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Sample records for nuclear transcription factor-kappab

  1. Propanil inhibits tumor necrosis factor-alpha production by reducing nuclear levels of the transcription factor nuclear factor-kappab in the macrophage cell line ic-21.

    PubMed

    Frost, L L; Neeley, Y X; Schafer, R; Gibson, L F; Barnett, J B

    2001-05-01

    Tumor necrosis factor-alpha (TNF-alpha) is an essential proinflammatory cytokine whose production is normally stimulated by bacterial cell wall components, such as lipopolysaccharide (LPS), during an infection. Macrophages stimulated with LPS in vitro produce several cytokines, including TNF-alpha. LPS-stimulated primary mouse macrophages produced less TNF-alpha protein and message after treatment with the herbicide propanil (Xie et al., Toxicol. Appl. Pharmacol. 145, 184-191, 1997). Nuclear factor-kappaB (NF-kappaB) tightly regulates TNF-alpha transcription. Therefore, as a step toward understanding the mechanism of the effect of propanil on TNF-alpha transcription, IC-21 cells were transfected with a TNF-alpha promoter-luciferase construct, and the effect of propanil on luciferase activity was measured. Cells transfected with promoter constructs containing a kappaB site showed decreased luciferase activity relative to controls after propanil treatment. These observations implicated NF-kappaB binding as an intracellular target of propanil. Further studies demonstrated a marked reduction in the nuclear levels of the stimulatory p65 subunit of NF-kappaB after propanil treatment, as measured by fluorescence confocal microscopy and Western blot analysis. The p50 subunit of NF-kappaB was not found to be reduced after propanil exposure by Western blot. Electrophoretic mobility gel shift assays showed decreased DNA binding of both p65/p50 heterodimers and p50/p50 homodimers to the kappaB3 site of the TNF-alpha promoter of propanil-treated cells. The marked reduction in nuclear p65/p50 NF-kappaB levels and diminished binding to the TNF-alpha promoter in propanil-treated cells are consistent with reduced TNF-alpha levels induced by LPS. PMID:11312646

  2. Nuclear actin levels as an important transcriptional switch

    PubMed Central

    Huet, Guillaume; Skarp, Kari-Pekka; Vartiainen, Maria K.

    2012-01-01

    Nuclear actin levels have recently been linked to different cellular fates, suggesting that actin could act as a switch between altered transcriptional states. Here we discuss our latest results on the mechanisms by which nuclear actin levels are regulated and their implications to the functional significance of nuclear actin. PMID:22771994

  3. Actin-based modeling of a transcriptionally competent nuclear substructure induced by transcription inhibition

    SciTech Connect

    Wang, I-F.; Chang, H.-Y.; James Shen, C.-K. . E-mail: ckshen@ccvax.sinica.edu.tw

    2006-11-15

    During transcription inactivation, the nuclear bodies in the mammalian cells often undergo reorganization. In particular, the interchromatin granule clusters, or IGCs, become colocalized with RNA polymerase II (RNAP II) upon treatment with transcription inhibitors. This colocalization has also been observed in untreated but transcriptionally inactive cells. We report here that the reorganized IGC domains are unique substructure consisting of outer shells made of SC35, ERK2, SF2/ASF, and actin. The apparently hollow holes of these domains contain clusters of RNAP II, mostly phosphorylated, and the splicing regulator SMN. This class of complexes are also the sites where prominent transcription activities are detected once the inhibitors are removed. Furthermore, actin polymerization is required for reorganization of the IGCs. In connection with this, immunoprecipitation and immunostaining experiments showed that nuclear actin is associated with IGCs and the reorganized IGC domains. The study thus provides further evidence for the existence of an actin-based nuclear skeleton structure in association with the dynamic reorganization processes in the nucleus. Overall, our data suggest that mammalian cells have adapted to utilize the reorganized, uniquely shaped IGC domains as the temporary storage sites of RNAP II transcription machineries in response to certain transient states of transcription inactivation.

  4. Nuclear positional control of HIV transcription in 4D

    PubMed Central

    Dhir, Somdutta; Dieudonné, Mariacarolina

    2010-01-01

    Retroviruses integrate their genome into the chromatin of the host cell and are subject to the same control mechanisms governing transcription in the nucleus. There is increasing evidence that the spatial position of a gene within the nucleus in time affects its activity. Therefore it becomes important to study the chromatin environment in space and time of the HIV-1 provirus, particularly in cells where a tight transcriptional control allows the virus to hide away from antiviral treatment and immune response. We recently showed that the HIV-1 provirus is found at the nuclear periphery of latently infected lymphocytes associated in trans with centromeric heterochromatin. After induction of transcription, this association was lost, although the location of the transcribing provirus remained peripheral. Our results reveal a novel mechanism of transcriptional silencing involved in HIV-1 post-transcriptional latency and open wider perspectives for the general organization of chromatin in the nucleus. PMID:21327098

  5. Regulation of mammalian transcription and splicing by Nuclear RNAi

    PubMed Central

    Kalantari, Roya; Chiang, Cheng-Ming; Corey, David R.

    2016-01-01

    RNA interference (RNAi) is well known as a mechanism for controlling mammalian mRNA translation in the cytoplasm, but what would be the consequences if it also functions in cell nuclei? Although RNAi has also been found in nuclei of plants, yeast, and other organisms, there has been relatively little progress towards understanding the potential involvement of mammalian RNAi factors in nuclear processes including transcription and splicing. This review summarizes evidence for mammalian RNAi factors in cell nuclei and mechanisms that might contribute to the control of gene expression. When RNAi factors bind small RNAs, they form ribonucleoprotein complexes that can be selective for target sequences within different classes of nuclear RNA substrates. The versatility of nuclear RNAi may supply a previously underappreciated layer of regulation to transcription, splicing, and other nuclear processes. PMID:26612865

  6. Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

    SciTech Connect

    Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha; Jeong, Jae Hoon; Pak, Youngmi Kim

    2014-07-18

    Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated that TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations.

  7. The Basal Transcription Complex Component TAF3 Transduces Changes in Nuclear Phosphoinositides into Transcriptional Output

    PubMed Central

    Stijf-Bultsma, Yvette; Sommer, Lilly; Tauber, Maria; Baalbaki, Mai; Giardoglou, Panagiota; Jones, David R.; Gelato, Kathy A.; van Pelt, Jason; Shah, Zahid; Rahnamoun, Homa; Toma, Clara; Anderson, Karen E.; Hawkins, Philip; Lauberth, Shannon M.; Haramis, Anna-Pavlina G.; Hart, Daniel; Fischle, Wolfgang; Divecha, Nullin

    2015-01-01

    Summary Phosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers. PMID:25866244

  8. The basal transcription complex component TAF3 transduces changes in nuclear phosphoinositides into transcriptional output.

    PubMed

    Stijf-Bultsma, Yvette; Sommer, Lilly; Tauber, Maria; Baalbaki, Mai; Giardoglou, Panagiota; Jones, David R; Gelato, Kathy A; van Pelt, Jason; Shah, Zahid; Rahnamoun, Homa; Toma, Clara; Anderson, Karen E; Hawkins, Philip; Lauberth, Shannon M; Haramis, Anna-Pavlina G; Hart, Daniel; Fischle, Wolfgang; Divecha, Nullin

    2015-05-01

    Phosphoinositides (PI) are important signaling molecules in the nucleus that influence gene expression. However, if and how nuclear PI directly affects the transcriptional machinery is not known. We report that the lipid kinase PIP4K2B regulates nuclear PI5P and the expression of myogenic genes during myoblast differentiation. A targeted screen for PI interactors identified the PHD finger of TAF3, a TATA box binding protein-associated factor with important roles in transcription regulation, pluripotency, and differentiation. We show that the PI interaction site is distinct from the known H3K4me3 binding region of TAF3 and that PI binding modulates association of TAF3 with H3K4me3 in vitro and with chromatin in vivo. Analysis of TAF3 mutants indicates that TAF3 transduces PIP4K2B-mediated alterations in PI into changes in specific gene transcription. Our study reveals TAF3 as a direct target of nuclear PI and further illustrates the importance of basal transcription components as signal transducers. PMID:25866244

  9. RNA transcription modulates phase transition-driven nuclear body assembly

    PubMed Central

    Berry, Joel; Weber, Stephanie C.; Vaidya, Nilesh; Haataja, Mikko; Brangwynne, Clifford P.

    2015-01-01

    Nuclear bodies are RNA and protein-rich, membraneless organelles that play important roles in gene regulation. The largest and most well-known nuclear body is the nucleolus, an organelle whose primary function in ribosome biogenesis makes it key for cell growth and size homeostasis. The nucleolus and other nuclear bodies behave like liquid-phase droplets and appear to condense from the nucleoplasm by concentration-dependent phase separation. However, nucleoli actively consume chemical energy, and it is unclear how such nonequilibrium activity might impact classical liquid–liquid phase separation. Here, we combine in vivo and in vitro experiments with theory and simulation to characterize the assembly and disassembly dynamics of nucleoli in early Caenorhabditis elegans embryos. In addition to classical nucleoli that assemble at the transcriptionally active nucleolar organizing regions, we observe dozens of “extranucleolar droplets” (ENDs) that condense in the nucleoplasm in a transcription-independent manner. We show that growth of nucleoli and ENDs is consistent with a first-order phase transition in which late-stage coarsening dynamics are mediated by Brownian coalescence and, to a lesser degree, Ostwald ripening. By manipulating C. elegans cell size, we change nucleolar component concentration and confirm several key model predictions. Our results show that rRNA transcription and other nonequilibrium biological activity can modulate the effective thermodynamic parameters governing nucleolar and END assembly, but do not appear to fundamentally alter the passive phase separation mechanism. PMID:26351690

  10. Nuclear dot antigens may specify transcriptional domains in the nucleus.

    PubMed

    Xie, K; Lambie, E J; Snyder, M

    1993-10-01

    A bank of 892 human autoimmune serum samples was screened by indirect immunofluorescence on human tissue culture HT-29 cells. Seven serum samples that stain 4 to 10 bright dots in cell lines of several different mammals, including humans, monkeys, rats, and pigs, were identified. Immunofluorescence experiments indicate that these antigens, called nuclear dot (ND) antigens, are distinct from splicing complexes, kinetochores, and other known nuclear structures. An ND antigen recognized by these sera was cloned by immunoscreening a human lambda gt11 expression library. Analysis of seven cDNA clones for the ND antigen indicates that several mRNAs exist, perhaps derived through alternative splicing mechanisms. One major form of the message has an open reading frame of 1,440 bp capable of encoding a 53,000-M(r) protein. Treatment of cells with detergent, salt, or RNase A fails to remove the ND antigen from the nucleus. However, incubation with DNase I obliterates ND staining, indicating that the ND protein directly or indirectly associates with nuclear DNA. Fusion of the ND protein to a LexA DNA binding domain activates transcription in Saccharomyces cerevisiae. A 75-amino-acid domain that activates transcription in both yeast and primate cells has been identified. We suggest that ND antigens may participate in the activation of transcription of specific regions of the genome. PMID:8413218

  11. RNA transcription modulates phase transition-driven nuclear body assembly.

    PubMed

    Berry, Joel; Weber, Stephanie C; Vaidya, Nilesh; Haataja, Mikko; Brangwynne, Clifford P

    2015-09-22

    Nuclear bodies are RNA and protein-rich, membraneless organelles that play important roles in gene regulation. The largest and most well-known nuclear body is the nucleolus, an organelle whose primary function in ribosome biogenesis makes it key for cell growth and size homeostasis. The nucleolus and other nuclear bodies behave like liquid-phase droplets and appear to condense from the nucleoplasm by concentration-dependent phase separation. However, nucleoli actively consume chemical energy, and it is unclear how such nonequilibrium activity might impact classical liquid-liquid phase separation. Here, we combine in vivo and in vitro experiments with theory and simulation to characterize the assembly and disassembly dynamics of nucleoli in early Caenorhabditis elegans embryos. In addition to classical nucleoli that assemble at the transcriptionally active nucleolar organizing regions, we observe dozens of "extranucleolar droplets" (ENDs) that condense in the nucleoplasm in a transcription-independent manner. We show that growth of nucleoli and ENDs is consistent with a first-order phase transition in which late-stage coarsening dynamics are mediated by Brownian coalescence and, to a lesser degree, Ostwald ripening. By manipulating C. elegans cell size, we change nucleolar component concentration and confirm several key model predictions. Our results show that rRNA transcription and other nonequilibrium biological activity can modulate the effective thermodynamic parameters governing nucleolar and END assembly, but do not appear to fundamentally alter the passive phase separation mechanism. PMID:26351690

  12. Dynamic Encounters of Genes and Transcripts with the Nuclear Pore.

    PubMed

    Ben-Yishay, Rakefet; Ashkenazy, Asaf J; Shav-Tal, Yaron

    2016-07-01

    Transcribed mRNA molecules must reach the cytoplasm to undergo translation. Technological developments in imaging have placed mRNAs under the spotlight, allowing the quantitative study of the spatial and temporal dynamics of the nucleocytoplasmic mRNA export process. Here, we discuss studies that have used such experimental approaches to demonstrate that gene tethering at the nuclear pore complex (NPC) regulates mRNA expression, and to characterize mRNA dynamics during transport in real time. The paths taken by mRNAs as they move from their sites of transcription and travel through the nucleoplasm, in between chromatin domains, and finally through the NPC, can now be observed in detail. PMID:27185238

  13. Transcriptional cross talk between orphan nuclear receptor ERRγ and transmembrane transcription factor ATF6α coordinates endoplasmic reticulum stress response

    PubMed Central

    Misra, Jagannath; Kim, Don-Kyu; Choi, Woogyun; Koo, Seung-Hoi; Lee, Chul-Ho; Back, Sung-Hoon; Kaufman, Randal J.; Choi, Hueng-Sik

    2013-01-01

    Orphan nuclear receptor ERRγ is a member of nuclear receptor superfamily that regulates several important cellular processes including hepatic glucose and alcohol metabolism. However, mechanistic understanding of transcriptional regulation of the ERRγ gene remains to be elucidated. Here, we report that activating transcription factor 6α (ATF6α), an endoplasmic reticulum (ER)-membrane–bound basic leucine zipper (bZip) transcription factor, directly regulates ERRγ gene expression in response to ER stress. ATF6α binds to ATF6α responsive element in the ERRγ promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6α and PGC1α on the ERRγ promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6α and PGC1α binding site. Of interest, ERRγ along with PGC1α induce ATF6α gene transcription upon ER stress. ERRγ binds to an ERRγ responsive element in the ATF6α promoter. ChIP assay confirmed that both ERRγ and PGC1α bind to a site in the ATF6α promoter that exhibits histone H3 and H4 acetylation. Overall, for the first time our data show a novel pathway of cross talk between nuclear receptors and ER-membrane–bound transcription factors and suggest a positive feed-forward loop regulates ERRγ and ATF6α gene transcription. PMID:23716639

  14. Transcriptional cross talk between orphan nuclear receptor ERRγ and transmembrane transcription factor ATF6α coordinates endoplasmic reticulum stress response.

    PubMed

    Misra, Jagannath; Kim, Don-Kyu; Choi, Woogyun; Koo, Seung-Hoi; Lee, Chul-Ho; Back, Sung-Hoon; Kaufman, Randal J; Choi, Hueng-Sik

    2013-08-01

    Orphan nuclear receptor ERRγ is a member of nuclear receptor superfamily that regulates several important cellular processes including hepatic glucose and alcohol metabolism. However, mechanistic understanding of transcriptional regulation of the ERRγ gene remains to be elucidated. Here, we report that activating transcription factor 6α (ATF6α), an endoplasmic reticulum (ER)-membrane-bound basic leucine zipper (bZip) transcription factor, directly regulates ERRγ gene expression in response to ER stress. ATF6α binds to ATF6α responsive element in the ERRγ promoter. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) is required for this transactivation. Chromatin immunoprecipitation (ChIP) assay confirmed the binding of both ATF6α and PGC1α on the ERRγ promoter. ChIP assay demonstrated histone H3 and H4 acetylation occurs at the ATF6α and PGC1α binding site. Of interest, ERRγ along with PGC1α induce ATF6α gene transcription upon ER stress. ERRγ binds to an ERRγ responsive element in the ATF6α promoter. ChIP assay confirmed that both ERRγ and PGC1α bind to a site in the ATF6α promoter that exhibits histone H3 and H4 acetylation. Overall, for the first time our data show a novel pathway of cross talk between nuclear receptors and ER-membrane-bound transcription factors and suggest a positive feed-forward loop regulates ERRγ and ATF6α gene transcription. PMID:23716639

  15. Nur77 forms novel nuclear structures upon DNA damage that cause transcriptional arrest

    SciTech Connect

    Leseleuc, Louis de; Denis, Francois . E-mail: francois.denis@iaf.inrs.ca

    2006-05-15

    The orphan nuclear receptor Nur77 has been implicated in both growth and apoptosis, and its function and activity can be modulated by cellular redistribution. Green fluorescent protein-tagged Nur77 was used to evaluate the role of Nur77 intracellular redistribution in response to genotoxic stress. Selected DNA damaging agents and transcription inhibition lead to rapid redistribution of Nur77 into nuclear structures distinct from conventional nuclear bodies. These nuclear bodies formed transiently were tightly bound to the nuclear matrix and conditions that lead to their appearance were associated with Nur77 transcriptional inhibition. The formation of Nur77 nuclear bodies might be involved in programmed cell death modulation upon exposure to DNA damaging agents that inhibit transcription by sequestrating this proapoptotic factor in dense nuclear structures.

  16. Multiple Nuclear Localization Signals Mediate Nuclear Localization of the GATA Transcription Factor AreA

    PubMed Central

    Hunter, Cameron C.; Siebert, Kendra S.; Downes, Damien J.; Wong, Koon Ho; Kreutzberger, Sara D.; Fraser, James A.; Clarke, David F.; Hynes, Michael J.; Davis, Meryl A.

    2014-01-01

    The Aspergillus nidulans GATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in the Saccharomyces cerevisiae functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA mutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes. Deletion of all five classical NLSs individually or collectively did not affect utilization of nitrogen sources or AreA-dependent gene expression and did not prevent AreA nuclear localization. Mutation of the bipartite NLS conferred the inability to utilize alternative nitrogen sources and abolished AreA-dependent gene expression likely due to effects on DNA binding but did not prevent AreA nuclear localization. Mutation of all six NLSs simultaneously prevented AreA nuclear accumulation. The bipartite NLS alone strongly directed GFP to the nucleus, whereas the classical NLSs collaborated to direct GFP to the nucleus. Therefore, AreA contains multiple conserved NLSs, which show redundancy and together function to mediate nuclear import. The noncanonical bipartite NLS is conserved in GATA factors from Aspergillus, yeast, and mammals, indicating an ancient origin. PMID:24562911

  17. Exosome Cofactors Connect Transcription Termination to RNA Processing by Guiding Terminated Transcripts to the Appropriate Exonuclease within the Nuclear Exosome.

    PubMed

    Kim, Kyumin; Heo, Dong-Hyuk; Kim, Iktae; Suh, Jeong-Yong; Kim, Minkyu

    2016-06-17

    The yeast Nrd1 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNApII) through its CTD-interacting domain (CID) and also associates with the nuclear exosome, thereby acting as both a transcription termination and RNA processing factor. Previously, we found that the Nrd1 CID is required to recruit the nuclear exosome to the Nrd1 complex, but it was not clear which exosome subunits were contacted. Here, we show that two nuclear exosome cofactors, Mpp6 and Trf4, directly and competitively interact with the Nrd1 CID and differentially regulate the association of Nrd1 with two catalytic subunits of the exosome. Importantly, Mpp6 promotes the processing of Nrd1-terminated transcripts preferentially by Dis3, whereas Trf4 leads to Rrp6-dependent processing. This suggests that Mpp6 and Trf4 may play a role in choosing a particular RNA processing route for Nrd1-terminated transcripts within the exosome by guiding the transcripts to the appropriate exonuclease. PMID:27076633

  18. Nuclear Matrix protein SMAR1 represses HIV-1 LTR mediated transcription through chromatin remodeling

    SciTech Connect

    Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep; Sinha, Surajit; Dash, Prasanta K.; Siddappa, Nagadenahalli B.; Ranga, Udaykumar; Mitra, Debashis; Chattopadhyay, Samit

    2010-04-25

    Nuclear Matrix and MARs have been implicated in the transcriptional regulation of host as well as viral genes but their precise role in HIV-1 transcription remains unclear. Here, we show that > 98% of HIV sequences contain consensus MAR element in their promoter. We show that SMAR1 binds to the LTR MAR and reinforces transcriptional silencing by tethering the LTR MAR to nuclear matrix. SMAR1 associated HDAC1-mSin3 corepressor complex is dislodged from the LTR upon cellular activation by PMA/TNFalpha leading to an increase in the acetylation and a reduction in the trimethylation of histones, associated with the recruitment of RNA Polymerase II on the LTR. Overexpression of SMAR1 lead to reduction in LTR mediated transcription, both in a Tat dependent and independent manner, resulting in a decreased virion production. These results demonstrate the role of SMAR1 in regulating viral transcription by alternative compartmentalization of LTR between the nuclear matrix and chromatin.

  19. Nuclear reprogramming: the zygotic transcription program is established through an "erase-and-rebuild" strategy.

    PubMed

    Sun, Feng; Fang, Haiyan; Li, Ruizhen; Gao, Tianlong; Zheng, Junke; Chen, Xuejin; Ying, Wenqin; Sheng, Hui Z

    2007-02-01

    Oocytes display a maternal-specific gene expression profile, which is switched to a zygotic profile when a haploid set of chromatin is passed on to the fertilized egg that develops into an embryo. The mechanism underlying this transcription reprogramming is currently unknown. Here we demonstrate that by the time when transcription is shut down in germinal vesicle oocytes, a range of general transcription factors and transcriptional regulators are dissociated from the chromatin. The global dissociation of chromatin factors (CFs) disrupts physical contacts between the chromatin and CFs and leads to erasure of the maternal transcription program at the functional level. Critical transcription factors and regulators remain separated from chromatin for a prolonged period, and become re-associated with chromatin shortly after pronuclear formation. This is followed temporally by the re-establishment of nuclear functions such as DNA replication and transcription. We propose that the maternal transcription program is erased during oogenesis to generate a relatively naïve chromatin and the zygotic transcription program is rebuilt de novo after fertilization. This process is termed as the "erase-and-rebuild" process, which is used to reset the transcription program, and most likely other nuclear processes as well, from a maternal one to that of the embryo. We further show in the accompanying paper (Gao T, et al., Cell Res 2007; 17: 135-150.) that the same strategy is also employed to reprogram transcriptional profiles in somatic cell nuclear transfer and parthenogenesis, suggesting that this model is universally applicable to all forms of transcriptional reprogramming during early embryogenesis. Displacement of CFs from chromatin also offers an explanation for the phenomenon of transcription silence during the maternal to zygotic transition. PMID:17287829

  20. piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire.

    PubMed

    Senti, Kirsten-André; Jurczak, Daniel; Sachidanandam, Ravi; Brennecke, Julius

    2015-08-15

    PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ∼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. PMID:26302790

  1. piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire

    PubMed Central

    Senti, Kirsten-André; Jurczak, Daniel; Sachidanandam, Ravi; Brennecke, Julius

    2015-01-01

    PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ∼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. PMID:26302790

  2. Telomere-surrounding regions are transcription-permissive 3D nuclear compartments in human cells

    SciTech Connect

    Quina, Ana Sofia; Parreira, Leonor . E-mail: lparreir@igc.gulbenkian.pt

    2005-07-01

    Positioning of genes relative to nuclear heterochromatic compartments is thought to help regulate their transcriptional activity. Given that human subtelomeric regions are rich in highly expressed genes, we asked whether human telomeres are related to transcription-permissive nuclear compartments. To address this question, we investigated in the nuclei of normal human lymphocytes the spatial relations of two constitutively expressed genes (ACTB and RARA) and three nuclear transcripts (ACTB, IL2RA and TCRB) to telomeres and centromeres, as a function of gene activity and transcription levels. We observed that genes and gene transcripts locate close to telomere clusters and away from chromocenters upon activation of transcription. These findings, together with the observation that SC35 domains, which are enriched in pre-mRNA processing factors, are in close proximity to telomeres, indicate that telomere-neighboring regions are permissive to gene expression in human cells. Therefore, the associations of telomeres observed in the interphase nucleus might contribute, as opposed to chromocenters, for the establishment of transcription-permissive 3D nuclear compartments.

  3. Nuclear RhoA signaling regulates MRTF-dependent SMC-specific transcription

    PubMed Central

    Staus, Dean P.; Weise-Cross, Laura; Mangum, Kevin D.; Medlin, Matt D.; Mangiante, Lee; Taylor, Joan M.

    2014-01-01

    We have previously shown that RhoA-mediated actin polymerization stimulates smooth muscle cell (SMC)-specific transcription by regulating the nuclear localization of the myocardin-related transcription factors (MRTFs). On the basis of the recent demonstration that nuclear G-actin regulates MRTF nuclear export and observations from our laboratory and others that the RhoA effector, mDia2, shuttles between the nucleus and cytoplasm, we investigated whether nuclear RhoA signaling plays a role in regulating MRTF activity. We identified sequences that control mDia2 nuclear-cytoplasmic shuttling and used mDia2 variants to demonstrate that the ability of mDia2 to fully stimulate MRTF nuclear accumulation and SMC-specific gene transcription was dependent on its localization to the nucleus. To test whether RhoA signaling promotes nuclear actin polymerization, we established a fluorescence recovery after photobleaching (FRAP)-based assay to measure green fluorescent protein-actin diffusion in the nuclear compartment. Nuclear actin FRAP was delayed in cells expressing nuclear-targeted constitutively active mDia1 and mDia2 variants and in cells treated with the polymerization inducer, jasplakinolide. In contrast, FRAP was enhanced in cells expressing a nuclear-targeted variant of mDia that inhibits both mDia1 and mDia2. Treatment of 10T1/2 cells with sphingosine 1-phosphate induced RhoA activity in the nucleus and forced nuclear localization of RhoA or the Rho-specific guanine nucleotide exchange factor (GEF), leukemia-associated RhoGEF, enhanced the ability of these proteins to stimulate MRTF activity. Taken together, these data support the emerging idea that RhoA-dependent nuclear actin polymerization has important effects on transcription and nuclear structure. PMID:24906914

  4. Transcriptional regulation via nuclear receptor crosstalk required for the Drosophila circadian clock.

    PubMed

    Jaumouillé, Edouard; Machado Almeida, Pedro; Stähli, Patrick; Koch, Rafael; Nagoshi, Emi

    2015-06-01

    Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and CLOCK/CYCLE (CLK/CYC) (in Drosophila) drive the expression of the period (per) family genes. The PER-containing complexes inhibit the activity of CLOCK/BMAL1 or CLK/CYC, thereby forming a negative feedback loop [1]. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation [2]. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. We show here that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor UNF are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and UNF bind to per regulatory sequences and act together to enhance the CLK/CYC-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. Our results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks. PMID:26004759

  5. Transcriptional Regulation via Nuclear Receptor Crosstalk Required for the Drosophila Circadian Clock

    PubMed Central

    Jaumouillé, Edouard; Machado Almeida, Pedro; Stähli, Patrick; Koch, Rafael; Nagoshi, Emi

    2015-01-01

    Summary Circadian clocks in large part rely on transcriptional feedback loops. At the core of the clock machinery, the transcriptional activators CLOCK/BMAL1 (in mammals) and CLOCK/CYCLE (CLK/CYC) (in Drosophila) drive the expression of the period (per) family genes. The PER-containing complexes inhibit the activity of CLOCK/BMAL1 or CLK/CYC, thereby forming a negative feedback loop [1]. In mammals, the ROR and REV-ERB family nuclear receptors add positive and negative transcriptional regulation to this core negative feedback loop to ensure the generation of robust circadian molecular oscillation [2]. Despite the overall similarities between mammalian and Drosophila clocks, whether comparable mechanisms via nuclear receptors are required for the Drosophila clock remains unknown. We show here that the nuclear receptor E75, the fly homolog of REV-ERB α and REV-ERB β, and the NR2E3 subfamily nuclear receptor UNF are components of the molecular clocks in the Drosophila pacemaker neurons. In vivo assays in conjunction with the in vitro experiments demonstrate that E75 and UNF bind to per regulatory sequences and act together to enhance the CLK/CYC-mediated transcription of the per gene, thereby completing the core transcriptional feedback loop necessary for the free-running clockwork. Our results identify a missing link in the Drosophila clock and highlight the significance of the transcriptional regulation via nuclear receptors in metazoan circadian clocks. PMID:26004759

  6. Bmal1 is a direct transcriptional target of the orphan nuclear receptor, NR2F1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Orphan nuclear receptor NR2F1 (also known as COUP-TFI, Chicken Ovalbumin Upstream Promoter Transcription Factor I) is a highly conserved member of the nuclear receptor superfamily. NR2F1 plays a critical role during embryonic development, particularly in the central and peripheral nervous systems a...

  7. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression

    PubMed Central

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the “status” of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  8. Rho, nuclear actin, and actin-binding proteins in the regulation of transcription and gene expression.

    PubMed

    Rajakylä, Eeva Kaisa; Vartiainen, Maria K

    2014-01-01

    Actin cytoskeleton is one of the main targets of Rho GTPases, which act as molecular switches on many signaling pathways. During the past decade, actin has emerged as an important regulator of gene expression. Nuclear actin plays a key role in transcription, chromatin remodeling, and pre-mRNA processing. In addition, the "status" of the actin cytoskeleton is used as a signaling intermediate by at least the MKL1-SRF and Hippo-pathways, which culminate in the transcriptional regulation of cytoskeletal and growth-promoting genes, respectively. Rho GTPases may therefore regulate gene expression by controlling either cytoplasmic or nuclear actin dynamics. Although the regulation of nuclear actin polymerization is still poorly understood, many actin-binding proteins, which are downstream effectors of Rho, are found in the nuclear compartment. In this review, we discuss the possible mechanisms and key proteins that may mediate the transcriptional regulation by Rho GTPases through actin. PMID:24603113

  9. Mapping neural circuits with activity-dependent nuclear import of a transcription factor.

    PubMed

    Masuyama, Kaoru; Zhang, Yi; Rao, Yi; Wang, Jing W

    2012-03-01

    Abstract: Nuclear factor of activated T cells (NFAT) is a calcium-responsive transcription factor. We describe here an NFAT-based neural tracing method-CaLexA (calcium-dependent nuclear import of LexA)-for labeling active neurons in behaving animals. In this system, sustained neural activity induces nuclear import of the chimeric transcription factor LexA-VP16-NFAT, which in turn drives green fluorescent protein (GFP) reporter expression only in active neurons. We tested this system in Drosophila and found that volatile sex pheromones excite specific neurons in the olfactory circuit. Furthermore, complex courtship behavior associated with multi-modal sensory inputs activated neurons in the ventral nerve cord. This method harnessing the mechanism of activity-dependent nuclear import of a transcription factor can be used to identify active neurons in specific neuronal population in behaving animals. PMID:22236090

  10. Transcriptional regulation of human small nuclear RNA genes

    PubMed Central

    Jawdekar, Gauri W.; Henry, R. William

    2009-01-01

    The products of human snRNA genes have been frequently described as performing housekeeping functions and their synthesis refractory to regulation. However, recent studies have emphasized that snRNA and other related non-coding RNA molecules control multiple facets of the central dogma, and their regulated expression is critical to cellular homeostasis during normal growth and in response to stress. Human snRNA genes contain compact and yet powerful promoters that are recognized by increasingly well-characterized transcription factors, thus providing a premier model system to study gene regulation. This review summarizes many recent advances deciphering the mechanism by which the transcription of human snRNA and related genes are regulated. PMID:18442490

  11. “Co-transcriptionality” - the transcription elongation complex as a nexus for nuclear transactions

    PubMed Central

    Perales, Roberto; Bentley, David

    2009-01-01

    Much of the complex process of RNP biogenesis takes place at the gene, co-transcriptionally. The target for RNA binding and processing factors is therefore not a solitary RNA molecule, but a transcription elongation complex (TEC) comprising the growing nascent RNA and RNA polymerase traversing a chromatin template with associated passenger proteins. RNA maturation factors are not the only nuclear machines whose work is organized co-transcriptionally around the TEC scaffold. In addition DNA repair, covalent chromatin modification, “gene gating” at the nuclear pore, Ig gene hypermutation, and sister chromosome cohesion have all been demonstrated or suggested to involve a co-transcriptional component. From this perspective, TEC’s can be viewed as potent “community organizers” within the nucleus. PMID:19854129

  12. Direct transcriptional regulation by nuclear microRNAs.

    PubMed

    Salmanidis, Marika; Pillman, Katherine; Goodall, Gregory; Bracken, Cameron

    2014-09-01

    The function of microRNAs is well characterized in the cytoplasm, where they direct an Argonaute-containing complex to target and repress mRNAs. More recently, regulatory roles for microRNAs and Argonaute have also been reported in the nucleus where microRNAs guide Argonaute to target gene promoters and directly regulate transcription in either a positive or a negative manner. Deep sequencing has revealed a high abundance of endogenous microRNAs within the nucleus, and in silico target prediction suggests thousands of potential microRNA:promoter interaction sites. The predicted high frequency of miRNA:promoter interactions is supported by chromatin immunoprecipitation, indicating the microRNA-dependent recruitment of Argonaute to thousands of transcriptional start sites and the subsequent regulation of RNA polymerase-II occupancy and chromatin modifiers. In this review we discuss the evidence for, and mechanisms associated with, direct transcriptional regulation by microRNAs which may represent a significant and largely unexplored aspect of microRNA function. This article is part of a Directed Issue entitled: The non-coding RNA revolution. PMID:24680896

  13. Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import.

    PubMed Central

    Luo, C; Shaw, K T; Raghavan, A; Aramburu, J; Garcia-Cozar, F; Perrino, B A; Hogan, P G; Rao, A

    1996-01-01

    The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8799126

  14. Transcriptional competence of the integrated HIV-1 provirus at the nuclear periphery

    PubMed Central

    Dieudonné, Mariacarolina; Maiuri, Paolo; Biancotto, Chiara; Knezevich, Anna; Kula, Anna; Lusic, Marina; Marcello, Alessandro

    2009-01-01

    Spatial distribution of genes within the nucleus contributes to transcriptional control, allowing optimal gene expression as well as constitutive or regulated gene repression. Human immunodeficiency virus type 1 (HIV-1) integrates into host chromatin to transcribe and replicate its genome. Lymphocytes harbouring a quiescent but inducible provirus are a challenge to viral eradication in infected patients undergoing antiviral therapy. Therefore, our understanding of the contribution of sub-nuclear positioning to viral transcription may also have far-reaching implications in the pathology of the infection. To gain an insight into the conformation of chromatin at the site of HIV-1 integration, we investigated lymphocytes carrying a single latent provirus. In the silenced state, the provirus was consistently found at the nuclear periphery, associated in trans with a pericentromeric region of chromosome 12 in a significant number of quiescent cells. After induction of the transcription, this association was lost, although the location of the transcribing provirus remained peripheral. These results, extended to several other cell clones, unveil a novel mechanism of transcriptional silencing involved in HIV-1 post-transcriptional latency and reinforce the notion that gene transcription may also occur at the nuclear periphery. PMID:19478796

  15. Glucose repression of yeast mitochondrial transcription: kinetics of derepression and role of nuclear genes.

    PubMed Central

    Ulery, T L; Jang, S H; Jaehning, J A

    1994-01-01

    Yeast mitochondrial transcript and gene product abundance has been observed to increase upon release from glucose repression, but the mechanism of regulation of this process has not been determined. We report a kinetic analysis of this phenomenon, which demonstrates that the abundance of all classes of mitochondrial RNA changes slowly relative to changes observed for glucose-repressed nuclear genes. Several cell doublings are required to achieve the 2- to 20-fold-higher steady-state levels observed after a shift to a nonrepressing carbon source. Although we observed that in some yeast strains the mitochondrial DNA copy number also increases upon derepression, this does not seem to play the major role in increased RNA abundance. Instead we found that three- to sevenfold increases in RNA synthesis rates, measured by in vivo pulse-labelling experiments, do correlate with increased transcript abundance. We found that mutations in the SNF1 and REG1 genes, which are known to affect the expression of many nuclear genes subject to glucose repression, affect derepression of mitochondrial transcript abundance. These genes do not appear to regulate mitochondrial transcript levels via regulation of the nuclear genes RPO41 and MTF1, which encode the subunits of the mitochondrial RNA polymerase. We conclude that a nuclear gene-controlled factor(s) in addition to the two RNA polymerase subunits must be involved in glucose repression of mitochondrial transcript abundance. Images PMID:8289797

  16. Isolation of Catharanthus roseus (L.) G. Don Nuclei and Measurement of Rate of Tryptophan decarboxylase Gene Transcription Using Nuclear Run-On Transcription Assay

    PubMed Central

    Kumar, Santosh; Bhatia, Sabhyata

    2015-01-01

    Background An accurate assessment of transcription ‘rate’ is often desired to describe the promoter activity. In plants, isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and therefore limit an accurate measurement of gene transcription ‘rate’. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA) pathway and therefore serves as a ‘molecular hub’ to understand gene expression profiles. Results The protocols presented here streamline, adapt and optimize the existing methods of nuclear run-on assay for use in C. roseus. Here, we fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of TDC gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA). However, the relative transcript abundance measured parallel through qRT-PCR was found to be inconsistent with the synthesis rate indicating that some post transcriptional events might have a role in transcript stability in response to stimuli. Conclusions Our study provides an optimized, efficient and inexpensive method of isolation of intact nuclei and nuclear ‘run-on’ transcription assay to carry out in-situ measurement of gene transcription rate in Catharanthus roseus. This would be valuable in investigating the transcriptional and post transcriptional response of other TIA pathway genes in C. roseus. Isolated nuclei may also provide a resource that could be used for performing the chip assay as well as serve as the source of nuclear proteins for in-vitro EMSA studies. Moreover, nascent nuclear run

  17. Nuclear actin polymerization from faster growing ends in the initial activation of Hox gene transcription are nuclear speckles involved?

