosynthesis of polyketide natural products.« less

Id2 Complexes with the SNAG Domain of Snai1 Inhibiting Snai1-Mediated Repression of Integrin β4

PubMed Central

Chang, Cheng; Yang, Xiaofang; Pursell, Bryan

2013-01-01

The epithelial-mesenchymal transition (EMT) is a fundamental process that underlies development and cancer. Although the EMT involves alterations in the expression of specific integrins that mediate stable adhesion to the basement membrane, such as α6β4, the mechanisms involved are poorly understood. Here, we report that Snai1 inhibits β4 transcription by increasing repressive histone modification (trimethylation of histone H3 at K27 [H3K27Me3]). Surprisingly, Snai1 is expressed and localized in the nucleus in epithelial cells, but it does not repress β4. We resolved this paradox by discovering that Id2 complexes with the SNAG domain of Snai1 on the β4 promoter and constrains the repressive function of Snai1. Disruption of the complex by depleting Id2 resulted in Snai1-mediated β4 repression with a concomitant increase in H3K27Me3 modification on the β4 promoter. These findings establish a novel function for Id2 in regulating Snai1 that has significant implications for the regulation of epithelial gene expression. PMID:23878399

PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li

2013-02-01

Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently ofmore » its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.« less

RYBP stimulates PRC1 to shape chromatin-based communication between Polycomb repressive complexes

PubMed Central

Rose, Nathan R; King, Hamish W; Blackledge, Neil P; Fursova, Nadezda A; Ember, Katherine JI; Fischer, Roman; Kessler, Benedikt M; Klose, Robert J

2016-01-01

Polycomb group (PcG) proteins function as chromatin-based transcriptional repressors that are essential for normal gene regulation during development. However, how these systems function to achieve transcriptional regulation remains very poorly understood. Here, we discover that the histone H2AK119 E3 ubiquitin ligase activity of Polycomb repressive complex 1 (PRC1) is defined by the composition of its catalytic subunits and is highly regulated by RYBP/YAF2-dependent stimulation. In mouse embryonic stem cells, RYBP plays a central role in shaping H2AK119 mono-ubiquitylation at PcG targets and underpins an activity-based communication between PRC1 and Polycomb repressive complex 2 (PRC2) which is required for normal histone H3 lysine 27 trimethylation (H3K27me3). Without normal histone modification-dependent communication between PRC1 and PRC2, repressive Polycomb chromatin domains can erode, rendering target genes susceptible to inappropriate gene expression signals. This suggests that activity-based communication and histone modification-dependent thresholds create a localized form of epigenetic memory required for normal PcG chromatin domain function in gene regulation. DOI: http://dx.doi.org/10.7554/eLife.18591.001 PMID:27705745

Necdin, a Prader-Willi syndrome candidate gene, regulates gonadotropin-releasing hormone neurons during development.

PubMed

Miller, Nichol L G; Wevrick, Rachel; Mellon, Pamela L

2009-01-15

Prader-Willi syndrome (PWS) is a complex genetic disorder characterized by hyperphagia, obesity and hypogonadotrophic hypogonadism, all highly suggestive of hypothalamic dysfunction. The NDN gene, encoding the MAGE family protein, necdin, maps to the PWS chromosome region and is highly expressed in mature hypothalamic neurons. Adult mice lacking necdin have reduced numbers of gonadotropin-releasing hormone (GnRH) neurons, but the mechanism for this reduction is unknown. Herein, we show that, although necdin is not expressed in an immature, migratory GnRH neuronal cell line (GN11), high levels are present in a mature GnRH neuronal cell line (GT1-7). Furthermore, overexpression of necdin activates GnRH transcription through cis elements bound by the homeodomain repressor Msx that are located in the enhancer and promoter of the GnRH gene, and knock-down of necdin expression reduces GnRH gene expression. In fact, overexpression of Necdin relieves Msx repression of GnRH transcription through these elements and necdin co-immunoprecipitates with Msx from GnRH neuronal cells, indicating that necdin may activate GnRH gene expression by preventing repression of GnRH gene expression by Msx. Finally, necdin is necessary for generation of the full complement of GnRH neurons during mouse development and extension of GnRH axons to the median eminence. Together, these results indicate that lack of necdin during development likely contributes to the hypogonadotrophic hypogonadal phenotype in individuals with PWS.

Necdin, a Prader–Willi syndrome candidate gene, regulates gonadotropin-releasing hormone neurons during development

PubMed Central

Miller, Nichol L.G.; Wevrick, Rachel; Mellon, Pamela L.

2009-01-01

Prader–Willi syndrome (PWS) is a complex genetic disorder characterized by hyperphagia, obesity and hypogonadotrophic hypogonadism, all highly suggestive of hypothalamic dysfunction. The NDN gene, encoding the MAGE family protein, necdin, maps to the PWS chromosome region and is highly expressed in mature hypothalamic neurons. Adult mice lacking necdin have reduced numbers of gonadotropin-releasing hormone (GnRH) neurons, but the mechanism for this reduction is unknown. Herein, we show that, although necdin is not expressed in an immature, migratory GnRH neuronal cell line (GN11), high levels are present in a mature GnRH neuronal cell line (GT1-7). Furthermore, overexpression of necdin activates GnRH transcription through cis elements bound by the homeodomain repressor Msx that are located in the enhancer and promoter of the GnRH gene, and knock-down of necdin expression reduces GnRH gene expression. In fact, overexpression of Necdin relieves Msx repression of GnRH transcription through these elements and necdin co-immunoprecipitates with Msx from GnRH neuronal cells, indicating that necdin may activate GnRH gene expression by preventing repression of GnRH gene expression by Msx. Finally, necdin is necessary for generation of the full complement of GnRH neurons during mouse development and extension of GnRH axons to the median eminence. Together, these results indicate that lack of necdin during development likely contributes to the hypogonadotrophic hypogonadal phenotype in individuals with PWS. PMID:18930956

L(3)mbt and the LINT complex safeguard cellular identity in the Drosophila ovary.

PubMed

Coux, Rémi-Xavier; Teixeira, Felipe Karam; Lehmann, Ruth

2018-04-04

Maintenance of cellular identity is essential for tissue development and homeostasis. At the molecular level, cell identity is determined by the coordinated activation and repression of defined sets of genes. The tumor suppressor L(3)mbt has been shown to secure cellular identity in Drosophila larval brains by repressing germline-specific genes. Here, we interrogate the temporal and spatial requirements for L(3)mbt in the Drosophila ovary, and show that it safeguards the integrity of both somatic and germline tissues. l(3)mbt mutant ovaries exhibit multiple developmental defects, which we find to be largely caused by the inappropriate expression of a single gene, nanos , a key regulator of germline fate, in the somatic ovarian cells. In the female germline, we find that L(3)mbt represses testis-specific and neuronal genes. At the molecular level, we show that L(3)mbt function in the ovary is mediated through its co-factor Lint-1 but independently of the dREAM complex. Together, our work uncovers a more complex role for L(3)mbt than previously understood and demonstrates that L(3)mbt secures tissue identity by preventing the simultaneous expression of original identity markers and tissue-specific misexpression signatures. © 2018. Published by The Company of Biologists Ltd.

Epigenetic regulation of normal human mammary cell type-specific miRNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vrba, Lukas; Garbe, James C.; Stampfer, Martha R.

2011-08-26

Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linkedmore » to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.« less

Effect of beta2-adrenoceptor agonists and other cAMP-elevating agents on inflammatory gene expression in human ASM cells: a role for protein kinase A.

PubMed

Kaur, Manminder; Holden, Neil S; Wilson, Sylvia M; Sukkar, Maria B; Chung, Kian Fan; Barnes, Peter J; Newton, Robert; Giembycz, Mark A

2008-09-01

In diseases such as asthma, airway smooth muscle (ASM) cells play a synthetic role by secreting inflammatory mediators such as granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, or IL-8 and by expressing surface adhesion molecules, including ICAM-1. In the present study, PGE(2), forskolin, and short-acting (salbutamol) and long-acting (salmeterol and formoterol) beta(2)-adrenoceptor agonists reduced the expression of ICAM-1 and the release of GM-CSF evoked by IL-1beta in ASM cells. IL-1beta-induced IL-8 release was also repressed by PGE(2) and forskolin, whereas the beta(2)-adrenoceptor agonists were ineffective. In each case, repression of these inflammatory indexes was prevented by adenoviral overexpression of PKIalpha, a highly selective PKA inhibitor. These data indicate a PKA-dependent mechanism of repression and suggest that agents that elevate intracellular cAMP, and thereby activate PKA, may have a widespread anti-inflammatory effect in ASM cells. Since ICAM-1 and GM-CSF are highly NF-kappaB-dependent genes, we used an adenoviral-delivered NF-kappaB-dependent luciferase reporter to examine the effects of forskolin and the beta(2)-adrenoceptor agonists on NF-kappaB activation. There was no effect on luciferase activity measured in the presence of forskolin or beta(2)-adrenoceptor agonists. This finding is consistent with the observation that IL-1beta-induced expression of IL-6, a known NF-kappaB-dependent gene in ASM, was also unaffected by beta(2)-adrenoceptor agonists, forskolin, PGE(2), 8-bromo-cAMP, or rolipram. Collectively, these results indicate that repression of IL-1beta-induced ICAM-1 expression and GM-CSF release by cAMP-elevating agents, including beta(2)-adrenoceptor agonists, may not occur through a generic effect on NF-kappaB.

Leukemia/lymphoma-related factor, a POZ domain-containing transcriptional repressor, interacts with histone deacetylase-1 and inhibits cartilage oligomeric matrix protein gene expression and chondrogenesis.

PubMed

Liu, Chuan-ju; Prazak, Lisa; Fajardo, Marc; Yu, Shuang; Tyagi, Neetu; Di Cesare, Paul E

2004-11-05

Mutations in the human cartilage oligomeric matrix protein (COMP) gene have been linked to the development of pseudoachondroplasia and multiple epiphyseal dysplasia. We previously cloned the promoter region of the COMP gene and delineated a minimal negative regulatory element (NRE) that is both necessary and sufficient to repress its promoter (Issack, P. S., Fang, C. H., Leslie, M. P., and Di Cesare, P. E. (2000) J. Orthop. Res. 18, 345-350; Issack, P. S., Liu, C. J., Prazak, L., and Di Cesare, P. E. (2004) J. Orthop. Res. 22, 751-758). In this study, a yeast one-hybrid screen for proteins that associate with the NRE led to the identification of the leukemia/lymphoma-related factor (LRF), a transcriptional repressor that contains a POZ (poxvirus zinc finger) domain, as an NRE-binding protein. LRF bound directly to the NRE both in vitro and in living cells. Nine nucleotides (GAGGGTCCC) in the 30-bp NRE are essential for binding to LRF. LRF showed dose-dependent inhibition of COMP-specific reporter gene activity, and exogenous overexpression of LRF repressed COMP gene expression in both rat chondrosarcoma cells and bone morphogenetic protein-2-treated C3H10T1/2 progenitor cells. In addition, LRF also inhibited bone morphogenetic protein-2-induced chondrogenesis in high density micromass cultures of C3H10T1/2 cells, as evidenced by lack of expression of other chondrocytic markers, such as aggrecan and collagen types II, IX, X, and XI, and by Alcian blue staining. LRF associated with histone deacetylase-1 (HDAC1), and experiments utilizing the HDAC inhibitor trichostatin A revealed that LRF-mediated repression requires deacetylase activity. LRF is the first transcription factor found to bind directly to the COMP gene promoter, to recruit HDAC1, and to regulate both COMP gene expression and chondrogenic differentiation.

Direct Role for the Rpd3 Complex in Transcriptional Induction of the Anaerobic DAN/TIR Genes in Yeast▿‡

PubMed Central

Sertil, Odeniel; Vemula, Arvind; Salmon, Sharon L.; Morse, Randall H.; Lowry, Charles V.

2007-01-01

Saccharomyces cerevisiae adapts to hypoxia by expressing a large group of “anaerobic” genes. Among these, the eight DAN/TIR genes are regulated by the repressors Rox1 and Mot3 and the activator Upc2/Mox4. In attempting to identify factors recruited by the DNA binding repressor Mot3 to enhance repression of the DAN/TIR genes, we found that the histone deacetylase and global repressor complex, Rpd3-Sin3-Sap30, was not required for repression. Strikingly, the complex was instead required for activation. In addition, the histone H3 and H4 amino termini, which are targets of Rpd3, were also required for DAN1 expression. Epistasis tests demonstrated that the Rpd3 complex is not required in the absence of the repressor Mot3. Furthermore, the Rpd3 complex was required for normal function and stable binding of the activator Upc2 at the DAN1 promoter. Moreover, the Swi/Snf chromatin remodeling complex was strongly required for activation of DAN1, and chromatin immunoprecipitation analysis showed an Rpd3-dependent reduction in DAN1 promoter-associated nucleosomes upon induction. Taken together, these data provide evidence that during anaerobiosis, the Rpd3 complex acts at the DAN1 promoter to antagonize the chromatin-mediated repression caused by Mot3 and Rox1 and that chromatin remodeling by Swi/Snf is necessary for normal expression. PMID:17210643

A repressive role of enhancer of zeste homolog 2 in 11β-hydroxysteroid dehydrogenase type 2 expression in the human placenta.

PubMed

Zuo, Rujuan; Liu, Xiaohui; Wang, Wangsheng; Li, Wenjiao; Ying, Hao; Sun, Kang

2017-05-05

The expression of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2), which acts as a placental glucocorticoid barrier, is silenced in cytotrophoblasts but substantially up-regulated during syncytialization. However, the repressive mechanism of 11β-HSD2 expression before syncytialization and how this repression is lifted during syncytialization remain mostly unresolved. Here we found that enhancer of zeste homolog 2 (EZH2) accounts for the silence of 11β-HSD2 expression via trimethylation of histone H3 lysine 27 at the promoter of the 11β-HSD2 gene. Further studies revealed that, upon syncytialization, human chorionic gonadotropin reduced the phosphorylation of retinoblastoma protein (pRB) via activation of the cAMP/PKA pathway, which sequesters E2F transcription factor 1 (E2F1), the transcription factor for EZH2 expression. As a result of inactivation of the pRB-E2F1-EZH2 pathway, the repressive marker trimethylation of histone H3 lysine 27 at the 11β-HSD2 promoter is removed, which leads to the robust expression of 11β-HSD2 during syncytialization. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

FH535, a β-catenin pathway inhibitor, represses pancreatic cancer xenograft growth and angiogenesis

PubMed Central

Gong, Fei-Ran; Zhou, Binhua P.; Lian, Lian; Shen, Bairong; Chen, Kai; Duan, Weiming; Wu, Meng-Yao; Tao, Min; Li, Wei

2016-01-01

The WNT/β-catenin pathway plays an important role in pancreatic cancer carcinogenesis. We evaluated the correlation between aberrant β-catenin pathway activation and the prognosis pancreatic cancer, and the potential of applying the β-catenin pathway inhibitor FH535 to pancreatic cancer treatment. Meta-analysis and immunohistochemistry showed that abnormal β-catenin pathway activation was associated with unfavorable outcome. FH535 repressed pancreatic cancer xenograft growth in vivo. Gene Ontology (GO) analysis of microarray data indicated that target genes responding to FH535 participated in stemness maintenance. Real-time PCR and flow cytometry confirmed that FH535 downregulated CD24 and CD44, pancreatic cancer stem cell (CSC) markers, suggesting FH535 impairs pancreatic CSC stemness. GO analysis of β-catenin chromatin immunoprecipitation sequencing data identified angiogenesis-related gene regulation. Immunohistochemistry showed that higher microvessel density correlated with elevated nuclear β-catenin expression and unfavorable outcome. FH535 repressed the secretion of the proangiogenic cytokines vascular endothelial growth factor (VEGF), interleukin (IL)-6, IL-8, and tumor necrosis factor-α, and also inhibited angiogenesis in vitro and in vivo. Protein and mRNA microarrays revealed that FH535 downregulated the proangiogenic genes ANGPT2, VEGFR3, IFN-γ, PLAUR, THPO, TIMP1, and VEGF. FH535 not only represses pancreatic CSC stemness in vitro, but also remodels the tumor microenvironment by repressing angiogenesis, warranting further clinical investigation. PMID:27323403

Zhx2 (zinc fingers and homeoboxes 2) regulates major urinary protein gene expression in the mouse liver

PubMed Central

Jiang, Jieyun; Creasy, Kate Townsend; Purnell, Justin; Peterson, Martha L.; Spear, Brett T.

2017-01-01

The mouse major urinary proteins (Mups) are encoded by a large family of highly related genes clustered on chromosome 4. Mups, synthesized primarily and abundantly in the liver and secreted through the kidneys, exhibit male-biased expression. Mups bind a variety of volatile ligands; these ligands, and Mup proteins themselves, influence numerous behavioral traits. Although urinary Mup protein levels vary between inbred mouse strains, this difference is most pronounced in BALB/cJ mice, which have dramatically low urinary Mup levels; this BALB/cJ trait had been mapped to a locus on chromosome 15. We previously identified Zhx2 (zinc fingers and homeoboxes 2) as a regulator of numerous liver-enriched genes. Zhx2 is located on chromosome 15, and a natural hypomorphic mutation in the BALB/cJ Zhx2 allele dramatically reduces Zhx2 expression. Based on these data, we hypothesized that reduced Zhx2 levels are responsible for lower Mup expression in BALB/cJ mice. Using both transgenic and knock-out mice along with in vitro assays, our data show that Zhx2 binds Mup promoters and is required for high levels of Mup expression in the adult liver. In contrast to previously identified Zhx2 targets that appear to be repressed by Zhx2, Mup genes are positively regulated by Zhx2. These data identify Zhx2 as a novel regulator of Mup expression and indicate that Zhx2 activates as well as represses expression of target genes. PMID:28258223

Genome-wide identification and characterization of TCP genes involved in ovule development of Phalaenopsis equestris.

PubMed

Lin, Yu-Fu; Chen, You-Yi; Hsiao, Yu-Yun; Shen, Ching-Yu; Hsu, Jui-Ling; Yeh, Chuan-Ming; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Liu, Zhong-Jian; Tsai, Wen-Chieh

2016-09-01

TEOSINTE-BRANCHED/CYCLOIDEA/PCF (TCP) proteins are plant-specific transcription factors known to have a role in multiple aspects of plant growth and development at the cellular, organ and tissue levels. However, there has been no related study of TCPs in orchids. Here we identified 23 TCP genes from the genome sequence of Phalaenopsis equestris Phylogenetic analysis distinguished two homology classes of PeTCP transcription factor families: classes I and II. Class II was further divided into two subclasses, CIN and CYC/TB1. Spatial and temporal expression analysis showed that PePCF10 was predominantly expressed in ovules at early developmental stages and PeCIN8 had high expression at late developmental stages in ovules, with overlapping expression at day 16 after pollination. Subcellular localization and protein-protein interaction analyses revealed that PePCF10 and PeCIN8 could form homodimers and localize in the nucleus. However, PePCF10 and PeCIN8 could not form heterodimers. In transgenic Arabidopsis thaliana plants (overexpression and SRDX, a super repression motif derived from the EAR-motif of the repression domain of tobacco ETHYLENE-RESPONSIVE ELEMENT-BINDING FACTOR 3 and SUPERMAN, dominantly repressed), the two genes helped regulate cell proliferation. Together, these results suggest that PePCF10 and PeCIN8 play important roles in orchid ovule development by modulating cell division. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

MicroRNA-193b represses cell proliferation and regulates cyclin D1 in melanoma.

PubMed

Chen, Jiamin; Feilotter, Harriet E; Paré, Geneviève C; Zhang, Xiao; Pemberton, Joshua G W; Garady, Cherif; Lai, Dulcie; Yang, Xiaolong; Tron, Victor A

2010-05-01

Cutaneous melanoma is an aggressive form of human skin cancer characterized by high metastatic potential and poor prognosis. To better understand the role of microRNAs (miRNAs) in melanoma, the expression of 470 miRNAs was profiled in tissue samples from benign nevi and metastatic melanomas. We identified 31 miRNAs that were differentially expressed (13 up-regulated and 18 down-regulated) in metastatic melanomas relative to benign nevi. Notably, miR-193b was significantly down-regulated in the melanoma tissues examined. To understand the role of miR-193b in melanoma, functional studies were undertaken. Overexpression of miR-193b in melanoma cell lines repressed cell proliferation. Gene expression profiling identified 314 genes down-regulated by overexpression of miR-193b in Malme-3M cells. Eighteen of these down-regulated genes, including cyclin D1 (CCND1), were also identified as putative miR-193b targets by TargetScan. Overexpression of miR-193b in Malme-3M cells down-regulated CCND1 mRNA and protein by > or = 50%. A luciferase reporter assay confirmed that miR-193b directly regulates CCND1 by binding to the 3'untranslated region of CCND1 mRNA. These studies indicate that miR-193b represses cell proliferation and regulates CCND1 expression and suggest that dysregulation of miR-193b may play an important role in melanoma development.

MicroRNA-193b Represses Cell Proliferation and Regulates Cyclin D1 in Melanoma

PubMed Central

Chen, Jiamin; Feilotter, Harriet E.; Paré, Geneviève C.; Zhang, Xiao; Pemberton, Joshua G.W.; Garady, Cherif; Lai, Dulcie; Yang, Xiaolong; Tron, Victor A.

2010-01-01

Cutaneous melanoma is an aggressive form of human skin cancer characterized by high metastatic potential and poor prognosis. To better understand the role of microRNAs (miRNAs) in melanoma, the expression of 470 miRNAs was profiled in tissue samples from benign nevi and metastatic melanomas. We identified 31 miRNAs that were differentially expressed (13 up-regulated and 18 down-regulated) in metastatic melanomas relative to benign nevi. Notably, miR-193b was significantly down-regulated in the melanoma tissues examined. To understand the role of miR-193b in melanoma, functional studies were undertaken. Overexpression of miR-193b in melanoma cell lines repressed cell proliferation. Gene expression profiling identified 314 genes down-regulated by overexpression of miR-193b in Malme-3M cells. Eighteen of these down-regulated genes, including cyclin D1 (CCND1), were also identified as putative miR-193b targets by TargetScan. Overexpression of miR-193b in Malme-3M cells down-regulated CCND1 mRNA and protein by ≥50%. A luciferase reporter assay confirmed that miR-193b directly regulates CCND1 by binding to the 3′untranslated region of CCND1 mRNA. These studies indicate that miR-193b represses cell proliferation and regulates CCND1 expression and suggest that dysregulation of miR-193b may play an important role in melanoma development. PMID:20304954

Divergent Expression Patterns and Function Implications of Four nanos Genes in a Hermaphroditic Fish, Epinephelus coioides

PubMed Central

Sun, Zhi-Hui; Wang, Yang; Lu, Wei-Jia; Li, Zhi; Liu, Xiao-Chun; Li, Shui-Sheng; Zhou, Li; Gui, Jian-Fang

2017-01-01

Multiple nanos genes have been characterized in several fishes, but the functional implications of their various expression patterns remain unclear. In this study, we identified and characterized four nanos genes from a hermaphroditic fish orange-spotted grouper, Epinephelus coioides. Ecnanos1a and Ecnanos1b show divergent expression patterns, and the dynamic expression change of Ecnanos1a in pituitaries during sex change is associated with testis differentiation and spermatogenesis. Ecnanos2 and Ecnanos3 might be germline stem cells (GSCs) and primordial germ cells (PGCs)-specific markers, respectively. Significantly, Ecnanos3 3′-untranslated region (UTR) is necessary for PGC specific expression, where a non-canonical “GCACGTTT” sequence is required for miR-430-mediated repression of Ecnanos3 RNA. Furthermore, grouper Dead end (Dnd) can relieve miR-430 repression in PGCs by associating with a 23 bp U-rich region (URR) in Ecnanos3 3′-UTR. The current study revealed the functional association of multiple nanos genes with PGC formation and germ cell development in orange-spotted grouper, and opened up new possibilities for developing biotechnologies through utilizing the associations between Ecnanos3 and PGCs or between Ecnanos2 and GSCs in the hermaphroditic fish. PMID:28333083

A Screen in Mice Uncovers Repression of Lipoprotein Lipase by MicroRNA-29a as a Mechanism for Lipid Distribution Away From the Liver

PubMed Central

Mattis, Aras N.; Song, Guisheng; Hitchner, Kelly; Kim, Roy Y.; Lee, Andrew Y.; Sharma, Amar D.; Malato, Yann; McManus, Michael T.; Esau, Christine C.; Koller, Erich; Koliwad, Suneil; Lim, Lee P.; Maher, Jacquelyn J.; Raffai, Robert L.; Willenbring, Holger

2015-01-01

Identification of microRNAs (miRNAs) that regulate lipid metabolism is important to advance the understanding and treatment of some of the most common human diseases. In the liver, a few key miRNAs have been reported that regulate lipid metabolism, but since many genes contribute to hepatic lipid metabolism, we hypothesized that other such miRNAs exist. To identify genes repressed by miRNAs in mature hepatocytes in vivo, we injected adult mice carrying floxed Dicer1 alleles with an adenoassociated viral vector expressing Cre recombinase specifically in hepatocytes. By inactivating Dicer in adult quiescent hepatocytes we avoided the hepatocyte injury and regeneration observed in previous mouse models of global miRNA deficiency in hepatocytes. Next, we combined gene and miRNA expression profiling to identify candidate gene/miRNA interactions involved in hepatic lipid metabolism, and validated their function in vivo using antisense oligonucleotides. A candidate gene that emerged from our screen was lipoprotein lipase (Lpl), which encodes an enzyme that facilitates cellular uptake of lipids from the circulation. Unlike in energy-dependent cells like myocytes, Lpl is normally repressed in adult hepatocytes. We identified miR-29a as the miRNA responsible for repressing Lpl in hepatocytes, and found that decreasing hepatic miR-29a levels causes lipids to accumulate in mouse livers. Conclusion Our screen suggests several new miRNAs are regulators of hepatic lipid metabolism. We show that one of these, miR-29a, contributes to physiological lipid distribution away from the liver and protects hepatocytes from steatosis. Our results, together with miR-29a’s known anti-fibrotic effect, suggest miR-29a is a therapeutic target in fatty liver disease. PMID:25131933

Stanniocalcin 2 is an estrogen-responsive gene coexpressed with the estrogen receptor in human breast cancer.

PubMed

Bouras, Toula; Southey, Melissa C; Chang, Andy C; Reddel, Roger R; Willhite, Dorian; Glynne, Richard; Henderson, Michael A; Armes, Jane E; Venter, Deon J

2002-03-01

Differences in gene expression are likely to explain the phenotypic variation between hormone-responsive and hormone-unresponsive breast cancers. In this study, DNA microarray analysis of approximately 10,000 known genes and 25,000 expressed sequence tag clusters was performed to identify genes induced by estrogen and repressed by the pure antiestrogen ICI 182 780 in vitro that correlated with estrogen receptor (ER) expression in primary breast carcinomas in vivo. Stanniocalcin (STC) 2 was identified as one of the genes that fulfilled these criteria. DNA microarray hybridization showed a 3-fold induction of STC2 mRNA expression in MCF-7 cells in < or = 3 h of estrogen exposure and a 3-fold repression in the presence of antiestrogen (one-way ANOVA, P < 0.0005). In 13 ER-positive and 12 ER-negative breast carcinomas, the microarray-derived mRNA levels observed for STC2 correlated with tumor ER mRNA (Pearson's correlation, r = 0.85; P < 0.0001) and ER protein status (Spearman's rank correlation, r = 0.73; P < 0.0001). The expression profile of STC2 was further confirmed by in situ hybridization and immunohistochemistry on a larger cohort of 236 unselected breast carcinomas using tissue microarrays. STC2 mRNA and protein expression were found to be associated with tumor ER status (Fisher's exact test, P < 0.005). The related gene, STC1, was also examined and shown to be associated with ER status in breast carcinomas (Fisher's exact test, P < 0.05). This study demonstrates the feasibility of using global gene expression data derived from an in vitro model to pinpoint novel estrogen-responsive genes of potential clinical relevance.

The master regulator PhoP coordinates phosphate and nitrogen metabolism, respiration, cell differentiation and antibiotic biosynthesis: comparison in Streptomyces coelicolor and Streptomyces avermitilis.

PubMed

Martín, Juan F; Rodríguez-García, Antonio; Liras, Paloma

2017-05-01

Phosphate limitation is important for production of antibiotics and other secondary metabolites in Streptomyces. Phosphate control is mediated by the two-component system PhoR-PhoP. Following phosphate depletion, PhoP stimulates expression of genes involved in scavenging, transport and mobilization of phosphate, and represses the utilization of nitrogen sources. PhoP reduces expression of genes for aerobic respiration and activates nitrate respiration genes. PhoP activates genes for teichuronic acid formation and reduces expression of genes for phosphate-rich teichoic acid biosynthesis. In Streptomyces coelicolor, PhoP repressed several differentiation and pleiotropic regulatory genes, which affects development and indirectly antibiotic biosynthesis. A new bioinformatics analysis of the putative PhoP-binding sequences in Streptomyces avermitilis was made. Many sequences in S. avermitilis genome showed high weight values and were classified according to the available genetic information. These genes encode phosphate scavenging proteins, phosphate transporters and nitrogen metabolism genes. Among of the genes highlighted in the new studies was aveR, located in the avermectin gene cluster, encoding a LAL-type regulator, and afsS, which is regulated by PhoP and AfsR. The sequence logo for S. avermitilis PHO boxes is similar to that of S. coelicolor, with differences in the weight value for specific nucleotides in the sequence.