    PubMed

    Naum-Onganía, Gabriela; Díaz, Víctor M; Blasi, Francesco; Rivera-Pomar, Rolando

    2013-01-01

    The HoxB cluster expression is activated by retinoic acid and transcribed in a collinear manner. The DNA-binding Pknox1-Pbx1 complex modulates Hox protein activity. Here, NT2-D1 teratocarcinoma cells -a model of Hox gene expression- were used to show that upon retinoic acid induction, Pknox1 co-localizes with polymeric nuclear actin. We have found that globular actin aggregates, polymeric actin, the elongating RNA polymerase II and THOC match euchromatic regions corresponding to nuclear speckles. Moreover, RNA polymerase II, N-WASP, and transcription/splicing factors p54(nrb) and PSF were validated as Pknox1 interactors by tandem affinity purification. PSF pulled down with THOC and nuclear actin, both of which co-localize in nuclear speckles. Although latrunculin A slightly decreases the general level of HoxB gene expression, inhibition of nuclear actin polymerization by cytochalasin D blocks the expression of HoxB transcripts in a collinear manner. Thus, our results support the hypothesis that nuclear actin polymerization is involved in the activation of HoxB gene expression by means of nuclear speckles. PMID:24406343

  18. Simultaneous live imaging of the transcription and nuclear position of specific genes

    PubMed Central

    Ochiai, Hiroshi; Sugawara, Takeshi; Yamamoto, Takashi

    2015-01-01

    The relationship between genome organization and gene expression has recently been established. However, the relationships between spatial organization, dynamics, and transcriptional regulation of the genome remain unknown. In this study, we developed a live-imaging method for simultaneous measurements of the transcriptional activity and nuclear position of endogenous genes, which we termed the ‘Real-time Observation of Localization and EXpression (ROLEX)’ system. We demonstrated that ROLEX is highly specific and does not affect the expression level of the target gene. ROLEX enabled detection of sub-genome-wide mobility changes that depended on the state of Nanog transactivation in embryonic stem cells. We believe that the ROLEX system will become a powerful tool for exploring the relationship between transcription and nuclear dynamics in living cells. PMID:26092696

  19. Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly.

    PubMed

    Sawamura, N; Ando, T; Maruyama, Y; Fujimuro, M; Mochizuki, H; Honjo, K; Shimoda, M; Toda, H; Sawamura-Yamamoto, T; Makuch, L A; Hayashi, A; Ishizuka, K; Cascella, N G; Kamiya, A; Ishida, N; Tomoda, T; Hai, T; Furukubo-Tokunaga, K; Sawa, A

    2008-12-01

    Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription. PMID:18762802

  20. Dependence of Enhancer-Mediated Transcription of the Immunoglobulin μ Gene on Nuclear Matrix Attachment Regions

    NASA Astrophysics Data System (ADS)

    Forrester, William C.; van Genderen, Courtney; Jenuwein, Thomas; Grosschedl, Rudolf

    1994-08-01

    Transcription of the immunoglobulin μ heavy chain locus is regulated by an intronic enhancer that is flanked on both sides by nuclear matrix attachment regions (MARs). These MARs have now been shown to be essential for transcription of a rearranged μ gene in transgenic B lymphocytes, but they were not required in stably transfected tissue culture cells. Normal rates of transcriptional initiation at a variable region promoter and the formation of an extended deoxyribonuclease I (DNase I)-sensitive chromatin domain were dependent on MARs, although DNase I hypersensitivity at the enhancer was detected in the absence of MARs. Thus, transcriptional activation of the μ gene during normal lymphoid development requires a synergistic collaboration between the enhancer and flanking MARs.

  1. Role of zinc finger structure in nuclear localization of transcription factor Sp1

    SciTech Connect

    Ito, Tatsuo; Azumano, Makiko; Uwatoko, Chisana; Itoh, Kohji Kuwahara, Jun

    2009-02-27

    Transcription factor Sp1 is localized in the nucleus and regulates gene expression. Our previous study demonstrated that the carboxyl terminal region of Sp1 containing 3-zinc finger region as DNA binding domain can also serve as nuclear localization signal (NLS). However, the nuclear transport mechanism of Sp1 has not been well understood. In this study, we performed a gene expression study on mutant Sp1 genes causing a set of amino acid substitutions in zinc finger domains to elucidate nuclear import activity. Nuclear localization of the GFP-fused mutant Sp1 proteins bearing concomitant substitutions in the first and third zinc fingers was highly inhibited. These mutant Sp1 proteins had also lost the binding ability as to the GC box sequence. The results suggest that the overall tertiary structure formed by the three zinc fingers is essential for nuclear localization of Sp1 as well as dispersed basic amino acids within the zinc fingers region.

  2. Nuclear Glycolytic Enzyme Enolase of Toxoplasma gondii Functions as a Transcriptional Regulator

    PubMed Central

    Mouveaux, Thomas; Oria, Gabrielle; Werkmeister, Elisabeth; Slomianny, Christian; Fox, Barbara A.; Bzik, David J.; Tomavo, Stanislas

    2014-01-01

    Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii. PMID:25153525

  3. Nuclear glycolytic enzyme enolase of Toxoplasma gondii functions as a transcriptional regulator.

    PubMed

    Mouveaux, Thomas; Oria, Gabrielle; Werkmeister, Elisabeth; Slomianny, Christian; Fox, Barbara A; Bzik, David J; Tomavo, Stanislas

    2014-01-01

    Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5' untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii. PMID:25153525

  4. A strain-dependent diffusivity model to study the nuclear import of mechanobiological transcription factors.

    PubMed

    Nava, Michele M; Fedele, Roberto; Raimondi, Manuela T

    2015-08-01

    Nuclear spreading plays a crucial role in stem cell fate determination. In previous works, we reported evidence of multipotency maintenance of mesenchymal stromal cells cultured on three-dimensional engineered niche substrates fabricated via two-photon laser polymerization (2PP). We correlated multipotency maintenance to a more roundish nuclear morphology of cells cultured in the 2PP-fabricated niches, with respect to those on flat substrates. To interpret these findings, here we present a multiphysics model coupling nuclear strains induced by cell adhesion to diffusive transport across the cell nucleus. We reconstructed the cell nuclear geometry from confocal Z-stack images of 2PP-cultured cells, and we estimated the volume, surface and shape factors. The levels of nuclear spreading significantly varied depending on the cell localization within the niche architecture. We assumed the cell diffusivity as a function of the local volumetric strain. The computational model also indicate that the larger the nuclear deformation (e.g. in spread nuclei), the higher the nuclear flux of small solutes such as transcription factors through the nuclear membrane. Our results point towards nuclear deformation as a primary mechanism by which the stem cell translates its shape into a fate decision, i.e. through a strain-dependent amplification of the diffusive flow of signaling molecules into the nucleus. PMID:26736643

  5. Nuclear Pore Proteins Nup153 and Megator Define Transcriptionally Active Regions in the Drosophila Genome

    PubMed Central

    Miura, Kota; Luscombe, Nicholas M.; Akhtar, Asifa

    2010-01-01

    Transcriptional regulation is one of the most important processes for modulating gene expression. Though much of this control is attributed to transcription factors, histones, and associated enzymes, it is increasingly apparent that the spatial organization of chromosomes within the nucleus has a profound effect on transcriptional activity. Studies in yeast indicate that the nuclear pore complex might promote transcription by recruiting chromatin to the nuclear periphery. In higher eukaryotes, however, it is not known whether such regulation has global significance. Here we establish nucleoporins as a major class of global regulators for gene expression in Drosophila melanogaster. Using chromatin-immunoprecipitation combined with microarray hybridisation, we show that Nup153 and Megator (Mtor) bind to 25% of the genome in continuous domains extending 10 kb to 500 kb. These Nucleoporin-Associated Regions (NARs) are dominated by markers for active transcription, including high RNA polymerase II occupancy and histone H4K16 acetylation. RNAi–mediated knock-down of Nup153 alters the expression of ∼5,700 genes, with a pronounced down-regulatory effect within NARs. We find that nucleoporins play a central role in coordinating dosage compensation—an organism-wide process involving the doubling of expression of the male X chromosome. NARs are enriched on the male X chromosome and occupy 75% of this chromosome. Furthermore, Nup153-depletion abolishes the normal function of the male-specific dosage compensation complex. Finally, by extensive 3D imaging, we demonstrate that NARs contribute to gene expression control irrespective of their sub-nuclear localization. Therefore, we suggest that NAR–binding is used for chromosomal organization that enables gene expression control. PMID:20174442

  6. Malondialdehyde inhibits an AMPK-mediated nuclear translocation and repression activity of ALDH2 in transcription

    SciTech Connect

    Choi, Ji-Woong; Kim, Jae-Hwan; Cho, Sung-Chun; Ha, Moon-Kyung; Song, Kye-Yong; Youn, Hong-Duk; Park, Sang Chul

    2011-01-07

    Research highlights: {yields} ALDH2 is an MDA-modified protein in old rat kidney tissues. {yields} AMPK associates with ALDH2 and triggers the nuclear localization of ALDH2. {yields} ALDH2 serves as a general transcriptional repressor by associating with HDACs. {yields} MDA inhibits the AMPK-mediated translocation of ALDH2 and its repression activity. -- Abstract: Aging process results from deleterious damages by reactive oxygen species, in particular, various metabolic aldehydes. Aldehyde dehydrogenase 2 (ALDH2) is one of metabolic enzymes detoxifying various aldehydes under oxidative conditions. AMP-activated protein kinase (AMPK) plays a key role in controlling metabolic process. However, little was known about the relationship of ALDH2 with AMPK under oxidative conditions. Here, we, by using MDA-specific monoclonal antibody, screened the tissues of young and old rats for MDA-modified proteins and identified an ALDH2 as a prominent MDA-modified protein band in the old rat kidney tissue. ALDH2 associates with AMPK and is phosphorylated by AMPK. In addition, AICAR, an activator of AMP-activated protein kinase, induces the nuclear translocation of ALDH2. ALDH2 in nucleus is involved in general transcription repression by association with histone deacetylases. Furthermore, MDA modification inhibited the translocation of ALDH2 and the association with AMPK, and ultimately led to de-repression of transcription in the reporter system analysis. In this study, we have demonstrated that ALDH2 acts as a transcriptional repressor in response to AMPK activation, and MDA modifies ALDH2 and inhibits repressive activity of ALDH2 in general transcription. We thus suggest that increasing amount of MDA during aging process may interrupt the nuclear function of ALDH2, modulated by AMPK.

  7. A role for Sp and nuclear receptor transcription factors in a cardiac hypertrophic growth program

    PubMed Central

    Sack, Michael N.; Disch, Dennis L.; Rockman, Howard A.; Kelly, Daniel P.

    1997-01-01

    During cardiac hypertrophy, the chief myocardial energy source switches from fatty acid β-oxidation (FAO) to glycolysis—a reversion to fetal metabolism. The expression of genes encoding myocardial FAO enzymes was delineated in a murine ventricular pressure overload preparation to characterize the molecular regulatory events involved in the alteration of energy substrate utilization during cardiac hypertrophy. Expression of genes involved in the thioesterification, mitochondrial import, and β-oxidation of fatty acids was coordinately down-regulated after 7 days of right ventricular (RV) pressure overload. Results of RV pressure overload studies in mice transgenic for the promoter region of the gene encoding human medium-chain acyl-CoA dehydrogenase (MCAD, which catalyzes a rate-limiting step in the FAO cycle) fused to a chloramphenicol acetyltransferase reporter confirmed that repression of MCAD gene expression in the hypertrophied ventricle occurred at the transcriptional level. Electrophoretic mobility-shift assays performed with MCAD promoter fragments and nuclear protein extracts prepared from hypertrophied and control RV identified pressure overload-induced protein/DNA interactions at a regulatory unit shown previously to confer control of MCAD gene transcription during cardiac development. Antibody “supershift” studies demonstrated that members of the Sp (Sp1, Sp3) and nuclear hormone receptor [chicken ovalbumin upstream promoter transcription factor (COUP-TF)/erbA-related protein 3] families interact with the pressure overload-responsive unit. Cardiomyocyte transfection studies confirmed that COUP-TF repressed the transcriptional activity of the MCAD promoter. The DNA binding activities and nuclear expression of Sp1/3 and COUP-TF in normal fetal mouse heart were similar to those in the hypertrophied adult heart. These results identify a transcriptional regulatory mechanism involved in the reinduction of a fetal metabolic program during pressure

  8. Serine phosphorylation by SYK is critical for nuclear localization and transcription factor function of Ikaros

    PubMed Central

    Uckun, Fatih M.; Ma, Hong; Zhang, Jian; Ozer, Zahide; Dovat, Sinisa; Mao, Cheney; Ishkhanian, Rita; Goodman, Patricia; Qazi, Sanjive

    2012-01-01

    Ikaros is a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis through transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation. Functional deficiency of Ikaros has been implicated in the pathogenesis of acute lymphoblastic leukemia, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros activity is considered of paramount importance, but the operative molecular mechanisms responsible for its regulation remain largely unknown. Here we provide multifaceted genetic and biochemical evidence for a previously unknown function of spleen tyrosine kinase (SYK) as a partner and posttranslational regulator of Ikaros. We demonstrate that SYK phoshorylates Ikaros at unique C-terminal serine phosphorylation sites S358 and S361, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Mechanistically, we establish that SYK-induced Ikaros activation is essential for its nuclear localization and optimal transcription factor function. PMID:23071339

  9. The A- and B-type nuclear lamin networks: microdomains involved in chromatin organization and transcription

    PubMed Central

    Shimi, Takeshi; Pfleghaar, Katrin; Kojima, Shin-ichiro; Pack, Chan-Gi; Solovei, Irina; Goldman, Anne E.; Adam, Stephen A.; Shumaker, Dale K.; Kinjo, Masataka; Cremer, Thomas; Goldman, Robert D.

    2008-01-01

    The nuclear lamins function in the regulation of replication, transcription, and epigenetic modifications of chromatin. However, the mechanisms responsible for these lamin functions are poorly understood. We demonstrate that A- and B-type lamins form separate, but interacting, stable meshworks in the lamina and have different mobilities in the nucleoplasm as determined by fluorescence correlation spectroscopy (FCS). Silencing lamin B1 (LB1) expression dramatically increases the lamina meshwork size and the mobility of nucleoplasmic lamin A (LA). The changes in lamina mesh size are coupled to the formation of LA/C-rich nuclear envelope blebs deficient in LB2. Comparative genomic hybridization (CGH) analyses of microdissected blebs, fluorescence in situ hybridization (FISH), and immunofluorescence localization of modified histones demonstrate that gene-rich euchromatin associates with the LA/C blebs. Enrichment of hyperphosphorylated RNA polymerase II (Pol II) and histone marks for active transcription suggest that blebs are transcriptionally active. However, in vivo labeling of RNA indicates that transcription is decreased, suggesting that the LA/C-rich microenvironment induces promoter proximal stalling of Pol II. We propose that different lamins are organized into separate, but interacting, microdomains and that LB1 is essential for their organization. Our evidence suggests that the organization and regulation of chromatin are influenced by interconnections between these lamin microdomains. PMID:19141474

  10. The nuclear factor SPBP contains different functional domains and stimulates the activity of various transcriptional activators.

    PubMed

    Rekdal, C; Sjøttem, E; Johansen, T

    2000-12-22

    SPBP (stromelysin-1 platelet-derived growth factor-responsive element binding protein) was originally cloned from a cDNA expression library by virtue of its ability to bind to a platelet-derived growth factor-responsive element in the human stromelysin-1 promoter. A 937-amino acid-long protein was deduced from a 3995-nucleotide murine cDNA sequence. By analyses of both human and murine cDNAs, we now show that SPBP is twice as large as originally found. The human SPBP gene contains six exons and is located on chromosome 22q13.1-13.3. Two isoforms differing in their C termini are expressed due to alternative splicing. PCR analyses of multitissue cDNA panels showed that SPBP is expressed in most tissues except for ovary and prostate. Functional mapping revealed that SPBP is a nuclear, multidomain protein containing an N-terminal region with transactivating ability, a novel type of DNA-binding domain containing an AT hook motif, and a bipartite nuclear localization signal as well as a C-terminal zinc finger domain. This type of zinc finger domain is also found in the trithorax family of chromatin-based transcriptional regulator proteins. Using cotransfection experiments, we find that SPBP enhances the transcriptional activity of various transcription factors such as c-Jun, Ets1, Sp1, and Pax6. Hence, SPBP seems to act as a transcriptional coactivator. PMID:10995766

  11. Transcriptional regulation of the human TR2 orphan receptor gene by nuclear factor 1-A

    SciTech Connect

    Lin, Y.-L.; Wang, Y.-H.; Lee, H.-J. . E-mail: hjlee@mail.ndhu.edu.tw

    2006-11-17

    The human testicular receptor 2 (TR2), a member of the nuclear hormone receptor superfamily, has no identified ligand yet. Previous evidence demonstrated that a 63 bp DNA fragment, named the promoter activating cis-element (PACE), has been identified as a positive regulatory region in the 5' promoter region of the human TR2 gene. In the present report, the human nuclear factor 1-A (NF1-A) was identified as a transcriptional activator to recognize the center of the PACE, called the PACE-C. NF1-A could bind to the 18 bp PACE-C region, and enhance about 13- to 17-fold of the luciferase reporter gene activity via the PACE-C in dose-dependent and orientation-independent manners. This transcriptional activation was further confirmed by real-time RT-PCR assay. In conclusion, our results indicated that NF1-A transcription factor plays an important role in the transcriptional activation of the TR2 gene expression via the PACE-C in the minimal promoter region.

  12. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    Many individual chloroplast genes require the products of a collection of nuclear genes for their successful expression. These nuclear gene products apparently work with great specificity, each committed to the expression of a single chloroplast gene. We have chosen as a model nuclear mutants of Chlamydomonas affected in different stages in the expression of the chloroplast encoded Photosystem II polypeptide, D2. We have made the progress in understanding how nuclear gene products affect the translation of the D2 encoding MRNA. Two nuclear genes are required for this process which have been mapped genetically. In contrast to other examples of nuclear control of translation in the chloroplast, these nuclear gene products appear to be required either for specific stages in translation elongation or for the post-translational stabilization of the nascent D2 protein. Pseudoreversion analysis has led us to a locus which may be directly involved in D2 expression. We have made considerable progress in pursuing the molecular basis of psbd MRNA stabilization. psbD 5' UTR specific transcripts have been synthesized in vitro and used in gel mobility shift assays. UV-crosslinking studies are underway to identify the transacting factors which bind to these sequences. The continued examination of these mutants will help us to understand how nuclear gene products work in this specific case of chloroplast gene expression, and will elucidate how two distinct genomes can interact generally.

  13. tRNAs Promote Nuclear Import of HIV-1 Intracellular Reverse Transcription Complexes

    PubMed Central

    Zaitseva, Lyubov; Myers, Richard; Fassati, Ariberto

    2006-01-01

    Infection of non-dividing cells is a biological property of HIV-1 crucial for virus transmission and AIDS pathogenesis. This property depends on nuclear import of the intracellular reverse transcription and pre-integration complexes (RTCs/PICs). To identify cellular factors involved in nuclear import of HIV-1 RTCs, cytosolic extracts were fractionated by chromatography and import activity examined by the nuclear import assay. A near-homogeneous fraction was obtained, which was active in inducing nuclear import of purified and labeled RTCs. The active fraction contained tRNAs, mostly with defective 3′ CCA ends. Such tRNAs promoted HIV-1 RTC nuclear import when synthesized in vitro. Active tRNAs were incorporated into and recovered from virus particles. Mutational analyses indicated that the anticodon loop mediated binding to the viral complex whereas the T-arm may interact with cellular factors involved in nuclear import. These tRNA species efficiently accumulated into the nucleus on their own in a energy- and temperature-dependent way. An HIV-1 mutant containing MLV gag did not incorporate tRNA species capable of inducing HIV-1 RTC nuclear import and failed to infect cell cycle–arrested cells. Here we provide evidence that at least some tRNA species can be imported into the nucleus of human cells and promote HIV-1 nuclear import. PMID:17020411

  14. Transcriptional activation of nuclear estrogen receptor and progesterone receptor and its regulation.

    PubMed

    Xin, Qi-Liang; Qiu, Jing-Tao; Cui, Sheng; Xia, Guo-Liang; Wang, Hai-Bin

    2016-08-25

    Estrogen receptor (ER) and progesterone receptor (PR) are two important members of steroid receptors family, an evolutionarily conserved family of transcription factors. Upon binding to their ligands, ER and PR enter cell nucleus to interact with specific DNA element in the context of chromatin to initiate the transcription of diverse target genes, which largely depends on the timely recruitment of a wide range of cofactors. Moreover, the interactions between steroid hormones and their respective receptors also trigger post-translational modifications on these receptors to fine-tune their transcriptional activities. Besides the well-known phosphorylation modifications on tyrosine and serine/threonine residues, recent studies have identified several other covalent modifications, such as ubiquitylation and sumoylation. These post-translational modifications of steroid receptors affect its stability, subcellular localization, and/or cofactor recruitment; eventually influence the duration and extent of transcriptional activation. This review is to focus on the recent research progress on the transcriptional activation of nuclear ER and PR as well as their physiological functions in early pregnancy, which may help us to better understand related female reproductive diseases. PMID:27546504

  15. Transcription-dependent nuclear localization of DAZAP1 requires an N-terminal signal

    SciTech Connect

    Lin, Yi-Tzu; Wen, Wan-Ching; Yen, Pauline H.

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer DAZAP1 shuttles between the nucleus and the cytoplasm. Black-Right-Pointing-Pointer DAZAP1 accumulates in the cytoplasm when the nuclear transcription is inhibited. Black-Right-Pointing-Pointer DAZAP1's transcription-dependent nuclear localization requires N-terminal N42. Black-Right-Pointing-Pointer SLIRP binds to N42 and may be involved in the process. -- Abstract: Deleted in Azoospermia Associated Protein 1 (DAZAP1) is a ubiquitous hnRNP protein required for normal development and spermatogenesis. It resides predominantly in the nucleus and moves between the nucleus and the cytoplasm via a ZNS shuttling signal at its C-terminus. DAZAP1 accumulates in the cytoplasm when RNA polymerase II activity is inhibited by actinomycin D. Here we report the mapping of a 42-amino acid segment (N42) at the N-terminus of DAZAP1 that is both necessary and sufficient for its transcription-dependent nuclear localization. In addition, using a yeast two-hybrid system, we have identified SLIRP as a N42-binding protein which may regulate DAZAP1 subcellular localization.

  16. Acetylation-deacetylation of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) regulates its transcriptional activity and nucleocytoplasmic localization.

    PubMed

    Kawai, Yumiko; Garduño, Lakisha; Theodore, Melanie; Yang, Jianqi; Arinze, Ifeanyi J

    2011-03-01

    Activation of Nrf2 by covalent modifications that release it from its inhibitor protein Keap1 has been extensively documented. In contrast, covalent modifications that may regulate its action after its release from Keap1 have received little attention. Here we show that CREB-binding protein induced acetylation of Nrf2, increased binding of Nrf2 to its cognate response element in a target gene promoter, and increased Nrf2-dependent transcription from target gene promoters. Heterologous sirtuin 1 (SIRT1) decreased acetylation of Nrf2 as well as Nrf2-dependent gene transcription, and its effects were overridden by dominant negative SIRT1 (SIRT1-H355A). The SIRT1-selective inhibitors EX-527 and nicotinamide stimulated Nrf2-dependent gene transcription, whereas resveratrol, a putative activator of SIRT1, was inhibitory, mimicking the effect of SIRT1. Mutating lysine to alanine or to arginine at Lys(588) and Lys(591) of Nrf2 resulted in decreased Nrf2-dependent gene transcription and abrogated the transcription-activating effect of CREB-binding protein. Furthermore, SIRT1 had no effect on transcription induced by these mutants, indicating that these sites are acetylation sites. Microscope imaging of GFP-Nrf2 in HepG2 cells as well as immunoblotting for Nrf2 showed that acetylation conditions resulted in increased nuclear localization of Nrf2, whereas deacetylation conditions enhanced its cytoplasmic rather than its nuclear localization. We posit that Nrf2 in the nucleus undergoes acetylation, resulting in binding, with basic-region leucine zipper protein(s), to the antioxidant response element and consequently in gene transcription, whereas deacetylation disengages it from the antioxidant response element, thereby resulting in transcriptional termination and subsequently in its nuclear export. PMID:21196497

  17. Multiple phosphorylation events control chicken ovalbumin upstream promoter transcription factor I orphan nuclear receptor activity.

    PubMed

    Gay, Frédérique; Baráth, Peter; Desbois-Le Péron, Christine; Métivier, Raphaël; Le Guével, Rémy; Birse, Darcy; Salbert, Gilles

    2002-06-01

    Chicken ovalbumin upstream promoter transcription factor I (COUP-TFI) is an orphan member of the nuclear hormone receptor superfamily that comprises key regulators of many biological functions, such as embryonic development, metabolism, homeostasis, and reproduction. Although COUP-TFI can both actively silence gene transcription and antagonize the functions of various other nuclear receptors, the COUP-TFI orphan receptor also acts as a transcriptional activator in certain contexts. Moreover, COUP-TFI has recently been shown to serve as an accessory factor for some ligand-bound nuclear receptors, suggesting that it may modulate, both negatively and positively, a wide range of hormonal responses. In the absence of any identified cognate ligand, the mechanisms involved in the regulation of COUP-TFI activity remain unclear. The elucidation of several putative phosphorylation sites for MAPKs, PKC, and casein kinase II within the sequence of this orphan receptor led us to investigate phosphorylation events regulating the various COUP-TFI functions. After showing that COUP-TFI is phosphorylated in vivo, we provide evidence that in vivo inhibition of either MAPK or PKC signaling pathway leads to a specific and pronounced decrease in COUP-TFI-dependent transcriptional activation of the vitronectin gene promoter. Focusing on the molecular mechanisms underlying the MAPK- and PKC-mediated regulation of COUP-TFI activity, we show that COUP-TFI can be directly targeted by PKC and MAPK. These phosphorylation events differentially modulate COUP-TFI functions: PKC-mediated phosphorylation enhances COUP-TFI affinity for DNA and MAPK-mediated phosphorylation positively regulates the transactivation function of COUP-TFI, possibly through enhancing specific coactivator recruitment. These data provide evidence that COUP-TFI is likely to integrate distinct signaling pathways and raise the possibility that multiple extracellular signals influence biological processes controlled by COUP

  18. O-GlcNAc-glycosylation of {beta}-catenin regulates its nuclear localization and transcriptional activity

    SciTech Connect

    Sayat, Ria; Leber, Brian; Grubac, Vanja; Wiltshire, Lesley; Persad, Sujata

    2008-09-10

    {beta}-catenin plays a role in intracellular adhesion and regulating gene expression. The latter role is associated with its oncogenic properties. Phosphorylation of {beta}-catenin controls its intracellular expression but mechanism/s that regulates the nuclear localization of {beta}-catenin is unknown. We demonstrate that O-GlcNAc glycosylation (O-GlcNAcylation) of {beta}-catenin negatively regulates its levels in the nucleus. We show that normal prostate cells (PNT1A) have significantly higher amounts of O-GlcNAcylated {beta}-catenin compared to prostate cancer (CaP) cells. The total nuclear levels of {beta}-catenin are higher in the CaP cells than PNT1A but only a minimal fraction of the nuclear {beta}-catenin in the CaP cells are O-GlcNAcylated. Increasing the levels of O-GlcNAcylated {beta}-catenin in the CaP cells with PUGNAc (O- (2-acetamido-2-deoxy-D-gluco-pyranosylidene) amino-N-phenylcarbamate) treatment is associated with a progressive decrease in the levels of {beta}-catenin in the nucleus. TOPFlash reporter assay and mRNA expressions of {beta}-catenin's target genes indicate that O-GlcNAcylation of {beta}-catenin results in a decrease in its transcriptional activity. We define a novel modification of {beta}-catenin that regulates its nuclear localization and transcriptional function.

  19. Transcriptional regulation of the phosphoenolpyruvate carboxykinase gene by cooperation between hepatic nuclear factors.

    PubMed Central

    Yanuka-Kashles, O; Cohen, H; Trus, M; Aran, A; Benvenisty, N; Reshef, L

    1994-01-01

    To study the transcriptional regulation of the liver gluconeogenic phenotype, the underdifferentiated mouse Hepa-1c1c7 (Hepa) hepatoma cell line was used. These cells mimicked the fetal liver by appreciably expressing the alpha-fetoprotein and albumin genes but not the phosphoenolpyruvate carboxykinase (PEPCK) gene. Unlike the fetal liver, however, Hepa cells failed to express the early-expressed factors hepatocyte nuclear factor 1 alpha (HNF-1 alpha) and HNF-4 and the late-expressed factor C/EBP alpha, thereby providing a suitable system for examining possible cooperation between these factors in the transcriptional regulation of the PEPCK gene. Transient transfection assays of a chimeric PEPCK-chloramphenicol acetyltransferase construct showed a residual PEPCK promoter activity in the Hepa cell line, which was slightly stimulated by cotransfection with a single transcription factor from either the C/EBP family or HNF-1 alpha but not at all affected by cotransfection of HNF-4. In contrast, cotransfection of the PEPCK construct with members from the C/EBP family plus HNF-1 alpha resulted in a synergistic stimulation of the PEPCK promoter activity. This synergistic effect depended on the presence in the PEPCK promoter region of the HNF-1 recognition sequence and on the presence of two C/EBP recognition sequences. The results demonstrate a requirement for coexistence and cooperation between early and late liver-enriched transcription factors in the transcriptional regulation of the PEPCK gene. In addition, the results suggest redundancy between members of the C/EBP family of transcription factors in the regulation of PEPCK gene expression. Images PMID:7935427

  20. A global comparison between nuclear and cytosolic transcriptomes reveals differential compartmentalization of alternative transcript isoforms

    PubMed Central

    Chen, Liang

    2010-01-01

    Transcriptome analyses have typically disregarded nucleocytoplasmic differences. This approach has ignored some post-transcriptional regulations and their effect on the ultimate protein expression levels. Despite a longstanding interest in the differences between the nuclear and cytosolic transcriptomes, it is only recently that data have become available to study such differences and their associated features on a genome-wide scale. Here, we compared the nuclear and cytosolic transcriptomes of HepG2 and HeLa cells. HepG2 and HeLa cells vary significantly in the differential compartmentalization of their transcript isoforms, indicating that nucleocytoplasmic compartmentalization is a cell-specific characteristic. The differential compartmentalization is manifested at the transcript isoform level instead of the gene level because alternative isoforms of one gene can display different nucleocytoplasmic distributions. The isoforms enriched in the cytosol tend to have more introns and longer introns in their pre-mRNAs. They have more functional RNA folds and unique exons in the 3′ regions. These isoforms are more conserved than the isoforms enriched in the nucleus. Surprisingly, the presence of microRNAs does not have a significant impact on the nucleocytoplasmic distribution of their target isoforms. In contrast, nonsense-mediated decay is significantly more associated with the isoforms enriched in the nucleus than those enriched in the cytosol. PMID:19969546

  1. Nuclear receptor-induced transcription is driven by spatially and timely restricted waves of ROS

    PubMed Central

    Perillo, Bruno; Di Santi, Annalisa; Cernera, Gustavo; Ombra, Maria Neve; Castoria, Gabriella; Migliaccio, Antimo

    2014-01-01

    Gene expression is governed by chromatin mainly through posttranslational modifications at the N-terminal tails of nucleosomal histone proteins. According to the histone code theory, peculiar sets of such modifications (marks) give rise to reproducible final effects on transcription and, very recently, a further level of complexity has been highlighted in binary switches between specific marks at adjacent residues. In particular, disappearance of dimethyl-lysine 9 in histone H3 is faced by phosphorylation of the following serine during activation of gene expression. Demethylation of lysine 9 by the lysine-specific demethylase 1 (LSD1) is a pre-requisite for addition of the phosphoryl mark to serine 10 and an essential step in the transcriptional control by estrogens. It generates a local burst of oxygen reactive species (ROS) that induce oxidation of nearby nucleotides and recruitment of repair enzymes with a consequent formation of single or double stranded nicks on DNA that modify chromatin flexibility in order to allow correct assembly of the transcriptional machinery.   We describe here the molecular mechanism by which members of the family of nuclear receptors prevent the potential damage to DNA during transcription of target genes elicited by the use of ROS to shape chromatin. The mechanism is based on the presence of phosphorylated serine 10 in histone H3 to prevent unbalanced DNA oxidation waves. We also discuss the opportunities raised by the use of voluntary derangement of this servo system to induce selective death in hormone-responsive transformed cells. PMID:25482200

  2. Mammalian Maf1 is a negative regulator of transcription by all three nuclear RNA polymerases.

    PubMed

    Johnson, Sandra S; Zhang, Cheng; Fromm, Jody; Willis, Ian M; Johnson, Deborah L

    2007-05-11

    Most eukaryotic transcriptional regulators act in an RNA polymerase (Pol)-selective manner. Here we show that the human Maf1 protein negatively regulates transcription by all three nuclear Pols. Changes in Maf1 expression affect Pol I- and Pol III-dependent transcription in human glioblastoma lines. These effects are mediated, in part, through the ability of Maf1 to repress transcription of the TATA binding protein, TBP. Maf1 targets an Elk-1-binding site in the TBP promoter, and its occupancy of this region is reciprocal with that of Elk-1. Similarly, Maf1 occupancy of Pol III genes is inversely correlated with that of the initiation factor TFIIIB and Pol III. The phenotypic consequences of reducing Maf1 expression include changes in cell morphology and the accumulation of actin stress fibers, whereas Maf1 overexpression suppresses anchorage-independent growth. Together with the ability of Maf1 to reduce biosynthetic capacity, these findings support the idea that Maf1 regulates the transformation state of cells. PMID:17499043

  3. Phosphatidylinositol 3-kinase/Akt signaling enhances nuclear localization and transcriptional activity of BRCA1

    PubMed Central

    Hinton, Cimona V.; Fitzgerald, Latricia D.; Thompson, Marilyn E.