CHES1/FOXN3 regulates cell proliferation by repressing PIM2 and protein biosynthesis

PubMed Central

Huot, Geneviève; Vernier, Mathieu; Bourdeau, Véronique; Doucet, Laurent; Saint-Germain, Emmanuelle; Gaumont-Leclerc, Marie-France; Moro, Alejandro; Ferbeyre, Gerardo

2014-01-01

The expression of the forkhead transcription factor checkpoint suppressor 1 (CHES1), also known as FOXN3, is reduced in many types of cancers. We show here that CHES1 decreases protein synthesis and cell proliferation in tumor cell lines but not in normal fibroblasts. Conversely, short hairpin RNA–mediated depletion of CHES1 increases tumor cell proliferation. Growth suppression depends on the CHES1 forkhead DNA-binding domain and correlates with the nuclear localization of CHES1. CHES1 represses the expression of multiple genes, including the kinases PIM2 and DYRK3, which regulate protein biosynthesis, and a number of genes in cilium biogenesis. CHES1 binds directly to the promoter of PIM2, and in cells expressing CHES1 the levels of PIM2 are reduced, as well as the phosphorylation of the PIM2 target 4EBP1. Overexpression of PIM2 or eIF4E partially reverses the antiproliferative effect of CHES1, indicating that PIM2 and protein biosynthesis are important targets of the antiproliferative effect of CHES1. In several human hematopoietic cancers, CHES1 and PIM2 expressions are inversely correlated, suggesting that repression of PIM2 by CHES1 is clinically relevant. PMID:24403608

CHES1/FOXN3 regulates cell proliferation by repressing PIM2 and protein biosynthesis.

PubMed

Huot, Geneviève; Vernier, Mathieu; Bourdeau, Véronique; Doucet, Laurent; Saint-Germain, Emmanuelle; Gaumont-Leclerc, Marie-France; Moro, Alejandro; Ferbeyre, Gerardo

2014-03-01

The expression of the forkhead transcription factor checkpoint suppressor 1 (CHES1), also known as FOXN3, is reduced in many types of cancers. We show here that CHES1 decreases protein synthesis and cell proliferation in tumor cell lines but not in normal fibroblasts. Conversely, short hairpin RNA-mediated depletion of CHES1 increases tumor cell proliferation. Growth suppression depends on the CHES1 forkhead DNA-binding domain and correlates with the nuclear localization of CHES1. CHES1 represses the expression of multiple genes, including the kinases PIM2 and DYRK3, which regulate protein biosynthesis, and a number of genes in cilium biogenesis. CHES1 binds directly to the promoter of PIM2, and in cells expressing CHES1 the levels of PIM2 are reduced, as well as the phosphorylation of the PIM2 target 4EBP1. Overexpression of PIM2 or eIF4E partially reverses the antiproliferative effect of CHES1, indicating that PIM2 and protein biosynthesis are important targets of the antiproliferative effect of CHES1. In several human hematopoietic cancers, CHES1 and PIM2 expressions are inversely correlated, suggesting that repression of PIM2 by CHES1 is clinically relevant.

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 {yields} S transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay

2011-07-01

Highlights: {yields} TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. {yields} TCP4 expression in yeast retards cell division by blocking G1 {yields} S transition. {yields} Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, theirmore » exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 {yields} S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 {yields} S arrest is discussed.« less

Epigenetic Regulation of Vitamin D 24-Hydroxylase/CYP24A1 in Human Prostate Cancer

PubMed Central

Luo, Wei; Karpf, Adam R.; Deeb, Kristin K.; Muindi, Josephia R.; Morrison, Carl D.; Johnson, Candace S.; Trump, Donald L.

2010-01-01

Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. ChIP-qPCR reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of VDR to the CYP24A1 promoter. RT-PCR analysis of paired human prostate samples reveals that CYP24A1 expression is down-regulated in prostate malignant lesions compared to adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared to matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications. PMID:20587525

Epstein-Barr Virus (EBV) Latent Protein EBNA3A Directly Targets and Silences the STK39 Gene in B Cells Infected by EBV.

PubMed

Bazot, Quentin; Paschos, Kostas; Allday, Martin J

2018-04-01

Epstein-Barr virus (EBV) establishes latent infection in human B cells and is associated with a wide range of cancers. The EBV nuclear antigen 3 (EBNA3) family proteins are critical for B cell transformation and function as transcriptional regulators. It is well established that EBNA3A and EBNA3C cooperate in the regulation of cellular genes. Here, we demonstrate that the gene STK39 is repressed only by EBNA3A. This is the first example of a gene regulated only by EBNA3A in EBV-transformed lymphoblastoid cell lines (LCLs) without the help of EBNA3C. This was demonstrated using a variety of LCLs carrying either knockout, revertant, or conditional EBNA3 recombinants. Investigating the kinetics of EBNA3A-mediated changes in STK39 expression showed that STK39 becomes derepressed quickly after EBNA3A inactivation. This derepression is reversible as EBNA3A reactivation represses STK39 in the same cells expressing a conditional EBNA3A. STK39 is silenced shortly after primary B cell infection by EBV, and no STK39 -encoded protein (SPAK) is detected 3 weeks postinfection. Chromatin immunoprecipitation (ChIP) analysis indicates that EBNA3A directly binds to a regulatory region downstream of the STK39 transcription start site. For the first time, we demonstrated that the polycomb repressive complex 2 with the deposition of the repressive mark H3K27me3 is not only important for the maintenance of an EBNA3A target gene ( STK39 ) but is also essential for the initial establishment of its silencing. Finally, we showed that DNA methyltransferases are involved in the EBNA3A-mediated repression of STK39 IMPORTANCE EBV is well known for its ability to transform B lymphocytes to continuously proliferating lymphoblastoid cell lines. This is achieved in part by the reprogramming of cellular gene transcription by EBV transcription factors, including the EBNA3 proteins that play a crucial role in this process. In the present study, we found that EBNA3A epigenetically silences STK39 This is the first gene where EBNA3A has been found to exert its repressive role by itself, without needing its coregulators EBNA3B and EBNA3C. Furthermore, we demonstrated that the polycomb repressor complex is essential for EBNA3A-mediated repression of STK39 Findings in this study provide new insights into the regulation of cellular genes by the transcription factor EBNA3A. Copyright © 2018 Bazot et al.

CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level

PubMed Central

El Mouali, Youssef; Gaviria-Cantin, Tania; Gibert, Marta; Westermann, Alexander J.; Vogel, Jörg

2018-01-01

Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression. PMID:29879120

Two Distinct Repressive Mechanisms for Histone 3 Lysine 4 Methylation through Promoting 3′-End Antisense Transcription

PubMed Central

Margaritis, Thanasis; Oreal, Vincent; Brabers, Nathalie; Maestroni, Laetitia; Vitaliano-Prunier, Adeline; Benschop, Joris J.; van Hooff, Sander; van Leenen, Dik

2012-01-01

Histone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3′-end, indicating that repression is coupled to 3′-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3′-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3′-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3′-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3′-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3′-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms. PMID:23028359

The B-cell identity factor Pax5 regulates distinct transcriptional programmes in early and late B lymphopoiesis

PubMed Central

Revilla-i-Domingo, Roger; Bilic, Ivan; Vilagos, Bojan; Tagoh, Hiromi; Ebert, Anja; Tamir, Ido M; Smeenk, Leonie; Trupke, Johanna; Sommer, Andreas; Jaritz, Markus; Busslinger, Meinrad

2012-01-01

Pax5 controls the identity and development of B cells by repressing lineage-inappropriate genes and activating B-cell-specific genes. Here, we used genome-wide approaches to identify Pax5 target genes in pro-B and mature B cells. In these cell types, Pax5 bound to 40% of the cis-regulatory elements defined by mapping DNase I hypersensitive (DHS) sites, transcription start sites and histone modifications. Although Pax5 bound to 8000 target genes, it regulated only 4% of them in pro-B and mature B cells by inducing enhancers at activated genes and eliminating DHS sites at repressed genes. Pax5-regulated genes in pro-B cells account for 23% of all expression changes occurring between common lymphoid progenitors and committed pro-B cells, which identifies Pax5 as an important regulator of this developmental transition. Regulated Pax5 target genes minimally overlap in pro-B and mature B cells, which reflects massive expression changes between these cell types. Hence, Pax5 controls B-cell identity and function by regulating distinct target genes in early and late B lymphopoiesis. PMID:22669466

Argonaute2 and LaminB modulate gene expression by controlling chromatin topology

PubMed Central

Nazer, Ezequiel; Dale, Ryan K.; Chinen, Madoka; Radmanesh, Behram

2018-01-01

Drosophila Argonaute2 (AGO2) has been shown to regulate expression of certain loci in an RNA interference (RNAi)-independent manner, but its genome-wide function on chromatin remains unknown. Here, we identified the nuclear scaffolding protein LaminB as a novel interactor of AGO2. When either AGO2 or LaminB are depleted in Kc cells, similar transcription changes are observed genome-wide. In particular, changes in expression occur mainly in active or potentially active chromatin, both inside and outside LaminB-associated domains (LADs). Furthermore, we identified a somatic target of AGO2 transcriptional repression, no hitter (nht), which is immersed in a LAD located within a repressive topologically-associated domain (TAD). Null mutation but not catalytic inactivation of AGO2 leads to ectopic expression of nht and downstream spermatogenesis genes. Depletion of either AGO2 or LaminB results in reduced looping interactions within the nht TAD as well as ectopic inter-TAD interactions, as detected by 4C-seq analysis. Overall, our findings reveal coordination of AGO2 and LaminB function to dictate genome architecture and thereby regulate gene expression. PMID:29529026

Transcription profiling of the chilling requirement for bud break in apples: a putative role for FLC-like genes.

PubMed

Porto, Diogo Denardi; Bruneau, Maryline; Perini, Pâmela; Anzanello, Rafael; Renou, Jean-Pierre; dos Santos, Henrique Pessoa; Fialho, Flávio Bello; Revers, Luís Fernando

2015-05-01

Apple production depends on the fulfilment of a chilling requirement for bud dormancy release. Insufficient winter chilling results in irregular and suboptimal bud break in the spring, with negative impacts on apple yield. Trees from apple cultivars with contrasting chilling requirements for bud break were used to investigate the expression of the entire set of apple genes in response to chilling accumulation in the field and controlled conditions. Total RNA was analysed on the AryANE v.1.0 oligonucleotide microarray chip representing 57,000 apple genes. The data were tested for functional enrichment, and differential expression was confirmed by real-time PCR. The largest number of differentially expressed genes was found in samples treated with cold temperatures. Cold exposure mostly repressed expression of transcripts related to photosynthesis, and long-term cold exposure repressed flavonoid biosynthesis genes. Among the differentially expressed selected candidates, we identified genes whose annotations were related to the circadian clock, hormonal signalling, regulation of growth, and flower development. Two genes, annotated as FLOWERING LOCUS C-like and MADS AFFECTING FLOWERING, showed strong differential expression in several comparisons. One of these two genes was upregulated in most comparisons involving dormancy release, and this gene's chromosomal position co-localized with the confidence interval of a major quantitative trait locus for the timing of bud break. These results indicate that photosynthesis and auxin transport are major regulatory nodes of apple dormancy and unveil strong candidates for the control of bud dormancy. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

WRKY6 Transcription Factor Restricts Arsenate Uptake and Transposon Activation in Arabidopsis[W

PubMed Central

Castrillo, Gabriel; Sánchez-Bermejo, Eduardo; de Lorenzo, Laura; Crevillén, Pedro; Fraile-Escanciano, Ana; TC, Mohan; Mouriz, Alfonso; Catarecha, Pablo; Sobrino-Plata, Juan; Olsson, Sanna; Leo del Puerto, Yolanda; Mateos, Isabel; Rojo, Enrique; Hernández, Luis E.; Jarillo, Jose A.; Piñeiro, Manuel; Paz-Ares, Javier; Leyva, Antonio

2013-01-01

Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing. PMID:23922208

The POU Transcription Factor Oct-1 Represses Virus-Induced Interferon A Gene Expression

PubMed Central

Mesplède, Thibault; Island, Marie-Laure; Christeff, Nicolas; Petek, Fahrettin; Doly, Janine; Navarro, Sébastien

2005-01-01

Alpha interferon (IFN-α) and IFN-β are able to interfere with viral infection. They exert a vast array of biologic functions, including growth arrest, cell differentiation, and immune system regulation. This regulation extends from innate immunity to cellular and humoral adaptive immune responses. A strict control of expression is needed to prevent detrimental effects of unregulated IFN. Multiple IFN-A subtypes are coordinately induced in human and mouse cells infected by virus and exhibit differences in expression of their individual mRNAs. We demonstrated that the weakly expressed IFN-A11 gene is negatively regulated after viral infection, due to a distal negative regulatory element, binding homeoprotein pituitary homeobox 1 (Pitx1). Here we show that the POU protein Oct-1 binds in vitro and in vivo to the IFN-A11 promoter and represses IFN-A expression upon interferon regulatory factor overexpression. Furthermore, we show that Oct-1-deficient MEFs exhibit increased in vivo IFN-A gene expression and increased antiviral activity. Finally, the IFN-A expression pattern is modified in Oct-1-deficient MEFs. The broad representation of effective and potent octamer-like sequences within IFN-A promoters suggests an important role for Oct-1 in IFN-A regulation. PMID:16166650

Effect of postharvest temperature and ethylene on carotenoid accumulation in the Flavedo and juice sacs of Satsuma Mandarin ( Citrus unshiu Marc.) fruit.

PubMed

Matsumoto, Hikaru; Ikoma, Yoshinori; Kato, Masaya; Nakajima, Naoko; Hasegawa, Yoshinori

2009-06-10

The effect of postharvest temperature (5, 20, and 30 degrees C) and ethylene at different temperatures (20 and 5 degrees C) on carotenoid content and composition and on the expression of the carotenoid biosynthesis-related genes was investigated in the flavedo and juice sacs of Satsuma mandarin ( Citrus unshiu Marc.) fruit. Under an ethylene-free atmosphere, storage at 20 degrees C rapidly increased the carotenoid content in flavedo and maintained the content in juice sacs. In contrast, storage at 5 and 30 degrees C gradually decreased the content in juice sacs but slowly increased that in flavedo. Under an ethylene atmosphere, storage at 20 degrees C enhanced the carotenoid accumulation in flavedo more dramatically than found under an ethylene-free atmosphere with distinct changes in the carotenoid composition but did not noticeably change the content and composition in juice sacs. In contrast, storage at 5 degrees C under an ethylene atmosphere repressed carotenoid accumulation with changes in the carotenoid composition in flavedo but did not clearly change the carotenoid content in juice sacs. Under an ethylene-free atmosphere, differences in the gene expression profile among the temperatures were observed but were not well-correlated with those in the carotenoid content in flavedo and juice sacs. Under an ethylene atmosphere, in flavedo, the gene expression of phytoene synthase (PSY) and phytoene desaturase (PDS) was slightly higher at 20 degrees C but lower at 5 degrees C than under an ethylene-free atmosphere. At 20 degrees C, the gene expression of several carotenoid biosynthetic enzymes promoted by ethylene seemed to be responsible for the enhanced accumulation of carotenoid in flavedo. In contrast, at 5 degrees C, the repressed gene expression of PSY and PDS by ethylene seemed to be primarily responsible for the repressed accumulation of carotenoid in flavedo. In juice sacs, the small response of the gene expression to ethylene seemed to be responsible for small changes in carotenoid accumulation under an ethylene atmosphere.

Feedback regulation of an Agrobacterium catalase gene katA involved in Agrobacterium-plant interaction.

PubMed

Xu, X Q; Li, L P; Pan, S Q

2001-11-01

Catalases are known to detoxify H2O2, a major component of oxidative stress imposed on a cell. An Agrobacterium tumefaciens catalase encoded by a chromosomal gene katA has been implicated as an important virulence factor as it is involved in detoxification of H2O2 released during Agrobacterium-plant interaction. In this paper, we report a feedback regulation pathway that controls the expression of katA in A. tumefaciens cells. We observed that katA could be induced by plant tissue sections and by acidic pH on a minimal medium, which resembles the plant environment that the bacteria encounter during the course of infection. This represents a new regulatory factor for catalase induction in bacteria. More importantly, a feedback regulation was observed when the katA-gfp expression was studied in different genetic backgrounds. We found that introduction of a wild-type katA gene encoding a functional catalase into A. tumefaciens cells could repress the katA-gfp expression over 60-fold. The katA gene could be induced by H2O2 and the encoded catalase could detoxify H2O2. In addition, the katA-gfp expression of one bacterial cell could be repressed by other surrounding catalase-proficient bacterial cells. Furthermore, mutation at katA caused a 10-fold increase of the intracellular H2O2 concentration in the bacteria grown on an acidic pH medium. These results suggest that the endogenous H2O2 generated during A. tumefaciens cell growth could serve as the intracellular and intercellular inducer for the katA gene expression and that the acidic pH could pose an oxidative stress on the bacteria. Surprisingly, one mutated KatA protein, exhibiting no significant catalase activity as a result of the alteration of two important residues at the putative active site, could partially repress the katA-gfp expression. The feedback regulation of the katA gene by both catalase activity and KatA protein could presumably maintain an appropriated level of catalase activity and H2O2 inside A. tumefaciens cells.

Genome-wide profiles of CtBP link metabolism with genome stability and epithelial reprogramming in breast cancer.

PubMed

Di, Li-Jun; Byun, Jung S; Wong, Madeline M; Wakano, Clay; Taylor, Tara; Bilke, Sven; Baek, Songjoon; Hunter, Kent; Yang, Howard; Lee, Maxwell; Zvosec, Cecilia; Khramtsova, Galina; Cheng, Fan; Perou, Charles M; Miller, C Ryan; Raab, Rachel; Olopade, Olufunmilayo I; Gardner, Kevin

2013-01-01

The C-terminal binding protein (CtBP) is a NADH-dependent transcriptional repressor that links carbohydrate metabolism to epigenetic regulation by recruiting diverse histone-modifying complexes to chromatin. Here global profiling of CtBP in breast cancer cells reveals that it drives epithelial-to-mesenchymal transition, stem cell pathways and genome instability. CtBP expression induces mesenchymal and stem cell-like features, whereas CtBP depletion or caloric restriction reverses gene repression and increases DNA repair. Multiple members of the CtBP-targeted gene network are selectively downregulated in aggressive breast cancer subtypes. Differential expression of CtBP-targeted genes predicts poor clinical outcome in breast cancer patients, and elevated levels of CtBP in patient tumours predict shorter median survival. Finally, both CtBP promoter targeting and gene repression can be reversed by small molecule inhibition. These findings define broad roles for CtBP in breast cancer biology and suggest novel chromatin-based strategies for pharmacologic and metabolic intervention in cancer.

Genetic Control of Amadori Product Degradation in Bacillus subtilis via Regulation of frlBONMD Expression by FrlR▿†

PubMed Central

Deppe, Veronika Maria; Klatte, Stephanie; Bongaerts, Johannes; Maurer, Karl-Heinz; O'Connell, Timothy; Meinhardt, Friedhelm

2011-01-01

Bacillus subtilis is capable of degrading fructosamines. The phosphorylation and the cleavage of the resulting fructosamine 6-phosphates is catalyzed by the frlD and frlB gene products, respectively. This study addresses the physiological importance of the frlBONMD genes (formerly yurPONML), revealing the necessity of their expression for growth on fructosamines and focusing on the complex regulation of the corresponding transcription unit. In addition to the known regulation by the global transcriptional regulator CodY, the frl genes are repressed by the convergently transcribed FrlR (formerly YurK). The latter causes repression during growth on substrates other than fructosamines. Additionally, we identified in the first intergenic region of the operon an FrlR binding site which is centrally located within a 38-bp perfect palindromic sequence. There is genetic evidence that this sequence, in combination with FrlR, contributes to the remarkable decrease in the transcription downstream of the first gene of the frl operon. PMID:21398478

Repression by PRDM13 is critical for generating precision in neuronal identity

PubMed Central

Kollipara, Rahul K; Ma, Zhenzhong; Borromeo, Mark D; Chang, Joshua C

2017-01-01

The mechanisms that activate some genes while silencing others are critical to ensure precision in lineage specification as multipotent progenitors become restricted in cell fate. During neurodevelopment, these mechanisms are required to generate the diversity of neuronal subtypes found in the nervous system. Here we report interactions between basic helix-loop-helix (bHLH) transcriptional activators and the transcriptional repressor PRDM13 that are critical for specifying dorsal spinal cord neurons. PRDM13 inhibits gene expression programs for excitatory neuronal lineages in the dorsal neural tube. Strikingly, PRDM13 also ensures a battery of ventral neural tube specification genes such as Olig1, Olig2 and Prdm12 are excluded dorsally. PRDM13 does this via recruitment to chromatin by multiple neural bHLH factors to restrict gene expression in specific neuronal lineages. Together these findings highlight the function of PRDM13 in repressing the activity of bHLH transcriptional activators that together are required to achieve precise neuronal specification during mouse development. PMID:28850031

Evolutionary origin and functional divergence of totipotent cell homeobox genes in eutherian mammals.

PubMed

Maeso, Ignacio; Dunwell, Thomas L; Wyatt, Chris D R; Marlétaz, Ferdinand; Vető, Borbála; Bernal, Juan A; Quah, Shan; Irimia, Manuel; Holland, Peter W H

2016-06-13

A central goal of evolutionary biology is to link genomic change to phenotypic evolution. The origin of new transcription factors is a special case of genomic evolution since it brings opportunities for novel regulatory interactions and potentially the emergence of new biological properties. We demonstrate that a group of four homeobox gene families (Argfx, Leutx, Dprx, Tprx), plus a gene newly described here (Pargfx), arose by tandem gene duplication from the retinal-expressed Crx gene, followed by asymmetric sequence evolution. We show these genes arose as part of repeated gene gain and loss events on a dynamic chromosomal region in the stem lineage of placental mammals, on the forerunner of human chromosome 19. The human orthologues of these genes are expressed specifically in early embryo totipotent cells, peaking from 8-cell to morula, prior to cell fate restrictions; cow orthologues have similar expression. To examine biological roles, we used ectopic gene expression in cultured human cells followed by high-throughput RNA-seq and uncovered extensive transcriptional remodelling driven by three of the genes. Comparison to transcriptional profiles of early human embryos suggest roles in activating and repressing a set of developmentally-important genes that spike at 8-cell to morula, rather than a general role in genome activation. We conclude that a dynamic chromosome region spawned a set of evolutionarily new homeobox genes, the ETCHbox genes, specifically in eutherian mammals. After these genes diverged from the parental Crx gene, we argue they were recruited for roles in the preimplantation embryo including activation of genes at the 8-cell stage and repression after morula. We propose these new homeobox gene roles permitted fine-tuning of cell fate decisions necessary for specification and function of embryonic and extra-embryonic tissues utilised in mammalian development and pregnancy.

Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster.

PubMed

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-07-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed D: rosophila, R: bf, E: 2F A: nd M: yb/ M: ulti-vulva class B: (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. Copyright © 2016 by the Genetics Society of America.

Roles for the Histone Modifying and Exchange Complex NuA4 in Cell Cycle Progression in Drosophila melanogaster

PubMed Central

Flegel, Kerry; Grushko, Olga; Bolin, Kelsey; Griggs, Ellen; Buttitta, Laura

2016-01-01

Robust and synchronous repression of E2F-dependent gene expression is critical to the proper timing of cell cycle exit when cells transition to a postmitotic state. Previously NuA4 was suggested to act as a barrier to proliferation in Drosophila by repressing E2F-dependent gene expression. Here we show that NuA4 activity is required for proper cell cycle exit and the repression of cell cycle genes during the transition to a postmitotic state in vivo. However, the delay of cell cycle exit caused by compromising NuA4 is not due to additional proliferation or effects on E2F activity. Instead NuA4 inhibition results in slowed cell cycle progression through late S and G2 phases due to aberrant activation of an intrinsic p53-independent DNA damage response. A reduction in NuA4 function ultimately produces a paradoxical cell cycle gene expression program, where certain cell cycle genes become derepressed in cells that are delayed during the G2 phase of the final cell cycle. Bypassing the G2 delay when NuA4 is inhibited leads to abnormal mitoses and results in severe tissue defects. NuA4 physically and genetically interacts with components of the E2F complex termed Drosophila, Rbf, E2F and Myb/Multi-vulva class B (DREAM/MMB), and modulates a DREAM/MMB-dependent ectopic neuron phenotype in the posterior wing margin. However, this effect is also likely due to the cell cycle delay, as simply reducing Cdk1 is sufficient to generate a similar phenotype. Our work reveals that the major requirement for NuA4 in the cell cycle in vivo is to suppress an endogenous DNA damage response, which is required to coordinate proper S and G2 cell cycle progression with differentiation and cell cycle gene expression. PMID:27184390

Left-right axis asymmetry determining human Cryptic gene is transcriptionally repressed by Snail.

PubMed

Gupta, Kartik; Pilli, Vijaya Satish Sekhar; Aradhyam, Gopala Krishna

2016-10-28

Establishment of the left-right axis is important for positioning organs asymmetrically in the developing vertebrate-embryo. A number of factors like maternally deposited molecules have emerged essential in initiating the specification of the axis; the downstream events, however, are regulated by signal-transduction and gene-expression changes identifying which remains a crucial challenge. The EGF-CFC family member Cryptic, that functions as a co-receptor for some TGF-beta ligands, is developmentally expressed in higher mammals and mutations in the gene cause loss or change in left-right axis asymmetry. Despite the strong phenotype, no transcriptional-regulator of this gene is known till date. Using promoter-analyses tools, we found strong evidence that the developmentally essential transcription factor Snail binds to the human Cryptic-promoter. We cloned the promoter-region of human Cryptic in a reporter gene and observed decreased Cryptic-promoter activation upon increasing Snail expression. Further, the expression of Cryptic is down-regulated upon exogenous Snail expression, validating the reporter assays and the previously identified role of Snail as a transcriptional repressor. Finally, we demonstrate using gel-shift assay that Snail in nuclear extract of PANC1 cells interacts with the promoter-construct bearing putative Snail binding sites and confirm this finding using chromatin immunoprecipitation assay. Snail represses the expression of human Cryptic and therefore, might affect the signaling via Nodal that has previously been demonstrated to specify the left-right axis using the EGF-CFC co-receptors.

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor.

PubMed

Ares, Miguel A; Fernández-Vázquez, José L; Pacheco, Sabino; Martínez-Santos, Verónica I; Jarillo-Quijada, Ma Dolores; Torres, Javier; Alcántar-Curiel, María D; González-Y-Merchand, Jorge A; De la Cruz, Miguel A

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae.

Additional regulatory activities of MrkH for the transcriptional expression of the Klebsiella pneumoniae mrk genes: Antagonist of H-NS and repressor

PubMed Central

Ares, Miguel A.; Fernández-Vázquez, José L.; Pacheco, Sabino; Martínez-Santos, Verónica I.; Jarillo-Quijada, Ma. Dolores; Torres, Javier; Alcántar-Curiel, María D.; González-y-Merchand, Jorge A.; De la Cruz, Miguel A.

2017-01-01

Klebsiella pneumoniae is a common opportunistic pathogen causing nosocomial infections. One of the main virulence determinants of K. pneumoniae is the type 3 pilus (T3P). T3P helps the bacterial interaction to both abiotic and biotic surfaces and it is crucial for the biofilm formation. T3P is genetically organized in three transcriptional units: the mrkABCDF polycistronic operon, the mrkHI bicistronic operon and the mrkJ gene. MrkH is a regulatory protein encoded in the mrkHI operon, which positively regulates the mrkA pilin gene and its own expression. In contrast, the H-NS nucleoid protein represses the transcriptional expression of T3P. Here we reported that MrkH and H-NS positively and negatively regulate mrkJ expression, respectively, by binding to the promoter of mrkJ. MrkH protein recognized a sequence located at position -63.5 relative to the transcriptional start site of mrkJ gene. Interestingly, our results show that, in addition to its known function as classic transcriptional activator, MrkH also positively controls the expression of mrk genes by acting as an anti-repressor of H-NS; moreover, our results support the notion that high levels of MrkH repress T3P expression. Our data provide new insights about the complex regulatory role of the MrkH protein on the transcriptional control of T3P in K. pneumoniae. PMID:28278272

Shoot to root communication is necessary to control the expression of iron-acquisition genes in Strategy I plants.