    2007-01-01

    Signaling pathways involved in regulating nuclear-cytoplasmic distribution of BRCA1 have not been previously reported. Here, we provide evidence that heregulin β1-induced activation of the Akt pathway increases the nuclear content of BRCA1. First, treatment of T47D breast cancer cells with heregulin β1 results in a two-fold increase in nuclear BRCA1 as assessed by FACS analysis, immunoblotting and immunofluorescence. This heregulin-induced increase in nuclear BRCA1 is blocked by siRNA-mediated down-regulation of Akt. Second, mutation of threonine 509 in BRCA1, the site of Akt phosphorylation, to an alanine, attenuates the ability of heregulin to induce BRCA1 nuclear accumulation. These data suggest that Akt-catalyzed phosphorylation of BRCA1 is required for the heregulin-regulated nuclear concentration of BRCA1. Because most functions ascribed to BRCA1 occur within the nucleus, we postulated that phosphorylation-dependent nuclear accumulation of BRCA1 would result in enhanced nuclear activity, specifically transcriptional activity, of BRCA1. This postulate is affirmed by our observation that the ability of BRCA1 to transactivate GADD45 promoter constructs was enhanced in T47D cells treated with heregulin β1. Furthermore, the heterologous expression of BRCA1 in HCC1937 human breast cancer cells, which have constitutively active Akt, also induces GADD45 promoter activity, whereas the expression of BRCA1 in which threonine 509 has been mutated to an alanine is able to only minimally induce promoter activity. These findings implicate Akt in upstream events leading to BRCA1 nuclear localization and function. PMID:17428466

  4. Phosphatidylinositol 3-kinase/Akt signaling enhances nuclear localization and transcriptional activity of BRCA1

    SciTech Connect

    Hinton, Cimona V.; Fitzgerald, Latricia D.; Thompson, Marilyn E. . E-mail: methompson@mmc.edu

    2007-05-15

    Signaling pathways involved in regulating nuclear-cytoplasmic distribution of BRCA1 have not been previously reported. Here, we provide evidence that heregulin {beta}1-induced activation of the Akt pathway increases the nuclear content of BRCA1. First, treatment of T47D breast cancer cells with heregulin {beta}1 results in a two-fold increase in nuclear BRCA1 as assessed by FACS analysis, immunoblotting and immunofluorescence. This heregulin-induced increase in nuclear BRCA1 is blocked by siRNA-mediated down-regulation of Akt. Second, mutation of threonine 509 in BRCA1, the site of Akt phosphorylation, to an alanine, attenuates the ability of heregulin to induce BRCA1 nuclear accumulation. These data suggest that Akt-catalyzed phosphorylation of BRCA1 is required for the heregulin-regulated nuclear concentration of BRCA1. Because most functions ascribed to BRCA1 occur within the nucleus, we postulated that phosphorylation-dependent nuclear accumulation of BRCA1 would result in enhanced nuclear activity, specifically transcriptional activity, of BRCA1. This postulate is affirmed by our observation that the ability of BRCA1 to transactivate GADD45 promoter constructs was enhanced in T47D cells treated with heregulin {beta}1. Furthermore, the heterologous expression of BRCA1 in HCC1937 human breast cancer cells, which have constitutively active Akt, also induces GADD45 promoter activity, whereas the expression of BRCA1 in which threonine 509 has been mutated to an alanine is able to only minimally induce promoter activity. These findings implicate Akt in upstream events leading to BRCA1 nuclear localization and function.

  5. Transcriptional activation by EBV nuclear antigen 1 is essential for the expression of EBV's transforming genes

    PubMed Central

    Altmann, Markus; Pich, Dagmar; Ruiss, Romana; Wang, Jindong; Sugden, Bill; Hammerschmidt, Wolfgang

    2006-01-01

    EBV is a paradigm for human tumor viruses because, although it infects most people benignly, it also can cause a variety of cancers. Both in vivo and in vitro, EBV infects B lymphocytes in G0, induces them to become blasts, and can maintain their proliferation in cell culture or in vivo as tumors. How EBV succeeds in these contrasting cellular environments in expressing its genes that control the host has not been explained. We have genetically dissected the EBV nuclear antigen 1 (EBNA1) gene that is required for replication of the viral genome, to elucidate its possible role in the transcription of viral genes. Strikingly, EBNA1 is essential to drive transcription of EBV's transforming genes after infection of primary B lymphocytes. PMID:16966603

  6. Nuclear speckles are detention centers for transcripts containing expanded CAG repeats.

    PubMed

    Urbanek, Martyna O; Jazurek, Magdalena; Switonski, Pawel M; Figura, Grzegorz; Krzyzosiak, Wlodzimierz J

    2016-09-01

    The human genetic disorders caused by CAG repeat expansions in the translated sequences of various genes are called polyglutamine (polyQ) diseases because of the cellular "toxicity" of the mutant proteins. The contribution of mutant transcripts to the pathogenesis of these diseases is supported by several observations obtained from cellular models of these disorders. Here, we show that the common feature of cell lines modeling polyQ diseases is the formation of nuclear CAG RNA foci. We performed qualitative and quantitative analyses of these foci in numerous cellular models endogenously and exogenously expressing mutant transcripts by fluorescence in situ hybridization (FISH). We compared the CAG RNA foci of polyQ diseases with the CUG foci of myotonic dystrophy type 1 and found substantial differences in their number and morphology. Smaller differences within the polyQ disease group were also revealed and included a positive correlation between the foci number and the CAG repeat length. We show that expanded CAA repeats, also encoding glutamine, did not trigger RNA foci formation and foci formation is independent of the presence of mutant polyglutamine protein. Using FISH combined with immunofluorescence, we demonstrated partial co-localization of CAG repeat foci with MBNL1 alternative splicing factor, which explains the mild deregulation of MBNL1-dependent genes. We also showed that foci reside within nuclear speckles in diverse cell types: fibroblasts, lymphoblasts, iPS cells and neuronal progenitors and remain dependent on integrity of these nuclear structures. PMID:27239700

  7. Cell-free production of transducible transcription factors for nuclear reprogramming.

    PubMed

    Yang, William C; Patel, Kedar G; Lee, Jieun; Ghebremariam, Yohannes T; Wong, H Edward; Cooke, John P; Swartz, James R

    2009-12-15

    Ectopic expression of a defined set of transcription factors chosen from Oct3/4, Sox2, c-Myc, Klf4, Nanog, and Lin28 can directly reprogram somatic cells to pluripotency. These reprogrammed cells are referred to as induced pluripotent stem cells (iPSCs). To date, iPSCs have been successfully generated using lentiviruses, retroviruses, adenoviruses, plasmids, transposons, and recombinant proteins. Nucleic acid-based approaches raise concerns about genomic instability. In contrast, a protein-based approach for iPSC generation can avoid DNA integration concerns as well as provide greater control over the concentration, timing, and sequence of transcription factor stimulation. Researchers recently demonstrated that polyarginine peptide conjugation can deliver recombinant protein reprogramming factor (RF) cargoes into cells and reprogram somatic cells into iPSCs. However, the protein-based approach requires a significant amount of protein for the reprogramming process. Producing fusion RFs in the large amounts required for this approach using traditional heterologous in vivo production methods is difficult and cumbersome since toxicity, product aggregation, and proteolysis by endogenous proteases limit yields. In this work, we show that cell-free protein synthesis (CFPS) is a viable option for producing soluble and functional transducible transcription factors for nuclear reprogramming. We used an E. coli-based CFPS system to express the above set of six human RFs as fusion proteins, each with a nona-arginine (R9) protein transduction domain. Using the flexibility offered by the CFPS platform, we successfully addressed proteolysis and protein solubility problems to produce full-length and soluble R9-RF fusions. We subsequently showed that R9-Oct3/4, R9-Sox2, and R9-Nanog exhibit cognate DNA-binding activities, R9-Nanog translocates across the plasma and nuclear membranes, and R9-Sox2 exerts transcriptional activity on a known downstream gene target. PMID:19718703

  8. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    The following is a review of research accomplished in the first two years of funding for the above mentioned project. The work performed is a molecular characterization of nuclear mutants of Chlamydomonas reinhardtii which are deficient in different stages in the post-transcriptional expression of a single chloroplast encoded polypeptide, the D2 protein of Photosystem II. Our long-term goals are to understand the molecular mechanisms by which nuclear gene products affect the expression of chloroplast genes. Specifically, we which to understand how specific nuclear gene products affect the turnover rate of the D2 encoding mRNA (psbD), how other nuclear encoded factors work to promote the translation of psbD mRNA and/or stabilize the D2 protein, and what the role of the D2 protein itself is in Photosystem II assembly and in the control of expression of other chloroplast genes. This progress report will be organized into four major sections concerning (I) The characterization of nuclear mutants affected in D2 translation/turnover, (II) The study of trans-acting factors which associate with the 5{prime} end of the psbD mRNA, (III) In vitro mutagenesis of the psbD gene, and (IV) Additional studies.

  9. Deacetylase-Independent Function of HDAC3 in Transcription and Metabolism Requires Nuclear Receptor Corepressor

    PubMed Central

    Sun, Zheng; Feng, Dan; Fang, Bin; Mullican, Shannon E.; You, Seo-Hee; Lim, Hee-Woong; Everett, Logan J.; Nabel, Christopher S.; Li, Yun; Selvakumaran, Vignesh; Won, Kyoung-Jae; Lazar, Mitchell A.

    2013-01-01

    Histone deacetylases (HDACs) are believed to regulate gene transcription by catalyzing deacetylation reactions. HDAC3 depletion in mouse liver upregulates lipogenic genes and results in severe hepatosteatosis. Here we show that pharmacologic HDAC inhibition in primary hepatocytes causes histone hyperacetylation but does not upregulate expression of HDAC3 target genes. Meanwhile, deacetylase-dead HDAC3 mutants can rescue hepatosteatosis and repress lipogenic genes expression in HDAC3-depleted mouse liver, demonstrating that histone acetylation is insufficient to activate gene transcription. Mutations abolishing interactions with the nuclear receptor corepressor (NCOR or SMRT) render HDAC3 nonfunctional in vivo. Additionally, liver-specific knockout of NCOR, but not SMRT, causes metabolic and transcriptomal alterations resembling those of mice without hepatic HDAC3, demonstrating that interaction with NCOR is essential for deacetylase-independent function of HDAC3. These findings highlight non-enzymatic roles of a major HDAC in transcriptional regulation in vivo and warrant reconsideration of the mechanism of action of HDAC inhibitors. PMID:24268577

  10. Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1

    PubMed Central

    Agbottah, Emmanuel T; Traviss, Christine; McArdle, James; Karki, Sambhav; St Laurent, Georges C; Kumar, Ajit

    2007-01-01

    Background Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression. Results Utilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction in vitro. Analysis of the effect of NF90ctv-TAR RNA interaction in vivo showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene. Conclusion Structural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1. PMID:17565699

  11. Modulation of Epstein–Barr Virus Nuclear Antigen 2-dependent transcription by protein arginine methyltransferase 5

    SciTech Connect

    Liu, Cheng-Der; Cheng, Chi-Ping; Fang, Jia-Shih; Chen, Ling-Chih; Zhao, Bo; Kieff, Elliott; Peng, Chih-Wen

    2013-01-18

    Highlights: ► Catalytic active PRMT5 substantially binds to the EBNA2 RG domain. ► PRMT5 augments the EBNA2-dependent transcription. ► PRMT5 triggers the symmetric dimethylation of the EBNA2 RG domain. ► PRMT5 enhances the promoter occupancy of EBNA2 on its target promoters. -- Abstract: Epstein–Barr Virus Nuclear Antigen (EBNA) 2 features an Arginine–Glycine repeat (RG) domain at amino acid positions 335–360, which is a known target for protein arginine methyltransferaser 5 (PRMT5). In this study, we performed protein affinity pull-down assays to demonstrate that endogenous PRMT5 derived from lymphoblastoid cells specifically associated with the protein bait GST-E2 RG. Transfection of a plasmid expressing PRMT5 induced a 2.5- to 3-fold increase in EBNA2-dependent transcription of both the LMP1 promoter in AKATA cells, which contain the EBV genome endogenously, and a Cp-Luc reporter plasmid in BJAB cells, which are EBV negative. Furthermore, we showed that there was a 2-fold enrichment of EBNA2 occupancy in target promoters in the presence of exogenous PRMT5. Taken together, we show that PRMT5 triggers the symmetric dimethylation of EBNA2 RG domain to coordinate with EBNA2-mediated transcription. This modulation suggests that PRMT5 may play a role in latent EBV infection.

  12. GABP Transcription Factor (Nuclear Respiratory Factor 2) Is Required for Mitochondrial Biogenesis

    PubMed Central

    Yang, Zhong-Fa; Drumea, Karen; Mott, Stephanie; Wang, Junling

    2014-01-01

    Mitochondria are membrane-bound cytoplasmic organelles that serve as the major source of ATP production in eukaryotic cells. GABP (also known as nuclear respiratory factor 2) is a nuclear E26 transformation-specific transcription factor (ETS) that binds and activates mitochondrial genes that are required for electron transport and oxidative phosphorylation. We conditionally deleted Gabpa, the DNA-binding component of this transcription factor complex, from mouse embryonic fibroblasts (MEFs) to examine the role of Gabp in mitochondrial biogenesis, function, and gene expression. Gabpα loss modestly reduced mitochondrial mass, ATP production, oxygen consumption, and mitochondrial protein synthesis but did not alter mitochondrial morphology, membrane potential, apoptosis, or the expression of several genes that were previously reported to be GABP targets. However, the expression of Tfb1m, a methyltransferase that modifies ribosomal rRNA and is required for mitochondrial protein translation, was markedly reduced in Gabpα-null MEFs. We conclude that Gabp regulates Tfb1m expression and plays an essential, nonredundant role in mitochondrial biogenesis. PMID:24958105

  13. Drosophila dany is essential for transcriptional control and nuclear architecture in spermatocytes.

    PubMed

    Trost, Martina; Blattner, Ariane C; Leo, Stefano; Lehner, Christian F

    2016-07-15

    The terminal differentiation of adult stem cell progeny depends on transcriptional control. A dramatic change in gene expression programs accompanies the transition from proliferating spermatogonia to postmitotic spermatocytes, which prepare for meiosis and subsequent spermiogenesis. More than a thousand spermatocyte-specific genes are transcriptionally activated in early Drosophila spermatocytes. Here we describe the identification and initial characterization of dany, a gene required in spermatocytes for the large-scale change in gene expression. Similar to tMAC and tTAFs, the known major activators of spermatocyte-specific genes, dany has a recent evolutionary origin, but it functions independently. Like dan and danr, its primordial relatives with functions in somatic tissues, dany encodes a nuclear Psq domain protein. Dany associates preferentially with euchromatic genome regions. In dany mutant spermatocytes, activation of spermatocyte-specific genes and silencing of non-spermatocyte-specific genes are severely compromised and the chromatin no longer associates intimately with the nuclear envelope. Therefore, as suggested recently for Dan/Danr, we propose that Dany is essential for the coordination of change in cell type-specific expression programs and large-scale spatial chromatin reorganization. PMID:27436041

  14. A Photo-Degradable Gene Delivery System for Enhanced Nuclear Gene Transcription

    PubMed Central

    Hoyoung, Lee; Yeji, Kim; Patrick G., Schweickert; Stephen F., Konieczny; You-Yeon, Won

    2013-01-01

    There currently exists a significant gap in our understanding of how the detailed chemical characteristics of polycation gene carriers influence their delivery performances in overcoming an important cellular-level transport barrier, i.e., intranuclear gene transcription. In this study, a UV-degradable gene carrier material (ENE4-1) was synthesized by crosslinking low molecular weight branched polyethylenimine (bPEI-2k) molecules using UV-cleavable o-nitrobenzyl urethane (NBU) as the linker molecule. NBU degrades upon exposure to mild UV irradiation. Therefore, this UV-degradable carrier allows us to control the chemical characteristics of the polymer/DNA complex (polyplex) particles at desired locations within the intracellular environment. By using this photolytic DNA carrier, we found that the exact timing of the UV degradation significantly influences the gene transfection efficiencies of ENE4-1/DNA(pGL2) polyplexes in HeLa cells. Interestingly, even if the polyplexes were UV-degraded at different intracellular locations/times, their nuclear entry efficiency was not influenced by the location/timing of UV degradation. The UV treatment did not influence the size or binding strength of the polyplexes. However, we confirmed that the degradation of the carrier molecules impacts the chemical characteristics of the polyplexes (it produces carbamic acid and nitrosobenzyl aldehyde groups on ENE4-1). We believe that these anionic acid groups enhance the interaction of the polyplexes with nuclear transcription proteins and thus the final gene expression levels; this effect was found to occur, even though UV irradiation itself has a general effect of reducing transfection efficiencies. Excess (uncomplexed) ENE4-1 polymers appear to not play any role in the UV-enhanced gene transcription phenomenon. PMID:24172855

  15. Transcription regulation of nuclear receptor PXR: Role of SUMO-1 modification and NDSM in receptor function.

    PubMed

    Priyanka; Kotiya, Deepak; Rana, Manjul; Subbarao, N; Puri, Niti; Tyagi, Rakesh K

    2016-01-15

    Pregnane & Xenobiotic Receptor (PXR) is one of the 48 members of the nuclear receptor superfamily of ligand-modulated transcription factors. PXR plays an important role in metabolism and elimination of diverse noxious endobiotics and xenobiotics. Like in case of some nuclear receptors its function may also be differentially altered, positively or negatively, by various post-translational modifications. In this context, regulation of PXR function by SUMOylation is the subject of present investigation. Here, we report that human PXR is modified by SUMO-1 resulting in its enhanced transcriptional activity. RT-PCR analysis showed that PXR SUMOylation in presence of rifampicin also enhances the endogenous expression levels of key PXR-regulated genes like CYP3A4, CYP2C9, MDR1 and UGT1A1. In addition, mammalian two-hybrid assay exhibited enhanced interaction between PXR and co-activator SRC-1. EMSA results revealed that SUMOylation has no influence on the DNA binding ability of PXR. In silico analysis suggested that PXR protein contains four putative SUMOylation sites, centered at K108, K129, K160 and K170. In addition to this, we identified the presence of NDSM (Negative charge amino acid Dependent SUMOylation Motif) in PXR. Substitution of all its four putative lysine residues along with NDSM abolished the effect of SUMO-1-mediated transactivation function of PXR. Furthermore, we show that interaction between PXR and E2-conjugation enzyme UBCh9, an important step for implementation of SUMOylation event, was reduced in case of NDSM mutant PXRD115A. Overall, our results suggest that SUMOylation at specific sites on PXR protein are involved in enhancement of transcription function of this receptor. PMID:26549688

  16. Inhibition of hepatitis B virus (HBV) by LNA-mediated nuclear interference with HBV DNA transcription

    SciTech Connect

    Sun, Zhen; Xiang, Wenqing; Guo, Yajuan; Chen, Zhi; Liu, Wei; Lu, Daru

    2011-06-10

    Highlights: {yields} LNA-modified oligonucleotides can pass through the plasma membrane of cultured cells even without using transfection machinery. {yields} LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. {yields} LNA-oligonucleotide designed to target nuclear HBV DNA efficiently suppresses HBV replication and transcription in cultured hepatic cells. -- Abstract: Silencing target genes with small regulatory RNAs is widely used to investigate gene function and therapeutic drug development. Recently, triplex-based approaches have provided another attractive means to achieve targeted gene regulation and gene manipulation at the molecular and cellular levels. Nuclear entry of oligonucleotides and enhancement of their affinity to the DNA targets are key points of such approaches. In this study, we developed lipid-based transport of a locked-nucleic-acid (LNA)-modified oligonucleotide for hepatitis B virus (HBV) DNA interference in human hepatocytes expressing HBV genomic DNA. In these cells, the LNA-modified oligonucleotides passed efficiently across the cell membrane, and lipid-coating facilitated translocation from the cytoplasm to the nucleus. The oligonucleotide specifically targeting HBV DNA clearly interfered with HBV DNA transcription as shown by a block in pregenomic RNA (pgRNA) production. The HBV DNA-targeted oligonucleotide suppressed HBV DNA replication and HBV protein production more efficiently than small interfering RNAs directed to the pgRNA. These results demonstrate that fusion with lipid can carry LNA-modified oligonucleotides to the nucleus where they regulate gene expression. Interfering with HBV DNA transcription by LNA-modified oligonucleotides has strong potential as a new strategy for HBV inhibition.

  17. Nuclear transcription factors: a new approach to enhancing cellular responses to ALA-mediated photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Maytin, Edward V.; Anand, Sanjay; Sato, Nobuyuki; Moore, Brian; Mack, Judith; Gasbarre, Christopher; Keevey, Samantha; Ortel, Bernhard; Sinha, Alok; Khachemoune, Amor

    2006-02-01

    Photodynamic therapy (PDT) using aminolevulinic acid (ALA) relies upon the uptake of ALA into cancer cells, where it is converted into a porphyrin intermediate, protoporphyrin IX (PpIX) that is highly photosensitizing. For large or resistant tumors, however, ALA/PDT is often not completely effective due to inadequate PpIX levels. Therefore, new approaches to enhance the intracellular production of PpIX are sought. Here, we describe a general approach to improve intracellular PpIX accumulation via manipulations that increase the expression of an enzyme, coproporphyrinogen oxidase (CPO), that is rate-determining for PpIX production. We show that nuclear hormones that promote terminal differentiation, e.g. vitamin D or androgens, can also increase the accumulation of PpIX and the amount of killing of the target cells upon exposure to light. These hormones bind to intracellular hormone receptors that translocate to the nucleus, where they act as transcription factors to increase the expression of target genes. We have found that several other transcription factors associated with terminal differentiation, including members of the CCAAT enhancer binding (C/EBP) family, and a homeobox protein named Hoxb13, are also capable of enhancing PpIX accumulation. These latter transcription factors appear to interact directly with the CPO gene promoter, resulting in enhanced CPO transcriptional activity. Our data in several different cell systems, including epithelial cells of the skin and prostate cancer cells, indicate that enhancement of CPO expression and PpIX accumulation represents a viable new approach toward improving the efficacy of ALA/PDT.

  18. Co-localization of the amyloid precursor protein and the Notch intracellular domains in nuclear transcription factories

    PubMed Central

    Konietzko, Uwe; Goodger, Zoë V.; Meyer, Michelle; Kohli, Bernhard M.; Bosset, Jérôme; Lahiri, Debomoy K.; Nitsch, Roger M.

    2009-01-01

    The β-amyloid precursor protein (APP) plays a major role in Alzheimer’s disease. The APP intracellular domain (AICD), together with Fe65 and Tip60, localizes to spherical nuclear AFT complexes that might represent sites of transcription. We now show that endogenous AICD is targeted to similar nuclear spots. AFT complexes were closely associated with Cajal and PML bodies but did not localize to nucleoli or splicing speckles. Live imaging revealed that AFT complexes were highly mobile within nuclei. Following pharmacological inhibition of transcription AFT complexes merged into a few large assemblies. We have previously shown that AICD regulates the expression of its own precursor APP. Transfection of APP promoter plasmids as substrates resulted in cytosolic AFT complex formation at the labeled APP promoter plasmids. In addition, identification of chromosomal APP or KAI1 gene loci by fluorescence in situ hybridization showed their close association with nuclear AFT complexes. The transcriptional activator Notch intracellular domain (NICD) localized to the same nuclear spots as occupied by AFT complexes, suggesting that these nuclear compartments correspond to transcription factories. Fe65 and Tip60 also co-localized with APP in the neurites of primary neurons. Pre-assembled AFT complexes may serve to assist fast nuclear signaling upon endoproteolytic APP cleavage. PMID:18403052

  19. Transcriptional activation of the cholesterol 7alpha-hydroxylase gene (CYP7A) by nuclear hormone receptors.

    PubMed

    Crestani, M; Sadeghpour, A; Stroup, D; Galli, G; Chiang, J Y

    1998-11-01

    The gene encoding cholesterol 7alpha-hydroxylase (CYP7A), the rate-limiting enzyme in bile acid synthesis, is transcriptionally regulated by bile acids and hormones. Previously, we have identified two bile acid response elements (BARE) in the promoter of the CYP7A gene. The BARE II is located in nt -149/-118 region and contains three hormone response element (HRE)-like sequences that form two overlapping nuclear receptor binding sites. One is a direct repeat separated by one nucleotide DR1 (-146- TGGACTtAGTTCA-134) and the other is a direct repeat separated by five nucleotides DR5 (-139-AGTTCAaggccGGG TAA-123). Mutagenesis of these HRE sequences resulted in lower transcriptional activity of the CYP7A promoter/reporter genes in transient transfection assay in HepG2 cells. The orphan nuclear receptor, hepatocyte nuclear factor 4 (HNF-4)1, binds to the DR1 sequence as assessed by electrophoretic mobility shift assay, and activates the CYP7A promoter/reporter activity by about 9-fold. Cotransfection of HNF-4 plasmid with another orphan nuclear receptor, chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), synergistically activated the CYP7A transcription by 80-fold. The DR5 binds the RXR/RAR heterodimer. A hepatocyte nuclear factor-3 (HNF-3) binding site (-175-TGTTTGTTCT-166) was identified. HNF-3 was required for both basal transcriptional activity and stimulation of the rat CYP7A promoter activity by retinoic acid. Combinatorial interactions and binding of these transcription factors to BAREs may modulate the promoter activity and also mediate bile acid repression of CYP7A gene transcription. PMID:9799805

  20. Herpes simplex virus type 1 protein IE63 affects the nuclear export of virus intron-containing transcripts.

    PubMed Central

    Phelan, A; Dunlop, J; Clements, J B

    1996-01-01

    Using in situ hybridization labelling methods, we have determined that the herpes simplex virus type 1 immediate-early protein IE63 (ICP27) affects the cellular localization of virus transcripts. Intronless transcripts from the IE63, UL38, and UL44 genes are rapidly exported to and accumulate in the cytoplasm throughout infection, in either the presence or absence of IE63 expression. The intron-containing transcripts from the IE110 and UL15 genes, while initially cytoplasmic, are increasingly retained in the nucleus in distinct clumps as infection proceeds, and the clumps colocalize with the redistributed small nuclear ribonucleoprotein particles. Infections with the IE63 mutant virus 27-lacZ demonstrated that in the absence of IE63 expression, nuclear retention of intron-containing transcripts was lost. The nuclear retention of UL15 transcripts, which demonstrated both nuclear and cytoplasmic label, was not as pronounced as that of the IE110 transcripts, and we propose that this is due to the late expression of UL15. Infections with the mutant virus 110C1, in which both introns of IE110 have been precisely removed (R.D. Everett, J. Gen. Virol. 72:651-659, 1991), demonstrated IE110 transcripts in both the nucleus and the cytoplasm; thus, exon definition sequences which regulate viral RNA transport are present in the IE110 transcript. By in situ hybridization a stable population of polyadenylated RNAs was found to accumulate in the nucleus in spots, most of which were separate from the small nuclear ribonucleoprotein particle clumps. The IE63 protein has an involvement, either direct or indirect, in the regulation of nucleocytoplasmic transport of viral transcripts, a function which contrasts with the recently proposed role of herpes simplex virus type 1 Us11 in promoting the nuclear export of partially spliced or unspliced transcripts (J.-J. Diaz, M. Duc Dodon, N. Schaerer-Uthurraly, D. Simonin, K. Kindbeiter, L. Gazzolo, and J.-J. Madjar, Nature [London] 379

  1. Transcription of Nrdp1 by the androgen receptor is regulated by nuclear filamin A in prostate cancer.

    PubMed

    Savoy, Rosalinda M; Chen, Liqun; Siddiqui, Salma; Melgoza, Frank U; Durbin-Johnson, Blythe; Drake, Christiana; Jathal, Maitreyee K; Bose, Swagata; Steele, Thomas M; Mooso, Benjamin A; D'Abronzo, Leandro S; Fry, William H; Carraway, Kermit L; Mudryj, Maria; Ghosh, Paramita M

    2015-06-01

    Prostate cancer (PCa) progression is regulated by the androgen receptor (AR); however, patients undergoing androgen-deprivation therapy (ADT) for disseminated PCa eventually develop castration-resistant PCa (CRPC). Results of previous studies indicated that AR, a transcription factor, occupies distinct genomic loci in CRPC compared with hormone-naïve PCa; however, the cause of this distinction was unknown. The E3 ubiquitin ligase Nrdp1 is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. Using Nrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximal Nrdp1 promoter contains an androgen response element (ARE); we demonstrated AR binding to this ARE in androgen-sensitive PCa. Analysis of hormone-naive human prostatectomy specimens revealed correlation between Nrdp1 and AR expression, supporting AR regulation of NRDP1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to the Nrdp1 promoter and Nrdp1 expression were suppressed in CRPC. Elucidation of the suppression mechanism demonstrated correlation of NRDP1 levels with nuclear localization of the scaffolding protein filamin A (FLNA) which, as we previously showed, is itself repressed following ADT in many CRPC tumors. Restoration of nuclear FLNA in CRPC stimulated AR binding to Nrdp1 ARE, increased its transcription, and augmented NRDP1 protein expression and responsiveness to ADT, indicating that nuclear FLNA controls AR-mediated androgen-sensitive Nrdp1 transcription. Expression of other AR-regulated genes lost in CRPC was also re-established by nuclear FLNA. Thus, our results indicate that nuclear FLNA promotes androgen-dependent AR-regulated transcription in PCa, while loss of nuclear FLNA in CRPC alters the AR-regulated transcription program. PMID:25759396

  2. Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function

    SciTech Connect

    Dement, Gregory A.; Maloney, Scott C.; Reeves, Raymond . E-mail: reevesr@mail.wsu.edu

    2007-01-01

    We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential ({delta}{psi}{sub m}). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7C cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression.

  3. Low ozone concentrations stimulate cytoskeletal organization, mitochondrial activity and nuclear transcription.

    PubMed

    Costanzo, M; Cisterna, B; Vella, A; Cestari, T; Covi, V; Tabaracci, G; Malatesta, M

    2015-01-01

    Ozone therapy is a modestly invasive procedure based on the regeneration capabilities of low ozone concentrations and used in medicine as an alternative/adjuvant treatment for different diseases. However, the cellular mechanisms accounting for the positive effects of mild ozonization are still largely unexplored. To this aim, in the present study the effects of low ozone concentrations (1 to 20 µg O3/mL O2) on structural and functional cell features have been investigated in vitro by using morphological, morphometrical, cytochemical and immunocytochemical techniques at bright field, fluorescence and transmission electron microscopy. Cells exposed to pure O2 or air served as controls. The results demonstrated that the effects of ozoneadministration are dependent on gas concentration, and the cytoskeletal organization, mitochondrial activity and nuclear transcription may be differently affected. This suggests that, to ensure effective and permanent metabolic cell activation, ozone treatments should take into account the cytological and cytokinetic features of the different tissues. PMID:26150162

  4. Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance

    PubMed Central

    Tsang, Chi Kwan; Liu, Yuan; Thomas, Janice; Zhang, Yanjie; Zheng, X. F. Steven

    2015-01-01

    Summary Superoxide dismutase 1 (Sod1) has been known for nearly half a century for catalysis of superoxide to hydrogen peroxide. Here we report a new Sod1 function in oxidative signaling: in response to elevated endogenous and exogenous reactive oxygen species (ROS), Sod1 rapidly relocates into the nucleus, which is important for maintaining genomic stability. Interestingly, H2O2 is sufficient to promote Sod1 nuclear localization, indicating that it is responding to general ROS rather than Sod1 substrate superoxide. ROS signaling is mediated by Mec1/ATM and its effector Dun1/Cds1 kinase, through Dun1 interaction with Sod1 and regulation of Sod1 by phosphorylation at S60, 99. In the nucleus, Sod1 binds to the promoters and regulates the expression of oxidative resistance and repair genes. Altogether, our study unravels an unorthodox function of Sod1 as a transcription factor and elucidates the regulatory mechanism for its localization. PMID:24647101

  5. Low Ozone Concentrations Stimulate Cytoskeletal Organization, Mitochondrial Activity and Nuclear Transcription

    PubMed Central

    Costanzo, M.; Cisterna, B.; Vella, A.; Cestari, T.; Covi, V.; Tabaracci, G.; Malatesta, M.

    2015-01-01

    Ozone therapy is a modestly invasive procedure based on the regeneration capabilities of low ozone concentrations and used in medicine as an alternative/adjuvant treatment for different diseases. However, the cellular mechanisms accounting for the positive effects of mild ozonization are still largely unexplored. To this aim, in the present study the effects of low ozone concentrations (1 to 20 µg O3/mL O2) on structural and functional cell features have been investigated in vitro by using morphological, morphometrical, cytochemical and immunocytochemical techniques at bright field, fluorescence and transmission electron microscopy. Cells exposed to pure O2 or air served as controls. The results demonstrated that the effects of ozone administration are dependent on gas concentration, and the cytoskeletal organization, mitochondrial activity and nuclear transcription may be differently affected. This suggests that, to ensure effective and permanent metabolic cell activation, ozone treatments should take into account the cytological and cytokinetic features of the different tissues. PMID:26150162

  6. Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription.

    PubMed

    Chambeyron, Séverine; Bickmore, Wendy A

    2004-05-15

    The colinearity of genes in Hox clusters suggests a role for chromosome structure in gene regulation. We reveal programmed changes in chromatin structure and nuclear organization upon induction of Hoxb expression by retinoic acid. There is an early increase in the histone modifications that are marks of active chromatin at both the early expressed gene Hoxb1, and also at Hoxb9 that is not expressed until much later. There is also a visible decondensation of the chromatin between Hoxb1 and Hoxb9 at this early stage. However, a further change in higher-order chromatin structure, looping out of genes from the chromosome territory, occurs in synchrony with the execution of the gene expression program. We suggest that higher-order chromatin structure regulates the expression of the HoxB cluster at several levels. Locus-wide changes in chromatin structure (histone modification and chromatin decondensation) may establish a transcriptionally poised state but are not sufficient for the temporal program of gene expression. The choreographed looping out of decondensed chromatin from chromosome territories may then allow for activation of high levels of transcription from the sequence of genes along the cluster. PMID:15155579

  7. Nuclear F-actin enhances the transcriptional activity of β-catenin by increasing its nuclear localization and binding to chromatin.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; de Lanerolle, Primal; Harata, Masahiko

    2016-04-01

    Actin plays multiple roles both in the cytoplasm and in the nucleus. Cytoplasmic actin, in addition to its structural role in the cytoskeleton, also contributes to the subcellular localization of transcription factors by interacting with them or their partners. The transcriptional cofactor β-catenin, which acts as an intracellular transducer of canonical Wnt signaling, indirectly associates with the cytoplasmic filamentous actin (F-actin). Recently, it has been observed that F-actin is transiently formed within the nucleus in response to serum stimulation and integrin signaling, and also during gene reprogramming. Despite these earlier observations, information about the function of nuclear F-actin is poorly defined. Here, by facilitating the accumulation of nuclear actin artificially, we demonstrate that polymerizing nuclear actin enhanced the nuclear accumulation and transcriptional function of β-catenin. Our results also show that the nuclear F-actin colocalizes with β-catenin and enhances the binding of β-catenin to the downstream target genes of the Wnt/β-catenin signaling pathway, including the genes for the cell cycle regulators c-myc and cyclin D, and the OCT4 gene. Nuclear F-actin itself also associated with these genes. Since Wnt/β-catenin signaling has important roles in cell differentiation and pluripotency, our observations suggest that nuclear F-actin formed during these biological processes is involved in regulating Wnt/β-catenin signaling. PMID:26900020

  8. The Nuclear Matrix Protein, NRP/B, Acts as a Transcriptional Repressor of E2F-mediated Transcriptional Activity

    PubMed Central

    Choi, Jina; Yang, Eun Sung; Cha, Kiweon; Whang, John; Choi, Woo-Jung; Avraham, Shalom; Kim, Tae-Aug

    2014-01-01

    Background: NRP/B, a family member of the BTB/Kelch repeat proteins, is implicated in neuronal and cancer development, as well as the regulation of oxidative stress responses in breast and brain cancer. Our previous studies indicate that the NRP/B-BTB/POZ domain is involved in the dimerization of NRP/B and in a complex formation with the tumor suppressor, retinoblastoma protein. Although much evidence supports the potential role of NRP/B as a tumor suppressor, the molecular mechanisms of NRP/B action on E2F transcription factors have not been elucidated. Methods: Three-dimensional modeling of NRP/B was used to generate point mutations in the BTB/Kelch domains. Tet-on inducible NRP/B expression was established. The NRP/B deficient breast cancer cell line, MDA-MB-231, was generated using lentiviral shNRP/B to evaluate the effect of NRP/B on cell proliferation, invasion and migration. Immunoprecipitation was performed to verify the interaction of NRP/B with E2F and histone deacetylase (HDAC-1), and the expression level of NRP/B protein was analyzed by Western blot analysis. Changes in cell cycle were determined by flow cytometry. Transcriptional activities of E2F transcription factors were measured by chloramphenicol acetyltransferase (CAT) activity. Results: Ectopic overexpression of NRP/B demonstrated that the NRP/B-BTB/POZ domain plays a critical role in E2F-mediated transcriptional activity. Point mutations within the BTB/POZ domain restored E2-promoter activity inhibited by NRP/B. Loss of NRP/B enhanced the proliferation and migration of breast cancer cells. Endogenous NRP/B interacted with E2F and HDAC1. Treatement with an HDAC inhibitor, trichostatin A (TSA), abolished the NRP/B-mediated suppression of E2-promoter activity. Gain or loss of NRP/B in HeLa cells confirmed the transcriptional repressive capability of NRP/B on the E2F target genes, Cyclin E and HsORC (Homo sapiens Origin Recognition Complex). Conclusions: The present study shows that NRP/B acts as a

  9. Preparation and characterization of yeast nuclear extracts for efficient RNA polymerase B (II)-dependent transcription in vitro.