PubMed

García, María J; Romera, Francisco J; Stacey, Minviluz G; Stacey, Gary; Villar, Eduardo; Alcántara, Esteban; Pérez-Vicente, Rafael

2013-01-01

Previous research showed that auxin, ethylene, and nitric oxide (NO) can activate the expression of iron (Fe)-acquisition genes in the roots of Strategy I plants grown with low levels of Fe, but not in plants grown with high levels of Fe. However, it is still an open question as to how Fe acts as an inhibitor and which pool of Fe (e.g., root, phloem, etc.) in the plant acts as the key regulator for gene expression control. To further clarify this, we studied the effect of the foliar application of Fe on the expression of Fe-acquisition genes in several Strategy I plants, including wild-type cultivars of Arabidopsis [Arabidopsis thaliana (L.) Heynh], pea [Pisum sativum L.], tomato [Solanum lycopersicon Mill.], and cucumber [Cucumis sativus L.], as well as mutants showing constitutive expression of Fe-acquisition genes when grown under Fe-sufficient conditions [Arabidopsis opt3-2 and frd3-3, pea dgl and brz, and tomato chln (chloronerva)]. The results showed that the foliar application of Fe blocked the expression of Fe-acquisition genes in the wild-type cultivars and in the frd3-3, brz, and chln mutants, but not in the opt3-2 and dgl mutants, probably affected in the transport of a Fe-related repressive signal in the phloem. Moreover, the addition of either ACC (ethylene precursor) or GSNO (NO donor) to Fe-deficient plants up-regulated the expression of Fe-acquisition genes, but this effect did not occur in Fe-deficient plants sprayed with foliar Fe, again suggesting the existence of a Fe-related repressive signal moving from leaves to roots.

Genome-wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma.

PubMed

Charlet, Jessica; Tomari, Ayumi; Dallosso, Anthony R; Szemes, Marianna; Kaselova, Martina; Curry, Thomas J; Almutairi, Bader; Etchevers, Heather C; McConville, Carmel; Malik, Karim T A; Brown, Keith W

2017-04-01

Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome-wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome-wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down-regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest-expressing tumors had reduced relapse-free survival. Our functional studies showed that knock-down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc.

Genome‐wide DNA methylation analysis identifies MEGF10 as a novel epigenetically repressed candidate tumor suppressor gene in neuroblastoma

PubMed Central

Charlet, Jessica; Tomari, Ayumi; Dallosso, Anthony R.; Szemes, Marianna; Kaselova, Martina; Curry, Thomas J.; Almutairi, Bader; Etchevers, Heather C.; McConville, Carmel; Malik, Karim T. A.

2016-01-01

Neuroblastoma is a childhood cancer in which many children still have poor outcomes, emphasising the need to better understand its pathogenesis. Despite recent genome‐wide mutation analyses, many primary neuroblastomas do not contain recognizable driver mutations, implicating alternate molecular pathologies such as epigenetic alterations. To discover genes that become epigenetically deregulated during neuroblastoma tumorigenesis, we took the novel approach of comparing neuroblastomas to neural crest precursor cells, using genome‐wide DNA methylation analysis. We identified 93 genes that were significantly differentially methylated of which 26 (28%) were hypermethylated and 67 (72%) were hypomethylated. Concentrating on hypermethylated genes to identify candidate tumor suppressor loci, we found the cell engulfment and adhesion factor gene MEGF10 to be epigenetically repressed by DNA hypermethylation or by H3K27/K9 methylation in neuroblastoma cell lines. MEGF10 showed significantly down‐regulated expression in neuroblastoma tumor samples; furthermore patients with the lowest‐expressing tumors had reduced relapse‐free survival. Our functional studies showed that knock‐down of MEGF10 expression in neuroblastoma cell lines promoted cell growth, consistent with MEGF10 acting as a clinically relevant, epigenetically deregulated neuroblastoma tumor suppressor gene. © 2016 The Authors. Molecular Carcinogenesis Published by Wiley Periodicals, Inc. PMID:27862318

Gene expression profiles of Vibrio parahaemolyticus in the early stationary phase.

PubMed

Meng, L; Alter, T; Aho, T; Huehn, S

2015-09-01

Vibrio (V.) parahaemolyticus is an aquatic bacterium capable of causing foodborne gastroenteritis. In the environment or the food chain, V. parahaemolyticus cells are usually forced into the stationary phase, the common phase for bacterial survival in the environment. So far, little is known about whole genomic expression of V. parahaemolyticus in the early stationary phase compared with the exponential growth phase. We performed whole transcriptomic profiling of V. parahaemolyticus cells in both phases (exponential and early stationary phase). Our data showed in total that 172 genes were induced in early stationary phase, while 61 genes were repressed in early stationary phase compared with the exponential phase. Three functional categories showed stable gene expression in the early stationary phase. Eleven functional categories showed that up-regulation of genes was dominant over down-regulation in the early stationary phase. Although genes related to endogenous metabolism were repressed in the early stationary phase, massive regulation of gene expression occurred in the early stationary phase, indicating the expressed gene set of V. parahaemolyticus in the early stationary phase impacts environmental survival. Vibrio (V.) parahaemolyticus is one of the main bacterial causes of foodborne intestinal infections. This bacterium usually is forced into stationary phase in the environment, which includes, e.g. seafood. When bacteria are in stationary phase, physiological changes can lead to a resistance to many stresses, including physical and chemical challenges during food processing. To the best of our knowledge, highlighting the whole genome expression changes in the early stationary phase compared with exponential phase, as well as the investigation of physiological changes of V. parahaemolyticus such as the survival mechanism in the stationary phase has been the very first study in this field. © 2015 The Society for Applied Microbiology.

The Human Papillomavirus E6 Oncogene Represses a Cell Adhesion Pathway and Disrupts Focal Adhesion through Degradation of TAp63β upon Transformation

PubMed Central

Ben Khalifa, Youcef; Teissier, Sébastien; Tan, Meng-Kwang Marcus; Phan, Quang Tien; Daynac, Mathieu; Wong, Wei Qi; Thierry, Françoise

2011-01-01

Cervical carcinomas result from cellular transformation by the human papillomavirus (HPV) E6 and E7 oncogenes which are constitutively expressed in cancer cells. The E6 oncogene degrades p53 thereby modulating a large set of p53 target genes as shown previously in the cervical carcinoma cell line HeLa. Here we show that the TAp63β isoform of the p63 transcription factor is also a target of E6. The p63 gene plays an essential role in skin homeostasis and is expressed as at least six isoforms. One of these isoforms, ΔNp63α, has been found overexpressed in squamous cell carcinomas and is shown here to be constitutively expressed in Caski cells associated with HPV16. We therefore explored the role of p63 in these cells by performing microarray analyses after repression of endogenous E6/E7 expression. Upon repression of the oncogenes, a large set of p53 target genes was found activated together with many p63 target genes related to cell adhesion. However, through siRNA silencing and ectopic expression of various p63 isoforms we demonstrated that TAp63β is involved in activation of this cell adhesion pathway instead of the constitutively expressed ΔNp63α and β. Furthermore, we showed in cotransfection experiments, combined with E6AP siRNA silencing, that E6 induces an accelerated degradation of TAp63β although not through the E6AP ubiquitin ligase used for degradation of p53. Repression of E6 transcription also induces stabilization of endogenous TAp63β in cervical carcinoma cells that lead to an increased concentration of focal adhesions at the cell surface. Consequently, TAp63β is the only p63 isoform suppressed by E6 in cervical carcinoma as demonstrated previously for p53. Down-modulation of focal adhesions through disruption of TAp63β therefore appears as a novel E6-dependent pathway in transformation. These findings identify a major physiological role for TAp63β in anchorage independent growth that might represent a new critical pathway in human carcinogenesis. PMID:21980285

Progesterone and the Repression of Myometrial Inflammation: The Roles of MKP-1 and the AP-1 System

PubMed Central

Lei, K.; Georgiou, E. X.; Chen, L.; Yulia, A.; Sooranna, S. R.; Brosens, J. J.; Bennett, P. R.

2015-01-01

Progesterone (P4) maintains uterine quiescence during pregnancy and its functional withdrawal is associated with increased prostaglandin synthesis and the onset of labor. In primary human myometrial cells, the glucocorticoid receptor (GR) rather than the P4 receptor mediates P4 antagonism of IL-1β-induced cyclooxygenase-2 (COX-2) expression, the rate-limiting enzyme in prostaglandin synthesis. We now report that P4 also acts via GR to induce MAPK phosphatase (MKP)-1 and knockdown of MKP-1 impairs the ability of P4 to repress IL-1β-dependent COX-2 induction. Microarray analysis revealed that P4 repressed preferentially activator protein-1-responsive genes in response to IL-1β. Consistent with these observations, we found that the ability of P4 to reduce c-Jun activation was lost upon GR as well as MKP-1 knockdown. Interestingly, c-Jun levels in human myometrial cells declined upon GR and MKP-1 knockdown, which suggests the presence of an activator protein-1 feedback loop. This is supported by our observation that c-Jun levels declined after an initial rise in primary myometrial cells treated with phorbol 12-myrisatate 13-acetate, a potent activator of c-Jun N-terminal kinase. Finally, we show that MKP-1 is an intermediate in P4-mediated repression of some but not all IL-1β-responsive genes. For example, P4 repression of IL11 and IRAK3 was maintained upon MKP-1 knockdown. Taken together, the data show that P4 acts via GR to drive MKP-1 expression, which in turn inhibits IL-1β-dependent c-Jun activation and COX-2 expression. PMID:26280733

CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum.

PubMed

Bryant, Jessica M; Regnault, Clément; Scheidig-Benatar, Christine; Baumgarten, Sebastian; Guizetti, Julien; Scherf, Artur

2017-07-11

Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes-the intron-in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression. Copyright © 2017 Bryant et al.

Gene array analysis reveals a common Runx transcriptional program controlling cell adhesion and survival

PubMed Central

Wotton, Sandy; Terry, Anne; Kilbey, Anna; Jenkins, Alma; Herzyk, Pawel; Cameron, Ewan; Neil, James C.

2008-01-01

The Runx genes play divergent roles in development and cancer, where they can act either as oncogenes or tumour suppressors. We compared the effects of ectopic Runx expression in established fibroblasts, where all three genes produce an indistinguishable phenotype entailing epithelioid morphology and increased cell survival under stress conditions. Gene array analysis revealed a strongly overlapping transcriptional signature, with no examples of opposing regulation of the same target gene. A common set of 50 highly regulated genes was identified after further filtering on regulation by inducible RUNX1-ER. This set revealed a strong bias towards genes with annotated roles in cancer and development, and a preponderance of targets encoding extracellular or surface proteins, reflecting the marked effects of Runx on cell adhesion. Furthermore, in silico prediction of resistance to glucocorticoid growth inhibition was confirmed in fibroblasts and lymphoid cells expressing ectopic Runx. The effects of fibroblast expression of common RUNX1 fusion oncoproteins (RUNX1-ETO, TEL-RUNX1, CBFB-MYH11) were also tested. While two direct Runx activation target genes were repressed (Ncam1, Rgc32), the fusion proteins appeared to disrupt regulation of down-regulated targets (Cebpd, Id2, Rgs2) rather than impose constitutive repression. These results elucidate the oncogenic potential of the Runx family and reveal novel targets for therapeutic inhibition. PMID:18560354

Interplay between EZH2 and G9a Regulates CXCL10 Gene Repression in Idiopathic Pulmonary Fibrosis.

PubMed

Coward, William R; Brand, Oliver J; Pasini, Alice; Jenkins, Gisli; Knox, Alan J; Pang, Linhua

2018-04-01

Selective repression of the antifibrotic gene CXCL10 contributes to tissue remodeling in idiopathic pulmonary fibrosis (IPF). We have previously reported that histone deacetylation and histone H3 lysine 9 (H3K9) methylation are involved in CXCL10 repression. In this study, we explored the role of H3K27 methylation and the interplay between the two histone lysine methyltransferases enhancer of zest homolog 2 (EZH2) and G9a in CXCL10 repression in IPF. By applying chromatin immunoprecipitation, Re-ChIP, and proximity ligation assays, we demonstrated that, like G9a-mediated H3K9 methylation, EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) was significantly enriched at the CXCL10 promoter in fibroblasts from IPF lungs (F-IPF) compared with fibroblasts from nonfibrotic lungs, and we also found that EZH2 and G9a physically interacted with each other. EZH2 knockdown reduced not only EZH2 and H3K27me3 but also G9a and H3K9me3, and G9a knockdown reduced not only G9 and H3K9me3 but also EZH2 and H3K27me3. Depletion and inhibition of EZH2 and G9a also reversed histone deacetylation and restored CXCL10 expression in F-IPF. Furthermore, treatment of fibroblasts from nonfibrotic lungs with the profibrotic cytokine transforming growth factor-β1 increased EZH2, G9a, H3K27me3, H3K9me3, and histone deacetylation at the CXCL10 promoter, similar to that observed in F-IPF, which was correlated with CXCL10 repression and was prevented by EZH2 and G9a knockdown. These findings suggest that a novel and functionally interdependent interplay between EZH2 and G9a regulates histone methylation-mediated epigenetic repression of the antifibrotic CXCL10 gene in IPF. This interdependent interplay may prove to be a target for epigenetic intervention to restore the expression of CXCL10 and other antifibrotic genes in IPF.

CCAAT/enhancer-binding protein β regulates the repression of type II collagen expression during the differentiation from proliferative to hypertrophic chondrocytes.

PubMed

Ushijima, Takahiro; Okazaki, Ken; Tsushima, Hidetoshi; Iwamoto, Yukihide

2014-01-31

CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that promotes hypertrophic differentiation by stimulating type X collagen and matrix metalloproteinase 13 during chondrocyte differentiation. However, the effect of C/EBPβ on proliferative chondrocytes is unclear. Here, we investigated whether C/EBPβ represses type II collagen (COL2A1) expression and is involved in the regulation of sex-determining region Y-type high mobility group box 9 (SOX9), a crucial factor for transactivation of Col2a1. Endogenous expression of C/EBPβ in the embryonic growth plate and differentiated ATDC5 cells were opposite to those of COL2A1 and SOX9. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked repression of Col2a1. The expression of Sox9 mRNA and nuclear protein was also repressed, resulting in decreased binding of SOX9 to the Col2a1 enhancer as shown by a ChIP assay. Knockdown of C/EBPβ by lentivirus expressing shRNA caused significant stimulation of these genes in ATDC5 cells. Reporter assays demonstrated that C/EBPβ repressed transcriptional activity of Col2a1. Deletion and mutation analysis showed that the C/EBPβ core responsive element was located between +2144 and +2152 bp within the Col2a1 enhancer. EMSA and ChIP assays also revealed that C/EBPβ directly bound to this region. Ex vivo organ cultures of mouse limbs transfected with C/EBPβ showed that the expression of COL2A1 and SOX9 was reduced upon ectopic C/EBPβ expression. Together, these results indicated that C/EBPβ represses the transcriptional activity of Col2a1 both directly and indirectly through modulation of Sox9 expression. This consequently promotes the phenotypic conversion from proliferative to hypertrophic chondrocytes during chondrocyte differentiation.

From genes to milk: genomic organization and epigenetic regulation of the mammary transcriptome.

PubMed

Lemay, Danielle G; Pollard, Katherine S; Martin, William F; Freeman Zadrowski, Courtneay; Hernandez, Joseph; Korf, Ian; German, J Bruce; Rijnkels, Monique

2013-01-01

Even in genomes lacking operons, a gene's position in the genome influences its potential for expression. The mechanisms by which adjacent genes are co-expressed are still not completely understood. Using lactation and the mammary gland as a model system, we explore the hypothesis that chromatin state contributes to the co-regulation of gene neighborhoods. The mammary gland represents a unique evolutionary model, due to its recent appearance, in the context of vertebrate genomes. An understanding of how the mammary gland is regulated to produce milk is also of biomedical and agricultural importance for human lactation and dairying. Here, we integrate epigenomic and transcriptomic data to develop a comprehensive regulatory model. Neighborhoods of mammary-expressed genes were determined using expression data derived from pregnant and lactating mice and a neighborhood scoring tool, G-NEST. Regions of open and closed chromatin were identified by ChIP-Seq of histone modifications H3K36me3, H3K4me2, and H3K27me3 in the mouse mammary gland and liver tissue during lactation. We found that neighborhoods of genes in regions of uniquely active chromatin in the lactating mammary gland, compared with liver tissue, were extremely rare. Rather, genes in most neighborhoods were suppressed during lactation as reflected in their expression levels and their location in regions of silenced chromatin. Chromatin silencing was largely shared between the liver and mammary gland during lactation, and what distinguished the mammary gland was mainly a small tissue-specific repertoire of isolated, expressed genes. These findings suggest that an advantage of the neighborhood organization is in the collective repression of groups of genes via a shared mechanism of chromatin repression. Genes essential to the mammary gland's uniqueness are isolated from neighbors, and likely have less tolerance for variation in expression, properties they share with genes responsible for an organism's survival.

Tightly regulated, high-level expression from controlled copy number vectors based on the replicon of temperate phage N15.

PubMed

Mardanov, Andrey V; Strakhova, Taisia S; Smagin, Vladimir A; Ravin, Nikolai V

2007-06-15

A new Escherichia coli host/vector system has been developed to allow a dual regulation of both the plasmid copy number and gene expression. The new pN15E vectors are low copy number plasmids based on the replicon of temperate phage N15, comprising the repA replicase gene and cB repressor gene, controlling the plasmid copy number. Regulation of pN15E copy number is achieved through arabinose-inducible expression of phage N15 antirepressor protein, AntA, whose gene was integrated into the chromosome of the host strain under control of the PBAD promoter. The host strain also carried phage N15 partition operon, sop, allowing stable inheritance of pN15E vectors in the absence of selection pressure. In the first vector, pN15E4, the same PBAD promoter controls expression of a cloned gene. The second vector, pN15E6, carries the phage T5 promoter with a double lac operator repression module thus allowing independent regulation of promoter activity and copy number. Using the lacZ gene to monitor expression in these vectors, we show that the ratio of induction/repression can be about 7600-fold for pN15E4 and more than 15,000-fold for pN15E6. The low copy number of these vectors ensures very low basal level of expression allowing cloning genes encoding toxic products that was demonstrated by the stable maintenance of a gene encoding a restriction endonuclease in pN15E4. The tight control of transcription and the potential to regulate gene activities quantitatively over wide ranges will open up new approaches in the study of gene function in vivo and controlled expression of heterologous genes.

From Genes to Milk: Genomic Organization and Epigenetic Regulation of the Mammary Transcriptome

PubMed Central

Lemay, Danielle G.; Pollard, Katherine S.; Martin, William F.; Freeman Zadrowski, Courtneay; Hernandez, Joseph; Korf, Ian; German, J. Bruce; Rijnkels, Monique

2013-01-01

Even in genomes lacking operons, a gene's position in the genome influences its potential for expression. The mechanisms by which adjacent genes are co-expressed are still not completely understood. Using lactation and the mammary gland as a model system, we explore the hypothesis that chromatin state contributes to the co-regulation of gene neighborhoods. The mammary gland represents a unique evolutionary model, due to its recent appearance, in the context of vertebrate genomes. An understanding of how the mammary gland is regulated to produce milk is also of biomedical and agricultural importance for human lactation and dairying. Here, we integrate epigenomic and transcriptomic data to develop a comprehensive regulatory model. Neighborhoods of mammary-expressed genes were determined using expression data derived from pregnant and lactating mice and a neighborhood scoring tool, G-NEST. Regions of open and closed chromatin were identified by ChIP-Seq of histone modifications H3K36me3, H3K4me2, and H3K27me3 in the mouse mammary gland and liver tissue during lactation. We found that neighborhoods of genes in regions of uniquely active chromatin in the lactating mammary gland, compared with liver tissue, were extremely rare. Rather, genes in most neighborhoods were suppressed during lactation as reflected in their expression levels and their location in regions of silenced chromatin. Chromatin silencing was largely shared between the liver and mammary gland during lactation, and what distinguished the mammary gland was mainly a small tissue-specific repertoire of isolated, expressed genes. These findings suggest that an advantage of the neighborhood organization is in the collective repression of groups of genes via a shared mechanism of chromatin repression. Genes essential to the mammary gland's uniqueness are isolated from neighbors, and likely have less tolerance for variation in expression, properties they share with genes responsible for an organism's survival. PMID:24086428

Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression.

PubMed

Scassa, María E; Guberman, Alejandra S; Ceruti, Julieta M; Cánepa, Eduardo T

2004-07-02

Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5'-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3beta or HNF3beta plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3beta is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3beta can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3beta binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.

Construction of new synthetic biology tools for the control of gene expression in the cyanobacterium Synechococcus sp. strain PCC 7002.

PubMed

Zess, Erin K; Begemann, Matthew B; Pfleger, Brian F

2016-02-01

Predictive control of gene expression is an essential tool for developing synthetic biological systems. The current toolbox for controlling gene expression in cyanobacteria is a barrier to more in-depth genetic analysis and manipulation. Towards relieving this bottleneck, this work describes the use of synthetic biology to construct an anhydrotetracycline-based induction system and adapt a trans-acting small RNA (sRNA) system for use in the cyanobacterium Synechococcus sp. strain PCC 7002. An anhydrotetracycline-inducible promoter was developed to maximize intrinsic strength and dynamic range. The resulting construct, PEZtet , exhibited tight repression and a maximum 32-fold induction upon addition of anhydrotetracycline. Additionally, a sRNA system based on the Escherichia coli IS10 RNA-IN/OUT regulator was adapted for use in Synechococcus sp. strain PCC 7002. This system exhibited 70% attenuation of target gene expression, providing a demonstration of the use of sRNAs for differential gene expression in cyanobacteria. These systems were combined to produce an inducible sRNA system, which demonstrated 59% attenuation of target gene expression. Lastly, the role of Hfq, a critical component of sRNA systems in E. coli, was investigated. Genetic studies showed that the Hfq homolog in Synechococcus sp. strain PCC 7002 did not impact repression by the engineered sRNA system. In summary, this work describes new synthetic biology tools that can be applied to physiological studies, metabolic engineering, or sRNA platforms in Synechococcus sp. strain PCC 7002. © 2015 Wiley Periodicals, Inc.

CIP, a cardiac Isl1-interacting protein, represses cardiomyocyte hypertrophy

PubMed Central

Huang, Zhan-Peng; Seok, Hee Young; Zhou, Bin; Chen, Jinghai; Chen, Jian-Fu; Tao, Yazhong; Pu, William T.; Wang, Da-Zhi

2012-01-01

Rationale Mammalian heart has minimal regenerative capacity. In response to mechanical or pathological stress, the heart undergoes cardiac remodeling. Pressure and volume overload in the heart cause increased size (hypertrophic growth) of cardiomyocytes. Whereas the regulatory pathways that activate cardiac hypertrophy have been well established, the molecular events that inhibit or repress cardiac hypertrophy are less known. Objective To identify and investigate novel regulators that modulate cardiac hypertrophy. Methods and Results Here, we report the identification, characterization and functional examination of CIP, a novel cardiac Isl1-interacting protein. CIP was identified from a bioinformatic search for novel cardiac-expressed genes in mouse embryonic hearts. CIP encodes a nuclear protein without recognizable motifs. Northern blotting, in situ hybridization and reporter gene tracing demonstrated that CIP is highly expressed in cardiomyocytes of developing and adult hearts. Yeast-two-hybrid screening identified Isl1, a LIM/homeodomain transcription factor essential for the specification of cardiac progenitor cells in the second heart field, as a co-factor of CIP. CIP directly interacted with Isl1 and we mapped the domains of these two proteins which mediate their interaction. We show that CIP represses the transcriptional activity of Isl1 in the activation of the MEF2C enhancer. The expression of CIP was dramatically reduced in hypertrophic cardiomyocytes. Most importantly, overexpression of CIP repressed agonist-induced cardiomyocyte hypertrophy. Conclusions Our studies therefore identify CIP a novel regulator of cardiac hypertrophy. PMID:22343712

Cyclo(valine-valine) inhibits Vibrio cholerae virulence gene expression.

PubMed

Vikram, Amit; Ante, Vanessa M; Bina, X Renee; Zhu, Qin; Liu, Xinyu; Bina, James E

2014-06-01

Vibrio cholerae has been shown to produce a cyclic dipeptide, cyclo(phenylalanine-proline) (cFP), that functions to repress virulence factor production. The objective of this study was to determine if heterologous cyclic dipeptides could repress V. cholerae virulence factor production. To that end, three synthetic cyclic dipeptides that differed in their side chains from cFP were assayed for virulence inhibitory activity in V. cholerae. The results revealed that cyclo(valine-valine) (cVV) inhibited virulence factor production by a ToxR-dependent process that resulted in the repression of the virulence regulator aphA. cVV-dependent repression of aphA was found to be independent of known aphA regulatory genes. The results demonstrated that V. cholerae was able to respond to exogenous cyclic dipeptides and implicated the hydrophobic amino acid side chains on both arms of the cyclo dipeptide scaffold as structural requirements for inhibitory activity. The results further suggest that cyclic dipeptides have potential as therapeutics for cholera treatment. © 2014 The Authors.

LHP1 Regulates H3K27me3 Spreading and Shapes the Three-Dimensional Conformation of the Arabidopsis Genome

PubMed Central

Ariel, Federico; Latrasse, David; Mariappan, Kiruthiga Gayathri; Kim, Soon-Kap; Crespi, Martin; Hirt, Heribert; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

2016-01-01

Precise expression patterns of genes in time and space are essential for proper development of multicellular organisms. Dynamic chromatin conformation and spatial organization of the genome constitute a major step in this regulation to modulate developmental outputs. Polycomb repressive complexes (PRCs) mediate stable or flexible gene repression in response to internal and environmental cues. In Arabidopsis thaliana, LHP1 co-localizes with H3K27me3 epigenetic marks throughout the genome and interacts with PRC1 and PRC2 members as well as with a long noncoding RNA. Here, we show that LHP1 is responsible for the spreading of H3K27me3 towards the 3’ end of the gene body. We also identified a subset of LHP1-activated genes and demonstrated that LHP1 shapes local chromatin topology in order to control transcriptional co-regulation. Our work reveals a general role of LHP1 from local to higher conformation levels of chromatin configuration to determine its accessibility to define gene expression patterns. PMID:27410265

Whole-Genome Microarray and Gene Deletion Studies Reveal Regulation of the Polyhydroxyalkanoate Production Cycle by the Stringent Response in Ralstonia eutropha H16

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brigham, CJ; Speth, DR; Rha, C

Poly(3-hydroxybutyrate) (PHB) production and mobilization in Ralstonia eutropha are well studied, but in only a few instances has PHB production been explored in relation to other cellular processes. We examined the global gene expression of wild-type R. eutropha throughout the PHB cycle: growth on fructose, PHB production using fructose following ammonium depletion, and PHB utilization in the absence of exogenous carbon after ammonium was resupplied. Our results confirm or lend support to previously reported results regarding the expression of PHB-related genes and enzymes. Additionally, genes for many different cellular processes, such as DNA replication, cell division, and translation, are selectivelymore » repressed during PHB production. In contrast, the expression levels of genes under the control of the alternative sigma factor sigma(54) increase sharply during PHB production and are repressed again during PHB utilization. Global gene regulation during PHB production is strongly reminiscent of the gene expression pattern observed during the stringent response in other species. Furthermore, a ppGpp synthase deletion mutant did not show an accumulation of PHB, and the chemical induction of the stringent response with DL-norvaline caused an increased accumulation of PHB in the presence of ammonium. These results indicate that the stringent response is required for PHB accumulation in R. eutropha, helping to elucidate a thus-far-unknown physiological basis for this process.« less

MyoR Modulates Cardiac Conduction by Repressing Gata4

PubMed Central

Harris, John P.; Bhakta, Minoti; Bezprozvannaya, Svetlana; Wang, Lin; Lubczyk, Christina; Olson, Eric N.

2014-01-01

The cardiac conduction system coordinates electrical activation through a series of interconnected structures, including the atrioventricular node (AVN), the central connection point that delays impulse propagation to optimize cardiac performance. Although recent studies have uncovered important molecular details of AVN formation, relatively little is known about the transcriptional mechanisms that regulate AV delay, the primary function of the mature AVN. We identify here MyoR as a novel transcription factor expressed in Cx30.2+ cells of the AVN. We show that MyoR specifically inhibits a Cx30.2 enhancer required for AVN-specific gene expression. Furthermore, we demonstrate that MyoR interacts directly with Gata4 to mediate transcriptional repression. Our studies reveal that MyoR contains two nonequivalent repression domains. While the MyoR C-terminal repression domain inhibits transcription in a context-dependent manner, the N-terminal repression domain can function in a heterologous context to convert the Hand2 activator into a repressor. In addition, we show that genetic deletion of MyoR in mice increases Cx30.2 expression by 50% and prolongs AV delay by 13%. Taken together, we conclude that MyoR modulates a Gata4-dependent regulatory circuit that establishes proper AV delay, and these findings may have wider implications for the variability of cardiac rhythm observed in the general population. PMID:25487574

Recruitment of CREB1 and Histone Deacetylase 2 (HDAC2) to the Mouse Ltbp-1 Promoter Regulates its Constitutive Expression in a Dioxin Receptor-dependent Manner

PubMed Central

Gomez-Duran, Aurea; Ballestar, Esteban; Carvajal-Gonzalez, Jose M.; Marlowe, Jennifer L.; Puga, Alvaro; Esteller, Manel; Fernandez-Salguero, Pedro M.

2010-01-01

Latent TGFβ-binding protein 1 (LTBP-1) is a key regulator of TGFβ targeting and activation in the extracellular matrix. LTBP-1 is recognized as a major docking molecule to localize, and possibly to activate, TGFβ in the extracellular matrix. Despite this relevant function, the molecular mechanisms regulating Ltbp-1 transcription remain largely unknown. Previous results from our laboratory revealed that mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR) had increased Ltbp-1 mRNA expression and elevated TGFβ activity, suggesting that AhR repressed Ltbp-1 transcription. Here, we have cloned the mouse Ltbp-1 gene promoter and analysed its mechanism of transcriptional repression by AhR. Reporter gene assays, AhR over-expression and site-directed mutagenesis showed that basal Ltbp-1 transcription is AhR-dependent. Chromatin immunoprecipitation (ChIP) and RNA interference (RNAi) revealed that AhR regulates Ltbp-1 transcription by a mechanism involving recruitment of co-activators such as CREB1 and co-repressors such as HDAC2 to the Ltbp-1 promoter. In AhR-expressing (AhR+/+) MEF cells, the recruitment of HDAC1, 2 and 4 correlated with decreased K8H4 acetylation and impaired binding of pCREBSer133 to the Ltbp-1 promoter, likely maintaining a constitutive repressed state. AhR−/− MEF cells had the opposite pattern of HDACs and pCREB1Ser133 binding to Ltbp-1 promoter, and therefore, over-expressed Ltbp-1 mRNA. In agreement, siRNA for HDAC2 increased Ltbp-1 expression and K8H4 acetylation in AhR+/+ but not in AhR−/− MEF cells. We suggest that HDAC2 binding keeps Ltbp-1 promoter repressed in AhR+/+ MEF cells, whereas in AhR-null MEF cells the absence of HDAC2 and the binding of pCREBSer133 allow Ltbp-1 transcription. Thus, epigenetics can contribute to constitutive Ltbp-1 repression by a mechanism requiring AhR activity. PMID:18508077

Ski represses BMP signaling in Xenopus and mammalian cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

kluo@lbl.gov

2001-05-16

The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells bymore » directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-{beta} family members.« less

Ski represses bone morphogenic protein signaling in Xenopus and mammalian cells

PubMed Central

Wang, Wei; Mariani, Francesca V.; Harland, Richard M.; Luo, Kunxin

2000-01-01

The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells by directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-β family members. PMID:11121043

Ski represses bone morphogenic protein signaling in Xenopus and mammalian cells.

PubMed

Wang, W; Mariani, F V; Harland, R M; Luo, K

2000-12-19

The bone morphogenic proteins (BMPs) play important roles in vertebrate development. In Xenopus, BMPs act as epidermal inducers and also as negative regulators of neurogenesis. Antagonism of BMP signaling results in neuralization. BMPs signal through the cell-surface receptors and downstream Smad molecules. Upon stimulation with BMP, Smad1, Smad5, and Smad8 are phosphorylated by the activated BMP receptors, form a complex with Smad4, and translocate into the nucleus, where they regulate the expression of BMP target genes. Here, we show that the Ski oncoprotein can block BMP signaling and the expression of BMP-responsive genes in both Xenopus and mammalian cells by directly interacting with and repressing the activity of BMP-specific Smad complexes. This ability to antagonize BMP signaling results in neuralization by Ski in the Xenopus embryo and blocking of osteoblast differentiation of murine W-20-17 cells. Thus, Ski is able to repress the activity of all receptor-associated Smads and may regulate vertebrate development by modulating the signaling activity of transforming growth factor-beta family members.

Allele-Specific DNA Methylation and Its Interplay with Repressive Histone Marks at Promoter-Mutant TERT Genes.

PubMed

Stern, Josh Lewis; Paucek, Richard D; Huang, Franklin W; Ghandi, Mahmoud; Nwumeh, Ronald; Costello, James C; Cech, Thomas R

2017-12-26

A mutation in the promoter of the Telomerase Reverse Transcriptase (TERT) gene is the most frequent noncoding mutation in cancer. The mutation drives unusual monoallelic expression of TERT, allowing immortalization. Here, we find that DNA methylation of the TERT CpG island (CGI) is also allele-specific in multiple cancers. The expressed allele is hypomethylated, which is opposite to cancers without TERT promoter mutations. The continued presence of Polycomb repressive complex 2 (PRC2) on the inactive allele suggests that histone marks of repressed chromatin may be causally linked to high DNA methylation. Consistent with this hypothesis, TERT promoter DNA containing 5-methyl-CpG has much increased affinity for PRC2 in vitro. Thus, CpG methylation and histone marks appear to collaborate to maintain the two TERT alleles in different epigenetic states in TERT promoter mutant cancers. Finally, in several cancers, DNA methylation levels at the TERT CGI correlate with altered patient survival. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Translation repression via modulation of the cytoplasmic poly(A)-binding protein in the inflammatory response

PubMed Central

Zhang, Xu; Chen, Xiaoli; Liu, Qiuying; Zhang, Shaojie; Hu, Wenqian

2017-01-01

Gene expression is precisely regulated during the inflammatory response to control infection and limit the detrimental effects of inflammation. Here, we profiled global mRNA translation dynamics in the mouse primary macrophage-mediated inflammatory response and identified hundreds of differentially translated mRNAs. These mRNAs’ 3’UTRs have enriched binding motifs for several RNA-binding proteins, which implies extensive translational regulatory networks. We characterized one such protein, Zfp36, as a translation repressor. Using primary macrophages from a Zfp36-V5 epitope tagged knock-in mouse generated by CRISPR/Cas9-mediated genome editing, we found that the endogenous Zfp36 directly interacts with the cytoplasmic poly(A)-binding protein. Importantly, this interaction is required for the translational repression of Zfp36’s target mRNAs in resolving inflammation. Altogether, these results uncovered critical roles of translational regulations in controlling appropriate gene expression during the inflammatory response and revealed a new biologically relevant molecular mechanism of translational repression via modulating the cytoplasmic poly(A)-binding protein. DOI: http://dx.doi.org/10.7554/eLife.27786.001 PMID:28635594

Anchoring of Heterochromatin to the Nuclear Lamina Reinforces Dosage Compensation-Mediated Gene Repression.

PubMed

Snyder, Martha J; Lau, Alyssa C; Brouhard, Elizabeth A; Davis, Michael B; Jiang, Jianhao; Sifuentes, Margarita H; Csankovszki, Györgyi

2016-09-01

Higher order chromosome structure and nuclear architecture can have profound effects on gene regulation. We analyzed how compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensation in the nematode C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, we found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. We propose a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression.

Anchoring of Heterochromatin to the Nuclear Lamina Reinforces Dosage Compensation-Mediated Gene Repression

PubMed Central

Brouhard, Elizabeth A.; Jiang, Jianhao; Sifuentes, Margarita H.

2016-01-01

Higher order chromosome structure and nuclear architecture can have profound effects on gene regulation. We analyzed how compartmentalizing the genome by tethering heterochromatic regions to the nuclear lamina affects dosage compensation in the nematode C. elegans. In this organism, the dosage compensation complex (DCC) binds both X chromosomes of hermaphrodites to repress transcription two-fold, thus balancing gene expression between XX hermaphrodites and XO males. X chromosome structure is disrupted by mutations in DCC subunits. Using X chromosome paint fluorescence microscopy, we found that X chromosome structure and subnuclear localization are also disrupted when the mechanisms that anchor heterochromatin to the nuclear lamina are defective. Strikingly, the heterochromatic left end of the X chromosome is less affected than the gene-rich middle region, which lacks heterochromatic anchors. These changes in X chromosome structure and subnuclear localization are accompanied by small, but significant levels of derepression of X-linked genes as measured by RNA-seq, without any observable defects in DCC localization and DCC-mediated changes in histone modifications. We propose a model in which heterochromatic tethers on the left arm of the X cooperate with the DCC to compact and peripherally relocate the X chromosomes, contributing to gene repression. PMID:27690361

[Subtractive gene cloning and gene-disruption for elucidation of pseudohyphal formation in Candida tropicalis].

PubMed

Suzuki, Takahito

2003-01-01

The dimorphic transition from yeast to pseudohyphae in the petroleum-assimilating yeast Candida tropicalis occurs following the addition of ethanol to glucose semi-defined medium. Subtractive gene cloning was performed on the cDNA from the yeast-growing control culture and on that from the ethanol-supplemented one (the ethanol culture). A homologue of Schizosaccharomyces pombe nmt1+ or Saccharomyces cerevisiae THI5 was isolated from the cDNA fraction as a preferentially expressed gene for the ethanol culture. This homologue was tentatively called Ctnmt1+, since exogenous thiamine repressed its expression in C. tropicalis growth media. The ethanol culture showed a biphasic pattern of growth phases and the expression of Ctnmt1+ occurred at the first growth phase. The supplementation of thiamine to the ethanol culture at the first phase was followed by repression of Ctnmt1+ expression and also delay of pseudohyphal growth: filamentous growth was inhibited and chains of yeast cells were formed. A Ctnmt1+ disruptant of this organism did not show thiamine auxotrophy and produced pseudohyphal filaments even in the control culture. The supplementation of oxythiamine, an analog of thiamine, to the control culture was followed by the appearance of pseudohyphal filaments, indicating the participation of thiamine during the process of pseudohyphal growth in this organism.

Genome-wide characterization reveals complex interplay between TP53 and TP63 in response to genotoxic stress

PubMed Central

McDade, Simon S.; Patel, Daksha; Moran, Michael; Campbell, James; Fenwick, Kerry; Kozarewa, Iwanka; Orr, Nicholas J.; Lord, Christopher J.; Ashworth, Alan A.; McCance, Dennis J.

2014-01-01

In response to genotoxic stress the TP53 tumour suppressor activates target gene expression to induce cell cycle arrest or apoptosis depending on the extent of DNA damage. These canonical activities can be repressed by TP63 in normal stratifying epithelia to maintain proliferative capacity or drive proliferation of squamous cell carcinomas, where TP63 is frequently overexpressed/amplified. Here we use ChIP-sequencing, integrated with microarray analysis, to define the genome-wide interplay between TP53 and TP63 in response to genotoxic stress in normal cells. We reveal that TP53 and TP63 bind to overlapping, but distinct cistromes of sites through utilization of distinctive consensus motifs and that TP53 is constitutively bound to a number of sites. We demonstrate that cisplatin and adriamycin elicit distinct effects on TP53 and TP63 binding events, through which TP53 can induce or repress transcription of an extensive network of genes by direct binding and/or modulation of TP63 activity. Collectively, this results in a global TP53-dependent repression of cell cycle progression, mitosis and DNA damage repair concomitant with activation of anti-proliferative and pro-apoptotic canonical target genes. Further analyses reveal that in the absence of genotoxic stress TP63 plays an important role in maintaining expression of DNA repair genes, loss of which results in defective repair. PMID:24823795

AMPKα2 regulates expression of estrogen-related receptor alpha, a metabolic transcription factor related to heart failure development

PubMed Central

Hu, Xinli; Xu, Xin; Lu, Zhongbing; Zhang, Ping; Fassett, John; Zhang, Ying; Xin, Yi; Hall, Jennifer L.; Viollet, Benoit; Bache, Robert J.; Huang, Yimin; Chen, Yingjie

2011-01-01

The normal expression of myocardial mitochondrial enzymes is essential to maintain the cardiac energy reserve and facilitate responses to stress, but the molecular mechanisms to maintain myocardial mitochondrial enzyme expression have been elusive. Here we report that congestive heart failure is associated with a significant decrease of myocardial Estrogen-Related Receptor alpha (ERRα), but not PPAR gamma coactivator-1 alpha (PGC1α), in human heart failure samples. In addition, chronic pressure overload in mice caused a decrease of ERRα expression that was significantly correlated to the degree of LV dysfunction, pulmonary congestion and decreases of a group of myocardial energy metabolism related genes. We found that the metabolic sensor AMP activated protein kinase (AMPK) regulates ERRα expression in vivo and in vitro. AMPKα2 KO decreased myocardial ERRα (both mRNA and protein) and its downstream targets under basal conditions, with no change in myocardial PGC1α expression. Using cultured rat neonatal cardiac myocytes, we found that overexpression of constitutively active AMPKα significantly induced ERRα mRNA, protein and promoter activity. Conversely, selective gene silencing of AMPKα2 repressed ERRα and its target gene levels, indicating that AMPKα2 is involved in the regulation of ERRα expression. In addition, over-expression of ERRα in AMPKα2 KO neonatal cardiac myocytes partially rescued the repressed expression of some energy metabolism related genes. These data support an important role for AMPKα2 in regulating the expression of myocardial ERRα and its downstream mitochondrial enzymes. PMID:21825219

The stretch responsive microRNA miR-148a-3p is a novel repressor of IKBKB, NF-κB signaling, and inflammatory gene expression in human aortic valve cells

PubMed Central

Patel, Vishal; Carrion, Katrina; Hollands, Andrew; Hinton, Andrew; Gallegos, Thomas; Dyo, Jeffrey; Sasik, Roman; Leire, Emma; Hardiman, Gary; Mohamed, Salah A.; Nigam, Sanjay; King, Charles C.; Nizet, Victor; Nigam, Vishal

2015-01-01

Bicuspid aortic valves calcify at a significantly higher rate than normal aortic valves, a process that involves increased inflammation. Because we have previously found that bicuspid aortic valve experience greater stretch, we investigated the potential connection between stretch and inflammation in human aortic valve interstitial cells (AVICs). Microarray, quantitative PCR (qPCR), and protein assays performed on AVICs exposed to cyclic stretch showed that stretch was sufficient to increase expression of interleukin and metalloproteinase family members by more than 1.5-fold. Conditioned medium from stretched AVICs was sufficient to activate leukocytes. microRNA sequencing and qPCR experiments demonstrated that miR-148a-3p was repressed in both stretched AVICs (43% repression) and, as a clinical correlate, human bicuspid aortic valves (63% reduction). miR-148a-3p was found to be a novel repressor of IKBKB based on data from qPCR, luciferase, and Western blot experiments. Furthermore, increasing miR-148a-3p levels in AVICs was sufficient to decrease NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling and NF-κB target gene expression. Our data demonstrate that stretch-mediated activation of inflammatory pathways is at least partly the result of stretch-repression of miR-148a-3p and a consequent failure to repress IKBKB. To our knowledge, we are the first to report that cyclic stretch of human AVICs activates inflammatory genes in a tissue-autonomous manner via a microRNA that regulates a central inflammatory pathway.—Patel, V., Carrion, K., Hollands, A., Hinton, A., Gallegos, T., Dyo, J., Sasik, R., Leire, E., Hardiman, G., Mohamed, S. A., Nigam, S., King, C. C., Nizet, V., Nigam V. The stretch responsive microRNA miR-148a-3p is a novel repressor of IKBKB, NF-κB signaling, and inflammatory gene expression in human aortic valve cells. PMID:25630970

Identification and consequences of miRNA-target interactions--beyond repression of gene expression.

PubMed

Hausser, Jean; Zavolan, Mihaela

2014-09-01

Comparative genomics analyses and high-throughput experimental studies indicate that a microRNA (miRNA) binds to hundreds of sites across the transcriptome. Although the knockout of components of the miRNA biogenesis pathway has profound phenotypic consequences, most predicted miRNA targets undergo small changes at the mRNA and protein levels when the expression of the miRNA is perturbed. Alternatively, miRNAs can establish thresholds in and increase the coherence of the expression of their target genes, as well as reduce the cell-to-cell variability in target gene expression. Here, we review the recent progress in identifying miRNA targets and the emerging paradigms of how miRNAs shape the dynamics of target gene expression.

Regulation of lac Operon Expression: Reappraisal of the Theory of Catabolite Repression

PubMed Central

Wanner, Barry L.; Kodaira, Ryoji; Neidhardt, Frederick C.

1978-01-01

The physiological state of Escherichia coli with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of β-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the lac gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type lac promoter showed an 18-fold variation in lac expression, and cells with the lacP37 (formerly lac-L37) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3′,5′-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of β-galactosidase synthesis from the wild-type lac promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased lac expression also occurred when the bacteria harbored the catabolite-insensitive lacP5 (formerly lacUV5) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism. PMID:214424

Curcumin differentially regulates endoplasmic reticulum stress through transcriptional corepressor SMILE (small heterodimer partner-interacting leucine zipper protein)-mediated inhibition of CREBH (cAMP responsive element-binding protein H).

PubMed

Misra, Jagannath; Chanda, Dipanjan; Kim, Don-kyu; Li, Tiangang; Koo, Seung-Hoi; Back, Sung-Hoon; Chiang, John Y L; Choi, Hueng-Sik

2011-12-09

Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), is a natural polyphenolic compound. Herein the effect of curcumin on endoplasmic reticulum (ER) stress responsive gene expression was investigated. We report that curcumin induces transcriptional corepressor small heterodimer partner-interacting leucine zipper protein (SMILE) gene expression through liver kinase B1 (LKB1)/adenosine monophosphate-activated kinase (AMPK) signaling pathway and represses ER stress-responsive gene transcription in an ER-bound transcription factor specific manner. cAMP responsive element-binding protein H (CREBH) and activating transcription factor 6 (ATF6) are both ER-bound bZIP family transcription factors that are activated upon ER stress. Of interest, we observed that both curcumin treatment and SMILE overexpression only represses CREBH-mediated transactivation of the target gene but not ATF6-mediated transactivation. Knockdown of endogenous SMILE significantly releases the inhibitory effect of curcumin on CREBH transactivation. Intrinsic repressive activity of SMILE is observed in the Gal4 fusion system, and the intrinsic repressive domain is mapped to the C terminus of SMILE spanning amino acid residues 203-269, corresponding to the basic region leucine zipper (bZIP) domain. In vivo interaction assay revealed that through its bZIP domain, SMILE interacts with CREBH and inhibits its transcriptional activity. Interestingly, we observed that SMILE does not interact with ATF6. Furthermore, competition between SMILE and the coactivator peroxisome proliferator-activated receptor α (PGC-1α) on CREBH transactivation has been demonstrated in vitro and in vivo. Finally, chromatin immunoprecipitation assays revealed that curcumin decreases the binding of PGC-1α and CREBH on target gene promoter in a SMILE-dependent manner. Overall, for the first time we suggest a novel phenomenon that the curcumin/LKB1/AMPK/SMILE/PGC1α pathway differentially regulates ER stress-mediated gene transcription.

Fine Time Course Expression Analysis Identifies Cascades of Activation and Repression and Maps a Putative Regulator of Mammalian Sex Determination

PubMed Central

Looger, Loren L.; Ohler, Uwe; Capel, Blanche

2013-01-01

In vertebrates, primary sex determination refers to the decision within a bipotential organ precursor to differentiate as a testis or ovary. Bifurcation of organ fate begins between embryonic day (E) 11.0–E12.0 in mice and likely involves a dynamic transcription network that is poorly understood. To elucidate the first steps of sexual fate specification, we profiled the XX and XY gonad transcriptomes at fine granularity during this period and resolved cascades of gene activation and repression. C57BL/6J (B6) XY gonads showed a consistent ∼5-hour delay in the activation of most male pathway genes and repression of female pathway genes relative to 129S1/SvImJ, which likely explains the sensitivity of the B6 strain to male-to-female sex reversal. Using this fine time course data, we predicted novel regulatory genes underlying expression QTLs (eQTLs) mapped in a previous study. To test predictions, we developed an in vitro gonad primary cell assay and optimized a lentivirus-based shRNA delivery method to silence candidate genes and quantify effects on putative targets. We provide strong evidence that Lmo4 (Lim-domain only 4) is a novel regulator of sex determination upstream of SF1 (Nr5a1), Sox9, Fgf9, and Col9a3. This approach can be readily applied to identify regulatory interactions in other systems. PMID:23874228

Retinoic acid signaling acts via Hox1 to establish the posterior limit of the pharynx in the chordate amphioxus.

PubMed

Schubert, Michael; Yu, Jr-Kai; Holland, Nicholas D; Escriva, Hector; Laudet, Vincent; Holland, Linda Z

2005-01-01

In the invertebrate chordate amphioxus, as in vertebrates, retinoic acid (RA) specifies position along the anterior/posterior axis with elevated RA signaling in the middle third of the endoderm setting the posterior limit of the pharynx. Here we show that AmphiHox1 is also expressed in the middle third of the developing amphioxus endoderm and is activated by RA signaling. Knockdown of AmphiHox1 function with an antisense morpholino oligonucleotide shows that AmphiHox1 mediates the role of RA signaling in setting the posterior limit of the pharynx by repressing expression of pharyngeal markers in the posterior foregut/midgut endoderm. The spatiotemporal expression of these endodermal genes in embryos treated with RA or the RA antagonist BMS009 indicates that Pax1/9, Pitx and Notch are probably more upstream than Otx and Nodal in the hierarchy of genes repressed by RA signaling. This work highlights the potential of amphioxus, a genomically simple, vertebrate-like invertebrate chordate, as a paradigm for understanding gene hierarchies similar to the more complex ones of vertebrates.

PADI4 acts as a coactivator of Tal1 by counteracting repressive histone arginine methylation

NASA Astrophysics Data System (ADS)

Kolodziej, Stephan; Kuvardina, Olga N.; Oellerich, Thomas; Herglotz, Julia; Backert, Ingo; Kohrs, Nicole; Buscató, Estel. La; Wittmann, Sandra K.; Salinas-Riester, Gabriela; Bonig, Halvard; Karas, Michael; Serve, Hubert; Proschak, Ewgenij; Lausen, Jörn

2014-05-01

The transcription factor Tal1 is a critical activator or repressor of gene expression in hematopoiesis and leukaemia. The mechanism by which Tal1 differentially influences transcription of distinct genes is not fully understood. Here we show that Tal1 interacts with the peptidylarginine deiminase IV (PADI4). We demonstrate that PADI4 can act as an epigenetic coactivator through influencing H3R2me2a. At the Tal1/PADI4 target gene IL6ST the repressive H3R2me2a mark triggered by PRMT6 is counteracted by PADI4, which augments the active H3K4me3 mark and thus increases IL6ST expression. In contrast, at the CTCF promoter PADI4 acts as a repressor. We propose that the influence of PADI4 on IL6ST transcription plays a role in the control of IL6ST expression during lineage differentiation of hematopoietic stem/progenitor cells. These results open the possibility to pharmacologically influence Tal1 in leukaemia.

Post-transcriptional gene expression control by NANOS is up-regulated and functionally important in pRb-deficient cells

PubMed Central

Miles, Wayne O; Korenjak, Michael; Griffiths, Lyra M; Dyer, Michael A; Provero, Paolo; Dyson, Nicholas J

2014-01-01

Inactivation of the retinoblastoma tumor suppressor (pRb) is a common oncogenic event that alters the expression of genes important for cell cycle progression, senescence, and apoptosis. However, in many contexts, the properties of pRb-deficient cells are similar to wild-type cells suggesting there may be processes that counterbalance the transcriptional changes associated with pRb inactivation. Therefore, we have looked for sets of evolutionary conserved, functionally related genes that are direct targets of pRb/E2F proteins. We show that the expression of NANOS, a key facilitator of the Pumilio (PUM) post-transcriptional repressor complex, is directly repressed by pRb/E2F in flies and humans. In both species, NANOS expression increases following inactivation of pRb/RBF1 and becomes important for tissue homeostasis. By analyzing datasets from normal retinal tissue and pRb-null retinoblastomas, we find a strong enrichment for putative PUM substrates among genes de-regulated in tumors. These include pro-apoptotic genes that are transcriptionally down-regulated upon pRb loss, and we characterize two such candidates, MAP2K3 and MAP3K1, as direct PUM substrates. Our data suggest that NANOS increases in importance in pRb-deficient cells and helps to maintain homeostasis by repressing the translation of transcripts containing PUM Regulatory Elements (PRE). PMID:25100735

Post-transcriptional gene expression control by NANOS is up-regulated and functionally important in pRb-deficient cells.

PubMed

Miles, Wayne O; Korenjak, Michael; Griffiths, Lyra M; Dyer, Michael A; Provero, Paolo; Dyson, Nicholas J

2014-10-01

Inactivation of the retinoblastoma tumor suppressor (pRb) is a common oncogenic event that alters the expression of genes important for cell cycle progression, senescence, and apoptosis. However, in many contexts, the properties of pRb-deficient cells are similar to wild-type cells suggesting there may be processes that counterbalance the transcriptional changes associated with pRb inactivation. Therefore, we have looked for sets of evolutionary conserved, functionally related genes that are direct targets of pRb/E2F proteins. We show that the expression of NANOS, a key facilitator of the Pumilio (PUM) post-transcriptional repressor complex, is directly repressed by pRb/E2F in flies and humans. In both species, NANOS expression increases following inactivation of pRb/RBF1 and becomes important for tissue homeostasis. By analyzing datasets from normal retinal tissue and pRb-null retinoblastomas, we find a strong enrichment for putative PUM substrates among genes de-regulated in tumors. These include pro-apoptotic genes that are transcriptionally down-regulated upon pRb loss, and we characterize two such candidates, MAP2K3 and MAP3K1, as direct PUM substrates. Our data suggest that NANOS increases in importance in pRb-deficient cells and helps to maintain homeostasis by repressing the translation of transcripts containing PUM Regulatory Elements (PRE). © 2014 The Authors.

An LXR–NCOA5 gene regulatory complex directs inflammatory crosstalk-dependent repression of macrophage cholesterol efflux

PubMed Central

Gillespie, Mark A; Gold, Elizabeth S; Ramsey, Stephen A; Podolsky, Irina; Aderem, Alan; Ranish, Jeffrey A

2015-01-01

LXR–cofactor complexes activate the gene expression program responsible for cholesterol efflux in macrophages. Inflammation antagonizes this program, resulting in foam cell formation and atherosclerosis; however, the molecular mechanisms underlying this antagonism remain to be fully elucidated. We use promoter enrichment-quantitative mass spectrometry (PE-QMS) to characterize the composition of gene regulatory complexes assembled at the promoter of the lipid transporter Abca1 following downregulation of its expression. We identify a subset of proteins that show LXR ligand- and binding-dependent association with the Abca1 promoter and demonstrate they differentially control Abca1 expression. We determine that NCOA5 is linked to inflammatory Toll-like receptor (TLR) signaling and establish that NCOA5 functions as an LXR corepressor to attenuate Abca1 expression. Importantly, TLR3–LXR signal crosstalk promotes recruitment of NCOA5 to the Abca1 promoter together with loss of RNA polymerase II and reduced cholesterol efflux. Together, these data significantly expand our knowledge of regulatory inputs impinging on the Abca1 promoter and indicate a central role for NCOA5 in mediating crosstalk between pro-inflammatory and anti-inflammatory pathways that results in repression of macrophage cholesterol efflux. PMID:25755249

sRNA antitoxins: more than one way to repress a toxin.

PubMed

Wen, Jia; Fozo, Elizabeth M

2014-08-04

Bacterial toxin-antitoxin loci consist of two genes: one encodes a potentially toxic protein, and the second, an antitoxin to repress its function or expression. The antitoxin can either be an RNA or a protein. For type I and type III loci, the antitoxins are RNAs; however, they have very different modes of action. Type I antitoxins repress toxin protein expression through interacting with the toxin mRNA, thereby targeting the mRNA for degradation or preventing its translation or both; type III antitoxins directly bind to the toxin protein, sequestering it. Along with these two very different modes of action for the antitoxin, there are differences in the functions of the toxin proteins and the mobility of these loci between species. Within this review, we discuss the major differences as to how the RNAs repress toxin activity, the potential consequences for utilizing different regulatory strategies, as well as the confirmed and potential biological roles for these loci across bacterial species.