    PubMed Central

    Verdier, J M; Stalder, R; Roberge, M; Amati, B; Sentenac, A; Gasser, S M

    1990-01-01

    We present a reproducible method for the preparation of nuclear extracts from the yeast Saccharomyces cerevisiae that support efficient RNA polymerase B (II)-dependent transcription. Extracts from both a crude nuclear fraction and Percoll-purified nuclei are highly active for site-specific initiation and transcription of a G-free cassette under the Adenovirus major late promoter. At optimal extract concentrations transcription is at least 5 times more efficient with the yeast extracts than with HeLa whole cell extracts. We show that the transcriptional activity is sensitive to alpha-amanitin and to depletion of factor(s) recognizing the TATA-box of the promoter. The in vitro reaction showed maximal activity after 45 min, was very sensitive to Cl-, but was not affected by high concentrations of potassium. We find that the efficiency of in vitro transcription in nuclear extracts is reproducibly high when spheroplasting is performed with a partially purified beta 1,3-glucanase (lyticase). Therefore a simplified method to isolate the lyticase from the supernatant of Oerskovia xanthineolytica is also presented. Images PMID:2263463

  10. Transcription-dependent nucleolar cap localization and possible nuclear function of DExH RNA helicase RHAU

    SciTech Connect

    Iwamoto, Fumiko; Stadler, Michael; Chalupnikova, Katerina; Oakeley, Edward; Nagamine, Yoshikuni

    2008-04-01

    RHAU (RNA helicase associated with AU-rich element) is a DExH protein originally identified as a factor accelerating AU-rich element-mediated mRNA degradation. The discovery that RHAU is predominantly localized in the nucleus, despite mRNA degradation occurring in the cytoplasm, prompted us to consider the nuclear functions of RHAU. In HeLa cells, RHAU was found to be localized throughout the nucleoplasm with some concentrated in nuclear speckles. Transcriptional arrest altered the localization to nucleolar caps, where RHAU is closely localized with RNA helicases p68 and p72, suggesting that RHAU is involved in transcription-related RNA metabolism in the nucleus. To see whether RHAU affects global gene expression transcriptionally or posttranscriptionally, we performed microarray analysis using total RNA from RHAU-depleted HeLa cell lines, measuring both steady-state mRNA levels and mRNA half-lives by actinomycin D chase. There was no change in the half-lives of most transcripts whose steady-state levels were affected by RHAU knockdown, suggesting that these transcripts are subjected to transcriptional regulation. We propose that RHAU has a dual function, being involved in both the synthesis and degradation of mRNA in different subcellular compartments.

  11. Function of steroidogenic factor 1 domains in nuclear localization, transactivation, and interaction with transcription factor TFIIB and c-Jun.

    PubMed

    Li, L A; Chiang, E F; Chen, J C; Hsu, N C; Chen, Y J; Chung, B C

    1999-09-01

    Normal endocrine development and function require nuclear hormone receptor SF-1 (steroidogenic factor 1). To understand the molecular mechanism of SF-1 action, we have investigated its domain function by mutagenesis and functional analyses. Our mutant studies show that the putative AF2 (activation function 2) helix located at the C-terminal end is indispensable for gene activation. SF-1 does not have an N-terminal AF1 domain. Instead, it contains a unique FP region, composed of the Ftz-F1 box and the proline cluster, after the zinc finger motif. The FP region interacts with transcription factor IIB (TFIIB) in vitro. This interaction requires residues 178-201 of TFIIB, a domain capable of binding several transcription factors. The FP region also mediates physical interaction with c-Jun, and this interaction greatly enhances SF-1 activity. The putative SF-1 ligand, 25-hydroxycholesterol, has no effects on these bindings. In addition, the Ftz-F1 box contains a bipartite nuclear localization signal (NLS). Removing the basic residues at either end of the key nuclear localization sequence NLS2.2 abolishes the nuclear transport. Expression of mutants containing only the FP region or lacking the AF2 domain blocks wild-type SF-1 activity in cells. By contrast, the mutant having a truncated nuclear localization signal lacks this dominant negative effect. These results delineate the importance of the FP and AF2 regions in nuclear localization, protein-protein interaction, and transcriptional activation. PMID:10478848

  12. Nuclear cereblon modulates transcriptional activity of Ikaros and regulates its downstream target, enkephalin, in human neuroblastoma cells.

    PubMed

    Wada, Takeyoshi; Asahi, Toru; Sawamura, Naoya

    2016-08-26

    The gene coding cereblon (CRBN) was originally identified in genetic linkage analysis of mild autosomal recessive nonsyndromic intellectual disability. CRBN has broad localization in both the cytoplasm and nucleus. However, the significance of nuclear CRBN remains unknown. In the present study, we aimed to elucidate the role of CRBN in the nucleus. First, we generated a series of CRBN deletion mutants and determined the regions responsible for the nuclear localization. Only CRBN protein lacking the N-terminal region was localized outside of the nucleus, suggesting that the N-terminal region is important for its nuclear localization. CRBN was also identified as a thalidomide-binding protein and component of the cullin-4-containing E3 ubiquitin ligase complex. Thalidomide has been reported to be involved in the regulation of the transcription factor Ikaros by CRBN-mediated degradation. To investigate the nuclear functions of CRBN, we performed co-immunoprecipitation experiments and evaluated the binding of CRBN to Ikaros. As a result, we found that CRBN was associated with Ikaros protein, and the N-terminal region of CRBN was required for Ikaros binding. In luciferase reporter gene experiments, CRBN modulated transcriptional activity of Ikaros. Furthermore, we found that CRBN modulated Ikaros-mediated transcriptional repression of the proenkephalin gene by binding to its promoter region. These results suggest that CRBN binds to Ikaros via its N-terminal region and regulates transcriptional activities of Ikaros and its downstream target, enkephalin. PMID:27329811

  13. Transcriptomic Changes Due to Cytoplasmic TDP-43 Expression Reveal Dysregulation of Histone Transcripts and Nuclear Chromatin

    PubMed Central

    Amlie-Wolf, Alexandre; Ryvkin, Paul; Tong, Rui; Dragomir, Isabelle; Suh, EunRan; Xu, Yan; Van Deerlin, Vivianna M.; Gregory, Brian D.; Kwong, Linda K.; Trojanowski, John Q.; Lee, Virginia M.-Y.; Wang, Li-San; Lee, Edward B.

    2015-01-01

    TAR DNA-binding protein 43 (TDP-43) is normally a nuclear RNA-binding protein that exhibits a range of functions including regulation of alternative splicing, RNA trafficking, and RNA stability. However, in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), TDP-43 is abnormally phosphorylated, ubiquitinated, and cleaved, and is mislocalized to the cytoplasm where it forms distinctive aggregates. We previously developed a mouse model expressing human TDP-43 with a mutation in its nuclear localization signal (ΔNLS-hTDP-43) so that the protein preferentially localizes to the cytoplasm. These mice did not exhibit a significant number of cytoplasmic aggregates, but did display dramatic changes in gene expression as measured by microarray, suggesting that cytoplasmic TDP-43 may be associated with a toxic gain-of-function. Here, we analyze new RNA-sequencing data from the ΔNLS-hTDP-43 mouse model, together with published RNA-sequencing data obtained previously from TDP-43 antisense oligonucleotide (ASO) knockdown mice to investigate further the dysregulation of gene expression in the ΔNLS model. This analysis reveals that the transcriptomic effects of the overexpression of the ΔNLS-hTDP-43 transgene are likely due to a gain of cytoplasmic function. Moreover, cytoplasmic TDP-43 expression alters transcripts that regulate chromatin assembly, the nucleolus, lysosomal function, and histone 3’ untranslated region (UTR) processing. These transcriptomic alterations correlate with observed histologic abnormalities in heterochromatin structure and nuclear size in transgenic mouse and human brains. PMID:26510133

  14. Transcriptomic Changes Due to Cytoplasmic TDP-43 Expression Reveal Dysregulation of Histone Transcripts and Nuclear Chromatin.

    PubMed

    Amlie-Wolf, Alexandre; Ryvkin, Paul; Tong, Rui; Dragomir, Isabelle; Suh, EunRan; Xu, Yan; Van Deerlin, Vivianna M; Gregory, Brian D; Kwong, Linda K; Trojanowski, John Q; Lee, Virginia M-Y; Wang, Li-San; Lee, Edward B

    2015-01-01

    TAR DNA-binding protein 43 (TDP-43) is normally a nuclear RNA-binding protein that exhibits a range of functions including regulation of alternative splicing, RNA trafficking, and RNA stability. However, in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), TDP-43 is abnormally phosphorylated, ubiquitinated, and cleaved, and is mislocalized to the cytoplasm where it forms distinctive aggregates. We previously developed a mouse model expressing human TDP-43 with a mutation in its nuclear localization signal (ΔNLS-hTDP-43) so that the protein preferentially localizes to the cytoplasm. These mice did not exhibit a significant number of cytoplasmic aggregates, but did display dramatic changes in gene expression as measured by microarray, suggesting that cytoplasmic TDP-43 may be associated with a toxic gain-of-function. Here, we analyze new RNA-sequencing data from the ΔNLS-hTDP-43 mouse model, together with published RNA-sequencing data obtained previously from TDP-43 antisense oligonucleotide (ASO) knockdown mice to investigate further the dysregulation of gene expression in the ΔNLS model. This analysis reveals that the transcriptomic effects of the overexpression of the ΔNLS-hTDP-43 transgene are likely due to a gain of cytoplasmic function. Moreover, cytoplasmic TDP-43 expression alters transcripts that regulate chromatin assembly, the nucleolus, lysosomal function, and histone 3' untranslated region (UTR) processing. These transcriptomic alterations correlate with observed histologic abnormalities in heterochromatin structure and nuclear size in transgenic mouse and human brains. PMID:26510133

  15. Depression of nuclear transcription and extension of mRNA half-life under anoxia in Artemia franciscana embryos.

    PubMed

    van Breukelen, F; Maier, R; Hand, S C

    2000-04-01

    Transcriptional activity, as assessed by nuclear run-on assays, was constant during 10 h of normoxic development for embryos of the brine shrimp Artemia franciscana. Exposure of embryos to only 4 h of anoxia resulted in a 79.3+/-1 % decrease in levels of in-vivo-initiated transcripts, and transcription was depressed by 88. 2+/-0.7 % compared with normoxic controls after 24 h of anoxia (means +/- s.e.m., N=3). Initiation of transcription was fully restored after 1 h of normoxic recovery. Artificially lowering the intracellular pH of aerobic embryos to the value reflective of anoxia (pH 6.7) showed that acidification alone explained over half the transcriptional arrest. Initiation of transcription was not rescued by application of 80 % carbon monoxide under anoxia, which suggests that heme-based oxygen sensing is not involved in this global arrest. When these transcriptional data are combined with the finding that mRNA levels are unchanged for at least 6 h of anoxia, it is clear that the half-life of mRNA is extended at least 8.5-fold compared with that in aerobic embryos. In contrast to the activation of compensatory mechanisms to cope with anoxia that occurs in mammalian cells, A. franciscana embryos enter a metabolically depressed state in which gene expression and mRNA turnover are cellular costs apparently not compatible with survival and in which extended tolerance supercedes the requirement for continued metabolic function. PMID:10708633

  16. Protein kinase A activation enhances β-catenin transcriptional activity through nuclear localization to PML bodies.

    PubMed

    Zhang, Mei; Mahoney, Emilia; Zuo, Tao; Manchanda, Parmeet K; Davuluri, Ramana V; Kirschner, Lawrence S

    2014-01-01

    The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of β-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of β-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on β-catenin were required for nuclear re-localization. Further, β-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between β-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/β-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of β-catenin and its differential protein-protein interaction that can be modulated by PKA signaling. PMID:25299576

  17. Lycopene activates antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers.

    PubMed

    Sahin, K; Orhan, C; Tuzcu, M; Sahin, N; Hayirli, A; Bilgili, S; Kucuk, O

    2016-05-01

    This study was conducted to evaluate the effects of dietary lycopene supplementation on growth performance, antioxidant status, and muscle nuclear transcription factor [Kelch like-ECH-associated protein 1 (Keap1) and (erythroid-derived 2)-like 2 (Nrf2)] expressions in broiler chickens exposed to heat stress (HS). A total of 180 one-day-old male broiler chicks (Ross 308) were assigned randomly to one of 2×3 factorially arranged treatments: two housing temperatures (22°C for 24 h/d; thermoneutral, TN or 34°C for 8 h/d HS) and three dietary lycopene levels (0, 200, or 400 mg/kg). Each treatment consisted of three replicates of 10 birds. Birds were reared to 42 d of age. Heat stress caused reductions in feed intake and weight gain by 12.2 and 20.7% and increased feed efficiency by 10.8% (P<0.0001 for all). Increasing dietary lycopene level improved performance in both environments. Birds reared under the HS environment had lower serum and muscle lycopene concentration (0.34 vs. 0.50 μg/mL and 2.80 vs. 2.13 μg/g), activities of superoxide dismutase (151 vs. 126 U/mL and 131 vs. 155 U/mg protein), glutathione peroxidase (184 vs. 154 U/mL and 1.39 vs. 1.74 U/mg protein), and higher malondialdehyde (MDA) concentration (0.53 vs. 0.83 μg/mL and 0.78 vs. 0.45 μg/ mg protein) than birds reared under the TN environment. Changes in levels of lycopene and MDA and activities of enzymes in serum and muscle varied by the environmental temperature as dietary lycopene level increased. Moreover, increasing dietary lycopene level suppressed muscle Keap1 expression and enhanced muscle Nrf2 expression, which had increased by 150% and decreased by 40%, respectively in response to HS. In conclusion, lycopene supplementation alleviates adverse effects of HS on performance through modulating expressions of stress-related nuclear transcription factors. PMID:26936958

  18. Transcriptional regulation of the hepatocyte growth factor gene by the nuclear receptors chicken ovalbumin upstream promoter transcription factor and estrogen receptor.

    PubMed

    Jiang, J G; Bell, A; Liu, Y; Zarnegar, R

    1997-02-14

    Hepatocyte growth factor (HGF) is a multifunctional cytokine that controls the growth and differentiation of various tissues. Previously, we described the existence of a negative cis-acting regulatory element(s) within the -1- to -0.7-kilobase pair (kb) portion of the 5'-flanking region of the mouse HGF promoter. In the present study, we show that the repressor element is located at position -872 to -860 base pairs and comprises an imperfect estrogen-responsive element 5'-AGGTCAGAAAGACCA-3'. We demonstrate that chicken ovalbumin upstream promoter transcription factor (COUP-TF), a nuclear orphan receptor belonging to the steroid/thyroid hormone receptor superfamily, through binding to this site effectively silences the transcriptional activity of the HGF promoter. We show that estrogen receptor, on the other hand, relieves the repressive action of COUP-TF, resulting in the induction of the HGF promoter. Using mice transgenic for either 2.7 or 0.7 kb of the HGF promoter region linked to the chloramphenicol acetyltransferase reporter gene, we found that injection of estradiol stimulates HGF promoter activity in tissues such as the mammary gland and ovary of mice harboring 2.7 but not 0.7 kb of the mouse HGF promoter region. Potential involvement of the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors in the regulation of HGF gene expression is also discussed. PMID:9020096

  19. The orphan nuclear receptor DAX-1 acts as a novel transcriptional corepressor of PPAR{gamma}

    SciTech Connect

    Kim, Gwang Sik; Lee, Gha Young; Nedumaran, Balachandar; Park, Yun-Yong; Kim, Kyung Tae; Park, Sang Chul; Lee, Young Chul; Kim, Jae Bum Choi, Hueng-Sik

    2008-05-30

    DAX-1 is an atypical nuclear receptor (NR) which functions primarily as a transcriptional corepressor of other NRs via heterodimerization. Peroxisome proliferator-activated receptor (PPAR) {gamma} is a ligand-dependent NR which performs a key function in adipogenesis. In this study, we evaluated a novel cross-talk mechanism between DAX-1 and PPAR{gamma}. Transient transfection assays demonstrated that DAX-1 inhibits the transactivity of PPAR{gamma} in a dose-dependent manner. DAX-1 directly competed with the PPAR{gamma} coactivator (PGC)-1{alpha} for binding to PPAR{gamma}. Endogenous levels of DAX-1 were significantly lower in differentiated 3T3-L1 adipocytes as compared to preadipocytes. Using a retroviral expression system, we demonstrated that DAX-1 overexpression downregulates the expression of PPAR{gamma} target genes, resulting in an attenuation of adipogenesis in 3T3-L1 cells. Our results suggest that DAX-1 acts as a corepressor of PPAR{gamma} and performs a potential function in the regulation of PPAR{gamma}-mediated cellular differentiation.

  20. Release of Positive Transcription Elongation Factor b (P-TEFb) from 7SK Small Nuclear Ribonucleoprotein (snRNP) Activates Hexamethylene Bisacetamide-inducible Protein (HEXIM1) Transcription*

    PubMed Central

    Liu, Pingyang; Xiang, Yanhui; Fujinaga, Koh; Bartholomeeusen, Koen; Nilson, Kyle A.; Price, David H.; Peterlin, B. Matija

    2014-01-01

    By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis. PMID:24515107

  1. Arsenic Trioxide Activate Transcription of Heme Oxygenase-1 by Promoting Nuclear Translocation of NFE2L2

    PubMed Central

    Yue, Zhen; Zhong, Lingzhi; Mou, Yan; Wang, Xiaotong; Zhang, Haiying; Wang, Yang; Xia, Jianxin; Li, Ronggui; Wang, Zonggui

    2015-01-01

    In a previous study, we found that induced expression of Heme Oxygenase-1 (HO-1) is responsible for the resistance of human osteosarcoma MG63 cells to the chemotherapeutic agent arsenic trioxide (ATO). The present study was aimed at investigating the molecular mechanisms underlying the induction of HO-1 that occurs after exposure of MG63 cells to ATO. First, using RT-QPCT and Western-blot, we found that ATO strongly induced the expression of heme oxygenase-1 (HO-1) in these human osteosarcoma cells. Then by analyzing HO-1 mRNA of MG63 cells exposed to ATO in the presence and absence of a transcription inhibitor Actinomycin-D (Act-D), we demonstrated that ATO activates HO-1 expression in MG63 cells by regulating the transcription of the gene. Finally, through the analysis of the NFE2L2 protein levels among the total cellular and nuclear proteins by Western-blot and Immunocytochemical staning, we determined that ATO enhanced the nuclear translocation of nuclear factor erythroid 2-like 2 (NFE2L2), also known as Nrf2. From these results we have concluded that transcription activation of HO-1 resulting from the nuclear translocation of NFE2L2 is the underlying molecular mechanism for its high induction, which, in turn, is responsible for the resistance of human osteosarcoma cells to ATO treatment. PMID:26283888

  2. Mitochondrial-Nuclear DNA Interactions Contribute to the Regulation of Nuclear Transcript Levels as Part of the Inter-Organelle Communication System

    PubMed Central

    Rodley, Chris D. M.; Grand, Ralph S.; Gehlen, Lutz R.; Greyling, Gary; Jones, M. Beatrix; O'Sullivan, Justin M.

    2012-01-01

    Nuclear and mitochondrial organelles must maintain a communication system. Loci on the mitochondrial genome were recently reported to interact with nuclear loci. To determine whether this is part of a DNA based communication system we used genome conformation capture to map the global network of DNA-DNA interactions between the mitochondrial and nuclear genomes (Mito-nDNA) in Saccharomyces cerevisiae cells grown under three different metabolic conditions. The interactions that form between mitochondrial and nuclear loci are dependent on the metabolic state of the yeast. Moreover, the frequency of specific mitochondrial - nuclear interactions (i.e. COX1-MSY1 and Q0182-RSM7) showed significant reductions in the absence of mitochondrial encoded reverse transcriptase machinery. Furthermore, these reductions correlated with increases in the transcript levels of the nuclear loci (MSY1 and RSM7). We propose that these interactions represent an inter-organelle DNA mediated communication system and that reverse transcription of mitochondrial RNA plays a role in this process. PMID:22292080

  3. Transcriptional Regulation by Nuclear Corepressors and PGC-1α: Implications for Mitochondrial Quality Control and Insulin Sensitivity

    PubMed Central

    Qi, Zhengtang; Ding, Shuzhe

    2012-01-01

    The peroxisome proliferator-activated receptors (PPARs) and estrogen-related receptor (ERRα) are ligand-activated nuclear receptors that coordinately regulate gene expression. Recent evidence suggests that nuclear corepressors, NCoR, RIP140, and SMRT, repress nuclear receptors-mediated transcriptional activity on specific promoters, and thus regulate insulin sensitivity, adipogenesis, mitochondrial number, and activity in vivo. Moreover, the coactivator PGC-1α that increases mitochondrial biogenesis during exercise and calorie restriction directly regulates autophagy in skeletal muscle and mitophagy in the pathogenesis of Parkinson's disease. In this paper, we discuss the PGC-1α's novel role in mitochondrial quality control and the role of nuclear corepressors in regulating insulin sensitivity and interacting with PGC-1α. PMID:23304112

  4. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products. Progress report, June 1, 1990--June 30, 1992

    SciTech Connect

    Kuchka, M.R.

    1992-08-01

    Many individual chloroplast genes require the products of a collection of nuclear genes for their successful expression. These nuclear gene products apparently work with great specificity, each committed to the expression of a single chloroplast gene. We have chosen as a model nuclear mutants of Chlamydomonas affected in different stages in the expression of the chloroplast encoded Photosystem II polypeptide, D2. We have made the progress in understanding how nuclear gene products affect the translation of the D2 encoding MRNA. Two nuclear genes are required for this process which have been mapped genetically. In contrast to other examples of nuclear control of translation in the chloroplast, these nuclear gene products appear to be required either for specific stages in translation elongation or for the post-translational stabilization of the nascent D2 protein. Pseudoreversion analysis has led us to a locus which may be directly involved in D2 expression. We have made considerable progress in pursuing the molecular basis of psbd MRNA stabilization. psbD 5` UTR specific transcripts have been synthesized in vitro and used in gel mobility shift assays. UV-crosslinking studies are underway to identify the transacting factors which bind to these sequences. The continued examination of these mutants will help us to understand how nuclear gene products work in this specific case of chloroplast gene expression, and will elucidate how two distinct genomes can interact generally.

  5. Morris Water Maze Training in Mice Elevates Hippocampal Levels of Transcription Factors Nuclear Factor (Erythroid-derived 2)-like 2 and Nuclear Factor Kappa B p65

    PubMed Central

    Snow, Wanda M.; Pahlavan, Payam S.; Djordjevic, Jelena; McAllister, Danielle; Platt, Eric E.; Alashmali, Shoug; Bernstein, Michael J.; Suh, Miyoung; Albensi, Benedict C.

    2015-01-01

    Research has identified several transcription factors that regulate activity-dependent plasticity and memory, with cAMP-response element binding protein (CREB) being the most well-studied. In neurons, CREB activation is influenced by the transcription factor nuclear factor kappa B (NF-κB), considered central to immunity but more recently implicated in memory. The transcription factor early growth response-2 (Egr-2), an NF-κB gene target, is also associated with learning and memory. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an antioxidant transcription factor linked to NF-κB in pathological conditions, has not been studied in normal memory. Given that numerous transcription factors implicated in activity-dependent plasticity demonstrate connections to NF-κB, this study simultaneously evaluated protein levels of NF-κB, CREB, Egr-2, Nrf2, and actin in hippocampi from young (1 month-old) weanling CD1 mice after training in the Morris water maze, a hippocampal-dependent spatial memory task. After a 6-day acquisition period, time to locate the hidden platform decreased in the Morris water maze. Mice spent more time in the target vs. non-target quadrants of the maze, suggestive of recall of the platform location. Western blot data revealed a decrease in NF-κB p50 protein after training relative to controls, whereas NF-κB p65, Nrf2 and actin increased. Nrf2 levels were correlated with platform crosses in nearly all tested animals. These data demonstrate that training in a spatial memory task results in alterations in and associations with particular transcription factors in the hippocampus, including upregulation of NF-κB p65 and Nrf2. Training-induced increases in actin protein levels caution against its use as a loading control in immunoblot studies examining activity-dependent plasticity, learning, and memory. PMID:26635523

  6. Two proteolytic fragments of menin coordinate the nuclear transcription and postsynaptic clustering of neurotransmitter receptors during synaptogenesis between Lymnaea neurons

    PubMed Central

    Getz, Angela M.; Visser, Frank; Bell, Erin M.; Xu, Fenglian; Flynn, Nichole M.; Zaidi, Wali; Syed, Naweed I.

    2016-01-01

    Synapse formation and plasticity depend on nuclear transcription and site-specific protein targeting, but the molecular mechanisms that coordinate these steps have not been well defined. The MEN1 tumor suppressor gene, which encodes the protein menin, is known to induce synapse formation and plasticity in the CNS. This synaptogenic function has been conserved across evolution, however the underlying molecular mechanisms remain unidentified. Here, using central neurons from the invertebrate Lymnaea stagnalis, we demonstrate that menin coordinates subunit-specific transcriptional regulation and synaptic clustering of nicotinic acetylcholine receptors (nAChR) during neurotrophic factor (NTF)-dependent excitatory synaptogenesis, via two proteolytic fragments generated by calpain cleavage. Whereas menin is largely regarded as a nuclear protein, our data demonstrate a novel cytoplasmic function at central synapses. Furthermore, this study identifies a novel synaptogenic mechanism in which a single gene product coordinates the nuclear transcription and postsynaptic targeting of neurotransmitter receptors through distinct molecular functions of differentially localized proteolytic fragments. PMID:27538741

  7. Two proteolytic fragments of menin coordinate the nuclear transcription and postsynaptic clustering of neurotransmitter receptors during synaptogenesis between Lymnaea neurons.

    PubMed

    Getz, Angela M; Visser, Frank; Bell, Erin M; Xu, Fenglian; Flynn, Nichole M; Zaidi, Wali; Syed, Naweed I

    2016-01-01

    Synapse formation and plasticity depend on nuclear transcription and site-specific protein targeting, but the molecular mechanisms that coordinate these steps have not been well defined. The MEN1 tumor suppressor gene, which encodes the protein menin, is known to induce synapse formation and plasticity in the CNS. This synaptogenic function has been conserved across evolution, however the underlying molecular mechanisms remain unidentified. Here, using central neurons from the invertebrate Lymnaea stagnalis, we demonstrate that menin coordinates subunit-specific transcriptional regulation and synaptic clustering of nicotinic acetylcholine receptors (nAChR) during neurotrophic factor (NTF)-dependent excitatory synaptogenesis, via two proteolytic fragments generated by calpain cleavage. Whereas menin is largely regarded as a nuclear protein, our data demonstrate a novel cytoplasmic function at central synapses. Furthermore, this study identifies a novel synaptogenic mechanism in which a single gene product coordinates the nuclear transcription and postsynaptic targeting of neurotransmitter receptors through distinct molecular functions of differentially localized proteolytic fragments. PMID:27538741

  8. Nuclear translocation of doublecortin-like protein kinase and phosphorylation of a transcription factor JDP2

    SciTech Connect

    Nagamine, Tadashi; Nomada, Shohgo; Onouchi, Takashi; Kameshita, Isamu; Sueyoshi, Noriyuki

    2014-03-28

    Highlights: • Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase. • In living cells, DCLK was cleaved into two functional fragments. • zDCLK(kinase) was translocated into the nucleus by osmotic stresses. • Jun dimerization protein 2 (JDP2) was identified as zDCLK(kinase)-binding protein. • JDP2 was efficiently phosphorylated by zDCLK(kinase) only when histone was present. - Abstract: Doublecortin-like protein kinase (DCLK) is a microtubule-associated protein kinase predominantly expressed in brain. In a previous paper, we reported that zebrafish DCLK2 (zDCLK) was cleaved into two functional fragments; the N-terminal zDCLK(DC + SP) with microtubule-binding activity and the C-terminal zDCLK(kinase) with a Ser/Thr protein kinase activity. In this study, we demonstrated that zDCLK(kinase) was widely distributed in the cytoplasm and translocated into the nucleus when the cells were treated under hyperosmotic conditions with NaCl or mannitol. By two-hybrid screening using the C-terminal domain of DCLK, Jun dimerization protein 2 (JDP2), a nuclear transcription factor, was identified as zDCLK(kinase)-binding protein. Furthermore, JDP2 served as an efficient substrate for zDCLK(kinase) only when histone was present. These results suggest that the kinase fragment of DCLK is translocated into the nucleus upon hyperosmotic stresses and that the kinase efficiently phosphorylates JDP2, a possible target in the nucleus, with the aid of histones.

  9. Rrp6: Integrated roles in nuclear RNA metabolism and transcription termination.

    PubMed

    Fox, Melanie J; Mosley, Amber L

    2016-01-01

    The yeast RNA exosome is a eukaryotic ribonuclease complex essential for RNA processing, surveillance, and turnover. It is comprised of a barrel-shaped core and cap as well as a 3'-5' ribonuclease known as Dis3 that contains both endo- and exonuclease domains. A second exonuclease, Rrp6, is added in the nucleus. Dis3 and Rrp6 have both shared and distinct roles in RNA metabolism, and this review will focus primarily on Rrp6 and the roles of the RNA exosome in the nucleus. The functions of the nuclear exosome are modulated by cofactors and interacting partners specific to each type of substrate. Generally, the cofactor TRAMP (Trf4/5-Air2/1-Mtr4 polyadenylation) complex helps unwind unstable RNAs, RNAs requiring processing such as rRNAs, tRNAs, or snRNAs or improperly processed RNAs and direct it toward the exosome. In yeast, Rrp6 interacts with Nrd1, the cap-binding complex, and RNA polymerase II to aid in nascent RNA processing, termination, and polyA tail length regulation. Recent studies have shown that proper termination and processing of short, noncoding RNAs by Rrp6 is particularly important for transcription regulation across the genome and has important implications for regulation of diverse processes at the cellular level. Loss of proper Rrp6 and exosome activity may contribute to various pathologies such as autoimmune disease, neurological disorders, and cancer. WIREs RNA 2016, 7:91-104. doi: 10.1002/wrna.1317 For further resources related to this article, please visit the WIREs website. PMID:26612606

  10. Nuclear envelope dystrophies show a transcriptional fingerprint suggesting disruption of Rb-MyoD pathways in muscle regeneration.

    PubMed

    Bakay, Marina; Wang, Zuyi; Melcon, Gisela; Schiltz, Louis; Xuan, Jianhua; Zhao, Po; Sartorelli, Vittorio; Seo, Jinwook; Pegoraro, Elena; Angelini, Corrado; Shneiderman, Ben; Escolar, Diana; Chen, Yi-Wen; Winokur, Sara T; Pachman, Lauren M; Fan, Chenguang; Mandler, Raul; Nevo, Yoram; Gordon, Erynn; Zhu, Yitan; Dong, Yibin; Wang, Yue; Hoffman, Eric P

    2006-04-01

    Mutations of lamin A/C (LMNA) cause a wide range of human disorders, including progeria, lipodystrophy, neuropathies and autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD). EDMD is also caused by X-linked recessive loss-of-function mutations of emerin, another component of the inner nuclear lamina that directly interacts with LMNA. One model for disease pathogenesis of LMNA and emerin mutations is cell-specific perturbations of the mRNA transcriptome in terminally differentiated cells. To test this model, we studied 125 human muscle biopsies from 13 diagnostic groups (125 U133A, 125 U133B microarrays), including EDMD patients with LMNA and emerin mutations. A Visual and Statistical Data Analyzer (VISDA) algorithm was used to statistically model cluster hierarchy, resulting in a tree of phenotypic classifications. Validations of the diagnostic tree included permutations of U133A and U133B arrays, and use of two probe set algorithms (MAS5.0 and MBEI). This showed that the two nuclear envelope defects (EDMD LMNA, EDMD emerin) were highly related disorders and were also related to fascioscapulohumeral muscular dystrophy (FSHD). FSHD has recently been hypothesized to involve abnormal interactions of chromatin with the nuclear envelope. To identify disease-specific transcripts for EDMD, we applied a leave-one-out (LOO) cross-validation approach using LMNA patient muscle as a test data set, with reverse transcription-polymerase chain reaction (RT-PCR) validations in both LMNA and emerin patient muscle. A high proportion of top-ranked and validated transcripts were components of the same transcriptional regulatory pathway involving Rb1 and MyoD during muscle regeneration (CRI-1, CREBBP, Nap1L1, ECREBBP/p300), where each was specifically upregulated in EDMD. Using a muscle regeneration time series (27 time points) we develop a transcriptional model for downstream consequences of LMNA and emerin mutations. We propose that key interactions between the nuclear

  11. Nuclear sequestration of COL1A1 mRNA transcript associated with type I osteogenesis imperfecta (OI)

    SciTech Connect

    Primorac, D.; Stover, M.L.; McKinstry, M.B.

    1994-09-01

    Previously we identified an OI type I patient with a splice donor mutation that resulted in intron 26 retention instead of exon skipping and sequestration of normal levels of the mutant transcript in the nuclear compartment. Intron retention was consistent with the exon definition hypothesis for splice site selection since the size of the exon-intron-exon unit was less than 300 bp. Furthermore, the retained intron contained in-frame stop codons which is thought to cause the mutant RNA to remain within the nucleus rather than appearing in the cytoplasm. To test these hypotheses, genomic fragments containing the normal sequence or the donor mutation were cloned into a collagen minigene and expressed in stably tansfected NIH 3T3 cells. None of the modifications to the normal intron altered the level of RNA that accumulated in the cytoplasm, as expected. However none of the modifications to the mutant intron allowed accumulation of normal levels of mRNA in the cytoplasm. Moreover, in contrast to our findings in the patient`s cells only low levels of mutant transcript were found in the nucleus; a fraction of the transcript did appear in the cytoplasm which had spliced the mutant donor site correctly. Nuclear run-on experiments demonstrated equal levels of transcription from each transgene. Expression of another donor mutation known to cause in-frame exon skipping in OI type IV was accurately reproduced in the minigene in transfected 3T3 cells. Our experience suggests that either mechanism can lead to formation of a null allele possibly related to the type of splicing events surrounding the potential stop codons. Understanding the rules governing inactivation of a collagen RNA transcript may be important in designing a strategy to inactivate a dominate negative mutation associated with the more severe forms of OI.

  12. Analysis of the Heat Shock Response in Mouse Liver Reveals Transcriptional Dependence on the Nuclear Receptor Peroxisome Proliferator-Activated Receptor alpha (PPARα)

    EPA Science Inventory

    BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by h...