CTCF-KDM4A complex correlates with histone modifications that negatively regulate CHD5 gene expression in cancer cell lines

PubMed Central

Guerra-Calderas, Lissania; González-Barrios, Rodrigo; Patiño, Carlos César; Alcaraz, Nicolás; Salgado-Albarrán, Marisol; de León, David Cantú; Hernández, Clementina Castro; Sánchez-Pérez, Yesennia; Maldonado-Martínez, Héctor Aquiles; De la Rosa-Velazquez, Inti A.; Vargas-Romero, Fernanda; Herrera, Luis A.; García-Carrancá, Alejandro; Soto-Reyes, Ernesto

2018-01-01

Histone demethylase KDM4A is involved in H3K9me3 and H3K36me3 demethylation, which are epigenetic modifications associated with gene silencing and RNA Polymerase II elongation, respectively. KDM4A is abnormally expressed in cancer, affecting the expression of multiple targets, such as the CHD5 gene. This enzyme localizes at the first intron of CHD5, and the dissociation of KDM4A increases gene expression. In vitro assays showed that KDM4A-mediated demethylation is enhanced in the presence of CTCF, suggesting that CTCF could increase its enzymatic activity in vivo, however the specific mechanism by which CTCF and KDM4A might be involved in the CHD5 gene repression is poorly understood. Here, we show that CTCF and KDM4A form a protein complex, which is recruited into the first intron of CHD5. This is related to a decrease in H3K36me3/2 histone marks and is associated with its transcriptional downregulation. Depletion of CTCF or KDM4A by siRNA, triggered the reactivation of CHD5 expression, suggesting that both proteins are involved in the negative regulation of this gene. Furthermore, the knockout of KDM4A restored the CHD5 expression and H3K36me3 and H3K36me2 histone marks. Such mechanism acts independently of CHD5 promoter DNA methylation. Our findings support a novel mechanism of epigenetic repression at the gene body that does not involve promoter silencing. PMID:29682202

Orphan nuclear receptor small heterodimer partner inhibits transforming growth factor-beta signaling by repressing SMAD3 transactivation.

PubMed

Suh, Ji Ho; Huang, Jiansheng; Park, Yun-Yong; Seong, Hyun-A; Kim, Dongwook; Shong, Minho; Ha, Hyunjung; Lee, In-Kyu; Lee, Keesook; Wang, Li; Choi, Hueng-Sik

2006-12-22

Orphan nuclear receptor small heterodimer partner (SHP) is an atypical member of the nuclear receptor superfamily; SHP regulates the nuclear receptor-mediated transcription of target genes but lacks a conventional DNA binding domain. In this study, we demonstrate that SHP represses transforming growth factor-beta (TGF-beta)-induced gene expression through a direct interaction with Smad, a transducer of TGF-beta signaling. Transient transfection studies demonstrate that SHP represses Smad3-induced transcription. In vivo and in vitro protein interaction assays revealed that SHP directly interacts with Smad2 and Smad3 but not with Smad4. Mapping of domains mediating the interaction between SHP and Smad3 showed that the entire N-terminal domain (1-159 amino acids) of SHP and the linker domain of Smad3 are involved in this interaction. In vitro glutathione S-transferase pulldown competition experiments revealed the SHP-mediated repression of Smad3 transactivation through competition with its co-activator p300. SHP also inhibits the activation of endogenous TGF-beta-responsive gene promoters, the p21, Smad7, and plasminogen activator inhibitor-1 (PAI-1) promoters. Moreover, adenovirus-mediated overexpression of SHP decreases PAI-1 mRNA levels, and down-regulation of SHP by a small interfering RNA increases both the transactivation of Smad3 and the PAI-1 mRNA levels. Finally, the PAI-1 gene is expressed in SHP(-/-) mouse hepatocytes at a higher level than in normal hepatocytes. Taken together, these data indicate that SHP is a novel co-regulator of Smad3, and this study provides new insights into regulation of TGF-beta signaling.

The prohibitin-repressive interaction with E2F1 is rapidly inhibited by androgen signalling in prostate cancer cells

PubMed Central

Koushyar, S; Economides, G; Zaat, S; Jiang, W; Bevan, C L; Dart, D A

2017-01-01

Prohibitin (PHB) is a tumour suppressor molecule with pleiotropic activities across several cellular compartments including mitochondria, cell membrane and the nucleus. PHB and the steroid-activated androgen receptor (AR) have an interplay where AR downregulates PHB, and PHB represses AR. Additionally, their cellular locations and chromatin interactions are in dynamic opposition. We investigated the mechanisms of cell cycle inhibition by PHB and how this is modulated by AR in prostate cancer. Using a prostate cancer cell line overexpressing PHB, we analysed the gene expression changes associated with PHB-mediated cell cycle arrest. Over 1000 gene expression changes were found to be significant and gene ontology analysis confirmed PHB-mediated repression of genes essential for DNA replication and synthesis, for example, MCMs and TK1, via an E2F1 regulated pathway—agreeing with its G1/S cell cycle arrest activity. PHB is known to inhibit E2F1-mediated transcription, and the PHB:E2F1 interaction was seen in LNCaP nuclear extracts, which was then reduced by androgen treatment. Upon two-dimensional western blot analysis, the PHB protein itself showed androgen-mediated charge differentiation (only in AR-positive cells), indicating a potential dephosphorylation event. Kinexus phosphoprotein array analysis indicated that Src kinase was the main interacting intracellular signalling hub in androgen-treated LNCaP cells, and that Src inhibition could reduce this AR-mediated charge differentiation. PHB charge change may be associated with rapid dissociation from chromatin and E2F1, allowing the cell cycle to proceed. The AR and androgens may deactivate the repressive functions of PHB upon E2F1 leading to cell cycle progression, and indicates a role for AR in DNA replication licensing. PMID:28504694

A single residue mutation in Hha preserving structure and binding to H-NS results in loss of H-NS mediated gene repression properties.

PubMed

Cordeiro, Tiago N; Garcia, Jesús; Pons, José-Ignacio; Aznar, Sonia; Juárez, Antonio; Pons, Miquel

2008-09-03

In this study, we report that a single mutation of cysteine 18 to isoleucine (C18I) in Escherichia coli Hha abolishes the repression of the hemolysin operon observed in the wild-type protein. The phenotype also includes a significant decrease in the growth rate of E. coli cells at low ionic strength. Other substitutions at this position (C18A, C18S) have no observable effects in E. coli growth or hemolysin repression. All mutants are stable and well folded and bind H-NS in vitro with similar affinities suggesting that Cys 18 is not directly involved in H-NS binding but this position is essential for the activity of the H-NS/Hha heterocomplexes in the regulation of gene expression.

Biochemical and Functional Interactions of Human Papillomavirus Proteins with Polycomb Group Proteins

PubMed Central

McLaughlin-Drubin, Margaret E.; Munger, Karl

2013-01-01

The role of enzymes involved in polycomb repression of gene transcription has been studied extensively in human cancer. Polycomb repressive complexes mediate oncogene-induced senescence, a principal innate cell-intrinsic tumor suppressor pathway that thwarts expansion of cells that have suffered oncogenic hits. Infections with human cancer viruses including human papillomaviruses (HPVs) and Epstein-Barr virus can trigger oncogene-induced senescence, and the viruses have evolved strategies to abrogate this response in order to establish an infection and reprogram their host cells to establish a long-term persistent infection. As a consequence of inhibiting polycomb repression and evading oncogene induced-senescence, HPV infected cells have an altered epigenetic program as evidenced by aberrant homeobox gene expression. Similar alterations are frequently observed in non-virus associated human cancers and may be harnessed for diagnosis and therapy. PMID:23673719

Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression

PubMed Central

Megger, Dominik A.; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko

2017-01-01

Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus. PMID:28959263

Deciphering of the Human Interferon-Regulated Proteome by Mass Spectrometry-Based Quantitative Analysis Reveals Extent and Dynamics of Protein Induction and Repression.

PubMed

Megger, Dominik A; Philipp, Jos; Le-Trilling, Vu Thuy Khanh; Sitek, Barbara; Trilling, Mirko

2017-01-01

Interferons (IFNs) are pleotropic cytokines secreted upon encounter of pathogens and tumors. Applying their antipathogenic, antiproliferative, and immune stimulatory capacities, recombinant IFNs are frequently prescribed as drugs to treat different diseases. IFNs act by changing the gene expression profile of cells. Due to characteristics such as rapid gene induction and signaling, IFNs also represent prototypical model systems for various aspects of biomedical research (e.g., signal transduction). In regard to the signaling and activated promoters, IFNs can be subdivided into two groups. Here, alterations of the cellular proteome of human cells treated with IFNα and IFNγ were elucidated in a time-resolved manner by quantitative proteome analysis. The majority of protein regulations were strongly IFN type and time dependent. In addition to the expected upregulation of IFN-responsive proteins, an astonishing number of proteins became profoundly repressed especially by IFNγ. Thus, our comprehensive analysis revealed important insights into the human IFN-regulated proteome and its dynamics of protein induction and repression. Interestingly, the new class of IFN-repressed genes comprises known host factors for highly relevant pathogens such as HIV, dengue virus, and hepatitis C virus.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yilin; Yang, Yang; Cai, Yanyan

Highlights: Black-Right-Pointing-Pointer We demonstrated that HBV represses MIA2 gene expression both invitro and in vivo. Black-Right-Pointing-Pointer The X protein of HBV plays a major role in such regulation. Black-Right-Pointing-Pointer Knock-down of MIA2 in HepG2 cells activates cell growth and proliferation. Black-Right-Pointing-Pointer HBx activates cell proliferation, over-expression of MIA2 impaired such regulation. Black-Right-Pointing-Pointer HBx activates hepatoma cell proliferation through repressing MIA2 expression. -- Abstract: Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer deaths globally. Chronic hepatitis B virus (HBV) infection accounts for over 75% of all HCC cases; however, the molecular pathogenesis of HCC is not well understood. Inmore » this study, we found that the expression of the newly identified gene melanoma inhibitory activity 2 (MIA2) was reduced by HBV infection invitro and invivo, and that HBV X protein (HBx) plays a major role in this regulation. Recent studies have revealed that MIA2 is a potential tumor suppressor, and that, in most HCCs, MIA2 expression is down-regulated or lost. We found that the knock-down of MIA2 in HepG2 cells activated cell growth and proliferation, suggesting that MIA2 inhibits HCC cell growth and proliferation. In addition, the over-expression of HBx alone induced cell proliferation, whereas MIA2 over-expression impaired the HBx-mediated induction of proliferation. Taken together, our results suggest that HBx activates hepatoma cell growth and proliferation through repression of the potential tumor suppressor MIA2.« less

A non-canonical Flt3ITD/NF-κB signaling pathway represses DAPK1 in acute myeloid leukemia (AML)

PubMed Central

Shanmugam, Rajasubramaniam; Sayar, Hamid; Suvannasankha, Attaya; Goswami, Chirayu; Li, Lang; Gupta, Sushil; Cardoso, Angelo A.; Baghdadi, Tareq Al; Sargent, Katie J.; Cripe, Larry D.; Kalvakolanu, Dhananjaya V.; Boswell, H. Scott

2014-01-01

Purpose DAPK1, a tumor suppressor, is a rate-limiting effector in an ER stress-dependent apoptotic pathway. Its expression is epigenetically suppressed in several tumors. A mechanistic basis for epigenetic/transcriptional repression of DAPK1 was investigated in certain forms of AML with poor prognosis, which lacked ER stress-induced apoptosis. Experimental Design Heterogeneous primary AMLs were screened to identify a subgroup with Flt3ITD in which repression of DAPK1, among NF-κB- and c- jun-responsive genes, was studied. RNAi knockdown studies were performed in Flt3ITD+ve cell line, MV-4-11, to establish genetic epistasis in the pathway Flt3ITD-TAK1-DAPK1 repression, and chromatin immunoprecipitations were performed to identify proximate effector proteins, including TAK1-activated p52NF-κB, at the DAPK1 locus. Results AMLs characterized by normal karyotype with Flt3ITD were found to have 10-100-fold lower DAPK1 transcripts normalized to the expression of c-jun, a transcriptional activator of DAPK1, as compared to a heterogeneous cytogenetic category. Meis1, a c-jun-responsive adverse AML prognostic gene signature was also measured as control. These Flt3ITD+ve AMLs over-express relB, a transcriptional repressor, which forms active heterodimers with p52NF-κB. Chromatin immunoprecipitation assays identified p52NF-κB binding to the DAPK1 promoter along with HDAC2 and HDAC6 in the Flt3ITD+ve human AML cell line MV-4-11. Knockdown of p52NF-κB or its upstream regulator, NIK, de-repressed DAPK1. DAPK1-repressed primary Flt3ITD+ve AMLs had selective nuclear activation of p52NF-κB. Conclusions Flt3ITD promotes a non-canonical pathway via TAK1 and p52NF-κB to suppress DAPK1 in association with HDACs, which explains DAPK1 repression in Flt3ITD+ve AML. PMID:22096027

Rev-erb beta regulates the Srebp-1c promoter and mRNA expression in skeletal muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramakrishnan, Sathiya N.; Lau, Patrick; Crowther, Lisa M.

2009-10-30

The nuclear hormone receptor, Rev-erb beta operates as a transcriptional silencer. We previously demonstrated that exogenous expression of Rev-erb{beta}{Delta}E in skeletal muscle cells increased Srebp-1c mRNA expression. We validated these in vitro observations by injection of an expression vector driving Rev-erb{beta}{Delta}E expression into mouse tibialis muscle that resulted in increased Srebp-1c mRNA expression. Paradoxically, Rev-erb{beta} siRNA expression in skeletal muscle cells repressed Srebp-1c expression, and indicated that Rev-erb{beta} expression was necessary for Srebp-1c expression. ChIP analysis demonstrated that Rev-erb{beta} was recruited to the Srebp-1c promoter. Moreover, Rev-erb{beta} trans-activated the Srebp-1c promoter, in contrast, Rev-erb{beta} efficiently repressed the Rev-erb{alpha} promoter, amore » previously characterized target gene. Finally, treatment with the Rev-erb agonist (hemin) (i) increased the trans-activation of the Srebp-1c promoter by Rev-erb{beta}; and (ii) increased Rev-erb{beta} and Srebp-1c mRNA expression. These data suggest that Rev-erb{beta} has the potential to activate gene expression, and is a positive regulator of Srebp-1c, a regulator of lipogenesis.« less

Epigenomic and functional analyses reveal roles of epialleles in the loss of photoperiod sensitivity during domestication of allotetraploid cottons.

PubMed

Song, Qingxin; Zhang, Tianzhen; Stelly, David M; Chen, Z Jeffrey

2017-05-31

Polyploidy is a pervasive evolutionary feature of all flowering plants and some animals, leading to genetic and epigenetic changes that affect gene expression and morphology. DNA methylation changes can produce meiotically stable epialleles, which are transmissible through selection and breeding. However, the relationship between DNA methylation and polyploid plant domestication remains elusive. We report comprehensive epigenomic and functional analyses, including ~12 million differentially methylated cytosines in domesticated allotetraploid cottons and their tetraploid and diploid relatives. Methylated genes evolve faster than unmethylated genes; DNA methylation changes between homoeologous loci are associated with homoeolog-expression bias in the allotetraploids. Significantly, methylation changes induced in the interspecific hybrids are largely maintained in the allotetraploids. Among 519 differentially methylated genes identified between wild and cultivated cottons, some contribute to domestication traits, including flowering time and seed dormancy. CONSTANS (CO) and CO-LIKE (COL) genes regulate photoperiodicity in Arabidopsis. COL2 is an epiallele in allotetraploid cottons. COL2A is hypermethylated and silenced, while COL2D is repressed in wild cottons but highly expressed due to methylation loss in all domesticated cottons tested. Inhibiting DNA methylation activates COL2 expression, and repressing COL2 in cultivated cotton delays flowering. We uncover epigenomic signatures of domestication traits during cotton evolution. Demethylation of COL2 increases its expression, inducing photoperiodic flowering, which could have contributed to the suitability of cotton for cultivation worldwide. These resources should facilitate epigenetic engineering, breeding, and improvement of polyploid crops.

Generation of genetic constructs that simultaneously express several shRNAs.

PubMed

Kretova, Olga V; Alembekov, Ildar R; Tchurikov, Nickolai A

2012-05-01

RNAi has potential as an antiviral gene therapy strategy. Cassette constructs simultaneously expressing several siRNAs could prove to be the most efficient technique in developing gene therapy approaches for highly mutable viruses such as HIV-1. Here we describe a rapid and cost-saving protocol to generate cassettes that simultaneously express three siRNAs for repression of HIV-1 and CCR5 transcripts. siRNA biological activity was tested in a non-viral system, and exhibited both efficiency and specificity. Our results suggest this protocol can be used to rapidly generate cassette constructs for antiviral gene therapy applications.

Arabidopsis CYP86A2 represses Pseudomonas syringae type III genes and is required for cuticle development

PubMed Central

Xiao, Fangming; Mark Goodwin, S; Xiao, Yanmei; Sun, Zhaoyu; Baker, Douglas; Tang, Xiaoyan; Jenks, Matthew A; Zhou, Jian-Min

2004-01-01

Pseudomonas syringae relies on type III secretion system to deliver effector proteins into the host cell for parasitism. Type III genes are induced in planta, but host factors affecting the induction are poorly understood. Here we report on the identification of an Arabidopsis mutant, att1 (for aberrant induction of type three genes), that greatly enhances the expression of bacterial type III genes avrPto and hrpL. att1 plants display enhanced disease severity to a virulent strain of P. syringae, suggesting a role of ATT1 in disease resistance. ATT1 encodes CYP86A2, a cytochrome P450 monooxygenase catalyzing fatty acid oxidation. The cutin content is reduced to 30% in att1, indicating that CYP86A2 plays a major role in the biosynthesis of extracellular lipids. att1 has a loose cuticle membrane ultrastructure and shows increased permeability to water vapor, demonstrating the importance of the cuticle membrane in controlling water loss. The enhanced avrPto-luc expression is specific to att1, but not another cuticle mutant, wax2. The results suggest that certain cutin-related fatty acids synthesized by CYP86A2 may repress bacterial type III gene expression in the intercellular spaces. PMID:15241470

The transcription factor Mlc promotes Vibrio cholerae biofilm formation through repression of phosphotransferase system components.

PubMed

Pickering, Bradley S; Lopilato, Jane E; Smith, Daniel R; Watnick, Paula I

2014-07-01

The phosphoenol phosphotransferase system (PTS) is a multicomponent signal transduction cascade that regulates diverse aspects of bacterial cellular physiology in response to the availability of high-energy sugars in the environment. Many PTS components are repressed at the transcriptional level when the substrates they transport are not available. In Escherichia coli, the transcription factor Mlc (for makes large colonies) represses transcription of the genes encoding enzyme I (EI), histidine protein (HPr), and the glucose-specific enzyme IIBC (EIIBC(Glc)) in defined media that lack PTS substrates. When glucose is present, the unphosphorylated form of EIIBC(Glc) sequesters Mlc to the cell membrane, preventing its interaction with DNA. Very little is known about Vibrio cholerae Mlc. We found that V. cholerae Mlc activates biofilm formation in LB broth but not in defined medium supplemented with either pyruvate or glucose. Therefore, we questioned whether V. cholerae Mlc functions differently than E. coli Mlc. Here we have shown that, like E. coli Mlc, V. cholerae Mlc represses transcription of PTS components in both defined medium and LB broth and that E. coli Mlc is able to rescue the biofilm defect of a V. cholerae Δmlc mutant. Furthermore, we provide evidence that Mlc indirectly activates transcription of the vps genes by repressing expression of EI. Because activation of the vps genes by Mlc occurs under only a subset of the conditions in which repression of PTS components is observed, we conclude that additional inputs present in LB broth are required for activation of vps gene transcription by Mlc. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction

PubMed Central

Goldfless, Stephen J.; Wagner, Jeffrey C.; Niles, Jacquin C.

2014-01-01

The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous, and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important out come given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum. PMID:25370483

The homeobox gene mirror links EGF signalling to embryonic dorso-ventral axis formation through notch activation.

PubMed

Jordan, K C; Clegg, N J; Blasi, J A; Morimoto, A M; Sen, J; Stein, D; McNeill, H; Deng, W M; Tworoger, M; Ruohola-Baker, H

2000-04-01

Recent studies in vertebrates and Drosophila melanogaster have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. Here we show that this may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis, at a distance. Ectopic Notch activation has a similar long-range effect on pip. Our results suggest that Mirror and Notch induce secretion of diffusible morphogens and we have identified TGF-beta (encoded by dpp) as such a molecule in germarium. We also found that mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and that mirr then represses pip expression in all but the ventral follicle cells, connecting EGFR activation in the dorsal follicle cells to repression of pip in the dorsal and lateral follicle cells. Our results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signalling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation.

A nodal signaling pathway regulates the laterality of neuroanatomical asymmetries in the zebrafish forebrain.

PubMed

Concha, M L; Burdine, R D; Russell, C; Schier, A F; Wilson, S W

2000-11-01

Animals show behavioral asymmetries that are mediated by differences between the left and right sides of the brain. We report that the laterality of asymmetric development of the diencephalic habenular nuclei and the photoreceptive pineal complex is regulated by the Nodal signaling pathway and by midline tissue. Analysis of zebrafish embryos with compromised Nodal signaling reveals an early role for this pathway in the repression of asymmetrically expressed genes in the diencephalon. Later signaling mediated by the EGF-CFC protein One-eyed pinhead and the forkhead transcription factor Schmalspur is required to overcome this repression. When expression of Nodal pathway genes is either absent or symmetrical, neuroanatomical asymmetries are still established but are randomized. This indicates that Nodal signaling is not required for asymmetric development per se but is essential to determine the laterality of the asymmetry.

Transcriptome Analysis of Human Injured Meniscus Reveals a Distinct Phenotype of Meniscus Degeneration with Aging

PubMed Central

Rai, Muhammad Farooq; Patra, Debabrata; Sandell, Linda J.; Brophy, Robert H.

2013-01-01

Objective Meniscus tears are associated with a heightened risk for osteoarthritis. We aimed to advance our understanding of the metabolic state of human injured meniscus at the time of arthroscopic partial meniscectomy through transcriptome-wide analysis of gene expression in relation to patient age and degree of cartilage chondrosis. Methods The degree of chondrosis of knee cartilage was recorded at the time of meniscectomy in symptomatic patients without radiographic osteoarthritis. RNA preparations from resected menisci (N=12) were subjected to transcriptome-wide microarray and QuantiGene Plex analyses. The relative changes in gene expression variation with age and chondrosis were analyzed and integrated biological processes were investigated computationally. Results We identified a set of genes in torn meniscus that were differentially expressed with age and chondrosis. There were 866 genes differentially regulated (≥1.5-fold; P<0.05) with age and 49 with chondrosis. In older patients, genes associated with cartilage and skeletal development and extracellular matrix synthesis were repressed while those involved in immune response, inflammation, cell cycle, and cellular proliferation were stimulated. With chondrosis, genes representing cell catabolism (cAMP catabolic process) and tissue and endothelial cell development were repressed and those involved in T cell differentiation and apoptosis were elevated. Conclusion Differences in age-related gene expression suggest that in older adults, meniscal cells might de-differentiate and initiate a proliferative phenotype. Conversely, meniscal cells in younger patients appear to respond to injury, but maintain the differentiated phenotype. Definitive molecular signatures identified in damaged meniscus could be segregated largely with age and, to a lesser extent, with chondrosis. PMID:23658108

runt Homology Domain Transcription Factors (Runx, Cbfa, and AML) Mediate Repression of the Bone Sialoprotein Promoter: Evidence for Promoter Context-Dependent Activity of Cbfa Proteins

PubMed Central

Javed, Amjad; Barnes, George L.; Jasanya, B. O.; Stein, Janet L.; Gerstenfeld, Louis; Lian, Jane B.; Stein, Gary S.

2001-01-01

Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2αA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediated repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not observed in BSP promoters in which Cbfa sites were deleted or mutated. Expression of the endogenous BSP gene in Gallus osteoblasts was similarly downregulated by forced expression of Cbfa factors. Our data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieved suppression of the BSP promoter. Our results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development. PMID:11283267

Overexpression of B-cell lymphoma 6 alters gene expression profile in a myeloma cell line and is associated with decreased DNA damage response.

PubMed

Tahara, Kenichi; Takizawa, Makiko; Yamane, Arito; Osaki, Yohei; Ishizaki, Takuma; Mitsui, Takeki; Yokohama, Akihiko; Saitoh, Takayuki; Tsukamoto, Norifumi; Matsumoto, Morio; Murakami, Hirokazu; Nojima, Yoshihisa; Handa, Hiroshi

2017-08-01

B-cell lymphoma 6 (BCL6) attenuates DNA damage response (DDR) through gene repression and facilitates tolerance to genomic instability during immunoglobulin affinity maturation in germinal center (GC) B cells. Although BCL6 expression is repressed through normal differentiation of GC B cells into plasma cells, a recent study showed the ectopic expression of BCL6 in primary multiple myeloma (MM) cells. However, the functional roles of BCL6 in MM cells are largely unknown. Here, we report that overexpression of BCL6 in a MM cell line, KMS12PE, induced transcriptional repression of ataxia telangiectasia mutated (ATM), a DDR signaling kinase, which was associated with a reduction in γH2AX formation after DNA damage. In contrast, transcription of known targets of BCL6 in GC B cells was not affected, suggesting a cell type-specific function of BCL6. To further investigate the effects of BCL6 overexpression on the MM cell line, we undertook mRNA sequence analysis and found an upregulation in the genomic mutator activation-induced cytidine deaminase (AID) with alteration in the gene expression profile, which is suggestive of de-differentiation from plasma cells. Moreover, interleukin-6 exposure to KMS12PE led to upregulation of BCL6 and AID, downregulation of ATM, and attenuation of DDR, which were consistent with the effects of BCL6 overexpression in this MM cell line. Taken together, these results indicated that overexpression of BCL6 alters gene expression profile and confers decreased DDR in MM cells. This phenotypic change could be reproduced by interleukin-6 stimulation, suggesting an important role of external stimuli in inducing genomic instability, which is a hallmark of MM cells. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Complex modulation of androgen responsive gene expression by methoxyacetic acid

PubMed Central

2011-01-01

Background Optimal androgen signaling is critical for testicular development and spermatogenesis. Methoxyacetic acid (MAA), the primary active metabolite of the industrial chemical ethylene glycol monomethyl ether, disrupts spermatogenesis and causes testicular atrophy. Transcriptional trans-activation studies have indicated that MAA can enhance androgen receptor activity, however, whether MAA actually impacts the expression of androgen-responsive genes in vivo, and which genes might be affected is not known. Methods A mouse TM3 Leydig cell line that stably expresses androgen receptor (TM3-AR) was prepared and analyzed by transcriptional profiling to identify target gene interactions between MAA and testosterone on a global scale. Results MAA is shown to have widespread effects on androgen-responsive genes, affecting processes ranging from apoptosis to ion transport, cell adhesion, phosphorylation and transcription, with MAA able to enhance, as well as antagonize, androgenic responses. Moreover, testosterone is shown to exert both positive and negative effects on MAA gene responses. Motif analysis indicated that binding sites for FOX, HOX, LEF/TCF, STAT5 and MEF2 family transcription factors are among the most highly enriched in genes regulated by testosterone and MAA. Notably, 65 FOXO targets were repressed by testosterone or showed repression enhanced by MAA with testosterone; these include 16 genes associated with developmental processes, six of which are Hox genes. Conclusions These findings highlight the complex interactions between testosterone and MAA, and provide insight into the effects of MAA exposure on androgen-dependent processes in a Leydig cell model. PMID:21453523

A novel interplay between HOTAIR and DNA methylation in osteosarcoma cells indicates a new therapeutic strategy.

PubMed

Li, Xingang; Lu, Hongming; Fan, Guilian; He, Miao; Sun, Yu; Xu, Kai; Shi, Fengjun

2017-11-01

Osteosarcoma (OS) is one of the most prevalent primary malignant bone tumors in adolescent. HOTAIR is highly expressed and associated with the epigenetic modifications, especially DNA methylation, in cancer. However, the regulation mechanism between HOTAIR and DNA methylation and the biological effects of them in the pathogenesis of osteosarcoma remains elusive. Through RNA-sequencing and computational analysis, followed by a variety of experimental validations, we report a novel interplay between HOTAIR, miR-126, and DNA methylation in OS. We found that HOTAIR is highly expressed in OS cells and the knockdown of HOTAIR leads to the down-regulation of DNMT1, as well as the decrease of global DNA methylation level. RNA-sequencing analysis of HOTAIR-regulated gene shows that CDKN2A is significantly repressed by HOTAIR. A series of experiments show that HOTAIR represses the expression of CDKN2A through inhibiting the promoter activity of CDKN2A by DNA hypermethylation. Further evidence shows that HOTAIR activates the expression of DNMT1 through repressing miR-126, which is the negative regulator of DNMT1. Functionally, HOTAIR depletion increases the sensibility of OS cells to DNMT1 inhibitor through regulating the viability and apoptosis of OS cells via HOTAIR-miR126-DNMT1-CDKN2A axis. These results not only enrich our understanding of the regulation relationship between non-coding RNA, DNA methylation, and gene expression, however, also provide a novel direction in developing more sophisticated therapeutic strategies for OS patients.

SNF5 Is an Essential Executor of Epigenetic Regulation during Differentiation

PubMed Central

You, Jueng Soo; De Carvalho, Daniel D.; Dai, Chao; Liu, Minmin; Pandiyan, Kurinji; Zhou, Xianghong J.; Liang, Gangning; Jones, Peter A.

2013-01-01

Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosome-depleted regions (NDRs) at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4-activated target genes such as OCT4 itself and NANOG. Furthermore, using loss- and gain-of-function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation. PMID:23637628

SNF5 is an essential executor of epigenetic regulation during differentiation.

PubMed

You, Jueng Soo; De Carvalho, Daniel D; Dai, Chao; Liu, Minmin; Pandiyan, Kurinji; Zhou, Xianghong J; Liang, Gangning; Jones, Peter A

2013-04-01

Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosome-depleted regions (NDRs) at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4-activated target genes such as OCT4 itself and NANOG. Furthermore, using loss- and gain-of-function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation.

Modulation of gene expression and cell cycle by botryosphaeran, a (1→3)(1→6)-β-d-glucan in human lymphocytes.

PubMed

Malini, Maressa; Souza, Marilesia Ferreira de; Oliveira, Marcelo Tempesta de; Antunes, Lusânia Maria Greggi; Figueiredo, Suely Gomes de; Barbosa, Aneli M; Dekker, Robert F H; Cólus, Ilce Mara de Syllos

2015-01-01

There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans. In the present study, the modulation of gene expression via RT-qPCR and cell cycle kinetics via flow cytometry were assessed in human normal and tumor (Jurkat) lymphocytes after treatment with botryosphaeran (a fungal (1→3)(1→6)-β-d-glucan) from Botryosphaeria rhodina MAMB-05. Cell cultures were treated with botryosphaeran either alone, or in combination with doxorubicin (DXR), in a post-treatment protocol. The expression of genes involved in immunomodulatory processes, apoptosis and cell cycle control, as well as β-d-glucans cell receptors were assessed. Flow cytometry analysis identified tetraploid Jurkat cells in G1 phase when treated with botryosphaeran combined with DXR. This antiproliferative effect in G1 may be associated with down-regulation of the expression of genes involved in the G1 checkpoint. The repression of the CCR5 gene following botryosphaeran treatment, either alone or in combination with DXR, in tumor lymphocytes indicates a possible affinity of this particular (1→3)(1→6)-β-d-glucan for the receptor CCR5. Therefore, botryosphaeran action appears to be involved in the repression of genes related to the G1 phase of the cell cycle and possibly in the interaction of the botryosphaeran, either alone, or in combination with DXR, with the CCR5 receptor. Copyright © 2015 Elsevier B.V. All rights reserved.

A tetracycline inducible expression vector for Corynebacterium glutamicum allowing tightly regulable gene expression.

PubMed

Lausberg, Frank; Chattopadhyay, Ava Rebecca; Heyer, Antonia; Eggeling, Lothar; Freudl, Roland

2012-09-01

Here we report on the construction of a tetracycline inducible expression vector that allows a tightly regulable gene expression in Corynebacterium glutamicum which is used in industry for production of small molecules such as amino acids. Using the green fluorescent protein (GFP) as a reporter protein we show that this vector, named pCLTON1, is characterized by tight repression under non-induced conditions as compared to a conventional IPTG inducible expression vector, and that it allows gradual GFP synthesis upon gradual increase of anhydrotetracycline addition. Copyright © 2012 Elsevier Inc. All rights reserved.

Functional characterization of EZH2β reveals the increased complexity of EZH2 isoforms involved in the regulation of mammalian gene expression

PubMed Central

2013-01-01

Background Histone methyltransferase enhancer of zeste homologue 2 (EZH2) forms an obligate repressive complex with suppressor of zeste 12 and embryonic ectoderm development, which is thought, along with EZH1, to be primarily responsible for mediating Polycomb-dependent gene silencing. Polycomb-mediated repression influences gene expression across the entire gamut of biological processes, including development, differentiation and cellular proliferation. Deregulation of EZH2 expression is implicated in numerous complex human diseases. To date, most EZH2-mediated function has been primarily ascribed to a single protein product of the EZH2 locus. Results We report that the EZH2 locus undergoes alternative splicing to yield at least two structurally and functionally distinct EZH2 methyltransferases. The longest protein encoded by this locus is the conventional enzyme, which we refer to as EZH2α, whereas EZH2β, characterized here, represents a novel isoform. We find that EZH2β localizes to the cell nucleus, complexes with embryonic ectoderm development and suppressor of zeste 12, trimethylates histone 3 at lysine 27, and mediates silencing of target promoters. At the cell biological level, we find that increased EZH2β induces cell proliferation, demonstrating that this protein is functional in the regulation of processes previously attributed to EZH2α. Biochemically, through the use of genome-wide expression profiling, we demonstrate that EZH2β governs a pattern of gene repression that is often ontologically redundant from that of EZH2α, but also divergent for a wide variety of specific target genes. Conclusions Combined, these results demonstrate that an expanded repertoire of EZH2 writers can modulate histone code instruction during histone 3 lysine 27-mediated gene silencing. These data support the notion that the regulation of EZH2-mediated gene silencing is more complex than previously anticipated and should guide the design and interpretation of future studies aimed at understanding the biochemical and biological roles of this important family of epigenomic regulators. PMID:23448518

A novel signal transduction pathway in Saccharomyces cerevisiae defined by Snf3-regulated expression of HXT6.

PubMed Central

Liang, H; Gaber, R F

1996-01-01

We show that cells deleted for SNF3, HXT1, HXT2, HXT3, HXT4, HXT6, and HXT7 do not take up glucose and cannot grow on media containing glucose as a sole carbon source. The expression of Hxt1, Hxt2, Hxt3, Hxt6, or Gal2 in these cells resulted in glucose transport and allowed growth on glucose media. In contrast, the expression of Snf3 failed to confer glucose uptake or growth on glucose. HXT6 is highly expressed on raffinose, low glucose, or nonfermentable carbon sources but is repressed in the presence of high concentrations of glucose. The maintenance of HXT6 glucose repression is strictly dependent on Snf3 and not on intracellular glucose. In snf3 delta cells expression of HXT6 is constitutive even when the entire repertoire of HXT genes is present and glucose uptake is abundant. In addition, glucose repression of HXT6 does not require glucose uptake by HXT1, HXT2, HXT3 or HXT4. We show that a signal transduction pathway defined by the Snf3-dependent hexose regulation of HXT6 is distinct from but also overlaps with general glucose regulation pathways in Saccharomyces cerevisiae. Finally, glucose repression of ADH2 and SUC2 is intact in snf3 delta hxt1 delta hxt2 delta hxt3 delta hxt4 delta hxt6 delta hxt7 delta gal2 cells, suggesting that the sensing and signaling mechanism for general glucose repression is independent from glucose uptake. Images PMID:8970157

BMPs regulate msx gene expression in the dorsal neuroectoderm of Drosophila and vertebrates by distinct mechanisms.

PubMed

Esteves, Francisco F; Springhorn, Alexander; Kague, Erika; Taylor, Erika; Pyrowolakis, George; Fisher, Shannon; Bier, Ethan

2014-09-01

In a broad variety of bilaterian species the trunk central nervous system (CNS) derives from three primary rows of neuroblasts. The fates of these neural progenitor cells are determined in part by three conserved transcription factors: vnd/nkx2.2, ind/gsh and msh/msx in Drosophila melanogaster/vertebrates, which are expressed in corresponding non-overlapping patterns along the dorsal-ventral axis. While this conserved suite of "neural identity" gene expression strongly suggests a common ancestral origin for the patterning systems, it is unclear whether the original regulatory mechanisms establishing these patterns have been similarly conserved during evolution. In Drosophila, genetic evidence suggests that Bone Morphogenetic Proteins (BMPs) act in a dosage-dependent fashion to repress expression of neural identity genes. BMPs also play a dose-dependent role in patterning the dorsal and lateral regions of the vertebrate CNS, however, the mechanism by which they achieve such patterning has not yet been clearly established. In this report, we examine the mechanisms by which BMPs act on cis-regulatory modules (CRMs) that control localized expression of the Drosophila msh and zebrafish (Danio rerio) msxB in the dorsal central nervous system (CNS). Our analysis suggests that BMPs act differently in these organisms to regulate similar patterns of gene expression in the neuroectoderm: repressing msh expression in Drosophila, while activating msxB expression in the zebrafish. These findings suggest that the mechanisms by which the BMP gradient patterns the dorsal neuroectoderm have reversed since the divergence of these two ancient lineages.

BMPs Regulate msx Gene Expression in the Dorsal Neuroectoderm of Drosophila and Vertebrates by Distinct Mechanisms

PubMed Central

Esteves, Francisco F.; Taylor, Erika; Pyrowolakis, George; Fisher, Shannon; Bier, Ethan

2014-01-01

In a broad variety of bilaterian species the trunk central nervous system (CNS) derives from three primary rows of neuroblasts. The fates of these neural progenitor cells are determined in part by three conserved transcription factors: vnd/nkx2.2, ind/gsh and msh/msx in Drosophila melanogaster/vertebrates, which are expressed in corresponding non-overlapping patterns along the dorsal-ventral axis. While this conserved suite of “neural identity” gene expression strongly suggests a common ancestral origin for the patterning systems, it is unclear whether the original regulatory mechanisms establishing these patterns have been similarly conserved during evolution. In Drosophila, genetic evidence suggests that Bone Morphogenetic Proteins (BMPs) act in a dosage-dependent fashion to repress expression of neural identity genes. BMPs also play a dose-dependent role in patterning the dorsal and lateral regions of the vertebrate CNS, however, the mechanism by which they achieve such patterning has not yet been clearly established. In this report, we examine the mechanisms by which BMPs act on cis-regulatory modules (CRMs) that control localized expression of the Drosophila msh and zebrafish (Danio rerio) msxB in the dorsal central nervous system (CNS). Our analysis suggests that BMPs act differently in these organisms to regulate similar patterns of gene expression in the neuroectoderm: repressing msh expression in Drosophila, while activating msxB expression in the zebrafish. These findings suggest that the mechanisms by which the BMP gradient patterns the dorsal neuroectoderm have reversed since the divergence of these two ancient lineages. PMID:25210771

P16INK4a MEDIATED SUPPRESSION OF TELOMERASE IN NORMAL AND MALIGNANT HUMAN BREAST CELLS

PubMed Central

Bazarov, Alexey V.; van Sluis, Marjolein; Hines, Curtis; Bassett, Ekaterina; Beliveau, Alain; Campeau, Eric; Mukhopadhyay, Rituparna; Lee, Won Jae; Melodyev, Sonya; Zaslavsky, Yuri; Lee, Leonard; Rodier, Francis; Chicas, Agustin; Lowe, Scott W.; Benhattar, Jean; Ren, Bing; Campisi, Judith; Yaswen, Paul

2010-01-01

Summary The cyclin-dependent kinase inhibitor p16INK4a (CDKN2A) is an important tumor-suppressor gene frequently inactivated in human tumors. p16 suppresses the development of cancer by triggering an irreversible arrest of cell proliferation termed cellular senescence. Here, we describe another anti-oncogenic function of p16 in addition to its ability to halt cell cycle progression. We show that transient expression of p16 stably represses the hTERT gene, encoding the catalytic subunit of telomerase, in both normal and malignant breast epithelial cells. Short-term p16 expression increases the amount of histone H3 trimethylated on lysine 27 (H3K27) bound to the hTERT promoter, resulting in transcriptional silencing, likely mediated by polycomb complexes. Our results indicate that transient p16 exposure may prevent malignant progression in dividing cells by irreversible repression of genes, such as hTERT, whose activity is necessary for extensive self-renewal. PMID:20569236

A polycomb repressive complex 2 gene regulates apogamy and gives evolutionary insights into early land plant evolution.

PubMed

Okano, Yosuke; Aono, Naoki; Hiwatashi, Yuji; Murata, Takashi; Nishiyama, Tomoaki; Ishikawa, Takaaki; Kubo, Minoru; Hasebe, Mitsuyasu

2009-09-22

Land plants have distinct developmental programs in haploid (gametophyte) and diploid (sporophyte) generations. Although usually the two programs strictly alternate at fertilization and meiosis, one program can be induced during the other program. In a process called apogamy, cells of the gametophyte other than the egg cell initiate sporophyte development. Here, we report for the moss Physcomitrella patens that apogamy resulted from deletion of the gene orthologous to the Arabidopsis thaliana CURLY LEAF (PpCLF), which encodes a component of polycomb repressive complex 2 (PRC2). In the deletion lines, a gametophytic vegetative cell frequently gave rise to a sporophyte-like body. This body grew indeterminately from an apical cell with the character of a sporophytic pluripotent stem cell but did not form a sporangium. Furthermore, with continued culture, the sporophyte-like body branched. Sporophyte branching is almost unknown among extant bryophytes. When PpCLF was expressed in the deletion lines once the sporophyte-like bodies had formed, pluripotent stem cell activity was arrested and a sporangium-like organ formed. Supported by the observed pattern of PpCLF expression, these results demonstrate that, in the gametophyte, PpCLF represses initiation of a sporophytic pluripotent stem cell and, in the sporophyte, represses that stem cell activity and induces reproductive organ development. In land plants, branching, along with indeterminate apical growth and delayed initiation of spore-bearing reproductive organs, were conspicuous innovations for the evolution of a dominant sporophyte plant body. Our study provides insights into the role of PRC2 gene regulation for sustaining evolutionary innovation in land plants.

Dt2 Is a Gain-of-Function MADS-Domain Factor Gene That Specifies Semideterminacy in Soybean[C][W

PubMed Central

Ping, Jieqing; Liu, Yunfeng; Sun, Lianjun; Zhao, Meixia; Li, Yinghui; She, Maoyun; Sui, Yi; Lin, Feng; Liu, Xiaodong; Tang, Zongxiang; Nguyen, Hanh; Tian, Zhixi; Qiu, Lijuan; Nelson, Randall L.; Clemente, Thomas E.; Specht, James E.; Ma, Jianxin

2014-01-01

Similar to Arabidopsis thaliana, the wild soybeans (Glycine soja) and many cultivars exhibit indeterminate stem growth specified by the shoot identity gene Dt1, the functional counterpart of Arabidopsis TERMINAL FLOWER1 (TFL1). Mutations in TFL1 and Dt1 both result in the shoot apical meristem (SAM) switching from vegetative to reproductive state to initiate terminal flowering and thus produce determinate stems. A second soybean gene (Dt2) regulating stem growth was identified, which, in the presence of Dt1, produces semideterminate plants with terminal racemes similar to those observed in determinate plants. Here, we report positional cloning and characterization of Dt2, a dominant MADS domain factor gene classified into the APETALA1/SQUAMOSA (AP1/SQUA) subfamily that includes floral meristem (FM) identity genes AP1, FUL, and CAL in Arabidopsis. Unlike AP1, whose expression is limited to FMs in which the expression of TFL1 is repressed, Dt2 appears to repress the expression of Dt1 in the SAMs to promote early conversion of the SAMs into reproductive inflorescences. Given that Dt2 is not the gene most closely related to AP1 and that semideterminacy is rarely seen in wild soybeans, Dt2 appears to be a recent gain-of-function mutation, which has modified the genetic pathways determining the stem growth habit in soybean. PMID:25005919

Oxygen-Dependent Transcriptional Regulator Hap1p Limits Glucose Uptake by Repressing the Expression of the Major Glucose Transporter Gene RAG1 in Kluyveromyces lactis▿

PubMed Central

Bao, Wei-Guo; Guiard, Bernard; Fang, Zi-An; Donnini, Claudia; Gervais, Michel; Passos, Flavia M. Lopes; Ferrero, Iliana; Fukuhara, Hiroshi; Bolotin-Fukuhara, Monique

2008-01-01

The HAP1 (CYP1) gene product of Saccharomyces cerevisiae is known to regulate the transcription of many genes in response to oxygen availability. This response varies according to yeast species, probably reflecting the specific nature of their oxidative metabolism. It is suspected that a difference in the interaction of Hap1p with its target genes may explain some of the species-related variation in oxygen responses. As opposed to the fermentative S. cerevisiae, Kluyveromyces lactis is an aerobic yeast species which shows different oxygen responses. We examined the role of the HAP1-equivalent gene (KlHAP1) in K. lactis. KlHap1p showed a number of sequence features and some gene targets (such as KlCYC1) in common with its S. cerevisiae counterpart, and KlHAP1 was capable of complementing the hap1 mutation. However, the KlHAP1 disruptant showed temperature-sensitive growth on glucose, especially at low glucose concentrations. At normal temperature, 28°C, the mutant grew well, the colony size being even greater than that of the wild type. The most striking observation was that KlHap1p repressed the expression of the major glucose transporter gene RAG1 and reduced the glucose uptake rate. This suggested an involvement of KlHap1p in the regulation of glycolytic flux through the glucose transport system. The ΔKlhap1 mutant showed an increased ability to produce ethanol during aerobic growth, indicating a possible transformation of its physiological property to Crabtree positivity or partial Crabtree positivity. Dual roles of KlHap1p in activating respiration and repressing fermentation may be seen as a basis of the Crabtree-negative physiology of K. lactis. PMID:18806211

Distinct Gene Expression Profiles in Peripheral Blood Mononuclear Cells from Patients Infected with Vaccinia Virus, Yellow Fever 17D Virus, or Upper Respiratory Infections Running Title: PBMC Expression Response to Viral Agents

PubMed Central

Scherer, Christina A.; Magness, Charles L.; Steiger, Kathryn V.; Poitinger, Nicholas D.; Caputo, Christine M.; Miner, Douglas G.; Winokur, Patricia L.; Klinzman, Donna; McKee, Janice; Pilar, Christine; Ward, Patricia A.; Gillham, Martha H.; Haulman, N. Jean; Stapleton, Jack T.; Iadonato, Shawn P.

2007-01-01

Gene expression in human peripheral blood mononuclear cells was systematically evaluated following smallpox and yellow fever vaccination, and naturally occurring upper respiratory infection (URI). All three infections were characterized by the induction of many interferon stimulated genes, as well as enhanced expression of genes involved in proteolysis and antigen presentation. Vaccinia infection was also characterized by a distinct expression signature composed of up-regulation of monocyte response genes, with repression of genes expressed by B and T-cells. In contrast, the yellow fever host response was characterized by a suppression of ribosomal and translation factors, distinguishing this infection from vaccinia and URI. No significant URI-specific signature was observed, perhaps reflecting greater heterogeneity in the study population and etiological agents. Taken together, these data suggest that specific host gene expression signatures may be identified that distinguish one or a small number of virus agents. PMID:17651872

A Role for Histone Deacetylases in the Cellular and Behavioral Mechanisms Underlying Learning and Memory

ERIC Educational Resources Information Center

Mahgoub, Melissa; Monteggia, Lisa M.

2014-01-01

Histone deacetylases (HDACs) are a family of chromatin remodeling enzymes that restrict access of transcription factors to the DNA, thereby repressing gene expression. In contrast, histone acetyltransferases (HATs) relax the chromatin structure allowing for an active chromatin state and promoting gene transcription. Accumulating data have…

Short-term transcriptional response of microbial communities to N-fertilization in pine forest soil

DOE Office of Scientific and Technical Information (OSTI.GOV)

Albright, Michaeline Burr Nelson; Johansen, Renee; Lopez, Deanna

Numerous studies have examined the long-term effect of experimental nitrogen (N) deposition in terrestrial ecosystems, however N-specific mechanistic markers are difficult to disentangle from responses to other environmental changes. The strongest picture of N-responsive mechanistic markers is likely to arise from measurements over a short (hours to days) timescale immediately after inorganic N deposition. Therefore, we assessed the short-term (3-day) transcriptional response of microbial communities in two soil strata from a pine forest to a high dose of N fertilization (c.a. 1mg/g of soil material) in laboratory microcosms. Here, we hypothesized that N fertilization would repress the expression of fungalmore » and bacterial genes linked to N-mining from plant litter. However, despite N-suppression of microbial respiration, the most pronounced differences in functional gene expression were between strata rather than in response to the N addition. Overall, ~4% of metabolic genes changed in expression with N addition, while three times as many (~12%) were significantly different across the different soil strata in the microcosms. In particular, we found little evidence of N changing expression levels of metabolic genes associated with complex carbohydrate degradation (CAZymes) or inorganic N utilization. This suggests that direct N repression of microbial functional gene expression is not the principle mechanism for reduced soil respiration immediately after N deposition. Instead, changes in expression with N addition occurred primarily in general cell maintenance areas, for example in ribosome-related transcripts. Transcriptional changes in functional gene abundance in response to N-addition observed in longer-term field studies likely results from changes in microbial composition.« less

Up-regulation of COUP-TFII gene expression in the nitrofen-induced hypoplastic lung.

PubMed

Doi, Takashi; Sugimoto, Kaoru; Puri, Prem

2009-02-01

Recent studies have suggested that the retinoid signaling pathway (RSP) is inhibited in the nitrofen-induced hypoplastic lung. The exact mechanism by which nitrofen acts in the RSP remains unclear. Targeted ablation of COUP-TFII, a gene encoding a transfactor regulated by the RSP, has been shown to cause Bochdalek-type congenital diaphragmatic hernia. It has been shown that COUP-TFII has 2 main roles in the RSP, (i) repressing the RSP by directly sequestering retinoid X receptors, thereby preventing heterodimerization to retinoid acid receptors and inhibiting gene transcription, and (ii) modulating the transcriptional activity of GATA proteins. We designed this study to investigate the gene expression of COUP-TFII in the nitrofen-induced hypoplastic lung. Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9 of gestation. Fetuses were harvested and lungs were dissected on day 15 (D15), D18, and D21 and divided into 2 groups: control (n = 9) and nitrofen (n = 9). Real-time reverse transcription-polymerase chain reaction was performed to evaluate the relative mRNA levels of COUP-TFII expression in the hypoplastic lung. The relative mRNA levels of COUP-TFII at D15 was significantly increased in the nitrofen group (0.76 +/- 0.53) compared to controls (0.45 +/- 0.05) (P < .01). The expression levels of COUP-TFII at D18 and D21 were not significantly different between the nitrofen group and controls. Our results provide evidence for the first time that the pulmonary gene expression of COUP-TFII is up-regulated in the early stages of lung development in the nitrofen-induced hypoplastic lung. We speculate that up-regulation of COUP-TFII gene expression during the stage of branching lung morphogenesis may cause pulmonary hypoplasia by repressing RSP.

Short-term transcriptional response of microbial communities to N-fertilization in pine forest soil

DOE PAGES

Albright, Michaeline Burr Nelson; Johansen, Renee; Lopez, Deanna; ...

2018-05-25

Numerous studies have examined the long-term effect of experimental nitrogen (N) deposition in terrestrial ecosystems, however N-specific mechanistic markers are difficult to disentangle from responses to other environmental changes. The strongest picture of N-responsive mechanistic markers is likely to arise from measurements over a short (hours to days) timescale immediately after inorganic N deposition. Therefore, we assessed the short-term (3-day) transcriptional response of microbial communities in two soil strata from a pine forest to a high dose of N fertilization (c.a. 1mg/g of soil material) in laboratory microcosms. Here, we hypothesized that N fertilization would repress the expression of fungalmore » and bacterial genes linked to N-mining from plant litter. However, despite N-suppression of microbial respiration, the most pronounced differences in functional gene expression were between strata rather than in response to the N addition. Overall, ~4% of metabolic genes changed in expression with N addition, while three times as many (~12%) were significantly different across the different soil strata in the microcosms. In particular, we found little evidence of N changing expression levels of metabolic genes associated with complex carbohydrate degradation (CAZymes) or inorganic N utilization. This suggests that direct N repression of microbial functional gene expression is not the principle mechanism for reduced soil respiration immediately after N deposition. Instead, changes in expression with N addition occurred primarily in general cell maintenance areas, for example in ribosome-related transcripts. Transcriptional changes in functional gene abundance in response to N-addition observed in longer-term field studies likely results from changes in microbial composition.« less

Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum.

PubMed

Li, Zhonghai; Yao, Guangshan; Wu, Ruimei; Gao, Liwei; Kan, Qinbiao; Liu, Meng; Yang, Piao; Liu, Guodong; Qin, Yuqi; Song, Xin; Zhong, Yaohua; Fang, Xu; Qu, Yinbo

2015-09-01

Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular β-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a "seesaw model" in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors.

Synergistic and Dose-Controlled Regulation of Cellulase Gene Expression in Penicillium oxalicum

PubMed Central

Li, Zhonghai; Yao, Guangshan; Wu, Ruimei; Gao, Liwei; Kan, Qinbiao; Liu, Meng; Yang, Piao; Liu, Guodong; Qin, Yuqi; Song, Xin; Zhong, Yaohua; Fang, Xu; Qu, Yinbo

2015-01-01

Filamentous fungus Penicillium oxalicum produces diverse lignocellulolytic enzymes, which are regulated by the combinations of many transcription factors. Here, a single-gene disruptant library for 470 transcription factors was constructed and systematically screened for cellulase production. Twenty transcription factors (including ClrB, CreA, XlnR, Ace1, AmyR, and 15 unknown proteins) were identified to play putative roles in the activation or repression of cellulase synthesis. Most of these regulators have not been characterized in any fungi before. We identified the ClrB, CreA, XlnR, and AmyR transcription factors as critical dose-dependent regulators of cellulase expression, the core regulons of which were identified by analyzing several transcriptomes and/or secretomes. Synergistic and additive modes of combinatorial control of each cellulase gene by these regulatory factors were achieved, and cellulase expression was fine-tuned in a proper and controlled manner. With one of these targets, the expression of the major intracellular β-glucosidase Bgl2 was found to be dependent on ClrB. The Bgl2-deficient background resulted in a substantial gene activation by ClrB and proved to be closely correlated with the relief of repression mediated by CreA and AmyR during cellulase induction. Our results also signify that probing the synergistic and dose-controlled regulation mechanisms of cellulolytic regulators and using it for reconstruction of expression regulation network (RERN) may be a promising strategy for cellulolytic fungi to develop enzyme hyper-producers. Based on our data, ClrB was identified as focal point for the synergistic activation regulation of cellulase expression by integrating cellulolytic regulators and their target genes, which refined our understanding of transcriptional-regulatory network as a “seesaw model” in which the coordinated regulation of cellulolytic genes is established by counteracting activators and repressors. PMID:26360497

Recruitment by the Repressor Freud-1 of Histone Deacetylase-Brg1 Chromatin Remodeling Complexes to Strengthen HTR1A Gene Repression.

PubMed

Souslova, Tatiana; Mirédin, Kim; Millar, Anne M; Albert, Paul R

2017-12-01

Five-prime repressor element under dual repression binding protein-1 (Freud-1)/CC2D1A is genetically linked to intellectual disability and implicated in neuronal development. Freud-1 represses the serotonin-1A (5-HT1A) receptor gene HTR1A by histone deacetylase (HDAC)-dependent or HDAC-independent mechanisms in 5-HT1A-negative (e.g., HEK-293) or 5-HT1A-expressing cells (SK-N-SH), respectively. To identify the underlying mechanisms, Freud-1-associated proteins were affinity-purified from HEK-293 nuclear extracts and members of the Brg1/SMARCCA chromatin remodeling and Sin3A-HDAC corepressor complexes were identified. Pull-down assays using recombinant proteins showed that Freud-1 interacts directly with the Brg1 carboxyl-terminal domain; interaction with Brg1 required the carboxyl-terminal of Freud-1. Freud-1 complexes in HEK-293 and SK-N-SH cells differed, with low levels of BAF170/SMARCC2 and BAF57/SMARCE1 in HEK-293 cells and low-undetectable BAF155/SMARCC1, Sin3A, and HDAC1/2 in SK-N-SH cells. Similarly, by quantitative chromatin immunoprecipitation, Brg1-BAF170/57 and Sin3A-HDAC complexes were observed at the HTR1A promoter in HEK-293 cells, whereas in SK-N-SH cells, Sin3A-HDAC proteins were not detected. Quantifying 5-HT1A receptor mRNA levels in cells treated with siRNA to Freud-1, Brg1, or both RNAs addressed the functional role of the Freud-1-Brg1 complex. In HEK-293 cells, 5-HT1A receptor mRNA levels were increased only when both Freud-1 and Brg1 were depleted, but in SK-N-SH cells, depletion of either protein upregulated 5-HT1A receptor RNA. Thus, recruitment by Freud-1 of Brg1, BAF155, and Sin3A-HDAC complexes appears to strengthen repression of the HTR1A gene to prevent its expression inappropriate cell types, while recruitment of the Brg1-BAF170/57 complex is permissive to 5-HT1A receptor expression. Alterations in Freud-1-Brg1 interactions in mutants associated with intellectual disability could impair gene repression leading to altered neuronal development.

Recruitment by the Repressor Freud-1 of Histone Deacetylase-Brg1 Chromatin Remodeling Complexes to Strengthen HTR1A Gene Repression

PubMed Central

Souslova, Tatiana; Mirédin, Kim; Millar, Anne M.