  13. Nuclear pore complex evolution: a trypanosome Mlp analogue functions in chromosomal segregation but lacks transcriptional barrier activity

    PubMed Central

    Holden, Jennifer M.; Koreny, Ludek; Obado, Samson; Ratushny, Alexander V.; Chen, Wei-Ming; Chiang, Jung-Hsien; Kelly, Steven; Chait, Brian T.; Aitchison, John D.; Rout, Michael P.; Field, Mark C.

    2014-01-01

    The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organization. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialized regions of active transcription, whereas Mlp2 associates with the mitotic spindle/NPC in a cell cycle–dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity of telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analogue of Mlp/Tpr proteins, and, together with the lamina analogue NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina. PMID:24600046

  14. Phosphatidic Acid Interacts with a MYB Transcription Factor and Regulates Its Nuclear Localization and Function in Arabidopsis[C][W

    PubMed Central

    Yao, Hongyan; Wang, Geliang; Guo, Liang; Wang, Xuemin

    2013-01-01

    Phosphatidic acid (PA) has emerged as a class of cellular mediators involved in various cellular and physiological processes, but little is known about its mechanism of action. Here we show that PA interacts with WEREWOLF (WER), a R2R3 MYB transcription factor involved in root hair formation. The PA-interacting region is confined to the end of the R2 subdomain. The ablation of the PA binding motif has no effect on WER binding to DNA, but abolishes its nuclear localization and its function in regulating epidermal cell fate. Inhibition of PA production by phospholipase Dζ also suppresses WER’s nuclear localization, root hair formation, and elongation. These results suggest a role for PA in promoting protein nuclear localization. PMID:24368785

  15. An in vitro investigation on the cytotoxic and nuclear receptor transcriptional activity of the mycotoxins fumonisin B1 and beauvericin.

    PubMed

    Fernández-Blanco, Celia; Frizzell, Caroline; Shannon, Maeve; Ruiz, Maria-Jose; Connolly, Lisa

    2016-08-22

    Fumonisin B1 (FB1) and beauvericin (BEA) are secondary metabolites of filamentous fungi, which under appropriate temperature and humidity conditions may develop on various foods and feeds. To date few studies have been performed to evaluate the toxicological and endocrine disrupting effects of FB1 and BEA. The present study makes use of various in vitro bioassays including; oestrogen, androgen, progestagen and glucocorticoid reporter gene assays (RGAs) for the study of nuclear receptor transcriptional activity, the thiazolyl blue tetrazolium bromide (MTT) assay to monitor cytotoxicity and high content analysis (HCA) for the detection of pre-lethal toxicity in the RGA and Caco-2 human colon adenocarcinoma cells. At the receptor level, 0.001-10μM BEA or FB1 did not induce any agonist responses in the RGAs. However at non-cytotoxic concentrations, an antagonistic effect was exhibited by FB1 on the androgen nuclear receptor transcriptional activity at 10μM and BEA on the progestagen and glucocorticoid receptors at 1μM. MTT analysis showed no decrease in cell viability at any concentration of FB1, whereas BEA showed a significant decrease in viability at 10μM. HCA analysis confirmed that the reduction in the progestagen receptor transcriptional activity at 1μM BEA was not due to pre-lethal toxicity. In addition, BEA (10μM) induced significant toxicity in both the TM-Luc (progestagen responsive) and Caco-2 cells. PMID:27234500

  16. Nuclear respiratory factor 2 regulates the transcription of AMPA receptor subunit GluA2 (Gria2).

    PubMed

    Priya, Anusha; Johar, Kaid; Nair, Bindu; Wong-Riley, Margaret T T

    2014-12-01

    Neuronal activity is highly dependent on energy metabolism. Nuclear respiratory factor 2 (NRF-2) tightly couples neuronal activity and energy metabolism by transcriptionally co-regulating all 13 subunits of an important energy-generating enzyme, cytochrome c oxidase (COX), as well as critical subunits of excitatory NMDA receptors. AMPA receptors are another major class of excitatory glutamatergic receptors that mediate most of the fast excitatory synaptic transmission in the brain. They are heterotetrameric proteins composed of various combinations of GluA1-4 subunits, with GluA2 being the most common one. We have previously shown that GluA2 (Gria2) is transcriptionally regulated by nuclear respiratory factor 1 (NRF-1) and specificity protein 4 (Sp4), which also regulate all subunits of COX. However, it was not known if NRF-2 also couples neuronal activity and energy metabolism by regulating subunits of the AMPA receptors. By means of multiple approaches, including electrophoretic mobility shift and supershift assays, chromatin immunoprecipitation, promoter mutations, real-time quantitative PCR, and western blot analysis, NRF-2 was found to functionally regulate the expression of Gria2, but not of Gria1, Gria3, or Gria4 genes in neurons. By regulating the GluA2 subunit of the AMPA receptor, NRF-2 couples energy metabolism and neuronal activity at the transcriptional level through a concurrent and parallel mechanism with NRF-1 and Sp4. PMID:25245478

  17. Chicken ovalbumin upstream promoter transcription factors act as auxiliary cofactors for hepatocyte nuclear factor 4 and enhance hepatic gene expression.

    PubMed Central

    Ktistaki, E; Talianidis, I

    1997-01-01

    Chicken ovalbumin upstream promoter transcription factors (COUP-TFs) strongly inhibit transcriptional activation mediated by nuclear hormone receptors, including hepatocyte nuclear factor 4 (HNF-4). COUP-TFs repress HNF-4-dependent gene expression by competition with HNF-4 for common binding sites found in several regulatory regions. Here we show that promoters, such as the HNF-1 promoter, which are recognized by HNF-4 but not by COUP-TFs are activated by COUP-TFI and COUP-TFII in conjunction with HNF-4 more than 100-fold above basal levels, as opposed to about 8-fold activation by HNF-4 alone. This enhancement was strictly dependent on an intact HNF-4 E domain. In vitro and in vivo evidence suggests that COUP-TFs enhance HNF-4 activity by a mechanism that involves their physical interaction with the amino acid 227 to 271 region of HNF-4. Our results indicate that in certain promoters, COUP-TFs act as auxiliary cofactors for HNF-4, orienting the HNF-4 activation domain in a more efficient configuration to achieve enhanced transcriptional activity. These findings provide new insights into the regulatory functions of COUP-TFs, suggesting their involvement in the initial activation and subsequent high-level expression of hepatic regulators, as well as in the positive and negative modulation of downstream target genes. PMID:9111350

  18. Chicken ovalbumin upstream promoter transcription factors act as auxiliary cofactors for hepatocyte nuclear factor 4 and enhance hepatic gene expression.

    PubMed

    Ktistaki, E; Talianidis, I

    1997-05-01

    Chicken ovalbumin upstream promoter transcription factors (COUP-TFs) strongly inhibit transcriptional activation mediated by nuclear hormone receptors, including hepatocyte nuclear factor 4 (HNF-4). COUP-TFs repress HNF-4-dependent gene expression by competition with HNF-4 for common binding sites found in several regulatory regions. Here we show that promoters, such as the HNF-1 promoter, which are recognized by HNF-4 but not by COUP-TFs are activated by COUP-TFI and COUP-TFII in conjunction with HNF-4 more than 100-fold above basal levels, as opposed to about 8-fold activation by HNF-4 alone. This enhancement was strictly dependent on an intact HNF-4 E domain. In vitro and in vivo evidence suggests that COUP-TFs enhance HNF-4 activity by a mechanism that involves their physical interaction with the amino acid 227 to 271 region of HNF-4. Our results indicate that in certain promoters, COUP-TFs act as auxiliary cofactors for HNF-4, orienting the HNF-4 activation domain in a more efficient configuration to achieve enhanced transcriptional activity. These findings provide new insights into the regulatory functions of COUP-TFs, suggesting their involvement in the initial activation and subsequent high-level expression of hepatic regulators, as well as in the positive and negative modulation of downstream target genes. PMID:9111350

  19. Global analysis of the nuclear processing of transcripts with unspliced U12-type introns by the exosome.

    PubMed

    Niemelä, Elina H; Oghabian, Ali; Staals, Raymond H J; Greco, Dario; Pruijn, Ger J M; Frilander, Mikko J

    2014-06-01

    U12-type introns are a rare class of introns in the genomes of diverse eukaryotes. In the human genome, they number over 700. A subset of these introns has been shown to be spliced at a slower rate compared to the major U2-type introns. This suggests a rate-limiting regulatory function for the minor spliceosome in the processing of transcripts containing U12-type introns. However, both the generality of slower splicing and the subsequent fate of partially processed pre-mRNAs remained unknown. Here, we present a global analysis of the nuclear retention of transcripts containing U12-type introns and provide evidence for the nuclear decay of such transcripts in human cells. Using SOLiD RNA sequencing technology, we find that, in normal cells, U12-type introns are on average 2-fold more retained than the surrounding U2-type introns. Furthermore, we find that knockdown of RRP41 and DIS3 subunits of the exosome stabilizes an overlapping set of U12-type introns. RRP41 knockdown leads to slower decay kinetics of U12-type introns and globally upregulates the retention of U12-type, but not U2-type, introns. Our results indicate that U12-type introns are spliced less efficiently and are targeted by the exosome. These characteristics support their role in the regulation of cellular mRNA levels. PMID:24848017

  20. Signal-dependent Regulation of Transcription by Histone Deacetylase 7 Involves Recruitment to Promyelocytic Leukemia Protein Nuclear Bodies

    PubMed Central

    Gao, Chengzhuo; Cheng, Xiwen; Lam, Minh; Liu, Yu; Liu, Qing; Chang, Kun-Sang

    2008-01-01

    Promyelocytic leukemia protein (PML) nuclear bodies (NBs) are dynamic subnuclear compartments that play roles in several cellular processes, including apoptosis, transcriptional regulation, and DNA repair. Histone deacetylase (HDAC) 7 is a potent corepressor that inhibits transcription by myocyte enhancer factor 2 (MEF2) transcription factors. We show here that endogenous HDAC7 and PML interact and partially colocalize in PML NBs. Tumor necrosis factor (TNF)-α treatment recruits HDAC7 to PML NBs and enhances association of HDAC7 with PML in human umbilical vein endothelial cells. Consequently, TNF-α promotes dissociation of HDAC7 from MEF2 transcription factors and the promoters of MEF2 target genes such as matrix metalloproteinase (MMP)-10, leading to accumulation of MMP-10 mRNA. Conversely, knockdown of PML enhances the association between HDAC7 and MEF2 and decreases MMP-10 mRNA accumulation. Accordingly, ectopic expression of PML recruits HDAC7 to PML NBs and leads to activation of MEF2 reporter activity. Notably, small interfering RNA knockdown of PML decreases basal and TNF-α-induced MMP-10 mRNA accumulation. Our results reveal a novel mechanism by which PML sequesters HDAC7 to relieve repression and up-regulate gene expression. PMID:18463162

  1. Nuclear Factor 1 and T-Cell Factor/LEF Recognition Elements Regulate Pitx2 Transcription in Pituitary Development▿

    PubMed Central

    Ai, Di; Wang, Jun; Amen, Melanie; Lu, Mei-Fang; Amendt, Brad A.; Martin, James F.

    2007-01-01

    Pitx2, a paired-related homeobox gene that is mutated in Rieger syndrome I, is the earliest known marker of oral ectoderm. Pitx2 was previously shown to be required for tooth, palate, and pituitary development in mice; however, the mechanisms regulating Pitx2 transcription in the oral ectoderm are poorly understood. Here we used an in vivo transgenic approach to investigate the mechanisms regulating Pitx2 transcription. We identified a 7-kb fragment that directs LacZ expression in oral ectoderm and in many of its derivatives. Deletion analysis of transgenic embryos reduced this fragment to a 520-bp region that directed LacZ activity to Rathke's pouch. A comparison of the mouse and human sequences revealed a conserved nuclear factor 1 (NF-1) recognition element near a consensus T-cell factor (TCF)/LEF binding site. The mutation of either site individually abolished LacZ activity in transgenic embryos, identifying Pitx2 as a direct target of Wnt signaling in pituitary development. These findings uncover a requirement for NF-1 and TCF factors in Pitx2 transcriptional regulation in the pituitary and provide insight into the mechanisms controlling region-specific transcription in the oral ectoderm and its derivatives. PMID:17562863

  2. Nucleolin is important for Epstein-Barr virus nuclear antigen 1-mediated episome binding, maintenance, and transcription.

    PubMed

    Chen, Ya-Lin; Liu, Cheng-Der; Cheng, Chi-Ping; Zhao, Bo; Hsu, Hao-Jen; Shen, Chih-Long; Chiu, Shu-Jun; Kieff, Elliott; Peng, Chih-wen

    2014-01-01

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for EBV episome maintenance, replication, and transcription. These effects are mediated by EBNA1 binding to cognate oriP DNA, which comprise 20 imperfect copies of a 30-bp dyad symmetry enhancer and an origin for DNA replication. To identify cell proteins essential for these EBNA1 functions, EBNA1 associated cell proteins were immune precipitated and analyzed by liquid chromatography-tandem mass spectrometry. Nucleolin (NCL) was identified to be EBNA1 associated. EBNA1's N-terminal 100 aa and NCL's RNA-binding domains were critical for EBNA1/NCL interaction. Lentivirus shRNA-mediated NCL depletion substantially reduced EBNA1 recruitment to oriP DNA, EBNA1-dependent transcription of an EBV oriP luciferase reporter, and EBV genome maintenance in lymphoblastoid cell lines. NCL RNA-binding domain K429 was critical for ATP and EBNA1 binding. NCL overexpression increased EBNA1 binding to oriP and transcription, whereas NCL K429A was deficient. Moreover, NCL silencing impaired lymphoblastoid cell line growth. These experiments reveal a surprisingly critical role for NCL K429 in EBNA1 episome maintenance and transcription, which may be a target for therapeutic intervention. PMID:24344309

  3. Myocyte nuclear factor, a novel winged-helix transcription factor under both developmental and neural regulation in striated myocytes.

    PubMed Central

    Bassel-Duby, R; Hernandez, M D; Yang, Q; Rochelle, J M; Seldin, M F; Williams, R S

    1994-01-01

    A sequence motif (CCAC box) within an upstream enhancer region of the human myoglobin gene is essential for transcriptional activity in both cardiac and skeletal muscle. A cDNA clone, myocyte nuclear factor (MNF), was isolated from a murine expression library on the basis of sequence-specific binding to the myoglobin CCAC box motif and was found to encode a novel member of the winged-helix or HNF-3/fork head family of transcription factors. Probes based on this sequence identify two mRNA species that are upregulated during myocyte differentiation, and antibodies raised against recombinant MNF identify proteins of approximately 90, 68, and 65 kDa whose expression is regulated following differentiation of myogenic cells in culture. In addition, the 90-kDa form of MNF is phosphorylated and is upregulated in intact muscles subjected to chronic motor nerve stimulation, a potent stimulus to myoglobin gene regulation. Amino acid residues 280 to 389 of MNF demonstrate 35 to 89% sequence identity to the winged-helix domain from other known members of this family, but MNF is otherwise divergent. A proline-rich amino-terminal region (residues 1 to 206) of MNF functions as a transcriptional activation domain. These studies provide the first evidence that members of the winged-helix family of transcription factors have a role in myogenic differentiation and in remodeling processes of adult muscles that occur in response to physiological stimuli. Images PMID:8007964

  4. The Epstein-Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins.

    PubMed

    Coppotelli, Giuseppe; Mughal, Nouman; Callegari, Simone; Sompallae, Ramakrishna; Caja, Laia; Luijsterburg, Martijn S; Dantuma, Nico P; Moustakas, Aristidis; Masucci, Maria G

    2013-03-01

    Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate-dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation. PMID:23358825

  5. Overexpression of the Transcriptional Repressor Complex BCL-6/BCoR Leads to Nuclear Aggregates Distinct from Classical Aggresomes

    PubMed Central

    Buchberger, Elisabeth; El Harchi, Miriam; Payrhuber, Dietmar; Zommer, Anna; Schauer, Dominic; Simonitsch-Klupp, Ingrid; Bilban, Martin; Brostjan, Christine

    2013-01-01

    Nuclear inclusions of aggregated proteins have primarily been characterized for molecules with aberrant poly-glutamine repeats and for mutated or structurally altered proteins. They were termed “nuclear aggresomes” and misfolding was shown to promote association with molecular chaperones and proteasomes. Here, we report that two components of a transcriptional repressor complex (BCL-6 and BCoR) of wildtype amino acid sequence can independently or jointly induce the formation of nuclear aggregates when overexpressed. The observation that the majority of cells rapidly downregulate BCL-6/BCoR levels, supports the notion that expression of these proteins is under tight control. The inclusions occur when BCL-6/BCoR expression exceeds 150-fold of endogenous levels. They preferentially develop in the nucleus by a gradual increase in aggregate size to form large, spheroid structures which are not associated with heat shock proteins or marked by ubiquitin. In contrast, we find the close association of BCL-6/BCoR inclusions with PML bodies and a reduction in aggregation upon the concomitant overexpression of histone deacetylases or heat shock protein 70. In summary, our data offer a perspective on nuclear aggregates distinct from classical “nuclear aggresomes”: Large complexes of spheroid structure can evolve in the nucleus without being marked by the cellular machinery for protein refolding and degradation. However, nuclear proteostasis can be restored by balancing the levels of chaperones. PMID:24146931

  6. Zinc fingers 1, 2, 5 and 6 of transcriptional regulator, PRDM4, are required for its nuclear localisation.

    PubMed

    Tunbak, Hale; Georgiou, Christiana; Guan, Cui; Richardson, William David; Chittka, Alexandra

    2016-05-27

    PRDM4 is a member of the PRDM family of transcriptional regulators which control various aspects of cellular differentiation and proliferation. PRDM proteins exert their biological functions both in the cytosol and the nucleus of cells. All PRDM proteins are characterised by the presence of two distinct structural motifs, the PR/SET domain and the zinc finger (ZF) motifs. We previously observed that deletion of all six zinc fingers found in PRDM4 leads to its accumulation in the cytosol, whereas overexpressed full length PRDM4 is found predominantly in the nucleus. Here, we investigated the requirements for single zinc fingers in the nuclear localisation of PRDM4. We demonstrate that ZF's 1, 2, 5 and 6 contribute to the accumulation of PRDM4 in the nucleus. Their effect is additive as deleting either ZF1-2 or ZF 5-6 redistributes PRDM4 protein from being almost exclusively nuclear to cytosolic and nuclear. We investigated the potential mechanism of nuclear shuttling of PRDM4 via the importin α/β-mediated pathway and find that PRDM4 nuclear targeting is independent of α/β-mediated nuclear import. PMID:27125459

  7. Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

    PubMed

    Horita, Henrick; Wysoczynski, Christina L; Walker, Lori A; Moulton, Karen S; Li, Marcella; Ostriker, Allison; Tucker, Rebecca; McKinsey, Timothy A; Churchill, Mair E A; Nemenoff, Raphael A; Weiser-Evans, Mary C M

    2016-01-01

    Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings. PMID:26940659

  8. Heterogeneous nuclear ribonuclear protein U associates with YAP and regulates its co-activation of Bax transcription.

    PubMed

    Howell, Michael; Borchers, Christoph; Milgram, Sharon L

    2004-06-18

    Although initially described as a cytosolic scaffolding protein, YAP (Yes-associated protein of 65 kDa) is known to associate with multiple transcription factors in the nucleus. Using affinity chromatography and mass spectrometry, we show that YAP interacts with heterogeneous nuclear ribonuclear protein U (hnRNP U), an RNA- and DNA-binding protein enriched in the nuclear matrix that also plays a role in the regulation of gene expression. hnRNP U interacts specifically with the proline-rich amino terminus of YAP, a region of YAP that is not found in the related protein TAZ. Although hnRNP U and YAP localize to both the nucleus and the cytoplasm, YAP does not translocate to the nucleus in an hnRNP U-dependent manner. Furthermore, hnRNP U and YAP only interact in the nucleus, suggesting that the association between the two proteins is regulated. Co-expression of hnRNP U attenuates the ability of YAP to increase the activity of a p73-driven Bax-luciferase reporter plasmid. In contrast, hnRNP U has no effect when co-expressed with a truncated YAP protein lacking the hnRNP U-binding site. Because YAP is distinguished from the homologue TAZ by its proline-rich amino terminus, the YAP-hnRNP U interaction may uniquely regulate the nuclear function(s) of YAP. The YAP-hnRNP U interaction provides another mechanism of YAP transcriptional regulation. PMID:15096513

  9. Thyroid transcription factor-1, hepatocyte nuclear factor-3β and surfactant protein A and B in the developing chick lung

    PubMed Central

    ZENG, XIN; YUTZEY, KATHERINE E.; WHITSETT, JEFFREY A.

    1998-01-01

    Expression of surfactant proteins SP-A, SP-B and the transcription factors TTF-1 and HNF-3β was identified by immunohistochemistry in the developing chicken. SP-B, a small hydrophobic peptide critical for lung function and surfactant homeostasis in mammals, was detected in the epithelial cells of parabronchi in embryonic chicken lung from the 15th day of incubation, prior to the onset of the breathing movements and was expressed at high levels in the posthatching chicken lung. SP-A, an abundant surfactant protein involved in innate defence of the mammalian lung, was detected in the chick embryo in subsets of epithelial cells in the mesobronchus, starting from d 15 and was detected in the posthatching chicken lung. The transcription factors hepatocyte nuclear factor 3β (HNF-3β) and thyroid transcription factor-1 (TTF-1), both regulators epithelial cell differentiation and gene expression in mammalian species, were detected at the onset of lung bud formation (d 4 of incubation) and throughout lung development. Abundant nuclear expression was detected in nuclei of respiratory epithelial cells of developing bronchial tubules for both transcription factors. In contrast to the surfactant proteins, expression of both TTF-1 and HNF-3β decreased markedly in posthatching chicken lung. The expression of SP-A and SP-B in chick lung demonstrates the conservation of surfactant proteins in vertebrates. The temporospatial pattern of TTF-1 and HNF-3β overlaps with that of SP-A and SP-B, supporting their potential roles in chick lung development and demonstrating the conservation of regulatory mechanisms contributing to gene expression in respiratory epithelial cells in vertebrates. PMID:9877295

  10. Signal transducers and activators of transcription 3 (STAT3) inhibits transcription of the inducible nitric oxide synthase gene by interacting with nuclear factor kappaB.

    PubMed Central

    Yu, Zhiyuan; Zhang, Wenzheng; Kone, Bruce C

    2002-01-01

    Prolific generation of NO by inducible nitric oxide synthase (iNOS) can cause unintended injury to host cells during glomerulonephritis and other inflammatory diseases. While much is known about the mechanisms of iNOS induction, few transcriptional repressors have been found. We explored the role of signal transducers and activators of transcription 3 (STAT3) proteins in interleukin (IL)-1beta- and lipopolysaccharide (LPS)+interferon (IFN)-gamma-mediated iNOS induction in murine mesangial cells. Both stimuli induced rapid phosphorylation of STAT3 and sequence-specific STAT3 DNA-binding activity. Supershift assays with a STAT3 element probe demonstrated that nuclear factor kappaB (NF-kappaB) p65 and p50 complexed with STAT3 in the DNA-protein complex. The direct interaction of STAT3 and NF-kappaB p65 was verified in vivo by co-immunoprecipitation and in vitro by pull-down assays with glutathione S-transferase-NF-kappaB p65 fusion protein and in vitro -translated STAT3alpha. Overexpression of STAT3 dramatically inhibited IL-1beta- or LPS+IFN-gamma-mediated induction of iNOS promoter-luciferase constructs that contained the wild-type iNOS promoter or ones harbouring mutated STAT-binding elements. In tests of indirect inhibitory effects of STAT3, overexpression of STAT3 dramatically inhibited the activity of an NF-kappaB-dependent promoter devoid of STAT-binding elements without affecting NF-kappaB DNA-binding activity. Thus STAT3, via direct interactions with NF-kappaB p65, serves as a dominant-negative inhibitor of NF-kappaB activity to suppress indirectly cytokine induction of the iNOS promoter in mesangial cells. These results provide a new model for the termination of NO production by activated iNOS following exposure to pro-inflammatory stimuli. PMID:12057007

  11. Release of Severe Acute Respiratory Syndrome Coronavirus Nuclear Import Block Enhances Host Transcription in Human Lung Cells

    PubMed Central

    Tilton, Susan C.; Menachery, Vineet D.; Gralinski, Lisa E.; Schäfer, Alexandra; Matzke, Melissa M.; Webb-Robertson, Bobbie-Jo M.; Chang, Jean; Luna, Maria L.; Long, Casey E.; Shukla, Anil K.; Bankhead, Armand R.; Burkett, Susan E.; Zornetzer, Gregory; Tseng, Chien-Te Kent; Metz, Thomas O.; Pickles, Raymond; McWeeney, Shannon; Smith, Richard D.; Katze, Michael G.; Waters, Katrina M.; Baric, Ralph S.

    2013-01-01

    The severe acute respiratory syndrome coronavirus accessory protein ORF6 antagonizes interferon signaling by blocking karyopherin-mediated nuclear import processes. Viral nuclear import antagonists, expressed by several highly pathogenic RNA viruses, likely mediate pleiotropic effects on host gene expression, presumably interfering with transcription factors, cytokines, hormones, and/or signaling cascades that occur in response to infection. By bioinformatic and systems biology approaches, we evaluated the impact of nuclear import antagonism on host expression networks by using human lung epithelial cells infected with either wild-type virus or a mutant that does not express ORF6 protein. Microarray analysis revealed significant changes in differential gene expression, with approximately twice as many upregulated genes in the mutant virus samples by 48 h postinfection, despite identical viral titers. Our data demonstrated that ORF6 protein expression attenuates the activity of numerous karyopherin-dependent host transcription factors (VDR, CREB1, SMAD4, p53, EpasI, and Oct3/4) that are critical for establishing antiviral responses and regulating key host responses during virus infection. Results were confirmed by proteomic and chromatin immunoprecipitation assay analyses and in parallel microarray studies using infected primary human airway epithelial cell cultures. The data strongly support the hypothesis that viral antagonists of nuclear import actively manipulate host responses in specific hierarchical patterns, contributing to the viral pathogenic potential in vivo. Importantly, these studies and modeling approaches not only provide templates for evaluating virus antagonism of nuclear import processes but also can reveal candidate cellular genes and pathways that may significantly influence disease outcomes following severe acute respiratory syndrome coronavirus infection in vivo. PMID:23365422

  12. Dexamethasone inhibits human interleukin 2 but not interleukin 2 receptor gene expression in vitro at the level of nuclear transcription.

    PubMed Central

    Boumpas, D T; Anastassiou, E D; Older, S A; Tsokos, G C; Nelson, D L; Balow, J E

    1991-01-01

    Glucocorticosteroids have an inhibitory effect on the expression of interleukin 2 (IL-2) and interleukin 2 receptor (IL-2R) genes. To determine the mechanisms of this inhibition, human T lymphocytes were stimulated with mitogens in the presence of dexamethasone. Nuclear transcription run-off assays showed that high doses of dexamethasone inhibited the transcription of the IL-2 gene but not that of the IL-2R gene. Post-transcriptionally, high doses of dexamethasone (10(-4) M) were required to inhibit IL-2R mRNA levels by 50%, whereas lower doses (10(-6) M) inhibited by greater than 70% the accumulation of IL-2 mRNA. IL-2 mRNA half-life decreased in the presence of dexamethasone (10(-6) M) by approximately 50%. At the protein product level, dexamethasone inhibited both IL-2 production, as well as cell surface and soluble forms of IL-2R. IL-2R gene expression was inhibited for at least 72 h after exposure of cells to dexamethasone. In the presence of exogenous IL-2, dexamethasone failed to exert a significant effect on the production of IL-2R protein. These data indicate that dexamethasone has a greater effect on the expression of the IL-2 gene than on the IL-2R gene. Dexamethasone both inhibits transcription of the IL-2 gene and decreases the stability of IL-2 mRNA. The effect of dexamethasone on the IL-2R gene is post-transcriptional and may result indirectly from decreased IL-2 production. Images PMID:2022743

  13. 7SK small nuclear RNA directly affects HMGA1 function in transcription regulation

    PubMed Central

    Eilebrecht, Sebastian; Brysbaert, Guillaume; Wegert, Thomas; Urlaub, Henning; Benecke, Bernd-Joachim; Benecke, Arndt

    2011-01-01

    Non-coding (nc) RNAs are increasingly recognized to play important regulatory roles in eukaryotic gene expression. The highly abundant and essential 7SK ncRNA has been shown to negatively regulate RNA Polymerase II transcription by inactivating the positive transcription elongation factor b (P-TEFb) in cellular and Tat-dependent HIV transcription. Here, we identify a more general, P-TEFb-independent role of 7SK RNA in directly affecting the function of the architectural transcription factor and chromatin regulator HMGA1. An important regulatory role of 7SK RNA in HMGA1-dependent cell differentiation and proliferation regulation is uncovered with the identification of over 1500 7SK-responsive HMGA1 target genes. Elevated HMGA1 expression is observed in nearly every type of cancer making the use of a 7SK substructure in the inhibition of HMGA1 activity, as pioneered here, potentially useful in therapy. The 7SK-HMGA1 interaction not only adds an essential facet to the comprehension of transcriptional plasticity at the coupling of initiation and elongation, but also might provide a molecular link between HIV reprogramming of cellular gene expression-associated oncogenesis. PMID:21087998

  14. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products. Progress report, June 1, 1991--May 31, 1992

    SciTech Connect

    Kuchka, M.R.

    1992-05-01

    The following is a review of research accomplished in the first two years of funding for the above mentioned project. The work performed is a molecular characterization of nuclear mutants of Chlamydomonas reinhardtii which are deficient in different stages in the post-transcriptional expression of a single chloroplast encoded polypeptide, the D2 protein of Photosystem II. Our long-term goals are to understand the molecular mechanisms by which nuclear gene products affect the expression of chloroplast genes. Specifically, we which to understand how specific nuclear gene products affect the turnover rate of the D2 encoding mRNA (psbD), how other nuclear encoded factors work to promote the translation of psbD mRNA and/or stabilize the D2 protein, and what the role of the D2 protein itself is in Photosystem II assembly and in the control of expression of other chloroplast genes. This progress report will be organized into four major sections concerning (I) The characterization of nuclear mutants affected in D2 translation/turnover, (II) The study of trans-acting factors which associate with the 5{prime} end of the psbD mRNA, (III) In vitro mutagenesis of the psbD gene, and (IV) Additional studies.

  15. The RNA Helicases AtMTR4 and HEN2 Target Specific Subsets of Nuclear Transcripts for Degradation by the Nuclear Exosome in Arabidopsis thaliana

    PubMed Central

    Lange, Heike; Zuber, Hélène; Sement, François M.; Chicher, Johana; Kuhn, Lauriane; Hammann, Philippe; Brunaud, Véronique; Bérard, Caroline; Bouteiller, Nathalie; Balzergue, Sandrine; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Vaucheret, Hervé; Gagliardi, Dominique

    2014-01-01

    The RNA exosome is the major 3′-5′ RNA degradation machine of eukaryotic cells and participates in processing, surveillance and turnover of both nuclear and cytoplasmic RNA. In both yeast and human, all nuclear functions of the exosome require the RNA helicase MTR4. We show that the Arabidopsis core exosome can associate with two related RNA helicases, AtMTR4 and HEN2. Reciprocal co-immunoprecipitation shows that each of the RNA helicases co-purifies with the exosome core complex and with distinct sets of specific proteins. While AtMTR4 is a predominantly nucleolar protein, HEN2 is located in the nucleoplasm and appears to be excluded from nucleoli. We have previously shown that the major role of AtMTR4 is the degradation of rRNA precursors and rRNA maturation by-products. Here, we demonstrate that HEN2 is involved in the degradation of a large number of polyadenylated nuclear exosome substrates such as snoRNA and miRNA precursors, incompletely spliced mRNAs, and spurious transcripts produced from pseudogenes and intergenic regions. Only a weak accumulation of these exosome substrate targets is observed in mtr4 mutants, suggesting that MTR4 can contribute, but plays rather a minor role for the degradation of non-ribosomal RNAs and cryptic transcripts in Arabidopsis. Consistently, transgene post-transcriptional gene silencing (PTGS) is marginally affected in mtr4 mutants, but increased in hen2 mutants, suggesting that it is mostly the nucleoplasmic exosome that degrades aberrant transgene RNAs to limit their entry in the PTGS pathway. Interestingly, HEN2 is conserved throughout green algae, mosses and land plants but absent from metazoans and other eukaryotic lineages. Our data indicate that, in contrast to human and yeast, plants have two functionally specialized RNA helicases that assist the exosome in the degradation of specific nucleolar and nucleoplasmic RNA populations, respectively. PMID:25144737

  16. Multiple NUCLEAR FACTOR Y transcription factors respond to abiotic stress in Brassica napus L.

    PubMed

    Xu, Li; Lin, Zhongyuan; Tao, Qing; Liang, Mingxiang; Zhao, Gengmao; Yin, Xiangzhen; Fu, Ruixin

    2014-01-01

    Members of the plant NUCLEAR FACTOR Y (NF-Y) family are composed of the NF-YA, NF-YB, and NF-YC subunits. In Brassica napus (canola), each of these subunits forms a multimember subfamily. Plant NF-Ys were reported to be involved in several abiotic stresses. In this study, we demonstrated that multiple members of thirty three BnNF-Ys responded rapidly to salinity, drought, or ABA treatments. Transcripts of five BnNF-YAs, seven BnNF-YBs, and two BnNF-YCs were up-regulated by salinity stress, whereas the expression of thirteen BnNF-YAs, ten BnNF-YBs, and four BnNF-YCs were induced by drought stress. Under NaCl treatments, the expression of one BnNF-YA10 and four NF-YBs (BnNF-YB3, BnNF-YB7, BnNF-YB10, and BnNF-YB14) were greatly increased. Under PEG treatments, the expression levels of four NF-YAs (BnNF-YA9, BnNF-YA10, BnNF-YA11, and BnNF-YA12) and five NF-YBs (BnNF-YB1, BnNF-YB8, BnNF-YB10, BnNF-YB13, and BnNF-YB14) were greatly induced. The expression profiles of 20 of the 27 salinity- or drought-induced BnNF-Ys were also affected by ABA treatment. The expression levels of six NF-YAs (BnNF-YA1, BnNF-YA7, BnNF-YA8, BnNF-YA9, BnNF-YA10, and BnNF-YA12) and seven BnNF-YB members (BnNF-YB2, BnNF-YB3, BnNF-YB7, BnNF-YB10, BnNF-YB11, BnNF-YB13, and BnNF-YB14) and two NF-YC members (BnNF-YC2 and BnNF-YC3) were greatly up-regulated by ABA treatments. Only a few BnNF-Ys were inhibited by the above three treatments. Several NF-Y subfamily members exhibited collinear expression patterns. The promoters of all stress-responsive BnNF-Ys harbored at least two types of stress-related cis-elements, such as ABRE, DRE, MYB, or MYC. The cis-element organization of BnNF-Ys was similar to that of Arabidopsis thaliana, and the promoter regions exhibited higher levels of nucleotide sequence identity with Brassica rapa than with Brassica oleracea. This work represents an entry point for investigating the roles of canola NF-Y proteins during abiotic stress responses and provides insight into

  17. Sensitive detection of transcription factors in cell nuclear extracts by using a molecular beacons based amplification strategy.