2017-01-01

Five-prime repressor element under dual repression binding protein-1 (Freud-1)/CC2D1A is genetically linked to intellectual disability and implicated in neuronal development. Freud-1 represses the serotonin-1A (5-HT1A) receptor gene HTR1A by histone deacetylase (HDAC)-dependent or HDAC-independent mechanisms in 5-HT1A-negative (e.g., HEK-293) or 5-HT1A-expressing cells (SK-N-SH), respectively. To identify the underlying mechanisms, Freud-1-associated proteins were affinity-purified from HEK-293 nuclear extracts and members of the Brg1/SMARCCA chromatin remodeling and Sin3A-HDAC corepressor complexes were identified. Pull-down assays using recombinant proteins showed that Freud-1 interacts directly with the Brg1 carboxyl-terminal domain; interaction with Brg1 required the carboxyl-terminal of Freud-1. Freud-1 complexes in HEK-293 and SK-N-SH cells differed, with low levels of BAF170/SMARCC2 and BAF57/SMARCE1 in HEK-293 cells and low-undetectable BAF155/SMARCC1, Sin3A, and HDAC1/2 in SK-N-SH cells. Similarly, by quantitative chromatin immuno-precipitation, Brg1-BAF170/57 and Sin3A-HDAC complexes were observed at the HTR1A promoter in HEK-293 cells, whereas in SK-N-SH cells, Sin3A-HDAC proteins were not detected. Quantifying 5-HT1A receptor mRNA levels in cells treated with siRNA to Freud-1, Brg1, or both RNAs addressed the functional role of the Freud-1-Brg1 complex. In HEK-293 cells, 5-HT1A receptor mRNA levels were increased only when both Freud-1 and Brg1 were depleted, but in SK-N-SH cells, depletion of either protein upregulated 5-HT1A receptor RNA. Thus, recruitment by Freud-1 of Brg1, BAF155, and Sin3A-HDAC complexes appears to strengthen repression of the HTR1A gene to prevent its expression inappropriate cell types, while recruitment of the Brg1-BAF170/57 complex is permissive to 5-HT1A receptor expression. Alterations in Freud-1-Brg1 interactions in mutants associated with intellectual disability could impair gene repression leading to altered neuronal development. PMID:27914010

Long noncoding RNA PANDA and scaffold-attachment-factor SAFA control senescence entry and exit.

PubMed

Puvvula, Pavan Kumar; Desetty, Rohini Devi; Pineau, Pascal; Marchio, Agnés; Moon, Anne; Dejean, Anne; Bischof, Oliver

2014-11-19

Cellular senescence is a stable cell cycle arrest that limits the proliferation of pre-cancerous cells. Here we demonstrate that scaffold-attachment-factor A (SAFA) and the long noncoding RNA PANDA differentially interact with polycomb repressive complexes (PRC1 and PRC2) and the transcription factor NF-YA to either promote or suppress senescence. In proliferating cells, SAFA and PANDA recruit PRC complexes to repress the transcription of senescence-promoting genes. Conversely, the loss of SAFA-PANDA-PRC interactions allows expression of the senescence programme. Accordingly, we find that depleting either SAFA or PANDA in proliferating cells induces senescence. However, in senescent cells where PANDA sequesters transcription factor NF-YA and limits the expression of NF-YA-E2F-coregulated proliferation-promoting genes, PANDA depletion leads to an exit from senescence. Together, our results demonstrate that PANDA confines cells to their existing proliferative state and that modulating its level of expression can cause entry or exit from senescence.

hnRNP-Q1 represses nascent axon growth in cortical neurons by inhibiting Gap-43 mRNA translation

PubMed Central

Williams, Kathryn R.; McAninch, Damian S.; Stefanovic, Snezana; Xing, Lei; Allen, Megan; Li, Wenqi; Feng, Yue; Mihailescu, Mihaela Rita; Bassell, Gary J.

2016-01-01

Posttranscriptional regulation of gene expression by mRNA-binding proteins is critical for neuronal development and function. hnRNP-Q1 is an mRNA-binding protein that regulates mRNA processing events, including translational repression. hnRNP-Q1 is highly expressed in brain tissue, suggesting a function in regulating genes critical for neuronal development. In this study, we have identified Growth-associated protein 43 (Gap-43) mRNA as a novel target of hnRNP-Q1 and have demonstrated that hnRNP-Q1 represses Gap-43 mRNA translation and consequently GAP-43 function. GAP-43 is a neuronal protein that regulates actin dynamics in growth cones and facilitates axonal growth. Previous studies have identified factors that regulate Gap-43 mRNA stability and localization, but it remains unclear whether Gap-43 mRNA translation is also regulated. Our results reveal that hnRNP-Q1 knockdown increased nascent axon length, total neurite length, and neurite number in mouse embryonic cortical neurons and enhanced Neuro2a cell process extension; these phenotypes were rescued by GAP-43 knockdown. Additionally, we have identified a G-quadruplex structure in the 5′ untranslated region of Gap-43 mRNA that directly interacts with hnRNP-Q1 as a means to inhibit Gap-43 mRNA translation. Therefore hnRNP-Q1–mediated repression of Gap-43 mRNA translation provides an additional mechanism for regulating GAP-43 expression and function and may be critical for neuronal development. PMID:26658614

Functional repression of PtSND2 represses growth and development by disturbing auxin biosynthesis, transport and signaling in transgenic poplar.

PubMed

Wang, Haihai; Tang, Renjie; Wang, Cuiting; Qi, Qi; Gai, Ying; Jiang, Xiangning; Zhang, Hongxia

2015-01-01

Using chimeric repressor silencing technology, we previously reported that functional repression of PtSND2 severely arrested wood formation in transgenic poplar (Populus). Here, we provide further evidence that auxin biosynthesis, transport and signaling were disturbed in these transgenic plants, leading to pleiotropic defects in their growth patterns, including inhibited leaf enlargement and vascular tissue development in the leaf central vein, suppressed cambial growth and fiber elongation in the stem, and arrested growth in the root system. Two transgenic lines, which displayed the most remarkable phenotypic deviation from the wild-type, were selected for detailed studies. In both transgenic lines, expression of genes for auxin biosynthesis, transport and signaling was down-regulated, and indole-3-acetic acid distribution was severely disturbed in the apical buds, leaves, stems and roots of field-grown transgenic plants. Transient transcription dual-luciferase assays of ProPtTYDC2::LUC, ProPttLAX2::LUC and ProPoptrIAA20.2::LUC in poplar protoplasts revealed that expression of auxin-related genes might be regulated by PtSND2 at the transcriptional level. All these results indicate that functional repression of PtSND2 altered auxin biosynthesis, transport and signaling, and thereby disturbed the normal growth and development of transgenic plants. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

A gene regulatory network armature for T-lymphocyte specification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fung, Elizabeth-sharon

Choice of a T-lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly-regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification; tests of the short-term Notchdependence of these gene expression changes; and perturbation analyses of the effects of overexpression of two essential transcription factors, namely PU.l and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through whichmore » T-cell precursors progress from primitive multipotency to T-lineage commitment. Distinct parts of the path reveal separate contributions of Notch signaling, GATA-3 activity, and downregulation of PU.l. Using BioTapestry, the results have been assembled into a draft gene regulatory network for the specification of T-cell precursors and the choice of T as opposed to myeloid dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfil against Egr-2 and of TCF-l against PU.l as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose-dependence of GATA-3 effects; the gene-specific modulation of PU.l activity based on Notch activity; the lack of direct opposition between PU.l and GATA-3; and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.« less

Development of a promoter shutoff system in Aspergillus oryzae using a sorbitol-sensitive promoter.

PubMed

Oda, Ken; Terado, Shiho; Toyoura, Rieko; Fukuda, Hisashi; Kawauchi, Moriyuki; Iwashita, Kazuhiro

2016-09-01

Promoter shutoff is a general method for analyzing essential genes, but in the fungus Aspergillus oryzae, no tightly repressed promoters have been reported. To overcome the current limitations of conditional promoters, we examined sorbitol- and galactose-responsive genes using microarrays to identify regulatable genes with only minor physiological and genetic effects. We identified two sorbitol-induced genes (designated as sorA and sorB), cloned their promoters, and built a regulated egfp and brlA expression system. Growth medium-dependent enhanced green fluorescence protein (EGFP) fluorescence and conidiation were confirmed for egfp and brlA under the control of their respective promoters. We also used this shutoff system to regulate the essential rhoA, which demonstrated the expected growth inhibition under repressed growth conditions. Our new sorbitol promoter shutoff system developed can serve as a valuable new tool for essential gene analyses of filamentous fungi.

Chromatin insulator elements: establishing barriers to set heterochromatin boundaries.

PubMed

Barkess, Gráinne; West, Adam G

2012-02-01

Epigenomic profiling has revealed that substantial portions of genomes in higher eukaryotes are organized into extensive domains of transcriptionally repressive chromatin. The boundaries of repressive chromatin domains can be fixed by DNA elements known as barrier insulators, to both shield neighboring gene expression and to maintain the integrity of chromosomal silencing. Here, we examine the current progress in identifying vertebrate barrier elements and their binding factors. We overview the design of the reporter assays used to define enhancer-blocking and barrier insulators. We look at the mechanisms vertebrate barrier proteins, such as USF1 and VEZF1, employ to counteract Polycomb- and heterochromatin-associated repression. We also undertake a critical analysis of whether CTCF could also act as a barrier protein. There is good evidence that barrier elements in vertebrates can form repressive chromatin domain boundaries. Future studies will determine whether barriers are frequently used to define repressive domain boundaries in vertebrates.

Gene expression profiling data of Schizosaccharomyces pombe under nitrosative stress using differential display.

PubMed

Biswas, Pranjal; Majumdar, Uddalak; Ghosh, Sanjay

2016-03-01

Excess production of nitric oxide (NO) and reactive nitrogen intermediates (RNIs) causes nitrosative stress on cells. Schizosaccharomyces pombe was used as a model to study nitrosative stress response. In the present data article, we have used differential display to identify the differentially expressed genes in the fission yeast under nitrosative stress conditions. We have used pure NO donor compound detaNONOate at final concentrations of 0.1 mM and 1 mM to treat the cells for 15 min alongside control before studying their gene expression profiles. At both the treated conditions, we identified genes which were commonly repressed while several genes were induced upon both 0.1 mM and 1 mM treatments. The differentially expressed genes were further analyzed in DAVID and categorized into several different pathways.

Kinetically-Defined Component Actions in Gene Repression

PubMed Central

Chow, Carson C.; Finn, Kelsey K.; Storchan, Geoffery B.; Lu, Xinping; Sheng, Xiaoyan; Simons, S. Stoney

2015-01-01

Gene repression by transcription factors, and glucocorticoid receptors (GR) in particular, is a critical, but poorly understood, physiological response. Among the many unresolved questions is the difference between GR regulated induction and repression, and whether transcription cofactor action is the same in both. Because activity classifications based on changes in gene product level are mechanistically uninformative, we present a theory for gene repression in which the mechanisms of factor action are defined kinetically and are consistent for both gene repression and induction. The theory is generally applicable and amenable to predictions if the dose-response curve for gene repression is non-cooperative with a unit Hill coefficient, which is observed for GR-regulated repression of AP1LUC reporter induction by phorbol myristate acetate. The theory predicts the mechanism of GR and cofactors, and where they act with respect to each other, based on how each cofactor alters the plots of various kinetic parameters vs. cofactor. We show that the kinetically-defined mechanism of action of each of four factors (reporter gene, p160 coactivator TIF2, and two pharmaceuticals [NU6027 and phenanthroline]) is the same in GR-regulated repression and induction. What differs is the position of GR action. This insight should simplify clinical efforts to differentially modulate factor actions in gene induction vs. gene repression. PMID:25816223

SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xia, Jun; Department of Respiratory Medicine, Jiangsu Provincial Hospital of Chinese Traditional Medicine; Wu, Xiaoyan

Highlights: Black-Right-Pointing-Pointer SIRT1 interacts with and deacetylates RFX5. Black-Right-Pointing-Pointer SIRT1 activation attenuates whereas SIRT1 inhibition enhances collagen repression by RFX5 in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 promotes cytoplasmic localization and proteasomal degradation of RFX5 and cripples promoter recruitment of RFX5. Black-Right-Pointing-Pointer IFN-{gamma} represses SIRT1 expression in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 agonist alleviates collagen repression by IFN-{gamma} in vascular smooth muscle cells. -- Abstract: Decreased expression of collagen by vascular smooth muscle cells (SMCs) within the atherosclerotic plaque contributes to the thinning of the fibrous cap and poses a great threat to plaque rupture. Elucidation of the mechanismmore » underlying repressed collagen type I (COL1A2) gene would potentially provide novel solutions that can prevent rupture-induced complications. We have previously shown that regulatory factor for X-box (RFX5) binds to the COL1A2 transcription start site and represses its transcription. Here we report that SIRT1, an NAD-dependent, class III deacetylase, forms a complex with RFX5. Over-expression of SIRT1 or NAMPT, which synthesizes NAD+ to activate SIRT1, or treatment with the SIRT1 agonist resveratrol decreases RFX5 acetylation and disrupts repression of the COL1A2 promoter activity by RFX5. On the contrary, knockdown of SIRT1 or treatment with SIRT1 inhibitors induces RFX5 acetylation and enhances the repression of collagen transcription. SIRT1 antagonizes RFX5 activity by promoting its nuclear expulsion and proteasomal degradation hence dampening its binding to the COL1A2 promoter. The pro-inflammatory cytokine IFN-{gamma} represses COL1A2 transcription by down-regulating SIRT1 expression in SMCs. Therefore, our data have identified as novel pathway whereby SIRT1 maintains collagen synthesis in SMCs by modulating RFX5 activity.« less

SHORT VEGETATIVE PHASE Up-Regulates TEMPRANILLO2 Floral Repressor at Low Ambient Temperatures1[OPEN

PubMed Central

Marín-González, Esther; Matías-Hernández, Luis; Aguilar-Jaramillo, Andrea E.; Lee, Jeong Hwan; Ahn, Ji Hoon; Suárez-López, Paula; Pelaz, Soraya

2015-01-01

Plants integrate day length and ambient temperature to determine the optimal timing for developmental transitions. In Arabidopsis (Arabidopsis thaliana), the floral integrator FLOWERING LOCUS T (FT) and its closest homolog TWIN SISTER OF FT promote flowering in response to their activator CONSTANS under long-day inductive conditions. Low ambient temperature (16°C) delays flowering, even under inductive photoperiods, through repression of FT, revealing the importance of floral repressors acting at low temperatures. Previously, we have reported that the floral repressors TEMPRANILLO (TEM; TEM1 and TEM2) control flowering time through direct regulation of FT at 22°C. Here, we show that tem mutants are less sensitive than the wild type to changes in ambient growth temperature, indicating that TEM genes may play a role in floral repression at 16°C. Moreover, we have found that TEM2 directly represses the expression of FT and TWIN SISTER OF FT at 16°C. In addition, the floral repressor SHORT VEGETATIVE PHASE (SVP) directly regulates TEM2 but not TEM1 expression at 16°C. Flowering time analyses of svp tem mutants indicate that TEM may act in the same genetic pathway as SVP to repress flowering at 22°C but that SVP and TEM are partially independent at 16°C. Thus, TEM2 partially mediates the temperature-dependent function of SVP at low temperatures. Taken together, our results indicate that TEM genes are also able to repress flowering at low ambient temperatures under inductive long-day conditions. PMID:26243615

Nitrogen Metabolite Repression of Metabolism and Virulence in the Human Fungal Pathogen Cryptococcus neoformans

PubMed Central

Lee, I. Russel; Chow, Eve W. L.; Morrow, Carl A.; Djordjevic, Julianne T.; Fraser, James A.

2011-01-01

Proper regulation of metabolism is essential to maximizing fitness of organisms in their chosen environmental niche. Nitrogen metabolite repression is an example of a regulatory mechanism in fungi that enables preferential utilization of easily assimilated nitrogen sources, such as ammonium, to conserve resources. Here we provide genetic, transcriptional, and phenotypic evidence of nitrogen metabolite repression in the human pathogen Cryptococcus neoformans. In addition to loss of transcriptional activation of catabolic enzyme-encoding genes of the uric acid and proline assimilation pathways in the presence of ammonium, nitrogen metabolite repression also regulates the production of the virulence determinants capsule and melanin. Since GATA transcription factors are known to play a key role in nitrogen metabolite repression, bioinformatic analyses of the C. neoformans genome were undertaken and seven predicted GATA-type genes were identified. A screen of these deletion mutants revealed GAT1, encoding the only global transcription factor essential for utilization of a wide range of nitrogen sources, including uric acid, urea, and creatinine—three predominant nitrogen constituents found in the C. neoformans ecological niche. In addition to its evolutionarily conserved role in mediating nitrogen metabolite repression and controlling the expression of catabolic enzyme and permease-encoding genes, Gat1 also negatively regulates virulence traits, including infectious basidiospore production, melanin formation, and growth at high body temperature (39°–40°). Conversely, Gat1 positively regulates capsule production. A murine inhalation model of cryptococcosis revealed that the gat1Δ mutant is slightly more virulent than wild type, indicating that Gat1 plays a complex regulatory role during infection. PMID:21441208

DACH1 inhibits transforming growth factor-beta signaling through binding Smad4.

PubMed

Wu, Kongming; Yang, Ying; Wang, Chenguang; Davoli, Maria A; D'Amico, Mark; Li, Anping; Cveklova, Kveta; Kozmik, Zbynek; Lisanti, Michael P; Russell, Robert G; Cvekl, Ales; Pestell, Richard G

2003-12-19

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, we hypothesized that DACH1 might play a similar cellular function. Herein, DACH1 was found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-beta (TGF-beta)-induced apoptosis. DACH1 repressed TGF-beta induction of AP-1 and Smad signaling in gene reporter assays and repressed endogenous TGF-beta-responsive genes by microarray analyses. DACH1 bound to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localized with NCoR in nuclear dotlike structures. NCoR enhanced DACH1 repression, and the repression of TGF-beta-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH was sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibited the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-beta signaling by interacting with NCoR and Smad4.

Regulation of Kruppel-like factor 4, 9, and 13 genes and the steroidogenic genes LDLR, StAR, and CYP11A in ovarian granulosa cells.

PubMed

Natesampillai, Sekar; Kerkvliet, Jason; Leung, Peter C K; Veldhuis, Johannes D

2008-02-01

Kruppel-like factors (KLFs) are important Sp1-like eukaryotic transcriptional proteins. The LDLR, StAR, and CYP11A genes exhibit GC-rich Sp1-like sites, which have the potential to bind KLFs in multiprotein complexes. We now report that KLF4, KLF9, and KLF13 transcripts are expressed in and regulate ovarian cells. KLF4 and 13, but not KLF9, mRNA expression was induced and then repressed over time (P < 0.001). Combined LH and IGF-I stimulation increased KLF4 mRNA at 2 h (P < 0.01), whereas LH decreased KLF13 mRNA at 6 h (P < 0.05), and IGF-I reduced KLF13 at 24 h (P < 0.01) compared with untreated control. KLF9 was not regulated by either hormone. Transient transfection of KLF4, KLF9, and KLF13 suppressed LDLR/luc, StAR/luc, and CYP11A/luc by 80-90% (P < 0.001). Histone-deacetylase (HDAC) inhibitors stimulated LDLR/luc five- to sixfold and StAR/luc and CYP11A/luc activity twofold (P < 0.001) and partially reversed suppression by all three KLFs (P < 0.001). Deletion of the zinc finger domain of KLF13 abrogated repression of LDLR/luc. Lentiviral overexpression of the KLF13 gene suppressed LDLR mRNA (P < 0.001) and CYP11A mRNA (P = 0.003) but increased StAR mRNA (P = 0.007). Collectively, these data suggest that KLFs may recruit inhibitory complexes containing HDAC corepressors, thereby repressing LDLR and CYP11A transcription. Conversely, KLF13 may recruit unknown coactivators or stabilize StAR mRNA, thereby explaining enhancement of in situ StAR gene expression. These data introduce new potent gonadal transregulators of genes encoding proteins that mediate sterol uptake and steroid biosynthesis.

Global alterations of the transcriptional landscape during yeast growth and development in the absence of Ume6-dependent chromatin modification

PubMed Central

Lardenois, Aurélie; Becker, Emmanuelle; Walther, Thomas; Law, Michael J.; Xie, Bingning; Demougin, Philippe; Strich, Randy

2017-01-01

Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MATa/α wild-type and ume6/ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes. PMID:25957495

Global alterations of the transcriptional landscape during yeast growth and development in the absence of Ume6-dependent chromatin modification.

PubMed

Lardenois, Aurélie; Becker, Emmanuelle; Walther, Thomas; Law, Michael J; Xie, Bingning; Demougin, Philippe; Strich, Randy; Primig, Michael

2015-10-01

Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MAT a/α wild-type and ume6/ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes.

Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads.

PubMed

Rombel, I T; McMorran, B J; Lamont, I L

1995-02-20

Many bacteria respond to a lack of iron in the environment by synthesizing siderophores, which act as iron-scavenging compounds. Fluorescent pseudomonads synthesize strain-specific but chemically related siderophores called pyoverdines or pseudobactins. We have investigated the mechanisms by which iron controls expression of genes involved in pyoverdine metabolism in Pseudomonas aeruginosa. Transcription of these genes is repressed by the presence of iron in the growth medium. Three promoters from these genes were cloned and the activities of the promoters were dependent on the amounts of iron in the growth media. Two of the promoters were sequenced and the transcriptional start site were identified by S1 nuclease analysis. Sequences similar to the consensus binding site for the Fur repressor protein, which controls expression of iron-repressible genes in several gram-negative species, were not present in the promoters, suggesting that they are unlikely to have a high affinity for Fur. However, comparison of the promoter sequences with those of iron-regulated genes from other Pseudomonas species and also the iron-regulated exotoxin gene of P. aeruginosa allowed identification of a shared sequence element, with the consensus sequence (G/C)CTAAAT-CCC, which is likely to act as a binding site for a transcriptional activator protein. Mutations in this sequence greatly reduced the activities of the promoters characterized here as well as those of other iron-regulated promoters. The requirement for this motif in the promoters of iron-regulated genes of different Pseudomonas species indicates that similar mechanisms are likely to be involved in controlling expression of a range of iron-regulated genes in pseudomonads.

Structural Insights into the Regulation of Foreign Genes in Salmonella by the Hha/H-NS Complex*

PubMed Central

Ali, Sabrina S.; Whitney, John C.; Stevenson, James; Robinson, Howard; Howell, P. Lynne; Navarre, William Wiley

2013-01-01

The bacterial nucleoid-associated proteins Hha and H-NS jointly repress horizontally acquired genes in Salmonella, including essential virulence loci encoded within Salmonella pathogenicity islands. Hha is known to interact with the N-terminal dimerization domain of H-NS; however, the manner in which this interaction enhances transcriptional silencing is not understood. To further understand this process, we solved the x-ray crystal structure of Hha in complex with the N-terminal dimerization domain of H-NS (H-NS(1–46)) to 3.2 Å resolution. Two monomers of Hha bind to symmetrical sites on either side of the H-NS(1–46) dimer. Disruption of the Hha/H-NS interaction by the H-NS site-specific mutation I11A results in increased expression of the Hha/H-NS co-regulated gene hilA without affecting the expression levels of proV, a target gene repressed by H-NS in an Hha-independent fashion. Examination of the structure revealed a cluster of conserved basic amino acids that protrude from the surface of Hha on the opposite side of the Hha/H-NS(1–46) interface. Hha mutants with a diminished positively charged surface maintain the ability to interact with H-NS but can no longer regulate hilA. Increased expression of the hilA locus did not correspond to significant depletion of H-NS at the promoter region in chromatin immunoprecipitation assays. However, in vitro, we find Hha improves H-NS binding to target DNA fragments. Taken together, our results show for the first time how Hha and H-NS interact to direct transcriptional repression and reveal that a positively charged surface of Hha enhances the silencing activity of H-NS nucleoprotein filaments. PMID:23515315

Global repression of host-associated genes of the Lyme disease spirochete through post-transcriptional modulation of the alternative sigma factor RpoS.

PubMed

Dulebohn, Daniel P; Hayes, Beth M; Rosa, Patricia A

2014-01-01

Borrelia burgdorferi, the agent of Lyme disease, is a vector-borne pathogen that transits between Ixodes ticks and vertebrate hosts. During the natural infectious cycle, spirochetes must globally adjust their transcriptome to survive in these dissimilar environments. One way B. burgdorferi accomplishes this is through the use of alternative sigma factors to direct transcription of specific genes. RpoS, one of only three sigma factors in B. burgdorferi, controls expression of genes required during tick-transmission and infection of the mammalian host. How spirochetes switch between different sigma factors during the infectious cycle has remained elusive. Here we establish a role for a novel protein, BBD18, in the regulation of the virulence-associated sigma factor RpoS. Constitutive expression of BBD18 repressed transcription of RpoS-dependent genes to levels equivalent to those observed in an rpoS mutant. Consistent with the global loss of RpoS-dependent transcripts, we were unable to detect RpoS protein. However, constitutive expression of BBD18 did not diminish the amount of rpoS transcript, indicating post-transcriptional regulation of RpoS by BBD18. Interestingly, BBD18-mediated repression of RpoS is independent of both the rpoS promoter and the 5' untranslated region, suggesting a mechanism of protein destabilization rather than translational control. We propose that BBD18 is a novel regulator of RpoS and its activity likely represents a first step in the transition from an RpoS-ON to an RpoS-OFF state, when spirochetes transition from the host to the tick vector.

Pheromone-Regulated Expression of Sex Pheromone Plasmid pAD1-Encoded Aggregation Substance Depends on at Least Six Upstream Genes and a cis-Acting, Orientation-Dependent Factor

PubMed Central

Muscholl-Silberhorn, Albrecht B.

2000-01-01

Conjugative transfer of Enterococcus faecalis-specific sex pheromone plasmids relies on an adhesin, called aggregation substance, to confer a tight cell-to-cell contact between the mating partners. To analyze the dependence of pAD1-encoded aggregation substance, Asa1, on pheromone induction, a variety of upstream fragments were fused to an α-amylase reporter gene, amyL, by use of a novel promoter probe vector, pAMY-em1. For pheromone-regulated α-amylase activity, a total of at least six genes, traB, traC, traA, traE1, orfY, and orf1, are required: TraB efficiently represses asa1 (by a mechanism unrelated to its presumptive function in pheromone shutdown, since a complete shutdown is observed exclusively in the presence of traC); only traC can relieve traB-mediated repression in a pheromone-dependent manner. In addition to traB, traA is required but not sufficient for negative control. Mutational inactivation of traE1, orfY, or orf1, respectively, results in a total loss of α-amylase activity for constructs normally mediating constitutive expression. Inversion of a fragment covering traA, P0, and traE1 without disrupting any gene or control element switches off amyL or asa1 expression, indicating the involvement of a cis-acting, orientation-dependent factor (as had been shown for plasmid pCF10). Unexpectedly, pAD1 represses all pAMY-em1 derivatives in trans, while its own pheromone-dependent functions are unaffected. The discrepancy between the new data and those of former studies defining TraE1 as a trans-acting positive regulator is discussed. PMID:10850999

Visualizing Changes in Cdkn1c Expression Links Early-Life Adversity to Imprint Mis-regulation in Adults.

PubMed

Van de Pette, Mathew; Abbas, Allifia; Feytout, Amelie; McNamara, Gráinne; Bruno, Ludovica; To, Wilson K; Dimond, Andrew; Sardini, Alessandro; Webster, Zoe; McGinty, James; Paul, Eleanor J; Ungless, Mark A; French, Paul M W; Withers, Dominic J; Uren, Anthony; Ferguson-Smith, Anne C; Merkenschlager, Matthias; John, Rosalind M; Fisher, Amanda G

2017-01-31

Imprinted genes are regulated according to parental origin and can influence embryonic growth and metabolism and confer disease susceptibility. Here, we designed sensitive allele-specific reporters to non-invasively monitor imprinted Cdkn1c expression in mice and showed that expression was modulated by environmental factors encountered in utero. Acute exposure to chromatin-modifying drugs resulted in de-repression of paternally inherited (silent) Cdkn1c alleles in embryos that was temporary and resolved after birth. In contrast, deprivation of maternal dietary protein in utero provoked permanent de-repression of imprinted Cdkn1c expression that was sustained into adulthood and occurred through a folate-dependent mechanism of DNA methylation loss. Given the function of imprinted genes in regulating behavior and metabolic processes in adults, these results establish imprinting deregulation as a credible mechanism linking early-life adversity to later-life outcomes. Furthermore, Cdkn1c-luciferase mice offer non-invasive tools to identify factors that disrupt epigenetic processes and strategies to limit their long-term impact. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Transcription factor-dependent chromatin remodeling of Il18r1 during Th1 and Th2 differentiation 1

PubMed Central

Yu, Qing; Chang, Hua-Chen; Ahyi, Ayele-Nati N.; Kaplan, Mark H.

2008-01-01

The IL-18Rα chain is expressed on Th1 but not Th2 cells. We have recently shown that Stat4 is an important component of programming the Il18r1 locus (encoding IL-18Rα) for maximal expression in Th1 cells. Il18r1 is reciprocally repressed during Th2 development. In this report we demonstrate that the establishment of DNase hypersensitivity patterns that are distinct among undifferentiated CD4 T cells, Th1 and Th2 cells. Stat6 is required for the repression of Il18r1 expression and in Stat6-deficient Th2 cultures, mRNA levels, histone acetylation and H3K4 methylation levels are intermediate between levels observed in Th1 and Th2 cells. Despite the repressive effects of IL-4 during Th2 differentiation, we observed only modest binding of Stat6 to the Il18r1 locus. In contrast, we observed robust GATA-3 binding to a central region of the locus where DNase hypersensitivity sites overlapped with conserved non-coding sequences in Il18r1 introns. Ectopic expression of GATA-3 in differentiated Th1 cells repressed Il18r1 mRNA and surface expression of IL-18Rα. These data provide further mechanistic insight into transcription factor dependent establishment of Th subset-specific patterns of gene expression. PMID:18714006

Orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) protein negatively regulates bone morphogenetic protein 2-induced osteoblast differentiation through suppressing runt-related gene 2 (Runx2) activity.

PubMed

Lee, Kkot-Nim; Jang, Won-Gu; Kim, Eun-Jung; Oh, Sin-Hye; Son, Hye-Ju; Kim, Sun-Hun; Franceschi, Renny; Zhang, Xiao-Kun; Lee, Shee-Eun; Koh, Jeong-Tae

2012-06-01

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway*

PubMed Central

Foda, Bardees M.; Singh, Upinder

2015-01-01

RNA interference (RNAi) is a fundamental biological process that plays a crucial role in regulation of gene expression in many organisms. Transcriptional gene silencing (TGS) is one of the important nuclear roles of RNAi. Our previous data show that Entamoeba histolytica has a robust RNAi pathway that links to TGS via Argonaute 2-2 (Ago2-2) associated 27-nucleotide small RNAs with 5′-polyphosphate termini. Here, we report the first repressive histone mark to be identified in E. histolytica, dimethylation of H3K27 (H3K27Me2), and demonstrate that it is enriched at genes that are silenced by RNAi-mediated TGS. An RNAi-silencing trigger can induce H3K27Me2 deposits at both episomal and chromosomal loci, mediating gene silencing. Our data support two phases of RNAi-mediated TGS: an active silencing phase where the RNAi trigger is present and both H3K27Me2 and Ago2-2 concurrently enrich at chromosomal loci; and an established silencing phase in which the RNAi trigger is removed, but gene silencing with H3K27Me2 enrichment persist independently of Ago2-2 deposition. Importantly, some genes display resistance to chromosomal silencing despite induction of functional small RNAs. In those situations, the RNAi-triggering plasmid that is maintained episomally gets partially silenced and has H3K27Me2 enrichment, but the chromosomal copy displays no repressive histone enrichment. Our data are consistent with a model in which H3K27Me2 is a repressive histone modification, which is strongly associated with transcriptional repression. This is the first example of an epigenetic histone modification that functions to mediate RNAi-mediated TGS in the deep-branching eukaryote E. histolytica. PMID:26149683

DAF-16 and TCER-1 Facilitate Adaptation to Germline Loss by Restoring Lipid Homeostasis and Repressing Reproductive Physiology in C. elegans

PubMed Central

Amrit, Francis Raj Gandhi; Steenkiste, Elizabeth Marie; Ratnappan, Ramesh; Chen, Shaw-Wen; McClendon, T. Brooke; Kostka, Dennis; Yanowitz, Judith; Olsen, Carissa Perez; Ghazi, Arjumand

2016-01-01

Elimination of the proliferating germline extends lifespan in C. elegans. This phenomenon provides a unique platform to understand how complex metazoans retain metabolic homeostasis when challenged with major physiological perturbations. Here, we demonstrate that two conserved transcription regulators essential for the longevity of germline-less adults, DAF-16/FOXO3A and TCER-1/TCERG1, concurrently enhance the expression of multiple genes involved in lipid synthesis and breakdown, and that both gene classes promote longevity. Lipidomic analyses revealed that key lipogenic processes, including de novo fatty acid synthesis, triglyceride production, desaturation and elongation, are augmented upon germline removal. Our data suggest that lipid anabolic and catabolic pathways are coordinately augmented in response to germline loss, and this metabolic shift helps preserve lipid homeostasis. DAF-16 and TCER-1 also perform essential inhibitory functions in germline-ablated animals. TCER-1 inhibits the somatic gene-expression program that facilitates reproduction and represses anti-longevity genes, whereas DAF-16 impedes ribosome biogenesis. Additionally, we discovered that TCER-1 is critical for optimal fertility in normal adults, suggesting that the protein acts as a switch supporting reproductive fitness or longevity depending on the presence or absence of the germline. Collectively, our data offer insights into how organisms adapt to changes in reproductive status, by utilizing the activating and repressive functions of transcription factors and coordinating fat production and degradation. PMID:26862916

Isolation of the MIG1 gene from Candida albicans and effects of its disruption on catabolite repression.

PubMed

Zaragoza, O; Rodríguez, C; Gancedo, C

2000-01-01

We have cloned a Candida albicans gene (CaMIG1) that encodes a protein homologous to the DNA-binding protein Mig1 from Saccharomyces cerevisiae (ScMig1). The C. albicans Mig1 protein (CaMig1) differs from ScMig1, in that, among other things, it lacks a putative phosphorylation site for Snf1 and presents several long stretches rich in glutamine or in asparagine, serine, and threonine and has the effector domain located at some distance (50 amino acids) from the carboxy terminus. Expression of CaMIG1 was low and was similar in glucose-, sucrose-, or ethanol-containing media. Disruption of the two CaMIG1 genomic copies had no effect in filamentation or infectivity. Levels of a glucose-repressible alpha-glucosidase, implicated in both sucrose and maltose utilization, were similar in wild-type or mig1/mig1 cells. Disruption of CaMIG1 had also no effect on the expression of the glucose-repressed gene CaGAL1. CaMIG1 was functional in S. cerevisiae, as judged by its ability to suppress the phenotypes produced by mig1 or tps1 mutations. In addition, CaMig1 formed specific complexes with the URS1 region of the S. cerevisiae FBP1 gene. The existence of a possible functional analogue of CaMIG1 in C. albicans was suggested by the results of band shift experiments.

Isolation of the MIG1 Gene from Candida albicans and Effects of Its Disruption on Catabolite Repression

PubMed Central

Zaragoza, Oscar; Rodríguez, Cristina; Gancedo, Carlos

2000-01-01

We have cloned a Candida albicans gene (CaMIG1) that encodes a protein homologous to the DNA-binding protein Mig1 from Saccharomyces cerevisiae (ScMig1). The C. albicans Mig1 protein (CaMig1) differs from ScMig1, in that, among other things, it lacks a putative phosphorylation site for Snf1 and presents several long stretches rich in glutamine or in asparagine, serine, and threonine and has the effector domain located at some distance (50 amino acids) from the carboxy terminus. Expression of CaMIG1 was low and was similar in glucose-, sucrose-, or ethanol-containing media. Disruption of the two CaMIG1 genomic copies had no effect in filamentation or infectivity. Levels of a glucose-repressible α-glucosidase, implicated in both sucrose and maltose utilization, were similar in wild-type or mig1/mig1 cells. Disruption of CaMIG1 had also no effect on the expression of the glucose-repressed gene CaGAL1. CaMIG1 was functional in S. cerevisiae, as judged by its ability to suppress the phenotypes produced by mig1 or tps1 mutations. In addition, CaMig1 formed specific complexes with the URS1 region of the S. cerevisiae FBP1 gene. The existence of a possible functional analogue of CaMIG1 in C. albicans was suggested by the results of band shift experiments. PMID:10629176

Functional analysis of sporophytic transcripts repressed by the female gametophyte in the ovule of Arabidopsis thaliana.

PubMed

Armenta-Medina, Alma; Huanca-Mamani, Wilson; Sanchez-León, Nidia; Rodríguez-Arévalo, Isaac; Vielle-Calzada, Jean-Philippe

2013-01-01

To investigate the genetic and molecular regulation that the female gametophyte could exert over neighboring sporophytic regions of the ovule, we performed a quantitative comparison of global expression in wild-type and nozzle/sporocyteless (spl) ovules of Arabidopsis thaliana (Arabidopsis), using Massively Parallel Signature Sequencing (MPSS). This comparison resulted in 1517 genes showing at least 3-fold increased expression in ovules lacking a female gametophyte, including those encoding 89 transcription factors, 50 kinases, 25 proteins containing a RNA-recognition motif (RRM), and 20 WD40 repeat proteins. We confirmed that eleven of these genes are either preferentially expressed or exclusive of spl ovules lacking a female gametophyte as compared to wild-type, and showed that six are also upregulated in determinant infertile1 (dif1), a meiotic mutant affected in a REC8-like cohesin that is also devoided of female gametophytes. The sporophytic misexpression of IOREMPTE, a WD40/transducin repeat gene that is preferentially expressed in the L1 layer of spl ovules, caused the arrest of female gametogenesis after differentiation of a functional megaspore. Our results show that in Arabidopsis, the sporophytic-gametophytic cross talk includes a negative regulation of the female gametophyte over specific genes that are detrimental for its growth and development, demonstrating its potential to exert a repressive control over neighboring regions in the ovule.

The H3K27me3-demethylase KDM6A is suppressed in breast cancer stem-like cells, and enables the resolution of bivalency during the mesenchymal-epithelial transition

PubMed Central

Taube, Joseph H.; Sphyris, Nathalie; Johnson, Kelsey S.; Reisenauer, Keighley N.; Nesbit, Taylor A.; Joseph, Robiya; Vijay, Geraldine V.; Sarkar, Tapasree R.; Bhangre, Neeraja A.; Song, Joon Jin; Chang, Jeffrey T.; Lee, Min Gyu; Soundararajan, Rama; Mani, Sendurai A.

2017-01-01

The deposition of the activating H3K4me3 and repressive H3K27me3 histone modifications within the same promoter, forming a so-called bivalent domain, maintains gene expression in a repressed but transcription-ready state. We recently reported a significantly increased incidence of bivalency following an epithelial-mesenchymal transition (EMT), a process associated with the initiation of the metastatic cascade. The reverse process, known as the mesenchymal-epithelial transition (MET), is necessary for efficient colonization. Here, we identify numerous genes associated with differentiation, proliferation and intercellular adhesion that are repressed through the acquisition of bivalency during EMT, and re-expressed following MET. The majority of EMT-associated bivalent domains arise through H3K27me3 deposition at H3K4me3-marked promoters. Accordingly, we show that the expression of the H3K27me3-demethylase KDM6A is reduced in cells that have undergone EMT, stem-like subpopulations of mammary cell lines and stem cell-enriched triple-negative breast cancers. Importantly, KDM6A levels are restored following MET, concomitant with CDH1/E-cadherin reactivation through H3K27me3 removal. Moreover, inhibition of KDM6A, using the H3K27me3-demethylase inhibitor GSK-J4, prevents the re-expression of bivalent genes during MET. Our findings implicate KDM6A in the resolution of bivalency accompanying MET, and suggest KDM6A inhibition as a viable strategy to suppress metastasis formation in breast cancer. PMID:29029452

Characterization and Use of Catabolite-Repressed Promoters from Gluconate Genes in Corynebacterium glutamicum†

PubMed Central

Letek, Michal; Valbuena, Noelia; Ramos, Angelina; Ordóñez, Efrén; Gil, José A.; Mateos, Luís M.

2006-01-01

The genes involved in gluconate catabolism (gntP and gntK) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis. In C. glutamicum, gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (PgntK and PgntP) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or egfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on PgntP and PgntK fragments was detected only in the presence of cAMP. PMID:16385030

Characterization and use of catabolite-repressed promoters from gluconate genes in Corynebacterium glutamicum.

PubMed

Letek, Michal; Valbuena, Noelia; Ramos, Angelina; Ordóñez, Efrén; Gil, José A; Mateos, Luís M

2006-01-01

The genes involved in gluconate catabolism (gntP and gntK) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis. In C. glutamicum, gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (PgntK and PgntP) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or egfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on PgntP and PgntK fragments was detected only in the presence of cAMP.

VE-Cadherin–Mediated Epigenetic Regulation of Endothelial Gene Expression

PubMed Central

Morini, Marco F.; Giampietro, Costanza; Corada, Monica; Pisati, Federica; Lavarone, Elisa; Cunha, Sara I.; Conze, Lei L.; O’Reilly, Nicola; Joshi, Dhira; Kjaer, Svend; George, Roger; Nye, Emma; Ma, Anqi; Jin, Jian; Mitter, Richard; Lupia, Michela; Cavallaro, Ugo; Pasini, Diego; Calado, Dinis P.

2018-01-01

Rationale: The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. Objective: We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. Methods and Results: We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5, vascular endothelial-protein tyrosine phosphatase (VE-PTP), and von Willebrand factor (vWf). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5, VE-PTP, and vWf. VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5, VE-PTP, and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. Conclusions: These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system. PMID:29233846

VE-Cadherin-Mediated Epigenetic Regulation of Endothelial Gene Expression.

PubMed

Morini, Marco F; Giampietro, Costanza; Corada, Monica; Pisati, Federica; Lavarone, Elisa; Cunha, Sara I; Conze, Lei L; O'Reilly, Nicola; Joshi, Dhira; Kjaer, Svend; George, Roger; Nye, Emma; Ma, Anqi; Jin, Jian; Mitter, Richard; Lupia, Michela; Cavallaro, Ugo; Pasini, Diego; Calado, Dinis P; Dejana, Elisabetta; Taddei, Andrea

2018-01-19

The mechanistic foundation of vascular maturation is still largely unknown. Several human pathologies are characterized by deregulated angiogenesis and unstable blood vessels. Solid tumors, for instance, get their nourishment from newly formed structurally abnormal vessels which present wide and irregular interendothelial junctions. Expression and clustering of the main endothelial-specific adherens junction protein, VEC (vascular endothelial cadherin), upregulate genes with key roles in endothelial differentiation and stability. We aim at understanding the molecular mechanisms through which VEC triggers the expression of a set of genes involved in endothelial differentiation and vascular stabilization. We compared a VEC-null cell line with the same line reconstituted with VEC wild-type cDNA. VEC expression and clustering upregulated endothelial-specific genes with key roles in vascular stabilization including claudin-5 , vascular endothelial-protein tyrosine phosphatase ( VE-PTP ), and von Willebrand factor ( vWf ). Mechanistically, VEC exerts this effect by inhibiting polycomb protein activity on the specific gene promoters. This is achieved by preventing nuclear translocation of FoxO1 (Forkhead box protein O1) and β-catenin, which contribute to PRC2 (polycomb repressive complex-2) binding to promoter regions of claudin-5 , VE-PTP , and vWf . VEC/β-catenin complex also sequesters a core subunit of PRC2 (Ezh2 [enhancer of zeste homolog 2]) at the cell membrane, preventing its nuclear translocation. Inhibition of Ezh2/VEC association increases Ezh2 recruitment to claudin-5 , VE-PTP , and vWf promoters, causing gene downregulation. RNA sequencing comparison of VEC-null and VEC-positive cells suggested a more general role of VEC in activating endothelial genes and triggering a vascular stability-related gene expression program. In pathological angiogenesis of human ovarian carcinomas, reduced VEC expression paralleled decreased levels of claudin-5 and VE-PTP. These data extend the knowledge of polycomb-mediated regulation of gene expression to endothelial cell differentiation and vessel maturation. The identified mechanism opens novel therapeutic opportunities to modulate endothelial gene expression and induce vascular normalization through pharmacological inhibition of the polycomb-mediated repression system. © 2017 The Authors.

Trithorax dependent changes in chromatin landscape at enhancer and promoter regions drive female puberty.

PubMed

Toro, Carlos A; Wright, Hollis; Aylwin, Carlos F; Ojeda, Sergio R; Lomniczi, Alejandro

2018-01-04

Polycomb group (PcG) proteins control the timing of puberty by repressing the Kiss1 gene in hypothalamic arcuate nucleus (ARC) neurons. Here we identify two members of the Trithorax group (TrxG) of modifiers, mixed-lineage leukemia 1 (MLL1), and 3 (MLL3), as central components of an activating epigenetic machinery that dynamically counteracts PcG repression. Preceding puberty, MLL1 changes the chromatin configuration at the promoters of Kiss1 and Tac3, two genes required for puberty to occur, from repressive to permissive. Concomitantly, MLL3 institutes a chromatin structure that changes the functional status of a Kiss1 enhancer from poised to active. RNAi-mediated, ARC-specific Mll1 knockdown reduced Kiss1 and Tac3 expression, whereas CRISPR-Cas9-directed epigenome silencing of the Kiss1 enhancer selectively reduced Kiss1 activity. Both interventions delay puberty and disrupt reproductive cyclicity. Our results demonstrate that an epigenetic switch from transcriptional repression to activation is crucial to the regulatory mechanism controlling the timing of mammalian puberty.

Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells.

PubMed

Zhang, Liyuan; Gu, Lingkun; Ringler, Patricia; Smith, Stanley; Rushton, Paul J; Shen, Qingxi J

2015-07-01

Members of the WRKY transcription factor superfamily are essential for the regulation of many plant pathways. Functional redundancy due to duplications of WRKY transcription factors, however, complicates genetic analysis by allowing single-mutant plants to maintain wild-type phenotypes. Our analyses indicate that three group I WRKY genes, OsWRKY24, -53, and -70, act in a partially redundant manner. All three showed characteristics of typical WRKY transcription factors: each localized to nuclei and yeast one-hybrid assays indicated that they all bind to W-boxes, including those present in their own promoters. Quantitative real time-PCR (qRT-PCR) analyses indicated that the expression levels of the three WRKY genes varied in the different tissues tested. Particle bombardment-mediated transient expression analyses indicated that all three genes repress the GA and ABA signaling in a dosage-dependent manner. Combination of all three WRKY genes showed additive antagonism of ABA and GA signaling. These results suggest that these WRKY proteins function as negative transcriptional regulators of GA and ABA signaling. However, different combinations of these WRKY genes can lead to varied strengths in suppression of their targets. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

An Examination of Dynamic Gene Expression Changes in the Mouse Brain During Pregnancy and the Postpartum Period.

PubMed

Ray, Surjyendu; Tzeng, Ruei-Ying; DiCarlo, Lisa M; Bundy, Joseph L; Vied, Cynthia; Tyson, Gary; Nowakowski, Richard; Arbeitman, Michelle N

2015-11-23

The developmental transition to motherhood requires gene expression changes that alter the brain to drive the female to perform maternal behaviors. We broadly examined the global transcriptional response in the mouse maternal brain, by examining four brain regions: hypothalamus, hippocampus, neocortex, and cerebellum, in virgin females, two pregnancy time points, and three postpartum time points. We find that overall there are hundreds of differentially expressed genes, but each brain region and time point shows a unique molecular signature, with only 49 genes differentially expressed in all four regions. Interestingly, a set of "early-response genes" is repressed in all brain regions during pregnancy and postpartum stages. Several genes previously implicated in underlying postpartum depression change expression. This study serves as an atlas of gene expression changes in the maternal brain, with the results demonstrating that pregnancy, parturition, and postpartum maternal experience substantially impact diverse brain regions. Copyright © 2016 Ray et al.

Distinct gene expression profiles in peripheral blood mononuclear cells from patients infected with vaccinia virus, yellow fever 17D virus, or upper respiratory infections.

PubMed

Scherer, Christina A; Magness, Charles L; Steiger, Kathryn V; Poitinger, Nicholas D; Caputo, Christine M; Miner, Douglas G; Winokur, Patricia L; Klinzman, Donna; McKee, Janice; Pilar, Christine; Ward, Patricia A; Gillham, Martha H; Haulman, N Jean; Stapleton, Jack T; Iadonato, Shawn P

2007-08-29

Gene expression in human peripheral blood mononuclear cells was systematically evaluated following smallpox and yellow fever vaccination, and naturally occurring upper respiratory infection (URI). All three infections were characterized by the induction of many interferon stimulated genes, as well as enhanced expression of genes involved in proteolysis and antigen presentation. Vaccinia infection was also characterized by a distinct expression signature composed of up-regulation of monocyte response genes, with repression of genes expressed by B and T-cells. In contrast, the yellow fever host response was characterized by a suppression of ribosomal and translation factors, distinguishing this infection from vaccinia and URI. No significant URI-specific signature was observed, perhaps reflecting greater heterogeneity in the study population and etiological agents. Taken together, these data suggest that specific host gene expression signatures may be identified that distinguish one or a small number of virus agents.

Genome engineering using a synthetic gene circuit in Bacillus subtilis.

PubMed

Jeong, Da-Eun; Park, Seung-Hwan; Pan, Jae-Gu; Kim, Eui-Joong; Choi, Soo-Keun

2015-03-31

Genome engineering without leaving foreign DNA behind requires an efficient counter-selectable marker system. Here, we developed a genome engineering method in Bacillus subtilis using a synthetic gene circuit as a counter-selectable marker system. The system contained two repressible promoters (B. subtilis xylA (Pxyl) and spac (Pspac)) and two repressor genes (lacI and xylR). Pxyl-lacI was integrated into the B. subtilis genome with a target gene containing a desired mutation. The xylR and Pspac-chloramphenicol resistant genes (cat) were located on a helper plasmid. In the presence of xylose, repression of XylR by xylose induced LacI expression, the LacIs repressed the Pspac promoter and the cells become chloramphenicol sensitive. Thus, to survive in the presence of chloramphenicol, the cell must delete Pxyl-lacI by recombination between the wild-type and mutated target genes. The recombination leads to mutation of the target gene. The remaining helper plasmid was removed easily under the chloramphenicol absent condition. In this study, we showed base insertion, deletion and point mutation of the B. subtilis genome without leaving any foreign DNA behind. Additionally, we successfully deleted a 2-kb gene (amyE) and a 38-kb operon (ppsABCDE). This method will be useful to construct designer Bacillus strains for various industrial applications. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

FLOWERING LOCUS C (FLC) regulates development pathways throughout the life cycle of Arabidopsis

PubMed Central

Deng, Weiwei; Ying, Hua; Helliwell, Chris A.; Taylor, Jennifer M.; Peacock, W. James; Dennis, Elizabeth S.

2011-01-01

FLOWERING LOCUS C (FLC) has a key role in the timing of the initiation of flowering in Arabidopsis. FLC binds and represses two genes that promote flowering, FT and SOC1. We show that FLC binds to many other genes, indicating that it has regulatory roles other than the repression of flowering. We identified 505 FLC binding sites, mostly located in the promoter regions of genes and containing at least one CArG box, the motif known to be associated with MADS-box proteins such as FLC. We examined 40 of the target genes, and 20 showed increased transcript levels in an flc mutant compared with the wild type. Five genes showed decreased expression in the mutant, indicating that FLC binding can result in either transcriptional repression or activation. The genes we identified as FLC targets are involved in developmental pathways throughout the life history of the plant, many of which are associated with reproductive development. FLC is also involved in vegetative development, as evidenced by its binding to SPL15, delaying the progression from juvenile to adult phase. Some of the FLC target genes are also bound by two other MADS-box proteins, AP1 and SEP3, suggesting that MADS-box genes may operate in a network of control at different stages of the life cycle, many ultimately contributing to the development of the reproductive phase of the plant. PMID:21464308

FLOWERING LOCUS C (FLC) regulates development pathways throughout the life cycle of Arabidopsis.

PubMed

Deng, Weiwei; Ying, Hua; Helliwell, Chris A; Taylor, Jennifer M; Peacock, W James; Dennis, Elizabeth S

2011-04-19

FLOWERING LOCUS C (FLC) has a key role in the timing of the initiation of flowering in Arabidopsis. FLC binds and represses two genes that promote flowering, FT and SOC1. We show that FLC binds to many other genes, indicating that it has regulatory roles other than the repression of flowering. We identified 505 FLC binding sites, mostly located in the promoter regions of genes and containing at least one CArG box, the motif known to be associated with MADS-box proteins such as FLC. We examined 40 of the target genes, and 20 showed increased transcript levels in an flc mutant compared with the wild type. Five genes showed decreased expression in the mutant, indicating that FLC binding can result in either transcriptional repression or activation. The genes we identified as FLC targets are involved in developmental pathways throughout the life history of the plant, many of which are associated with reproductive development. FLC is also involved in vegetative development, as evidenced by its binding to SPL15, delaying the progression from juvenile to adult phase. Some of the FLC target genes are also bound by two other MADS-box proteins, AP1 and SEP3, suggesting that MADS-box genes may operate in a network of control at different stages of the life cycle, many ultimately contributing to the development of the reproductive phase of the plant.

Differential in vivo gene expression of major Leptospira proteins in resistant or susceptible animal models.

PubMed

Matsui, Mariko; Soupé, Marie-Estelle; Becam, Jérôme; Goarant, Cyrille

2012-09-01

Transcripts of Leptospira 16S rRNA, FlaB, LigB, LipL21, LipL32, LipL36, LipL41, and OmpL37 were quantified in the blood of susceptible (hamsters) and resistant (mice) animal models of leptospirosis. We first validated adequate reference genes and then evaluated expression patterns in vivo compared to in vitro cultures. LipL32 expression was downregulated in vivo and differentially regulated in resistant and susceptible animals. FlaB expression was also repressed in mice but not in hamsters. In contrast, LigB and OmpL37 were upregulated in vivo. Thus, we demonstrated that a virulent strain of Leptospira differentially adapts its gene expression in the blood of infected animals.

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1→S transition.

PubMed

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay; Sarvepalli, Kavitha; Sadhale, Parag P; Nath, Utpal

2011-07-01

The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1→S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1→S arrest is discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Pax-3 expression in segmental mesoderm marks early stages in myogenic cell specification.

PubMed

Williams, B A; Ordahl, C P

1994-04-01

Specification of the myogenic lineage begins prior to gastrulation and culminates in the emergence of determined myogenic precursor cells from the somites. The myoD family (MDF) of transcriptional activators controls late step(s) in myogenic specification that are closely followed by terminal muscle differentiation. Genes expressed in myogenic specification at stages earlier than MDFs are unknown. The Pax-3 gene is expressed in all the cells of the caudal segmental plate, the early mesoderm compartment that contains the precursors of skeletal muscle. As somites form from the segmental plate and mature, Pax-3 expression is progressively modulated. Beginning at the time of segmentation, Pax-3 becomes repressed in the ventral half of the somite, leaving Pax-3 expression only in the dermomyotome. Subsequently, differential modulation of Pax-3 expression levels delineates the medial and lateral halves of the dermomyotome, which contain precursors of axial (back) muscle and limb muscle, respectively. Pax-3 expression is then repressed as dermomyotome-derived cells activate MDFs. Quail-chick chimera and ablation experiments confirmed that the migratory precursors of limb muscle continue to express Pax-3 during migration. Since limb muscle precursors do not activate MDFs until 2 days after they leave the somite, Pax-3 represents the first molecular marker for this migratory cell population. A null mutation of the mouse Pax-3 gene, Splotch, produces major disruptions in early limb muscle development (Franz, T., Kothary, R., Surani, M. A. H., Halata, Z. and Grim, M. (1993) Anat. Embryol. 187, 153-160; Goulding, M., Lumsden, A. and Paquette, A. (1994) Development 120, 957-971). We conclude, therefore, that Pax-3 gene expression in the paraxial mesoderm marks earlier stages in myogenic specification than MDFs and plays a crucial role in the specification and/or migration of limb myogenic precursors.

Spermine oxidase maintains basal skeletal muscle gene expression and fiber size and is strongly repressed by conditions that cause skeletal muscle atrophy

PubMed Central

Bongers, Kale S.; Fox, Daniel K.; Kunkel, Steven D.; Stebounova, Larissa V.; Murry, Daryl J.; Pufall, Miles A.; Ebert, Scott M.; Dyle, Michael C.; Bullard, Steven A.; Dierdorff, Jason M.

2014-01-01

Skeletal muscle atrophy is a common and debilitating condition that remains poorly understood at the molecular level. To better understand the mechanisms of muscle atrophy, we used mouse models to search for a skeletal muscle protein that helps to maintain muscle mass and is specifically lost during muscle atrophy. We discovered that diverse causes of muscle atrophy (limb immobilization, fasting, muscle denervation, and aging) strongly reduced expression of the enzyme spermine oxidase. Importantly, a reduction in spermine oxidase was sufficient to induce muscle fiber atrophy. Conversely, forced expression of spermine oxidase increased muscle fiber size in multiple models of muscle atrophy (immobilization, fasting, and denervation). Interestingly, the reduction of spermine oxidase during muscle atrophy was mediated by p21, a protein that is highly induced during muscle atrophy and actively promotes muscle atrophy. In addition, we found that spermine oxidase decreased skeletal muscle mRNAs that promote muscle atrophy (e.g., myogenin) and increased mRNAs that help to maintain muscle mass (e.g., mitofusin-2). Thus, in healthy skeletal muscle, a relatively low level of p21 permits expression of spermine oxidase, which helps to maintain basal muscle gene expression and fiber size; conversely, during conditions that cause muscle atrophy, p21 expression rises, leading to reduced spermine oxidase expression, disruption of basal muscle gene expression, and muscle fiber atrophy. Collectively, these results identify spermine oxidase as an important positive regulator of muscle gene expression and fiber size, and elucidate p21-mediated repression of spermine oxidase as a key step in the pathogenesis of skeletal muscle atrophy. PMID:25406264