    PubMed

    Zhang, Kai; Wang, Ke; Zhu, Xue; Xie, Minhao

    2016-03-15

    Monitoring transcription factor (TF) levels provides an important assessment of the state of cell populations. Unfortunately, traditional methods for monitoring TF concentration are generally cumbersome and time-consuming. We developed an ultrasensitive one-pot TF detection method that uses target-molecular beacons-dependent amplification (TMDA) fluorescence strategy to circumvent the aforementioned limitations in TF detection. In this assay, we employed a DNA1/DNA2 duplex as the reporting probe and a stem-loop DNA molecular beacon (MB) as the signaling probe. The integration of protein-DNA1/DNA2 duplex and exonuclease III (Exo III) digestion can convert the detection of transcription factors to the detection of reporter oligonucleotides. The subsequent hybridization of the reporter oligonucleotides with the molecular beacons opens the stem-loop structure. The formation of the DNA complex triggers amplification reaction and the recovery of the fluorescence. This assay exhibits high sensitivity with a detection limit of 2.2 pM and a detection range of 3 orders of magnitude, which is superior to most currently used methods for transcription factor detection. More importantly, this method is suitable for the direct detection of TFs in crude nuclear extracts of cancer cells. PMID:26410390

  18. Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function

    PubMed Central

    Gallardo-Montejano, Violeta I.; Saxena, Geetu; Kusminski, Christine M.; Yang, Chaofeng; McAfee, John L.; Hahner, Lisa; Hoch, Kathleen; Dubinsky, William; Narkar, Vihang A.; Bickel, Perry E.

    2016-01-01

    Dysfunctional cellular lipid metabolism contributes to common chronic human diseases, including type 2 diabetes, obesity, fatty liver disease and diabetic cardiomyopathy. How cells balance lipid storage and mitochondrial oxidative capacity is poorly understood. Here we identify the lipid droplet protein Perilipin 5 as a catecholamine-triggered interaction partner of PGC-1α. We report that during catecholamine-stimulated lipolysis, Perilipin 5 is phosphorylated by protein kinase A and forms transcriptional complexes with PGC-1α and SIRT1 in the nucleus. Perilipin 5 promotes PGC-1α co-activator function by disinhibiting SIRT1 deacetylase activity. We show by gain-and-loss of function studies in cells that nuclear Perilipin 5 promotes transcription of genes that mediate mitochondrial biogenesis and oxidative function. We propose that Perilipin 5 is an important molecular link that couples the coordinated catecholamine activation of the PKA pathway and of lipid droplet lipolysis with transcriptional regulation to promote efficient fatty acid catabolism and prevent mitochondrial dysfunction. PMID:27554864

  19. Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function.

    PubMed

    Gallardo-Montejano, Violeta I; Saxena, Geetu; Kusminski, Christine M; Yang, Chaofeng; McAfee, John L; Hahner, Lisa; Hoch, Kathleen; Dubinsky, William; Narkar, Vihang A; Bickel, Perry E

    2016-01-01

    Dysfunctional cellular lipid metabolism contributes to common chronic human diseases, including type 2 diabetes, obesity, fatty liver disease and diabetic cardiomyopathy. How cells balance lipid storage and mitochondrial oxidative capacity is poorly understood. Here we identify the lipid droplet protein Perilipin 5 as a catecholamine-triggered interaction partner of PGC-1α. We report that during catecholamine-stimulated lipolysis, Perilipin 5 is phosphorylated by protein kinase A and forms transcriptional complexes with PGC-1α and SIRT1 in the nucleus. Perilipin 5 promotes PGC-1α co-activator function by disinhibiting SIRT1 deacetylase activity. We show by gain-and-loss of function studies in cells that nuclear Perilipin 5 promotes transcription of genes that mediate mitochondrial biogenesis and oxidative function. We propose that Perilipin 5 is an important molecular link that couples the coordinated catecholamine activation of the PKA pathway and of lipid droplet lipolysis with transcriptional regulation to promote efficient fatty acid catabolism and prevent mitochondrial dysfunction. PMID:27554864

  20. Trichostatin A specifically improves the aberrant expression of transcription factor genes in embryos produced by somatic cell nuclear transfer

    PubMed Central

    Inoue, Kimiko; Oikawa, Mami; Kamimura, Satoshi; Ogonuki, Narumi; Nakamura, Toshinobu; Nakano, Toru; Abe, Kuniya; Ogura, Atsuo

    2015-01-01

    Although mammalian cloning by somatic cell nuclear transfer (SCNT) has been established in various species, the low developmental efficiency has hampered its practical applications. Treatment of SCNT-derived embryos with histone deacetylase (HDAC) inhibitors can improve their development, but the underlying mechanism is still unclear. To address this question, we analysed gene expression profiles of SCNT-derived 2-cell mouse embryos treated with trichostatin A (TSA), a potent HDAC inhibitor that is best used for mouse cloning. Unexpectedly, TSA had no effect on the numbers of aberrantly expressed genes or the overall gene expression pattern in the embryos. However, in-depth investigation by gene ontology and functional analyses revealed that TSA treatment specifically improved the expression of a small subset of genes encoding transcription factors and their regulatory factors, suggesting their positive involvement in de novo RNA synthesis. Indeed, introduction of one of such transcription factors, Spi-C, into the embryos at least partially mimicked the TSA-induced improvement in embryonic development by activating gene networks associated with transcriptional regulation. Thus, the effects of TSA treatment on embryonic gene expression did not seem to be stochastic, but more specific than expected, targeting genes that direct development and trigger zygotic genome activation at the 2-cell stage. PMID:25974394

  1. Trichostatin A specifically improves the aberrant expression of transcription factor genes in embryos produced by somatic cell nuclear transfer.

    PubMed

    Inoue, Kimiko; Oikawa, Mami; Kamimura, Satoshi; Ogonuki, Narumi; Nakamura, Toshinobu; Nakano, Toru; Abe, Kuniya; Ogura, Atsuo

    2015-01-01

    Although mammalian cloning by somatic cell nuclear transfer (SCNT) has been established in various species, the low developmental efficiency has hampered its practical applications. Treatment of SCNT-derived embryos with histone deacetylase (HDAC) inhibitors can improve their development, but the underlying mechanism is still unclear. To address this question, we analysed gene expression profiles of SCNT-derived 2-cell mouse embryos treated with trichostatin A (TSA), a potent HDAC inhibitor that is best used for mouse cloning. Unexpectedly, TSA had no effect on the numbers of aberrantly expressed genes or the overall gene expression pattern in the embryos. However, in-depth investigation by gene ontology and functional analyses revealed that TSA treatment specifically improved the expression of a small subset of genes encoding transcription factors and their regulatory factors, suggesting their positive involvement in de novo RNA synthesis. Indeed, introduction of one of such transcription factors, Spi-C, into the embryos at least partially mimicked the TSA-induced improvement in embryonic development by activating gene networks associated with transcriptional regulation. Thus, the effects of TSA treatment on embryonic gene expression did not seem to be stochastic, but more specific than expected, targeting genes that direct development and trigger zygotic genome activation at the 2-cell stage. PMID:25974394

  2. Epstein–Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1

    PubMed Central

    Bazot, Quentin; Deschamps, Thibaut; Tafforeau, Lionel; Siouda, Maha; Leblanc, Pascal; Harth-Hertle, Marie L.; Rabourdin-Combe, Chantal; Lotteau, Vincent; Kempkes, Bettina; Tommasino, Massimo; Gruffat, Henri; Manet, Evelyne

    2014-01-01

    The Epstein–Barr virus (EBV) nuclear antigen 3 family of protein is critical for the EBV-induced primary B-cell growth transformation process. Using a yeast two-hybrid screen we identified 22 novel cellular partners of the EBNA3s. Most importantly, among the newly identified partners, five are known to play direct and important roles in transcriptional regulation. Of these, the Myc-interacting zinc finger protein-1 (MIZ-1) is a transcription factor initially characterized as a binding partner of MYC. MIZ-1 activates the transcription of a number of target genes including the cell cycle inhibitor CDKN2B. Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus. Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A. Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1. PMID:25092922

  3. microRNA-365-targeted nuclear factor I/B transcriptionally represses cyclin-dependent kinase 6 and 4 to inhibit the progression of cutaneous squamous cell carcinoma.

    PubMed

    Zhou, Liang; Wang, Yinghui; Ou, Chengshan; Lin, Zhixiang; Wang, Jianyu; Liu, Hongxia; Zhou, Meijuan; Ding, Zhenhua

    2015-08-01

    Cyclin-dependent kinases are either post-transcriptionally regulated by interacting with cyclins and cyclin-dependent kinase inhibitors or are transcriptionally regulated by transcription factors, but the latter mechanism has not been extensively investigated. Dysregulated transcription factors resulting from aberrantly expressed microRNAs play critical roles in tumor development and progression. Our previous work identified miR-365 as an oncogenic microRNA that promotes the development of cutaneous squamous cell carcinoma via repression of cyclin-dependent kinase 6, while miR-365 also targets nuclear factor I/B. However, the underlying mechanism(s) of the interaction between nuclear factor I/B and cyclin-dependent kinase 6 are unclear. In this work, we demonstrate that miR-365-regulated nuclear factor I/B transcriptionally inhibits cyclin-dependent kinases 6 and 4 by binding to their promoter regions. In vivo and in vitro experiments demonstrate that the loss of nuclear factor I/B after miR-365 expression or treatment with small interfering RNAs results in the upregulation of cyclin-dependent kinases 6 and 4. This upregulation, in turn, enhances the phosphorylation of retinoblastoma protein and tumor progression. Characterizing this transcriptional repression of cyclin-dependent kinases 6 and 4 by nuclear factor I/B contributes to the understanding of the transcriptional regulation of cyclin-dependent kinases by transcription factors and also facilitates the development of new therapeutic regimens to improve the clinical treatment of cutaneous squamous cell carcinoma. PMID:26072217

  4. Mutations in nuclear genes alter post-transcriptional regulation of mitochondrial genes.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nuclear gene products are required for the expression of mitochondrial genes and elaboration of functional mitochondrial protein complexes. To better understand the roles of these nuclear genes, we exploited the mitochondrial encoded S-type of cytoplasmic male sterility (CMS-S) and developed a nove...

  5. The C175R mutation alters nuclear localization and transcriptional activity of the nephronophthisis NPHP7 gene product

    PubMed Central

    Ramachandran, Haribaskar; Yakulov, Toma A; Engel, Christina; Müller, Barbara; Walz, Gerd

    2016-01-01

    Nephronophthisis (NPH) is a rare autosomal ciliopathy, but the leading cause for hereditary end-stage renal disease in children. Most NPH family members form large protein networks, which appear to participate in structural elements of the cilium and/or function to restrict access of molecules to the ciliary compartment. The zinc-finger protein GLIS2/NPHP7 represents an exception as it has been implicated in transcriptional regulation; only two families with GLIS2/NPHP7 mutations and typical NPH manifestations have been identified so far. We describe here that the recently identified GLIS2/NPHP7C175R point mutation abolished the nuclear localization of GLIS2/NPHP7. Forced nuclear import did not rescue the transcriptional defects of GLIS2/NPHP7C175R, indicating additional defects as DNA-binding protein. We further observed that wild type, but not GLIS2/NPHP7C175R, prevented the cyst formation caused by depletion of nphp7 in zebrafish embryos. Taken together, our findings indicate that the C175R mutation affects both localization and function of GLIS2/NPHP7, supporting a role of this mutation in NPH, but questioning the direct involvement of GLIS2/NPHP7 in ciliary functions. PMID:26374130

  6. Analysis of premature termination in c-myc during transcription by RNA polymerase II in a HeLa nuclear extract.

    PubMed Central

    London, L; Keene, R G; Landick, R

    1991-01-01

    Transcriptional regulation of the human c-myc gene, an important aspect of cellular differentiation, occurs in part at the level of transcript elongation. In vivo, transcriptional arrest, due to either pausing or termination, occurs near the junction between the first exon and first intron and varies with the growth state of the cell. We have tested the transcription of c-myc templates in HeLa nuclear extracts. We did not observe significant arrest under standard conditions, but we found that a considerable fraction of transcription complexes stopped at the c-myc TII site (just past the first exon-intron junction) when the KCl concentration was raised to 400 mM during elongation. Transcriptional arrest at TII also was observed at KCl concentrations as low as 130 mM and when potassium acetate or potassium glutamate was substituted for KCl. Under these conditions, arrest occurred at the TII site when transcription was initiated at either the c-myc P2 promoter or the adenovirus 2 major late promoter. Further, the TII sequence itself, in forward but not reverse orientation, was sufficient to stop transcription in a HeLa nuclear extract. By separating the TII RNA from active transcription complexes by using gel filtration, we found that arrest at TII at 400 mM KCl resulted in transcript release and thus true transcriptional termination. The efficiency of termination at TII depended on the growth state of the cells from which the extracts were made, suggesting that some factor or factors control premature termination in c-myc. Images PMID:1715021

  7. Mitochondrial Dynamics Impacts Stem Cell Identity and Fate Decisions by Regulating a Nuclear Transcriptional Program.

    PubMed

    Khacho, Mireille; Clark, Alysen; Svoboda, Devon S; Azzi, Joelle; MacLaurin, Jason G; Meghaizel, Cynthia; Sesaki, Hiromi; Lagace, Diane C; Germain, Marc; Harper, Mary-Ellen; Park, David S; Slack, Ruth S

    2016-08-01

    Regulated mechanisms of stem cell maintenance are key to preventing stem cell depletion and aging. While mitochondrial morphology plays a fundamental role in tissue development and homeostasis, its role in stem cells remains unknown. Here, we uncover that mitochondrial dynamics regulates stem cell identity, self-renewal, and fate decisions by orchestrating a transcriptional program. Manipulation of mitochondrial structure, through OPA1 or MFN1/2 deletion, impaired neural stem cell (NSC) self-renewal, with consequent age-dependent depletion, neurogenesis defects, and cognitive impairments. Gene expression profiling revealed ectopic expression of the Notch self-renewal inhibitor Botch and premature induction of transcription factors that promote differentiation. Changes in mitochondrial dynamics regulate stem cell fate decisions by driving a physiological reactive oxygen species (ROS)-mediated process, which triggers a dual program to suppress self-renewal and promote differentiation via NRF2-mediated retrograde signaling. These findings reveal mitochondrial dynamics as an upstream regulator of essential mechanisms governing stem cell self-renewal and fate decisions through transcriptional programming. PMID:27237737

  8. Src tyrosine kinase signaling antagonizes nuclear localization of FOXO and inhibits its transcription factor activity.

    PubMed

    Bülow, Margret H; Bülow, Torsten R; Hoch, Michael; Pankratz, Michael J; Jünger, Martin A

    2014-01-01

    Biochemical experiments in mammalian cells have linked Src family kinase activity to the insulin signaling pathway. To explore the physiological link between Src and a central insulin pathway effector, we investigated the effect of different Src signaling levels on the Drosophila transcription factor dFOXO in vivo. Ectopic activation of Src42A in the starved larval fatbody was sufficient to drive dFOXO out of the nucleus. When Src signaling levels were lowered by means of loss-of-function mutations or pharmacological inhibition, dFOXO localization was shifted to the nucleus in growing animals, and transcription of the dFOXO target genes d4E-BP and dInR was induced. dFOXO loss-of-function mutations rescued the induction of dFOXO target gene expression and the body size reduction of Src42A mutant larvae, establishing dFOXO as a critical downstream effector of Src signaling. Furthermore, we provide evidence that the regulation of FOXO transcription factors by Src is evolutionarily conserved in mammalian cells. PMID:24513978

  9. Transcription Factor Hepatocyte Nuclear Factor-1β Regulates Renal Cholesterol Metabolism.

    PubMed

    Aboudehen, Karam; Kim, Min Soo; Mitsche, Matthew; Garland, Kristina; Anderson, Norma; Noureddine, Lama; Pontoglio, Marco; Patel, Vishal; Xie, Yang; DeBose-Boyd, Russell; Igarashi, Peter

    2016-08-01

    HNF-1β is a tissue-specific transcription factor that is expressed in the kidney and other epithelial organs. Humans with mutations in HNF-1β develop kidney cysts, and HNF-1β regulates the transcription of several cystic disease genes. However, the complete spectrum of HNF-1β-regulated genes and pathways is not known. Here, using chromatin immunoprecipitation/next generation sequencing and gene expression profiling, we identified 1545 protein-coding genes that are directly regulated by HNF-1β in murine kidney epithelial cells. Pathway analysis predicted that HNF-1β regulates cholesterol metabolism. Expression of dominant negative mutant HNF-1β or kidney-specific inactivation of HNF-1β decreased the expression of genes that are essential for cholesterol synthesis, including sterol regulatory element binding factor 2 (Srebf2) and 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr). HNF-1β mutant cells also expressed lower levels of cholesterol biosynthetic intermediates and had a lower rate of cholesterol synthesis than control cells. Additionally, depletion of cholesterol in the culture medium mitigated the inhibitory effects of mutant HNF-1β on the proteins encoded by Srebf2 and Hmgcr, and HNF-1β directly controlled the renal epithelial expression of proprotein convertase subtilisin-like kexin type 9, a key regulator of cholesterol uptake. These findings reveal a novel role of HNF-1β in a transcriptional network that regulates intrarenal cholesterol metabolism. PMID:26712526

  10. Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1.

    PubMed

    Sladek, F M; Ruse, M D; Nepomuceno, L; Huang, S M; Stallcup, M R

    1999-10-01

    Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events. Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants. In this study, transient transfection, yeast two-hybrid, and GST pulldown assays are used to show not only that nuclear receptor hepatocyte nuclear factor 4 alpha1 (HNF4alpha1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommonly large F domain in the C terminus of the receptor inhibits that interaction. In vitro, the F domain was found to obscure an AF-2-independent binding site for GRIP1 that did not map to nuclear receptor boxes II or III. The results also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4alpha2) abrogates that inhibition in vivo and in vitro. A series of protease digestion assays indicates that there may be structural differences between HNF4alpha1 and HNF4alpha2 in the F domain as well as in the ligand binding domain (LBD). The data also suggest that there is a direct physical contact between the F domain and the LBD of HNF4alpha1 and -alpha2 and that that contact is different in the HNF4alpha1 and HNF4alpha2 isoforms. Finally, we propose a model in which the F domain of HNF4alpha1 acts as a negative regulatory region for transactivation and in which the alpha2 insert ameliorates the negative effect of the F domain. A conserved repressor sequence in the F domains of HNF4alpha1 and -alpha2 suggests that this model may be relevant to other nuclear receptors as well. PMID:10490591

  11. Expression of Nuclear Transcription Factor Kappa B in Locally Advanced Human Cervical Cancer Treated With Definitive Chemoradiation

    SciTech Connect

    Garg, Amit K.; Jhingran, Anuja; Klopp, Ann H.; Aggarwal, Bharat B.; Kunnumakkara, Ajai B.; Broadus, Russell R.; Eifel, Patricia J.; Buchholz, Thomas A.

    2010-12-01

    Purpose: Nuclear factor kappa B (NF-{kappa}B), a transcriptional factor that has been shown to be constitutively active in cervical cancer, is part of an important pathway leading to treatment resistance in many tumor types. The purpose of our study was to determine whether expression of NF-{kappa}B in pretreatment specimens and specimens taken shortly after treatment initiation correlated with outcome in cervical cancer patients treated with definitive chemoradiation. Methods and Materials: Eighteen patients with locally advanced cervical cancer were enrolled in a study in which cervical biopsy specimens were obtained before radiation therapy and 48 h after treatment initiation. Matched biopsy specimens from 16 of these patients were available and evaluated for the nuclear expression of NF-{kappa}B protein by immunohistochemical staining. Results: After a median follow-up of 43 months, there were 9 total treatment failures. Nuclear staining for NF-{kappa}B was positive in 3 of 16 pretreatment biopsy specimens (19%) and 5 of 16 postradiation biopsy specimens (31%). Pretreatment expression of NF-{kappa}B nuclear staining correlated with increased rates of local-regional failure (100% vs. 23%, p = 0.01), distant failure (100% vs. 38%, p = 0.055), disease-specific mortality (100% vs. 31%, p = 0.03), and overall mortality (100% vs. 38%, p = 0.055). Conclusions: Our data suggest that pretreatment nuclear expression of NF-{kappa}B may be associated with a poor outcome for cervical cancer patients treated with chemoradiation. Although these data require validation in a larger group of patients, the results support the continued study of the relationship between NF-{kappa}B and outcome in patients treated for carcinoma of the cervix.

  12. Transcription-Dependent Nuclear-Cytoplasmic Trafficking Is Required for the Function of the von Hippel-Lindau Tumor Suppressor Protein

    PubMed Central

    Lee, Stephen; Neumann, Markus; Stearman, Robert; Stauber, Roland; Pause, Arnim; Pavlakis, George N.; Klausner, Richard D.

    1999-01-01

    Mutation of the von Hippel-Lindau tumor suppressor gene (vhl) causes the von Hippel-Lindau cancer syndrome as well as sporadic renal clear cell carcinoma. To pursue our study of the intracellular localization of VHL protein in relation to its function, we fused VHL to the green fluorescent protein (GFP) to produce the VHL-GFP fusion protein. Like VHL, VHL-GFP binds to elongins B and C and Cullin-2 and regulates target gene product levels, including levels of vascular endothelial growth factor and glucose transporter 1. VHL-GFP localizes predominantly to the cytoplasm, with some detectable nuclear signal. Inhibition of transcription by actinomycin D or 5,6-dichlorobenzimidazole riboside (DRB) causes VHL to be redistributed to the nucleus. A cellular fusion assay was used to demonstrate that inhibition of transcription induces a decrease in the nuclear export rate of VHL. The dependence of transcription for trafficking is lost with a deletion of exon 2, a region with a mutation causing a splice defect in the VHL gene in sporadic renal clear cell carcinoma. Addition of a strong nuclear export signal (NES) derived from the Rev protein results in complete nuclear exclusion and abrogates the redistribution of VHL-GFP-NES into the nucleus upon inhibition of transcription. Leptomycin B, which inhibits NES-mediated nuclear export, reverts the distribution of VHL-GFP-NES to that of VHL-GFP and restores sensitivity to actinomycin D and DRB. Uncoupling of VHL-GFP trafficking to transcription either by an exon 2 deletion or fusion to NES abolishes VHL function. We suggest that VHL function requires not only nuclear or cytoplasmic localization, but also exon 2-mediated transcription-dependent trafficking between these two cellular compartments. PMID:9891082

  13. Transcription Factor EB Is Selectively Reduced in the Nuclear Fractions of Alzheimer's and Amyotrophic Lateral Sclerosis Brains

    PubMed Central

    Wang, Hongjie

    2016-01-01

    Multiple studies suggest that autophagy is strongly dysregulated in Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), as evidenced by accumulation of numerous autophagosomes, lysosomes with discontinuous membranes, and aggregated proteins in the patients' brains. Transcription factor EB (TFEB) was recently discovered to be a master regulator of lysosome biogenesis and autophagy. To examine whether aberrant autophagy in AD and ALS is due to alterations in TFEB expression, we systematically quantified the levels of TFEB in these brains by immunoblotting. Interestingly, cytoplasmic fractions of AD brains showed increased levels of normalized (to tubulin) TFEB only at Braak stage IV (61%, p < 0.01). Most importantly, normalized (to lamin) TFEB levels in the nuclear fractions were consistently reduced starting from Braak stage IV (52%, p < 0.01), stage V (67%, p < 0.01), and stage VI (85%, p < 0.01) when compared to normal control (NC) brains. In the ALS brains also, nuclear TFEB levels were reduced by 62% (p < 0.001). These data suggest that nuclear TFEB is selectively lost in ALS as well as AD brains, in which TFEB reduction was Braak-stage-dependent. Taken together, the observed reductions in TFEB protein levels may be responsible for the widely reported autophagy defects in these disorders. PMID:27433468

  14. Nuclear factor of activated T cell (NFAT) transcription proteins regulate genes involved in adipocyte metabolism and lipolysis

    SciTech Connect

    Holowachuk, Eugene W. . E-mail: geneh@telenet.net

    2007-09-21

    NFAT involvement in adipocyte physiological processes was examined by treatment with CsA and/or GSK3{beta} inhibitors (Li{sup +} or TZDZ-8), which prevent or increase NFAT nuclear translocation, respectively. CsA treatment reduced basal and TNF{alpha}-induced rates of lipolysis by 50%. Adipocytes preincubated with Li{sup +} or TZDZ-8 prior to CsA and/or TNF{alpha}, exhibited enhanced basal rates of lipolysis and complete inhibition of CsA-mediated decreased rates of lipolysis. CsA treatment dramatically reduced the mRNA levels of adipocyte-specific genes (aP2, HSL, PPAR{gamma}, ACS and Adn), compared with control or TNF{alpha}-treatment, whereas Li{sup +} pretreatment blocked the inhibitory effects of CsA, and mRNA levels of aP2, HSL, PPAR{gamma}, and ACS were found at or above control levels. NFAT nuclear localization, assessed by EMSA, confirmed that CsA or Li{sup +} treatments inhibited or increased NFAT nuclear translocation, respectively. These results show that NFAT proteins in mature adipocytes participate in the transcriptional control of genes involved in adipocyte metabolism and lipolysis.

  15. Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR

    PubMed Central

    2013-01-01

    Nuclear receptors are integrators of hormonal and nutritional signals, mediating changes to metabolic pathways within the body. Given that modulation of lipid and glucose metabolism has been linked to diseases including type 2 diabetes, obesity and atherosclerosis, a greater understanding of pathways that regulate metabolism in physiology and disease is crucial. The liver X receptors (LXRs) and the farnesoid X receptors (FXRs) are activated by oxysterols and bile acids, respectively. Mounting evidence indicates that these nuclear receptors have essential roles, not only in the regulation of cholesterol and bile acid metabolism but also in the integration of sterol, fatty acid and glucose metabolism. PMID:22414897

  16. Global transcriptional analysis of nuclear reprogramming in the transition from MEFs to iPSCs.

    PubMed

    Dong, Fulu; Song, Zhenwei; Zhang, Jinping; Lu, Youde; Song, Chunlei; Jiang, BaoChun; Zhang, Baole; Cong, Peiqing; Sun, Hongyan; Shi, Fangxiong; Liu, Honglin

    2013-01-01

    Induced pluripotent stem cells (iPSCs) are flourishing in the investigation of cell reprogramming. However, we still know little about the sequential molecular mechanism during somatic cell reprogramming (SCR). Here, we first observed rapid generation of colonies whereas mouse embryonic fibroblasts (MEFs) were induced by OCT4, SOX2, KLF4 (OSK), and vitamin C for 7 days. The colony's global transcriptional profiles were analyzed using Affymetrix microarray. Microarray data confirmed that SCR was a process in which transcriptome got reversed and pluripotent genes expressed de novo. There were many changes, especially substantial growth expression of epigenetic factors, on transcriptome during the transition from Day 7 to iPSCs indicating that this period may provide 'flexibility' genome structure, chromatin remodeling, and epigenetic modifications to rebind to the transcriptional factors. Several biological processes such as viral immune response, apoptosis, cell fate specification, and cell communication were mainly involved before Day 7 whereas cell cycle, DNA methylation, and histone modification were mainly involved after Day 7. Furthermore, it was suggested that p53 signaling contributed to the transition 'hyperdynamic plastic' cell state and assembled cell niche for SCR, and small molecular compounds useful for chromatin remodeling can enhance iPSCs by exciting epigenetic modification rather than the exogenous expression of more TFs vectors. PMID:23231677

  17. Nuclear pyruvate kinase M2 complex serves as a transcriptional coactivator of arylhydrocarbon receptor

    PubMed Central

    Matsuda, Shun; Adachi, Jun; Ihara, Masaru; Tanuma, Nobuhiro; Shima, Hiroshi; Kakizuka, Akira; Ikura, Masae; Ikura, Tsuyoshi; Matsuda, Tomonari

    2016-01-01

    Pyruvate kinase M2 (PKM2) and pyruvate dehydrogenase complex (PDC) regulate production of acetyl-CoA, which functions as an acetyl donor in diverse enzymatic reactions, including histone acetylation. However, the mechanism by which the acetyl-CoA required for histone acetylation is ensured in a gene context-dependent manner is not clear. Here we show that PKM2, the E2 subunit of PDC and histone acetyltransferase p300 constitute a complex on chromatin with arylhydrocarbon receptor (AhR), a transcription factor associated with xenobiotic metabolism. All of these factors are recruited to the enhancer of AhR-target genes, in an AhR-dependent manner. PKM2 contributes to enhancement of transcription of cytochrome P450 1A1 (CYP1A1), an AhR-target gene, acetylation at lysine 9 of histone H3 at the CYP1A1 enhancer. Site-directed mutagenesis of PKM2 indicates that this enhancement of histone acetylation requires the pyruvate kinase activity of the enzyme. Furthermore, we reveal that PDC activity is present in nuclei. Based on these findings, we propose a local acetyl-CoA production system in which PKM2 and PDC locally supply acetyl-CoA to p300 from abundant PEP for histone acetylation at the gene enhancer, and our data suggest that PKM2 sensitizes AhR-mediated detoxification in actively proliferating cells such as cancer and fetal cells. PMID:26405201

  18. Structural and calorimetric studies demonstrate that the hepatocyte nuclear factor 1β (HNF1β) transcription factor is imported into the nucleus via a monopartite NLS sequence.

    PubMed

    Wiedmann, Mareike M; Aibara, Shintaro; Spring, David R; Stewart, Murray; Brenton, James D

    2016-09-01

    The transcription factor hepatocyte nuclear factor 1β (HNF1β) is ubiquitously overexpressed in ovarian clear cell carcinoma (CCC) and is a potential therapeutic target. To explore potential approaches that block HNF1β transcription we have identified and characterised extensively the nuclear localisation signal (NLS) for HNF1β and its interactions with the nuclear protein import receptor, Importin-α. Pull-down assays demonstrated that the DNA binding domain of HNF1β interacted with a spectrum of Importin-α isoforms and deletion constructs tagged with eGFP confirmed that the HNF1β (229)KKMRRNR(235) sequence was essential for nuclear localisation. We further characterised the interaction between the NLS and Importin-α using complementary biophysical techniques and have determined the 2.4Å resolution crystal structure of the HNF1β NLS peptide bound to Importin-α. The functional, biochemical, and structural characterisation of the nuclear localisation signal present on HNF1β and its interaction with the nuclear import protein Importin-α provide the basis for the development of compounds targeting transcription factor HNF1β via its nuclear import pathway. PMID:27346421

  19. Transcriptional Regulation of CYP2B6 Expression by Hepatocyte Nuclear Factor 3β in Human Liver Cells

    PubMed Central

    Li, Linhao; Li, Daochuan; Heyward, Scott; Wang, Hongbing

    2016-01-01

    CYP2B6 plays an increasingly important role in xenobiotic metabolism and detoxification. The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) have been established as predominant regulators for the inductive expression of CYP2B6 gene in human liver. However, there are dramatic interindividual variabilities in CYP2B6 expression that cannot be fully explained by the CAR/PXR-based modulation alone. Here, we show that expression level of CYP2B6 was correlated with that of hepatocyte nuclear factor 3β (HNF3β) in human primary hepatocytes prepared from 35 liver donors. Utilizing recombinant virus-mediated overexpression or knockdown of HNF3β in HepG2 cells, as well as constructs containing serial deletion and site-directed mutation of HNF3β binding motifs in CYP2B6 luciferase reporter assays, we demonstrated that the presence or lack of HNF3β expression markedly correlated with CYP2B6 gene expression and its promoter activity. Novel enhancer modules of HNF3β located upstream of the CYP2B6 gene transcription start site were identified and functionally validated as key elements governing HNF3β-mediated CYP2B6 expression. Chromatin immunoprecipitation assays in human primary hepatocytes and surface plasmon resonance binding affinity experiments confirmed the essential role of these enhancers in the recruitment of HNF3β to the promoter of CYP2B6 gene. Overall, these findings indicate that HNF3β represents a new liver enriched transcription factor that is involved in the transcription of CYP2B6 gene and contributes to the large interindividual variations of CYP2B6 expression in human population. PMID:26930610

  20. PUBLIC HEARING TRANSCRIPT: FEDERAL NON-NUCLEAR ENERGY RESEARCH AND DEVELOPMENT PROGRAM

    EPA Science Inventory

    This document presents the proceedings of three days of public hearings on the Federal Non-nuclear Energy Research and Development Program. The document is presented in three sections: (1) Future Energy Patterns and Levels of Coal Use, (2) Solar Energy and Conservation, and (3) O...

  1. An overview of the CCAAT-box binding factor in filamentous fungi: assembly, nuclear translocation, and transcriptional enhancement.

    PubMed

    Kato, Masashi

    2005-04-01

    Filamentous fungi are frequently used for the production of industrial enzymes, since they produce a variety of enzymes including polysaccharide-degrading enzymes. Among the many filamentous fungi, Aspergillus species, such as A. oryzae and A. niger, are known as strong producers of amylolytic enzymes. We have been studying on the regulatory mechanisms underlying the expression of A. oryzae amylolytic genes. Based on analyses using a hybrid model system of A. nidulans transformed by a gene encoding A. oryzae Taka-amylase A, the major amylase (taaG2), we have found that three factors, CCAAT-box binding protein, CreA, and AmyR, are involved in taaG2 gene expression and regulation. In this review, the focus is on the CCAAT-box binding protein of filamentous fungi. The assembly, nuclear translocation, and transcriptional enhancement mechanisms of the CCAAT-box binding protein are discussed. PMID:15849404

  2. Cynaropicrin from Cynara scolymus L. suppresses photoaging of skin by inhibiting the transcription activity of nuclear factor-kappa B.

    PubMed

    Tanaka, Yuka Tsuda; Tanaka, Kiyotaka; Kojima, Hiroyuki; Hamada, Tomoji; Masutani, Teruaki; Tsuboi, Makoto; Akao, Yukihiro

    2013-01-15

    Aging of skin is characterized by skin wrinkling, laxity, and pigmentation induced by several environmental stress factors. Histological changes during the photoaging of skin include hyperproliferation of keratinocytes and melanocytes causing skin wrinkles and pigmentation. Nuclear factor kappa B (NF-κB) is one of the representative transcription factors active in conjunction with inflammation. NF-κB is activated by stimulation such as ultraviolet rays and inflammatory cytokines and induces the expression of various genes such as those of basic fibroblast growth factor (bFGF) and matrix metalloprotease-1 (MMP-1). We screened several plant extracts for their possible inhibitory effect on the transcriptional activity of NF-κB. One of them, an extract from Cynara scolymus L., showed a greatest effect on the suppression of NF-κB transactivation. As a result, we found that cynaropicrin, which is a sesquiterpene lactone, inhibited the NF-κB-mediated transactivation of bFGF and MMP-1. Furthermore, it was confirmed that in an in vivo mouse model cynaropicrin prevented skin photoaging processes leading to the hyperproliferation of keratinocytes and melanocytes. These findings taken together indicate that cynaropicrin is an effective antiphotoaging agent that acts by inhibiting NF-κB-mediated transactivation. PMID:23232059

  3. Nuclear Import of the Parsley bZIP Transcription Factor CPRF2 Is Regulated by Phytochrome Photoreceptors

    PubMed Central

    Kircher, Stefan; Wellmer, Frank; Nick, Peter; Rügner, Alexander; Schäfer, Eberhard; Harter, Klaus

    1999-01-01

    In plants, light perception by photoreceptors leads to differential expression of an enormous number of genes. An important step for differential gene expression is the regulation of transcription factor activities. To understand these processes in light signal transduction we analyzed the three well-known members of the common plant regulatory factor (CPRF) family from parsley (Petroselinum crispum). Here, we demonstrate that these CPRFs, which belong to the basic- region leucine-zipper (bZIP) domain-containing transcription factors, are differentially distributed within parsley cells, indicating different regulatory functions within the regulatory networks of the plant cell. In particular, we show by cell fractionation and immunolocalization approaches that CPRF2 is transported from the cytosol into the nucleus upon irradiation due to action of phytochrome photoreceptors. Two NH2-terminal domains responsible for cytoplasmic localization of CPRF2 in the dark were characterized by deletion analysis using a set of CPRF2-green fluorescent protein (GFP) gene fusion constructs transiently expressed in parsley protoplasts. We suggest that light-induced nuclear import of CPRF2 is an essential step in phytochrome signal transduction. PMID:9922448

  4. Promoter-specific transactivation of hepatitis B virus transcription by a glutamine- and proline-rich domain of hepatocyte nuclear factor 1.

    PubMed Central

    Raney, A K; Easton, A J; Milich, D R; McLachlan, A

    1991-01-01

    The cloned transcription factor hepatocyte nuclear factor 1 (HNF1) transactivates transcription from the hepatitis B virus (HBV) large surface antigen promoter but does not influence the transcriptional activities of the other three HBV promoters. This indicates that this transcription factor can differentially influence the activities of the HBV promoter. By using a transient-transfection system, the major domain of the HNF1 polypeptide involved in transcriptional activation of the large surface antigen promoter in the human hepatoma cell line HepG2.1 has been mapped to a region that is rich in glutamine and proline residues (9 of 18) and is different from the previously identified regions of this factor responsible for in vitro transcriptional activation of a promoter containing human albumin promoter HNF1 binding sites. The human albumin promoter HNF1 binding site mediates transcriptional activation through the same HNF1 polypeptide domain as the HBV large surface antigen promoter HNF1 binding site in transient-transfection assays with HepG2.1 cells, suggesting that HNF1 may possess multiple transcriptional activation domains. Images PMID:1656070

  5. Hepatic nuclear factor 3 is an accessory factor required for the stimulation of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids.

    PubMed

    Wang, J C; Strömstedt, P E; O'Brien, R M; Granner, D K

    1996-07-01

    Transcription of the hepatic phosphoenolpyruvate carboxykinase gene is stimulated by glucocorticoids and inhibited by insulin. The glucocorticoid response is mediated by a complex glucocorticoid response unit that consists of two glucocorticoid receptor (GR)-binding sites (GR1 and GR2) and two accessory factor-binding sites (AF1 and AF2). The complete unit is required for the full glucocorticoid response. The dominant insulin effect is mediated in part through an insulin response sequence that is coincident with the AF2 element. Members of the hepatic nuclear factor 3 (HNF3) and CCAAT enhancer binding protein (C/EBP) families bind to the AF2 element; however, there is no correlation between binding of these factors and the ability of the AF2 element to mediate an insulin response. We show here that binding of HNF3 does correlate with the stimulation of the glucocorticoid response by the AF2 element and that C/EBP is apparently not involved in this effect. This requirement for HNF3 is quite specific since the substitution of elements known to enhance the action of the GR in other promoters fails to recapitulate AF2 accessory factor activity. By contrast, an HNF3-binding site from the transthyretin gene is able to substitute for the wild type AF2 sequence and elicit a maximal glucocorticoid response. Based on current and previous observations, the glucocorticoid response unit consists of four DNA elements that bind four different proteins. These are: AF1 (hepatic nuclear factor 4/chicken ovalbumin upstream promoter transcription factor), AF2 (HNF3), GR1 (GR), and GR2 (GR). PMID:8813720

  6. Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

    PubMed Central

    Tsai, Shang-Yi A.; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-fei; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

    2015-01-01

    The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER–mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal. PMID:26554014

  7. Nuclear-encoded factors involved in post-transcriptional processing and modification of mitochondrial tRNAs in human disease

    PubMed Central

    Powell, Christopher A.; Nicholls, Thomas J.; Minczuk, Michal

    2015-01-01

    The human mitochondrial genome (mtDNA) encodes 22 tRNAs (mt-tRNAs) that are necessary for the intraorganellar translation of the 13 mtDNA-encoded subunits of the mitochondrial respiratory chain complexes. Maturation of mt-tRNAs involves 5′ and 3′ nucleolytic excision from precursor RNAs, as well as extensive post-transcriptional modifications. Recent data suggest that over 7% of all mt-tRNA residues in mammals undergo post-transcriptional modification, with over 30 different modified mt-tRNA positions so far described. These processing and modification steps are necessary for proper mt-tRNA function, and are performed by dedicated, nuclear-encoded enzymes. Recent growing evidence suggests that mutations in these nuclear genes (nDNA), leading to incorrect maturation of mt-tRNAs, are a cause of human mitochondrial disease. Furthermore, mtDNA mutations in mt-tRNA genes, which may also affect mt-tRNA function, processing, and modification, are also frequently associated with human disease. In theory, all pathogenic mt-tRNA variants should be expected to affect only a single process, which is mitochondrial translation, albeit to various extents. However, the clinical manifestations of mitochondrial disorders linked to mutations in mt-tRNAs are extremely heterogeneous, ranging from defects of a single tissue to complex multisystem disorders. This review focuses on the current knowledge of nDNA coding for proteins involved in mt-tRNA maturation that have been linked to human mitochondrial pathologies. We further discuss the possibility that tissue specific regulation of mt-tRNA modifying enzymes could play an important role in the clinical heterogeneity observed for mitochondrial diseases caused by mutations in mt-tRNA genes. PMID:25806043

  8. Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope.

    PubMed

    Tsai, Shang-Yi A; Chuang, Jian-Ying; Tsai, Meng-Shan; Wang, Xiao-Fei; Xi, Zheng-Xiong; Hung, Jan-Jong; Chang, Wen-Chang; Bonci, Antonello; Su, Tsung-Ping

    2015-11-24

    The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER-mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal. PMID:26554014

  9. A pleiotropic element in the medium-chain acyl coenzyme A dehydrogenase gene promoter mediates transcriptional regulation by multiple nuclear receptor transcription factors and defines novel receptor-DNA binding motifs.

    PubMed Central

    Carter, M E; Gulick, T; Moore, D D; Kelly, D P

    1994-01-01

    We previously identified a complex regulatory element in the medium-chain acyl coenzyme A dehydrogenase gene promoter that confers transcriptional regulation by the retinoid receptors RAR and RXR and the orphan nuclear receptor HNF-4. In this study we demonstrate a trans-repressing regulatory function for the orphan receptor COUP-TF at this same nuclear receptor response element (NRRE-1). The transcriptional regulatory properties and receptor binding sequences of each nuclear receptor response element within NRRE-1 are also characterized. NRRE-1 consists of four potential nuclear hormone receptor hexamer binding sites, arranged as [<--1-(n)s-2-->-3-->(n)4<--4], three of which are used in alternative pairwise binding by COUP-TF and HNF-4 homodimers and by RAR-RXR heterodimers, as demonstrated by mobility shift assays and methylation interference analysis. Binding and transactivation studies with mutant NRRE-1 elements confirmed the existence of distinct retinoid, COUP-TF, and HNF-4 response elements that define novel receptor binding motifs: COUP-TF homodimers bound sites 1 and 3 (two hexamer repeat sequences arranged as an everted imperfect repeat separated by 14 bp or ER14), RAR-RXR heterodimers bound sites 1 and 2 (ER8), and HNF-4 homodimers bound sites 2 and 3 (imperfect DR0). Mixing cotransfection experiments demonstrated that the nuclear receptor dimers compete at NRRE-1 to modulate constitutive and ligand-mediated transcriptional activity. These data suggest a mechanism for the transcriptional modulation of genes encoding enzymes involved in cellular metabolism. Images PMID:8007945

  10. Mechanism of a transcriptional cross talk between transforming growth factor-beta-regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4.

    PubMed

    Chou, Wan-Chih; Prokova, Vassiliki; Shiraishi, Keiko; Valcourt, Ulrich; Moustakas, Aristidis; Hadzopoulou-Cladaras, Margarita; Zannis, Vassilis I; Kardassis, Dimitris

    2003-03-01

    We have shown previously that the transforming growth factor-beta (TGFbeta)-regulated Sma-Mad (Smad) protein 3 and Smad4 proteins transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone response element that binds the nuclear receptor hepatocyte nuclear factor 4 (HNF-4). In the present study, we show that Smad3 and Smad4 but not Smad2 physically interact with HNF-4 via their Mad homology 1 domains both in vitro and in vivo. The synergistic transactivation of target promoters by Smads and HNF-4 was shown to depend on the specific promoter context and did not require an intact beta-hairpin/DNA binding domain of the Smads. Using glutathione S-transferase interaction assays, we established that two regions of HNF-4, the N-terminal activation function 1 (AF-1) domain (aa 1-24) and the C-terminal F domain (aa 388-455) can mediate physical Smad3/HNF-4 interactions in vitro. In vivo, Smad3 and Smad4 proteins enhanced the transactivation function of various GAL4-HNF-4 fusion proteins via the AF-1 and the adjacent DNA binding domain, whereas a single tyrosine to alanine substitution in AF-1 abolished coactivation by Smads. The findings suggest that the transcriptional cross talk between the TGFbeta-regulated Smads and HNF-4 is mediated by specific functional domains in the two types of transcription factors. Furthermore, the specificity of this interaction for certain target promoters may play an important role in various hepatocyte functions, which are regulated by TGFbeta and the Smads. PMID:12631740

  11. Decreased expression of hepatocyte nuclear factor 3 alpha during the acute-phase response influences transthyretin gene transcription.

    PubMed Central

    Qian, X; Samadani, U; Porcella, A; Costa, R H

    1995-01-01

    Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (alpha, beta, and gamma) are known to regulate the transcription of numerous liver-specific genes. The HNF-3 proteins bind to DNA as monomers through a winged-helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic fork head (fkh) protein. We have previously characterized a strong-affinity HNF-3S site in the transthyretin (TTR) promoter region which is essential for expression in human hepatoma (HepG2) cells. In the current study, we identify an activating protein 1 (AP-1) site which partially overlaps the HNF-3S sequence in the TTR promoter. We show that in HepG2 cells the AP-1 sequence confers 12-O-tetradecanoylphorbol-13-acetate inducibility to the TTR promoter and contributes to normal TTR transcriptional activity. We also demonstrate that the HNF-3 proteins and AP-1 bind independently to the TTR AP-1-HNF-3 site, and cotransfection experiments suggest that they do not cooperate to activate an AP-1-HNF-3 reporter construct. In addition, 12-O-tetradecanoylphorbol-13-acetate exposure of HepG2 cells results in a reciprocal decrease in HNF-3 alpha and -3 gamma expression which may facilitate interaction of AP-1 with the TTR AP-1-HNF-3 site. In order to explore the role of HNF-3 in the liver, we have examined expression patterns of TTR and HNF-3 during the acute-phase response and liver regeneration. Partial hepatectomy produced minimal fluctuation in HNF-3 and TTR expression, suggesting that HNF-3 expression is not influenced by proliferative signals induced during liver regeneration. In acute-phase livers, we observed a dramatic reduction in HNF-3 alpha expression which correlates with a decrease in the expression of its target gene, the TTR gene. Furthermore, consistent with previous studies, the acute-phase livers are induced for c-jun but not c-fos expression. We propose that the reduction in TTR gene expression during the acute phase is likely due

  12. Transcription factor nuclear factor erythroid-2 mediates expression of the cytokine interleukin 8, a known predictor of inferior outcome in patients with myeloproliferative neoplasms

    PubMed Central

    Wehrle, Julius; Seeger, Thalia S.; Schwemmers, Sven; Pfeifer, Dietmar; Bulashevska, Alla; Pahl, Heike L.

    2013-01-01

    The transcription factor nuclear factor erythroid-2 is over-expressed in patients with myeloproliferative neoplasms irrespective of the presence of the JAK2V617F mutation. Our transgenic mouse model over-expressing nuclear factor erythroid-2, which recapitulates many features of myeloproliferative neoplasms including transformation to acute myeloid leukemia, clearly implicates this transcription factor in the pathophysiology of myeloproliferative neoplasms. Because the targets mediating nuclear factor erythroid-2 effects are not well characterized, we conducted microarray analysis of CD34+ cells lentivirally transduced to over-express nuclear factor erythroid-2 or to silence this transcription factor via shRNA, in order to identify novel target genes. Here, we report that the cytokine interleukin 8 is a novel target gene. Nuclear factor erythroid-2 directly binds the interleukin 8 promoter in vivo, and these binding sites are required for promoter activity. Serum levels of interleukin 8 are known to be elevated in both polycythemia vera and primary myelofibrosis patients. Recently, increased interleukin 8 levels have been shown to be predictive of inferior survival in primary myelofibrosis patients in multivariate analysis. Therefore, one of the mechanisms by which nuclear factor erythroid-2 contributes to myeloproliferative neoplasm pathology may be increased interleukin 8 expression. PMID:23445878

  13. Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription.

    PubMed

    Busch, S J; Martin, G A; Barnhart, R L; Mano, M; Cardin, A D; Jackson, R L

    1992-01-01

    Heparin blocks the phorbol ester-induced progression of nontransformed cells through the G0/G1 phase (Wright, T.C., L.A. Pukac, J.J. Castellot, M.J. Karnovsky, R.A. Levine, H.-Y. Kim-Park, and J. Campisi. 1989. Proc. Natl. Acad. Sci. USA. 86: 3199-3203) or G1 to S phase (Reilly, C. F., M. S. Kindy, K. E. Brown, R. D. Rosenberg, and G. E Sonenshein. 1989. J. Biol. Chem. 264:6990-6995) of the cell cycle. Cell cycle arrest was associated with decreased levels of stage-specific mRNAs suggesting transcriptional regulation of cell growth. In the present report, we show that heparin selectively repressed TPA-inducible AP-1-mediated gene expression. Heparin-induced trans-repression was observed in primary vascular smooth muscle cells, as well as in the transformed HeLa cell line and in nondifferentiated F9 teratocarcinoma cells. Inhibition of AP-1-mediated trans-activation occurred with heparin and pentosan polysulfate but not with chondroitin sulfate A or C. Heparin-binding peptides or heparitinase I addition to nuclear lysates of heparin-treated cells allowed enhanced recovery of endogenous AP-1-specific DNA binding activity. We propose a model in which nuclear glycosaminoglycans play a trans-regulatory role in altering the patterns of inducible gene expression. PMID:1730747

  14. Interaction of Sp1 zinc finger with transport factor in the nuclear localization of transcription factor Sp1

    SciTech Connect

    Ito, Tatsuo; Kitamura, Haruka; Uwatoko, Chisana; Azumano, Makiko; Itoh, Kohji; Kuwahara, Jun

    2010-12-10

    Research highlights: {yields} Sp1 zinc fingers themselves interact with importin {alpha}. {yields} Sp1 zinc finger domains play an essential role as a nuclear localization signal. {yields} Sp1 can be transported into the nucleus in an importin-dependent manner. -- Abstract: Transcription factor Sp1 is localized in the nucleus and regulates the expression of many cellular genes, but the nuclear transport mechanism of Sp1 is not well understood. In this study, we revealed that GST-fused Sp1 protein bound to endogenous importin {alpha} in HeLa cells via the Sp1 zinc finger domains, which comprise the DNA binding domain of Sp1. It was found that the Sp1 zinc finger domains directly interacted with a wide range of importin {alpha} including the armadillo (arm) repeat domain and the C-terminal acidic domain. Furthermore, it turned out that all three zinc fingers of Sp1 are essential for binding to importin {alpha}. Taken together, these results suggest that the Sp1 zinc finger domains play an essential role as a NLS and Sp1 can be transported into the nucleus in an importin-dependent manner even though it possesses no classical NLSs.

  15. Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells

    PubMed Central

    Miyazaki, Yusuke; Chen, Ling-chun; Chu, Bernard W; Swigut, Tomek; Wandless, Thomas J

    2015-01-01

    Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called ‘destabilizing domains’, whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies. DOI: http://dx.doi.org/10.7554/eLife.07687.001 PMID:26314864

  16. The ciliopathy disease protein NPHP9 promotes nuclear delivery and activation of the oncogenic transcriptional regulator TAZ.

    PubMed

    Habbig, Sandra; Bartram, Malte P; Sägmüller, Josef G; Griessmann, Anabel; Franke, Mareike; Müller, Roman-Ulrich; Schwarz, Ricarda; Hoehne, Martin; Bergmann, Carsten; Tessmer, Claudia; Reinhardt, H Christian; Burst, Volker; Benzing, Thomas; Schermer, Bernhard

    2012-12-15

    Nephronophthisis (NPH) is a genetically heterogenous kidney disease and represents the most common genetic cause for end-stage renal disease in children. It is caused by the mutation of genes encoding for the nephrocystin proteins (NPHPs) which localize to primary cilia or centrosomes, classifying this disease as a 'ciliopathy'. Recently, it has been shown that NPHP4 acts as a potent negative regulator of mammalian Hippo signalling by interacting with the Lats protein kinase and controlling the phosphorylation of the oncogenic transcriptional activator TAZ. Here, we demonstrate that NPHP9, another NPH family member, also controls TAZ activity by a distinct mechanism. NPHP9, which is also called NEK8, directly interacted with TAZ and induced nuclear translocation of the TAZ/NPHP9 protein complex. Binding of NPHP9 to TAZ was enhanced in a TAZ mutant that lost its ability to bind 14-3-3, suggesting that 14-3-3 and NPHP9 may compete for TAZ binding, with 14-3-3 favouring cytoplasmic retention and NPHP9 mediating nuclear delivery. Consistently, co-expression of NPHP4, which inhibits TAZ phosphorylation at the 14-3-3 binding site through the inhibition of Lats kinase activity, induced efficient nuclear delivery of the TAZ/NPHP9 protein pair. Consistent with a role for TAZ in controlling proliferation and tumorigenesis, the downregulation of NPHP9 inhibited the TAZ-dependent proliferation of hippo-responsive normal epithelial and also breast cancer cells. As NPHP9 has been shown to be upregulated in breast cancer, these data do not only support a critical role for TAZ/hippo signalling in the pathogenesis of NPH but may also imply a possible role for NPHP9 in TAZ-mediated tumorigenesis. PMID:23026745

  17. Virus-Dependent Phosphorylation of the IRF-3 Transcription Factor Regulates Nuclear Translocation, Transactivation Potential, and Proteasome-Mediated Degradation

    PubMed Central

    Lin, Rongtuan; Heylbroeck, Christophe; Pitha, Paula M.; Hiscott, John

    1998-01-01

    Ser-398 in IRF-3 abrogated its binding to CBP. These results are discussed in terms of a model in which virus-inducible, C-terminal phosphorylation of IRF-3 alters protein conformation to permit nuclear translocation, association with transcriptional partners, and primary activation of IFN- and IFN-responsive genes. PMID:9566918

  18. Transcriptional activation of cyclooxygenase-2 by tumor suppressor p53 requires nuclear factor-kappaB.

    PubMed

    Benoit, V; de Moraes, E; Dar, N A; Taranchon, E; Bours, V; Hautefeuille, A; Tanière, P; Chariot, A; Scoazec, J-Y; de Moura Gallo, C V; Merville, M-P; Hainaut, P

    2006-09-21

    Overexpression of cyclooxygenase-2 (Cox-2) is thought to exert antiapoptotic effects in cancer. Here we show that the tumor suppressor p53 upregulated Cox-2 in esophageal and colon cancer cell lines by inducing the binding of nuclear factor-kappaB (NF-kappaB) to its response element in the COX-2 promoter. Inhibition of NF-kappaB prevented p53 induction of Cox-2 expression. Cooperation between p53 and NF-kappaB was required for activation of COX-2 promoter in response to daunomycin, a DNA-damaging agent. Pharmacological inhibition of Cox-2 enhanced apoptosis in response to daunomycin, in particular in cells containing active p53. In esophageal cancer, there was a correlation between Cox-2 expression and wild-type TP53 in Barrett's esophagus (BE) and in adenocarcinoma, but not in squamous cell carcinoma (P<0.01). These results suggest that p53 and NF-kappaB cooperate in upregulating Cox-2 expression, promoting cell survival in inflammatory precursor lesions such as BE. PMID:16682957

  19. Inducible nuclear expression of newly synthesized I kappa B alpha negatively regulates DNA-binding and transcriptional activities of NF-kappa B.

    PubMed Central

    Arenzana-Seisdedos, F; Thompson, J; Rodriguez, M S; Bachelerie, F; Thomas, D; Hay, R T

    1995-01-01

    The transcription factor NF-kappa B is exploited by many viruses, including the human immunodeficiency virus, for expression of viral genes, but its primary role appears to be in the rapid induction of cellular genes during immune and inflammatory responses. The inhibitor protein I kappa B alpha maintains NF-kappa B in an inactive form in the cytoplasms of unstimulated cells, but upon cell activation, I kappa B alpha is rapidly degraded, leading to nuclear translocation of free NF-kappa B. However, NF-kappa B-dependent transcription of the I kappa B alpha gene leads to rapid resynthesis of the I kappa B alpha protein and inhibition of NF-kappa B-dependent transcription. Here we demonstrate a new regulatory function of I kappa B alpha exerted on NF-kappa B in the nuclear compartment. Although normally found in the cytoplasm, I kappa B alpha, newly synthesized in response to tumor necrosis factor or interleukin I, is transported to the nucleus. In the nucleus I kappa B alpha associates with the p50 and p65 subunits of NF-kappa B, inhibiting DNA binding of the transcription factor. Furthermore, nuclear expression of I kappa B alpha correlates with transcription termination of transfected NF-kappa B-dependent luciferase genes. Following the appearance of I kappa B alpha in the nuclei of activated cells, a dramatic reduction in the amount of nuclear p50 occurs, suggesting that NF-kappa B-I kappa B alpha complexes are cleared from the nucleus. PMID:7739549

  20. Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor

    PubMed Central

    2010-01-01

    Background CrMYC2 is an early jasmonate-responsive bHLH transcription factor involved in the regulation of the expression of the genes of the terpenic indole alkaloid biosynthesis pathway in Catharanthus roseus. In this paper, we identified the amino acid domains necessary for the nuclear targeting of CrMYC2. Findings We examined the intracellular localization of whole CrMYC2 and of various deletion mutants, all fused with GFP, using a transient expression assay in onion epidermal cells. Sequence analysis of this protein revealed the presence of four putative basic nuclear localization signals (NLS). Assays showed that none of the predicted NLS is active alone. Further functional dissection of CrMYC2 showed that the nuclear targeting of this transcription factor involves the cooperation of three domains located in the C-terminal region of the protein. The first two domains are located at amino acid residues 454-510 and 510-562 and contain basic classical monopartite NLSs; these regions are referred to as NLS3 (KRPRKR) and NLS4 (EAERQRREK), respectively. The third domain, between residues 617 and 652, is rich in basic amino acids that are well conserved in other phylogenetically related bHLH transcription factors. Our data revealed that these three domains are inactive when isolated but act cooperatively to target CrMYC2 to the nucleus. Conclusions This study identified three amino acid domains that act in cooperation to target the CrMYC2 transcription factor to the nucleus. Further fine structure/function analysis of these amino acid domains will allow the identification of new NLS domains and will allow the investigation of the related molecular mechanisms involved in the nuclear targeting of the CrMYC2 bHLH transcription factor. PMID:21073696

  1. Trafficking of the Transcription Factor Nrf2 to Promyelocytic Leukemia-Nuclear Bodies

    PubMed Central

    Malloy, Melanie Theodore; McIntosh, Deneshia J.; Walters, Treniqka S.; Flores, Andrea; Goodwin, J. Shawn; Arinze, Ifeanyi J.

    2013-01-01

    Ubiquitylation of Nrf2 by the Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex targets Nrf2 for proteasomal degradation in the cytoplasm and is an extensively studied mechanism for regulating the cellular level of Nrf2. Although mechanistic details are lacking, reports abound that Nrf2 can also be degraded in the nucleus. Here, we demonstrate that Nrf2 is a target for sumoylation by both SUMO-1 and SUMO-2. HepG2 cells treated with As2O3, which enhances attachment of SUMO-2/3 to target proteins, increased SUMO-2/3-modification (polysumoylation) of Nrf2. We show that Nrf2 traffics, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs). Cell fractions harboring key components of PML-NBs did not contain biologically active Keap1 but contained modified Nrf2 as well as RING finger protein 4 (RNF4), a poly-SUMO-specific E3 ubiquitin ligase. Overexpression of wild-type RNF4, but not the catalytically inactive mutant, decreased the steady-state levels of Nrf2, measured in the PML-NB-enriched cell fraction. The proteasome inhibitor MG-132 interfered with this decrease, resulting in elevated levels of polysumoylated Nrf2 that was also ubiquitylated. Wild-type RNF4 accelerated the half-life (t½) of Nrf2, measured in PML-NB-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf2 as a target for sumoylation and provides a novel mechanism for its degradation in the nucleus, independent of Keap1. PMID:23543742

  2. Genetic disruption of KSHV major latent nuclear antigen LANA enhances viral lytic transcriptional program

    SciTech Connect

    Li Qiuhua; Zhou Fuchun; Ye Fengchun; Gao Shoujiang

    2008-09-30

    Following primary infection, KSHV establishes a lifelong persistent latent infection in the host. The mechanism of KSHV latency is not fully understood. The latent nuclear antigen (LANA or LNA) encoded by ORF73 is one of a few viral genes expressed during KSHV latency, and is consistently detected in all KSHV-related malignancies. LANA is essential for KSHV episome persistence, and regulates the expression of viral lytic genes through epigenetic silencing, and inhibition of the expression and transactivation function of the key KSHV lytic replication initiator RTA (ORF50). In this study, we used a genetic approach to examine the role of LANA in regulating KSHV lytic replication program. Deletion of LANA did not affect the expression of its adjacent genes vCyclin (ORF72) and vFLIP (ORF71). In contrast, the expression levels of viral lytic genes including immediate-early gene RTA, early genes MTA (ORF57), vIL-6 (ORF-K2) and ORF59, and late gene ORF-K8.1 were increased before and after viral lytic induction with 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. This enhanced expression of viral lytic genes was also observed following overexpression of RTA with or without simultaneous chemical induction. Consistent with these results, the LANA mutant cells produced more infectious virions than the wild-type virus cells did. Furthermore, genetic repair of the mutant virus reverted the phenotypes to those of wild-type virus. Together, these results have demonstrated that, in the context of viral genome, LANA contributes to KSHV latency by regulating the expression of RTA and its downstream genes.

  3. Heterogeneous nuclear ribonucleoprotein K and nucleolin as transcriptional activators of the vascular endothelial growth factor promoter through interaction with secondary DNA structures

    PubMed Central

    Uribe, Diana J.; Guo, Kexiao; Shin, Yoon-Joo; Sun, Daekyu

    2011-01-01

    The human vascular endothelial growth factor (VEGF) promoter contains a polypurine/polypyrimidine (pPu/pPy) tract that is known to play a critical role in its transcriptional regulation. This pPu/pPy tract undergoes a conformational transition between B-DNA, single stranded DNA and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We studied the interaction of the cytosine-rich (C-rich) and guanine-rich (G-rich) strands of this tract with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of VEGF. Using chromatin immunoprecipitation (ChIP) assay for our in vivo studies and electrophoretic mobility shift assay (EMSA) for our in vitro studies, we demonstrated that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the VEGF promoter. The small interfering RNA (siRNA)-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within the pPu/pPy tract will provide new insight into mechanisms of transcriptional regulation of the VEGF gene. PMID:21466159

  4. Recombinant human ciliary neurotrophic factor reduces weight partly by regulating nuclear respiratory factor 1 and mitochondrial transcription factor A.

    PubMed

    Liu, Qing-Shan; Wang, Qiu-Juan; Du, Guan-Hua; Zhu, Shen-Yin; Gao, Mei; Zhang, Li; Zhu, Jun-Ming; Cao, Jian-Feng

    2007-06-01

    Ciliary neurotrophic factor (CNTF) can lead to weight loss by up-regulating energy metabolism and the expression of UCP-1 in mitochondria. To investigate the up-stream regulators of the expression of UCP-1, recombinant human CNTF (rhCNTF) (0.1, 0.3, 0.9 mg/kg/day s.c.) administered to KK-Ay mice for 30 days resulting in reductions in body weight and perirenal fat mass. In brown adipose tissues, the gene expressions of nuclear respiratory factor (NRF)-1, mitochondrial transcription factor A (TFam) and uncoupling protein (UCP)-1 were found up-regulated by rhCNTF. To the best of our knowledge, these effects represent new insights on the mechanisms of action of weight loss by rhCNTF. In addition, we also found that rhCNTF increased the activity of mitochondrial complex IV. The stimulation of NRF-1, TFam, UCP-1 and the enhanced activity of mitochondrial complex IV may be associated with remedying obesity. The result indicates that rhCNTF can enhance the expressions of NRF-1 and TFam, both of which can up-regulate the expression of UCP-1. PMID:17397829

  5. Activation of nuclear transcription factor-kappaB in mouse brain induced by a simulated microgravity environment

    NASA Technical Reports Server (NTRS)

    Wise, Kimberly C.; Manna, Sunil K.; Yamauchi, Keiko; Ramesh, Vani; Wilson, Bobby L.; Thomas, Renard L.; Sarkar, Shubhashish; Kulkarni, Anil D.; Pellis, Neil R.; Ramesh, Govindarajan T.

    2005-01-01

    Microgravity induces inflammatory responses and modulates immune functions that may increase oxidative stress. Exposure to a microgravity environment induces adverse neurological effects; however, there is little research exploring the etiology of these effects resulting from exposure to such an environment. It is also known that spaceflight is associated with increase in oxidative stress; however, this phenomenon has not been reproduced in land-based simulated microgravity models. In this study, an attempt has been made to show the induction of reactive oxygen species (ROS) in mice brain, using ground-based microgravity simulator. Increased ROS was observed in brain stem and frontal cortex with concomitant decrease in glutathione, on exposing mice to simulated microgravity for 7 d. Oxidative stress-induced activation of nuclear factor-kappaB was observed in all the regions of the brain. Moreover, mitogen-activated protein kinase kinase was phosphorylated equally in all regions of the brain exposed to simulated microgravity. These results suggest that exposure of brain to simulated microgravity can induce expression of certain transcription factors, and these have been earlier argued to be oxidative stress dependent.

  6. Transcription factors nuclear factor I and Sp1 interact with the murine collagen alpha 1 (I) promoter.

    PubMed Central

    Nehls, M C; Rippe, R A; Veloz, L; Brenner, D A

    1991-01-01

    The collagen alpha 1(I) promoter, which is efficiently transcribed in NIH 3T3 fibroblasts, contains four binding sites for trans-acting factors, as demonstrated by DNase I protection assays (D. A. Brenner, R. A. Rippe, and L. Veloz, Nucleic Acids Res. 17:6055-6064, 1989). This study characterizes the DNA-binding proteins that interact with the two proximal footprinted regions, both of which contain a reverse CCAAT box and a G + C-rich 12-bp direct repeat. Analysis by DNase I protection assays, mobility shift assays, competition with specific oligonucleotides, binding with recombinant proteins, and reactions with specific antisera showed that the transcriptional factors nuclear factor I (NF-I) and Sp1 bind to these two footprinted regions. Because of overlapping binding sites, NF-I binding and Sp1 binding appear to be mutually exclusive. Overexpression of NF-I in cotransfection experiments with the alpha 1(I) promoter in NIH 3T3 fibroblasts increased alpha 1(I) expression, while Sp1 overexpression reduced this effect, as well as basal promoter activity. The herpes simplex virus thymidine kinase promoter, which contains independent NF-I- and Sp1-binding sites, was stimulated by both factors. Therefore, expression of the collagen alpha 1(I) gene may depend on the relative activities of NF-I and Sp1. Images PMID:2072909

  7. Activation of nuclear transcription factor-kappaB in mouse brain induced by a simulated microgravity environment.

    PubMed

    Wise, Kimberly C; Manna, Sunil K; Yamauchi, Keiko; Ramesh, Vani; Wilson, Bobby L; Thomas, Renard L; Sarkar, Shubhashish; Kulkarni, Anil D; Pellis, Neil R; Ramesh, Govindarajan T

    2005-01-01

    Microgravity induces inflammatory responses and modulates immune functions that may increase oxidative stress. Exposure to a microgravity environment induces adverse neurological effects; however, there is little research exploring the etiology of these effects resulting from exposure to such an environment. It is also known that spaceflight is associated with increase in oxidative stress; however, this phenomenon has not been reproduced in land-based simulated microgravity models. In this study, an attempt has been made to show the induction of reactive oxygen species (ROS) in mice brain, using ground-based microgravity simulator. Increased ROS was observed in brain stem and frontal cortex with concomitant decrease in glutathione, on exposing mice to simulated microgravity for 7 d. Oxidative stress-induced activation of nuclear factor-kappaB was observed in all the regions of the brain. Moreover, mitogen-activated protein kinase kinase was phosphorylated equally in all regions of the brain exposed to simulated microgravity. These results suggest that exposure of brain to simulated microgravity can induce expression of certain transcription factors, and these have been earlier argued to be oxidative stress dependent. PMID:16029073

  8. Isolation of a Novel Family of C2H2 Zinc Finger Proteins Implicated in Transcriptional Repression Mediated by Chicken Ovalbumin Upstream Promoter Transcription Factor (COUP-TF) Orphan Nuclear Receptors*

    PubMed Central

    Avram, Dorina; Fields, Andrew; Top, Karen Pretty On; Nevrivy, Daniel J.; Ishmael, Jane E.; Leid, Mark

    2010-01-01

    Two novel and related C2H2 zinc finger proteins that are highly expressed in the brain, CTIP1 and CTIP2 (COUP TF-interacting proteins 1 and 2, respectively), were isolated and shown to interact with all members of the chicken ovalbumin upstream promoter transcription factor (COUP-TF) subfamily of orphan nuclear receptors. The interaction of CTIP1 with ARP1 was studied in detail, and CTIP1 was found to harbor two independent ARP1 interaction domains, ID1 and ID2, whereas the putative AF-2 of ARP1 was required for interaction with CTIP1. CTIP1, which exhibited a punctate staining pattern within the nucleus of transfected cells, recruited cotransfected ARP1 to these foci and potentiated ARP1-mediated transcriptional repression of a reporter construct. However, transcriptional repression mediated by ARP1 acting through CTIP1 did not appear to involve recruitment of a trichostatin A-sensitive histone deacetylase(s) to the template, suggesting that this repression pathway may be distinct from that utilized by several other nuclear receptors. PMID:10744719

  9. Inhibitors of oxygen sensing prolyl hydroxylases regulate nuclear localization of the transcription factors Smad2 and YAP/TAZ involved in CTGF synthesis.

    PubMed

    Preisser, Felix; Giehl, Klaudia; Rehm, Margot; Goppelt-Struebe, Margarete

    2016-08-01

    Pharmacological inhibition of oxygen sensing prolyl hydroxylase domain enzymes (PHDs) has been shown to preserve renal structure and function in various models of kidney disease. Since transforming growth factor β-1 (TGFβ-1) is one of the major mediators of kidney injury, we investigated if inhibition of PHDs with subsequent stabilization of hypoxia inducible transcription factors (HIF) might interfere with TGFβ-1 signaling with special emphasis on its target gene connective tissue growth factor (CTGF). Overnight incubation of human renal tubular cells, primary cells and cell lines, with the PDH inhibitor DMOG increased Smad3 expression, but barely affected Smad2. Both Smads were translocated into the nucleus upon activation of the cells with TGFβ-1. Interestingly, Smad3 nuclear localization was enhanced upon pretreatment of the cells with DMOG for several hours, whereas nuclear Smad2 was reduced. This differential localization was independent of Smad2/3 phosphorylation. Reduced nuclear Smad2 correlated with impaired CTGF secretion in DMOG-treated cells and transient downregulation of Smad2 interfered with TGFβ-1-induced CTGF synthesis. Furthermore, YAP was confirmed as indispensable transcription factor involved in CTGF synthesis. Nuclear localization of YAP and TAZ was reduced in DMOG-treated cells. Our data thus provide evidence for DMOG-mediated reduction of CTGF expression by regulating the nuclear localization of the transcription factors Smad2, YAP and TAZ. Prolonged inhibition of PHDs was necessary to achieve alterations in cellular localization suggesting an indirect HIF-mediated effect. This mechanism might be extended to other transcription factors and target genes, and may thus represent a novel mechanism of negative regulation of gene expression by PHD inhibition. PMID:27155083

  10. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells.

    PubMed

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT. PMID:26160345

  11. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells

    NASA Astrophysics Data System (ADS)

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B.

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.

  12. BZLF1, an Epstein-Barr virus immediate-early protein, induces p65 nuclear translocation while inhibiting p65 transcriptional function

    SciTech Connect

    Morrison, Thomas E.; Kenney, Shannon C. . E-mail: shann@med.unc.edu

    2004-10-25

    We have previously demonstrated that the Epstein-Barr virus immediate-early BZLF1 protein interacts with, and is inhibited by, the NF-{kappa}B family member p65. However, the effects of BZLF1 on NF-{kappa}B activity have not been intensively studied. Here we show that BZLF1 inhibits p65-dependent gene expression. BZLF1 inhibited the ability of IL-1, as well as transfected p65, to activate the expression of two different NF-{kappa}B-responsive genes, ICAM-1 and I{kappa}B-{alpha}. BZLF1 also reduced the constitutive level of I{kappa}B-{alpha} protein in HeLa and A549 cells, and increased the amount of nuclear NF-{kappa}B to a similar extent as tumor necrosis factor-alpha (TNF-{alpha}) treatment. In spite of this BZLF1-associated increase in the nuclear form of NF-{kappa}B, BZLF1 did not induce binding of NF-{kappa}B to NF-{kappa}B responsive promoters (as determined by chromatin immunoprecipitation assay) in vivo, although TNF-{alpha} treatment induced NF-{kappa}B binding as expected. Overexpression of p65 dramatically inhibited the lytic replication cycle of EBV in 293-EBV cells, confirming that NF-{kappa}B also inhibits BZLF1 transcriptional function. Our results are consistent with a model in which BZLF1 inhibits the transcriptional function of p65, resulting in decreased transcription of I{kappa}B-{alpha}, decreased expression of I{kappa}B-{alpha} protein, and subsequent translocation of NF-{kappa}B to the nucleus. This nuclear translocation of NF-{kappa}B may promote viral latency by negatively regulating BZLF1 transcriptional activity. In situations where p65 activity is limiting in comparison to BZLF1, the ability of BZLF1 to inhibit p65 transcriptional function may protect the virus from the host immune system during the lytic form of infection.

  13. Transcription of the Tollip gene is elevated in intestinal epithelial cells through impaired O-GlcNAcylation-dependent nuclear translocation of the negative regulator Elf-1

    SciTech Connect

    Sugi, Yutaka; Takahashi, Kyoko; Nakano, Kou; Hosono, Akira; Kaminogawa, Shuichi

    2011-09-09

    Highlights: {yields} Transcriptional activation of the Tollitip gene is higher in IECs than in monocytes. {yields} Nt -194/-186 region acts as a cis-element and is recognized by Elf-1. {yields} Elf-1 suppresses Tollip gene transcription in monocytes but not in IECs. {yields} O-GlcNAc modification is necessary for nuclear translocation of Elf-1. {yields} O-GlcNAcylation-dependent nuclear translocation of Elf-1 is impaired in IECs. -- Abstract: Intestinal epithelial cells (IECs) must be tolerant of the large number of commensal bacteria inhabiting the intestinal tract to avoid excessive inflammatory reactions. Toll-interacting protein (Tollip), a negative regulator of Toll-like receptor signaling, is known to be expressed at high levels in IECs, and to thereby contribute to the hyporesponsiveness of IECs to commensals. In this study, we analyzed the underlying mechanisms for elevated transcription of the Tollip gene in IECs using a human IEC line, Caco-2, and a human monocyte line, THP-1, as a control. Elf-1 was identified as a transcription factor that negatively regulates Tollip gene expression. The transcription factor Elf-1 was localized in the nucleus by O-linked N-acetylglucosamine (O-GlcNAc) modification, whereas the unmodified form was detected only in the cytoplasm. Comparison of Caco-2 and THP-1 cells revealed that O-GlcNAc modification of Elf-1 was significantly lower in IECs than in monocytes. Collectively, the results indicate that insufficient O-GlcNAc modification prevents Elf-1-mediated transcriptional repression and thereby upregulates Tollip gene expression in IECs.

  14. Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha

    PubMed Central

    Silvestrini, Lucia; Rossi, Beatrice; Gallmetzer, Andreas; Mathieu, Martine; Scazzocchio, Claudio; Berardi, Enrico; Strauss, Joseph

    2015-01-01

    A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems. PMID:26335797

  15. Transcription of the viral genome in cell lines transformed by simian virus 40. I. Mapping of virus-specific nuclear RNAs.

    PubMed Central

    Chumakov, P M

    1980-01-01

    Mapping of virus-specific nuclear transcripts was carried out in three lines of rat cells transformed by SV40. Each of these cell lines contained a single copy of integrated viral DNA with identified regions adjacent to cell DNA (1). The main virus-specific nuclear transcript in all of these cell lines was shown to be complementary to the minus strand of the early region in SV40 genome. Each cell nucleus contained approximately 50 copies of these RNAs. Transcripts complementary to both strands of the late region in viral genome were also detectable in all of these cell lines. Its content varied depending on the cell line and was 20-50-fold less than that of the main virus-specific transcript. All the regions of integrated SV40 genome in isolated nuclei of transformed cells were equally sensitive to pancreatic DNase I treatment suggesting that the whole viral genome served as a template for RNA synthesis in these cell lines. PMID:6243776

  16. The transcription factor-like nuclear regulator (TFNR) contains a novel 55-amino-acid motif repeated nine times and maps closely to SMN1.

    PubMed

    Kelter, A R; Herchenbach, J; Wirth, B

    2000-12-15

    The transcription factor-like nuclear regulator (TFNR) is a novel human gene that maps on 5q13, distal to the duplicated region that includes SMN1, the spinal muscular atrophy (SMA) determining gene. The location of TFNR allowed us to design an evolutionary model of the SMA region. The 9.5-kb TFNR transcript is highly expressed in cerebellum and weakly in all other tissues tested. TFNR encodes a protein of 2254 amino acids (aa) and contains nine repeats of a novel 55-aa motif, of yet unknown function. The coding region is organized in 32 exons. Alternative splicing of exon 15 results in a truncated protein of 796 aa. TFNR comprises a series of polypeptides that range from 55 to 250 kDa. Immunocytological studies showed that the TFNR protein is present exclusively in the nucleus, where it is concentrated in several nuclear structures. Amino acids 155-474 show significant homology to TFC5, a subunit of the yeast transcription factor TFIIIB, suggesting that TFNR is a putative transcription factor. Based on its proximity to SMN1 and its expression pattern, TFNR may be a candidate gene for atypical forms of SMA with cerebral atrophy and axonal neuropathy that have been shown to carry large deletions in the SMA region. PMID:11161782

  17. Structural protein 4.1R is integrally involved in nuclear envelope protein localization, centrosome–nucleus association and transcriptional signaling

    PubMed Central

    Meyer, Adam J.; Almendrala, Donna K.; Go, Minjoung M.; Krauss, Sharon Wald

    2011-01-01

    The multifunctional structural protein 4.1R is required for assembly and maintenance of functional nuclei but its nuclear roles are unidentified. 4.1R localizes within nuclei, at the nuclear envelope, and in cytoplasm. Here we show that 4.1R, the nuclear envelope protein emerin and the intermediate filament protein lamin A/C co-immunoprecipitate, and that 4.1R-specific depletion in human cells by RNA interference produces nuclear dysmorphology and selective mislocalization of proteins from several nuclear subcompartments. Such 4.1R-deficiency causes emerin to partially redistribute into the cytoplasm, whereas lamin A/C is disorganized at nuclear rims and displaced from nucleoplasmic foci. The nuclear envelope protein MAN1, nuclear pore proteins Tpr and Nup62, and nucleoplasmic proteins NuMA and LAP2α also have aberrant distributions, but lamin B and LAP2β have normal localizations. 4.1R-deficient mouse embryonic fibroblasts show a similar phenotype. We determined the functional effects of 4.1R-deficiency that reflect disruption of the association of 4.1R with emerin and A-type lamin: increased nucleus–centrosome distances, increased β-catenin signaling, and relocalization of β-catenin from the plasma membrane to the nucleus. Furthermore, emerin- and lamin-A/C-null cells have decreased nuclear 4.1R. Our data provide evidence that 4.1R has important functional interactions with emerin and A-type lamin that impact upon nuclear architecture, centrosome–nuclear envelope association and the regulation of β-catenin transcriptional co-activator activity that is dependent on β-catenin nuclear export. PMID:21486941

  18. Role of Calcineurin, hnRNPA2 and Akt in Mitochondrial Respiratory Stress-Mediated Transcription Activation of Nuclear Gene Targets

    PubMed Central

    Guha, Manti; Tang, Weigang; Sondheimer, Neal; Avadhani, Narayan G.

    2010-01-01

    Pathophysiological conditions causing mitochondrial dysfunction and altered transmembrane potential (Δψm) initiate a mitochondrial respiratory stress response, also known as mitochondrial retrograde response, in a variety of mammalian cells. An increase in the cytosolic Ca2+ [Ca2+]c as part of this signaling cascade activates Ca2+ responsive phosphatase, Calcineurin (Cn). Activation of IGF1R accompanied by increased glycolysis, invasiveness, and resistance to apoptosis are phenotypic hallmarks of C2C12 rhabdomyoblast cells subjected to this stress. The signaling is associated with activation and increased nuclear translocation of a number of transcription factors including a novel NFκB (cRel: p50) pathway, NFAT, CREB and C/EBPδ. This culminates in the upregulation of a number of nuclear genes including Cathepsin L, RyR1, Glut4 and Akt1. We observed that stress regulated transcription activation of nuclear genes involves a cooperative interplay between NFκB (cRel:p50), C/EBPδ, CREB, NFAT. Our results show that the functional synergy of these factors requires the stress-activated heterogeneous nuclear ribonucleoprotein, hnRNPA2 as a transcriptional co-activator. We report here that mitochondrial stress leads to induced expression and activation of serine threonine kinase Akt1. Interestingly, we observe that Akt1 phosphorylates hnRNPA2 under mitochondrial stress conditions, which is a crucial step for the recruitment of this coactivator to the stress target promoters and culmination in mitochondrial stress-mediated transcription activation of target genes. We propose that mitochondrial stress plays an important role in tumor progression and emergence of invasive phenotypes. PMID:20153290

  19. Induction of transcription within chromosomal DNA loops flanked by MAR elements causes an association of loop DNA with the nuclear matrix

    PubMed Central

    Iarovaia, Olga V.; Akopov, Sergey B.; Nikolaev, Lev G.; Sverdlov, Eugene D.; Razin, Sergey V.

    2005-01-01

    The spatial organization of an ∼170 kb region of human chromosome 19, including CD22 and GPR40–GPR43 genes, was studied using in situ hybridization of a set of cosmid and PAC probes with nuclear halos prepared from proliferating and differentiated HL60 cells. The whole region under study was found to be looped out into the nuclear halo in proliferating cells. It is likely that the loop observed was attached to the nuclear matrix via MAR elements present at the flanks of the area under study. Upon dimethyl sulfoxide-induced differentiation of the cells the looped fragment became associated with the nuclear matrix. This change in the spatial organization correlated with the activation of transcription of at least two (CD22 and GPR43) genes present within the loop. The data obtained are discussed in the framework of the hypothesis postulating that the spatial organization of chromosomal DNA is maintained via constitutive (basic) and facultative (transcription-related) interactions of the latter with the nuclear matrix. PMID:16049024

  20. Induction of transcription within chromosomal DNA loops flanked by MAR elements causes an association of loop DNA with the nuclear matrix.

    PubMed

    Iarovaia, Olga V; Akopov, Sergey B; Nikolaev, Lev G; Sverdlov, Eugene D; Razin, Sergey V

    2005-01-01

    The spatial organization of an approximately 170 kb region of human chromosome 19, including CD22 and GPR40-GPR43 genes, was studied using in situ hybridization of a set of cosmid and PAC probes with nuclear halos prepared from proliferating and differentiated HL60 cells. The whole region under study was found to be looped out into the nuclear halo in proliferating cells. It is likely that the loop observed was attached to the nuclear matrix via MAR elements present at the flanks of the area under study. Upon dimethyl sulfoxide-induced differentiation of the cells the looped fragment became associated with the nuclear matrix. This change in the spatial organization correlated with the activation of transcription of at least two (CD22 and GPR43) genes present within the loop. The data obtained are discussed in the framework of the hypothesis postulating that the spatial organization of chromosomal DNA is maintained via constitutive (basic) and facultative (transcription-related) interactions of the latter with the nuclear matrix. PMID:16049024

  1. The mitochondrial fatty acid synthesis (mtFASII) pathway is capable of mediating nuclear-mitochondrial cross talk through the PPAR system of transcriptional activation

    SciTech Connect

    Parl, Angelika; Mitchell, Sabrina L.; Clay, Hayley B.; Reiss, Sara; Li, Zhen; Murdock, Deborah G.

    2013-11-15

    Highlights: •The function of the mitochondria fatty acid synthesis pathway is partially unknown. •Overexpression of the pathway causes transcriptional activation through PPARs. •Knock down of the pathway attenuates that activation. •The last enzyme in the pathway regulates its own transcription. •Products of the mtFASII pathway are able to drive nuclear transcription. -- Abstract: Mammalian cells contain two fatty acid synthesis pathways, the cytosolic FASI pathway, and the mitochondrial FASII pathway. The selection behind the conservation of the mitochondrial pathway is not completely understood, given the presence of the cytosolic FAS pathway. In this study, we show through heterologous gene reporter systems and PCR-based arrays that overexpression of MECR, the last step in the mtFASII pathway, causes modulation of gene expression through the PPAR pathway. Electromobility shift assays (EMSAs) demonstrate that overexpression of MECR causes increased binding of PPARs to DNA, while cell fractionation and imaging studies show that MECR remains localized to the mitochondria. Interestingly, knock down of the mtFASII pathway lessens the effect of MECR on this transcriptional modulation. Our data are most consistent with MECR-mediated transcriptional activation through products of the mtFASII pathway, although we cannot rule out MECR acting as a coactivator. Further investigation into the physiological relevance of this communication will be necessary to better understand some of the phenotypic consequences of deficits in this pathway observed in animal models and human disease.

  2. Heat shock factor 1 upregulates transcription of Epstein-Barr Virus nuclear antigen 1 by binding to a heat shock element within the BamHI-Q promoter

    SciTech Connect

    Wang, Feng-Wei; Wu, Xian-Rui; Liu, Wen-Ju; Liao, Yi-Ji; Lin, Sheng; Zong, Yong-Sheng; Zeng, Mu-Sheng; Zeng, Yi-Xin; Mai, Shi-Juan; Xie, Dan

    2011-12-20

    Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for maintenance of the episome and establishment of latency. In this study, we observed that heat treatment effectively induced EBNA1 transcription in EBV-transformed B95-8 and human LCL cell lines. Although Cp is considered as the sole promoter used for the expression of EBNA1 transcripts in the lymphoblastoid cell lines, the RT-PCR results showed that the EBNA1 transcripts induced by heat treatment arise from Qp-initiated transcripts. Using bioinformatics, a high affinity and functional heat shock factor 1 (HSF1)-binding element within the - 17/+4 oligonucleotide of the Qp was found, and was determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Moreover, heat shock and exogenous HSF1 expression induced Qp activity in reporter assays. Further, RNA interference-mediated HSF1 gene silencing attenuated heat-induced EBNA1 expression in B95-8 cells. These results provide evidence that EBNA1 is a new target for the transcription factor HSF1.

  3. Interaction of Heat Shock Protein Cpn10 with the Cyclin E/Cdk2 Substrate Nuclear Protein Ataxia-Telangiectasia (NPAT) Is Involved in Regulating Histone Transcription*

    PubMed Central

    Ling Zheng, Li; Wang, Fei Ya; Cong, Xiao Xia; Shen, Yue; Rao, Xi Sheng; Huang, Dao Sheng; Fan, Wei; Yi, Peng; Wang, Xin Bao; Zheng, Lei; Zhou, Yi Ting; Luo, Yan

    2015-01-01

    Precise modulation of histone gene transcription is critical for cell cycle progression. As a direct substrate of Cyclin E/CDK2, nuclear protein ataxia-telangiectasia (NPAT) is a crucial factor in regulating histone transcription and cell cycle progression. Here we identified that Cpn10/HSPE, a 10-kDa heat shock protein, is a novel interacting partner of NPAT. A pool of Cpn10 is colocalized with NPAT foci during G1 and S phases in nuclei. Gain- and loss-of-function experiments unraveled an essential role of Cpn10 in histone transcription. A conserved DLFD motif within Cpn10 was critical for targeting NPAT and modulating histone transcription. More importantly, knockdown of Cpn10 disrupted the focus formation of both NPAT and FADD-like interleukin-1β-converting enzyme-associated huge protein without affecting Coilin-positive Cajal bodies. Finally, Cpn10 is important for S phase progression and cell proliferation. Taken together, our finding revealed a novel role of Cpn10 in the spatial regulation of NPAT signaling and disclosed a previously unappreciated link between the heat shock protein and histone transcription regulation. PMID:26429916

  4. Induction of megakaryocyte differentiation drives nuclear accumulation and transcriptional function of MKL1 via actin polymerization and RhoA activation

    PubMed Central

    Smith, Elenoe C.; Teixeira, Alexandra M.; Chen, Rachel C.; Wang, Lin; Gao, Yuan; Hahn, Katherine L.

    2013-01-01

    How components of the cytoskeleton regulate complex cellular responses is fundamental to understanding cellular function. Megakaryoblast leukemia 1 (MKL1), an activator of serum response factor (SRF) transcriptional activity, promotes muscle, neuron, and megakaryocyte differentiation. In muscle cells, where MKL1 subcellular localization is one mechanism by which cells control SRF activity, MKL1 translocation from the cytoplasm to the nucleus in response to actin polymerization is critical for its function as a transcriptional regulator. MKL1 localization is cell-type specific; it is predominantly cytoplasmic in unstimulated fibroblasts and some muscle cell types and is constitutively nuclear in neuronal cells. In the present study, we report that in megakaryocytes, subcellular localization and regulation of MKL1 is dependent on RhoA activity and actin organization. Induction of megakaryocytic differentiation of human erythroleukemia cells by 12-O-tetradecanoylphorbol-13-acetate and primary megakaryocytes by thrombopoietin promotes MKL1 nuclear localization. This MKL1 localization is blocked by drugs inhibiting RhoA activity or actin polymerization. We also show that nuclear-localized MKL1 activates the transcription of SRF target genes. This report broadens our knowledge of the molecular mechanisms regulating megakaryocyte differentiation. PMID:23243284

  5. BMAL1-dependent circadian oscillation of nuclear CLOCK: posttranslational events induced by dimerization of transcriptional activators of the mammalian clock system

    PubMed Central

    Kondratov, Roman V.; Chernov, Mikhail V.; Kondratova, Anna A.; Gorbacheva, Victoria Y.; Gudkov, Andrei V.; Antoch, Marina P.

    2003-01-01

    Mammalian CLOCK and BMAL1 are two members of bHLH-PAS-containing family of transcription factors that represent the positive elements of circadian autoregulatory feedback loop. In the form of a heterodimer, they drive transcription from E-box enhancer elements in the promoters of responsive genes. We have examined abundance, posttranslational modifications, cellular localization of endogenous and ectopically expressed CLOCK and BMAL1 proteins. Nuclear/cytoplasm distribution of CLOCK was found to be under circadian regulation. Analysis of subcellular localization of CLOCK in embryo fibroblasts of mice carrying different germ-line circadian mutations showed that circadian regulation of nuclear accumulation of CLOCK is BMAL1-dependent. Formation of CLOCK/BMAL1 complex following ectopic coexpression of both proteins is followed by their codependent phosphorylation, which is tightly coupled to CLOCK nuclear translocation and degradation. This binding-dependent coregulation is specific for CLOCK/BMAL1 interaction, as no other PAS domain protein that can form a complex with either CLOCK or BMAL1 was able to induce similar effects. Importantly, all posttranslational events described in our study are coupled with active transactivation complex formation, which argues for their significant functional role. Altogether, these results provide evidence for an additional level of circadian system control, which is based on regulation of transcriptional activity or/and availability of CLOCK/BMAL1 complex. PMID:12897057

  6. A Phylogenetically Conserved Group of Nuclear Factor-Y Transcription Factors Interact to Control Nodulation in Legumes1[OPEN

    PubMed Central

    Laloum, Tom; Lepage, Agnès; Ariel, Federico; Frances, Lisa; Gamas, Pascal; de Carvalho-Niebel, Fernanda

    2015-01-01

    The endosymbiotic association between legumes and soil bacteria called rhizobia leads to the formation of a new root-derived organ called the nodule in which differentiated bacteria convert atmospheric nitrogen into a form that can be assimilated by the host plant. Successful root infection by rhizobia and nodule organogenesis require the activation of symbiotic genes that are controlled by a set of transcription factors (TFs). We recently identified Medicago truncatula nuclear factor-YA1 (MtNF-YA1) and MtNF-YA2 as two M. truncatula TFs playing a central role during key steps of the Sinorhizobium meliloti-M. truncatula symbiotic interaction. NF-YA TFs interact with NF-YB and NF-YC subunits to regulate target genes containing the CCAAT box consensus sequence. In this study, using a yeast two-hybrid screen approach, we identified the NF-YB and NF-YC subunits able to interact with MtNF-YA1 and MtNF-YA2. In yeast (Saccharomyces cerevisiae) and in planta, we further demonstrated by both coimmunoprecipitation and bimolecular fluorescence complementation that these NF-YA, -B, and -C subunits interact and form a stable NF-Y heterotrimeric complex. Reverse genetic and chromatin immunoprecipitation-PCR approaches revealed the importance of these newly identified NF-YB and NF-YC subunits for rhizobial symbiosis and binding to the promoter of MtERN1 (for Ethylene Responsive factor required for Nodulation), a direct target gene of MtNF-YA1 and MtNF-YA2. Finally, we verified that a similar trimer is formed in planta by the common bean (Phaseolus vulgaris) NF-Y subunits, revealing the existence of evolutionary conserved NF-Y protein complexes to control nodulation in leguminous plants. This sheds light on the process whereby an ancient heterotrimeric TF mainly controlling cell division in animals has acquired specialized functions in plants. PMID:26432878

  7. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans

    PubMed Central

    Ahn, Jeong H.; Rechsteiner, Andreas; Strome, Susan; Kelly, William G.

    2016-01-01

    The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

  8. A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans.

    PubMed

    Ahn, Jeong H; Rechsteiner, Andreas; Strome, Susan; Kelly, William G

    2016-08-01

    The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3' end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

  9. Nuclear Localization of the Autism Candidate Gene Neurobeachin and Functional Interaction with the NOTCH1 Intracellular Domain Indicate a Role in Regulating Transcription

    PubMed Central

    Tuand, Krizia; Stijnen, Pieter; Volders, Karolien; Declercq, Jeroen; Nuytens, Kim; Meulemans, Sandra; Creemers, John

    2016-01-01

    Background Neurobeachin (NBEA) is an autism spectrum disorders (ASD) candidate gene. NBEA deficiency affects regulated secretion, receptor trafficking, synaptic architecture and protein kinase A (PKA)-mediated phosphorylation. NBEA is a large multidomain scaffolding protein. From N- to C-terminus, NBEA has a concanavalin A-like lectin domain flanked by armadillo repeats (ACA), an A-kinase anchoring protein domain that can bind to PKA, a domain of unknown function (DUF1088) and a BEACH domain, preceded by a pleckstrin homology-like domain and followed by WD40 repeats (PBW). Although most of these domains mediate protein-protein interactions, no interaction screen has yet been performed. Methods Yeast two-hybrid screens with the ACA and PBW domain modules of NBEA gave a list of interaction partners, which were analyzed for Gene Ontology (GO) enrichment. Neuro-2a cells were used for confocal microscopy and nuclear extraction analysis. NOTCH-mediated transcription was studied with luciferase reporter assays and qRT-PCR, combined with NBEA knockdown or overexpression. Results Both domain modules showed a GO enrichment for the nucleus. PBW almost exclusively interacted with transcription regulators, while ACA interacted with a number of PKA substrates. NBEA was partially localized in the nucleus of Neuro-2a cells, albeit much less than in the cytoplasm. A nuclear localization signal was found in the DUF1088 domain, which was shown to contribute to the nuclear localization of an EGFP-DPBW fusion protein. Yeast two-hybrid identified the Notch1 intracellular domain as a physical interactor of the PBW domain and a role for NBEA as a negative regulator in Notch-mediated transcription was demonstrated. Conclusion Defining novel interaction partners of conserved NBEA domain modules identified a role for NBEA as transcriptional regulator in the nucleus. The physical interaction of NBEA with NOTCH1 is most relevant for ASD pathogenesis because NOTCH signaling is essential for

  10. Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells

    PubMed Central

    Bach, Anne-Sophie; Derocq, Danielle; Laurent-Matha, Valérie; Montcourrier, Philippe; Salwa Sebti, Salwa; Orsetti, Béatrice; Theillet, Charles; Gongora, Céline; Pattingre, Sophie; Ibing, Eva; Roger, Pascal; Linares, Laetitia K.; Reinheckel, Thomas; Meurice, Guillaume; Kaiser, Frank J.; Gespach, Christian; Liaudet-Coopman, Emmanuelle

    2015-01-01

    The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner. PMID:26183398

  11. Nuclear cathepsin D enhances TRPS1 transcriptional repressor function to regulate cell cycle progression and transformation in human breast cancer cells.

    PubMed

    Bach, Anne-Sophie; Derocq, Danielle; Laurent-Matha, Valérie; Montcourrier, Philippe; Sebti, Salwa; Orsetti, Béatrice; Theillet, Charles; Gongora, Céline; Pattingre, Sophie; Ibing, Eva; Roger, Pascal; Linares, Laetitia K; Reinheckel, Thomas; Meurice, Guillaume; Kaiser, Frank J; Gespach, Christian; Liaudet-Coopman, Emmanuelle

    2015-09-29

    The lysosomal protease cathepsin D (Cath-D) is overproduced in breast cancer cells (BCC) and supports tumor growth and metastasis formation. Here, we describe the mechanism whereby Cath-D is accumulated in the nucleus of ERα-positive (ER+) BCC. We identified TRPS1 (tricho-rhino-phalangeal-syndrome 1), a repressor of GATA-mediated transcription, and BAT3 (Scythe/BAG6), a nucleo-cytoplasmic shuttling chaperone protein, as new Cath-D-interacting nuclear proteins. Cath-D binds to BAT3 in ER+ BCC and they partially co-localize at the surface of lysosomes and in the nucleus. BAT3 silencing inhibits Cath-D accumulation in the nucleus, indicating that Cath-D nuclear targeting is controlled by BAT3. Fully mature Cath-D also binds to full-length TRPS1 and they co-localize in the nucleus of ER+ BCC where they are associated with chromatin. Using the LexA-VP16 fusion co-activator reporter assay, we then show that Cath-D acts as a transcriptional repressor, independently of its catalytic activity. Moreover, microarray analysis of BCC in which Cath-D and/or TRPS1 expression were silenced indicated that Cath-D enhances TRPS1-mediated repression of several TRPS1-regulated genes implicated in carcinogenesis, including PTHrP, a canonical TRPS1 gene target. In addition, co-silencing of TRPS1 and Cath-D in BCC affects the transcription of cell cycle, proliferation and transformation genes, and impairs cell cycle progression and soft agar colony formation. These findings indicate that Cath-D acts as a nuclear transcriptional cofactor of TRPS1 to regulate ER+ BCC proliferation and transformation in a non-proteolytic manner. PMID:26183398

  12. {beta}-Catenin can act as a nuclear import receptor for its partner transcription factor, lymphocyte enhancer factor-1 (lef-1)

    SciTech Connect

    Asally, Munehiro; Yoneda, Yoshihiro . E-mail: yyoneda@anat3.med.osaka-u.ac.jp

    2005-08-15

    Nuclear accumulation of {beta}-catenin plays an important role in the Wnt signaling pathway. In the nucleus, {beta}-catenin acts as a transcriptional co-activator for TCF/LEF family of transcription factors. It has been shown that lef-1 contains a typical basic type nuclear localization signal (NLS) and is transported into the nucleus by the conventional import pathway. In this study, we found that a mutant lef-1 lacking the classical NLS accumulated in the nucleus of living cells, when {beta}-catenin was co-expressed. In addition, in a cell-free import assay, lef-1 migrated into the nucleus in the presence of {beta}-catenin alone without any other soluble factors. In contrast, another mutant lef-1 lacking the {beta}-catenin binding domain failed to migrate into the nucleus, even in the presence of {beta}-catenin. These findings indicate that {beta}-catenin alone can mediate the nuclear import of lef-1 through the direct binding. Collectively, we propose that there are two distinct pathways for the nuclear import of lef-1: importin {alpha}/{beta}-mediated and {beta}-catenin-mediated one, which provides a novel paradigm for Wnt signaling pathway.

  13. Nuclear respiratory factor 2 induces the expression of many but not all human proteins acting in mitochondrial DNA transcription and replication.

    PubMed

    Bruni, Francesco; Polosa, Paola Loguercio; Gadaleta, Maria Nicola; Cantatore, Palmiro; Roberti, Marina

    2010-02-01

    In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-gamma, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-gamma and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins. PMID:19951946

  14. The relationship between transcript expression levels of nuclear encoded (TFAM, NRF1) and mitochondrial encoded (MT-CO1) genes in single human oocytes during oocyte maturation

    PubMed Central

    Novin, M Ghaffari; Allahveisi, A; Noruzinia, M; Farhadifar, F; Yousefian, E; Fard, A Dehghani; Salimi, M

    2015-01-01

    In some cases of infertility in women, human oocytes fail to mature when they reach the metaphase II (MII) stage. Mitochondria plays an important role in oocyte maturation. A large number of mitochondrial DNA (mtDNA), copied in oocytes, is essential for providing adenosine triphosphate (ATP) during oocyte maturation. The purpose of this study was to identify the relationship between transcript expression levels of the mitochondrial encoded gene (MT-CO1) and two nuclear encoded genes, nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) in various stages of human oocyte maturation. Nine consenting patients, age 21–35 years old, with male factors were selected for ovarian stimulation and intracytoplasmic sperm injection (ICSI) procedures. mRNA levels of mitochondrial-related genes were performed by singlecell TaqMan® quantitative real-time polymerase chain reaction (qRT-PCR). There was no significant relationship between the relative expression levels in germinal vesicle (GV) stage oocytes (p = 0.62). On the contrary, a significant relationship was seen between the relative expression levels of TFAM and NRF1 and the MT-CO1 genes at the stages of metaphase I (MI) and MII (p = 0.03 and p = 0.002). A relationship exists between the transcript expression levels of TFAM and NRF1, and MT-CO1 genes in various stages of human oocyte maturation. PMID:26929904

  15. Testicular Nuclear Receptor 4 (TR4) Regulates UV Light-induced Responses via Cockayne Syndrome B Protein-mediated Transcription-coupled DNA Repair*

    PubMed Central

    Liu, Su; Yan, Shian-Jang; Lee, Yi-Fen; Liu, Ning-Chun; Ting, Huei-Ju; Li, Gonghui; Wu, Qiao; Chen, Lu-Min; Chang, Chawnshang

    2011-01-01

    UV irradiation is one of the major external insults to cells and can cause skin aging and cancer. In response to UV light-induced DNA damage, the nucleotide excision repair (NER) pathways are activated to remove DNA lesions. We report here that testicular nuclear receptor 4 (TR4), a member of the nuclear receptor family, modulates DNA repair specifically through the transcription-coupled (TC) NER pathway but not the global genomic NER pathway. The level of Cockayne syndrome B protein (CSB), a member of the TC-NER pathway, is 10-fold reduced in TR4-deficient mouse tissues, and TR4 directly regulates CSB at the transcriptional level. Moreover, restored CSB expression rescues UV hypersensitivity of TR4-deficient cells. Together, these results indicate that TR4 modulates UV sensitivity by promoting the TC-NER DNA repair pathway through transcriptional regulation of CSB. These results may lead to the development of new treatments for UV light-sensitive syndromes, skin cancer, and aging. PMID:21918225

  16. Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene.

    PubMed Central

    Wu, K L; Gannon, M; Peshavaria, M; Offield, M F; Henderson, E; Ray, M; Marks, A; Gamer, L W; Wright, C V; Stein, R

    1997-01-01

    The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdr-1 is expressed principally within insulin-secreting pancreatic islet beta cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb -4.5 to +8.2 was used to drive a beta-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp -2560 and -1880 (site 1), bp -1330 and -800 (site 2), and bp -260 and +180 (site 3), were identified within the 5'-flanking region of the endogenous pdx-1 gene. Pancreatic beta-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdr-1-herpes simplex virus thymidine kinase promoter expression constructs in transfected beta-cell and non-beta-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven beta-galactosidase reporter construct was directed to islet beta-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in beta-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides -2007 and -1996. Gel shift analysis indicated that HNF3beta present in islet beta cells binds to this element. Immunohistochemical studies revealed that HNF3beta was present within the nuclei of almost all islet beta cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3beta, a key regulator of endodermal cell lineage

  17. Transcription factor (TF)-like nuclear regulator, the 250-kDa form of Homo sapiens TFIIIB", is an essential component of human TFIIIC1 activity.

    PubMed

    Weser, Stephan; Gruber, Christin; Hafner, Heike M; Teichmann, Martin; Roeder, Robert G; Seifart, Klaus H; Meissner, Wolfgang

    2004-06-25

    The general human RNA polymerase III transcription factor (TF) IIIC1 has hitherto been ill defined with respect to the polypeptides required for reconstitution of its activity. Here we identify Homo sapiens TFIIIB" (HsBdp1) as an essential component of hTFIIIC1 and hTFIIIC1-like activities. Several forms of HsBdp1 are described. The 250-kDa form of HsBdp1, also designated the "transcription factor-like nuclear regulator," strictly co-eluted with TFIIIC1 activity over multiple chromatographic purification steps as revealed by Western blot with anti-HsBdp1 antibodies and by MALDI-TOF analysis. In addition, TFIIIC1 activity could be depleted from partially purified fractions with anti-HsBdp1 antibodies but not with control antibodies. Moreover, highly purified recombinant HsBdp1 could replace TFIIIC1 activity in reconstituted transcription of the VAI gene in vitro. Furthermore, smaller proteins of approximately 90-150 kDa that were recognized by anti-HsBdp1 antibodies co-eluted with TFIIIC1-like activity. Finally, cytoplasmic extracts from differentiated mouse F9 fibroblast cells that lacked TFIIIC1 activity could be made competent for transcription of the VA1 gene by the addition of TFIIIC1, TFIIIC1-like, or recombinant HsBdp1. These results suggest that HsBdp1 proteins represent essential components of TFIIIC1 and TFIIIC1-like activities. PMID:15096501

  18. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells.

    PubMed

    Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. PMID:23948303

  19. Evidence for the Involvement of Xenobiotic-responsive Nuclear Receptors in Transcriptional Effects Upon Perfluoroalkyl Acid Exposure in Diverse Species.

    EPA Science Inventory

    Humans and other species have detectable body burdens of a number of perfluorinated alkyl acids (PFAA) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). In mouse and rat liver these compounds elicit transcriptional and phenotypic effects similar to pe...

  20. Evidence for the Involvement of Xenobiotic-response Nuclear Receptors in Transcriptional Effects Upon Perfluroroalkyl Acid Exposure in Diverse Species

    EPA Science Inventory

    Humans and ecological species have been found to have detectable body burdens of a number of perfluorinated alkyl acids (PFAA) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). In mouse and rat liver these compounds elicit transcriptional and phenotyp...

  1. STAT6 Transcription Factor Is a Facilitator of the Nuclear Receptor PPARγ-Regulated Gene Expression in Macrophages and Dendritic Cells

    PubMed Central

    Szanto, Attila; Balint, Balint L.; Nagy, Zsuzsanna S.; Barta, Endre; Dezso, Balazs; Pap, Attila; Szeles, Lajos; Poliska, Szilard; Oros, Melinda; Evans, Ronald M.; Barak, Yaacov; Schwabe, John; Nagy, Laszlo

    2010-01-01

    Summary Peroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We demonstrate here a mechanism how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and signal transducer and activators of transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. Thus, STAT6 acts as a facilitating factor for PPARγ by promoting DNA binding and consequently increasing the number of regulated genes and the magnitude of responses. This interaction, underpinning cell type-specific responses, represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells. PMID:21093321

  2. Poly(ADP-ribose) Polymerase 1 Interacts with Nuclear Respiratory Factor 1 (NRF-1) and Plays a Role in NRF-1 Transcriptional Regulation*S⃞

    PubMed Central

    Hossain, Mohammad B.; Ji, Ping; Anish, Ramakrishnan; Jacobson, Raymond H.; Takada, Shinako

    2009-01-01

    Nuclear respiratory factor 1 (NRF-1) is one of the key transcriptional activators for nuclear-coded genes involved in mitochondrial biogenesis and function as well as for many housekeeping genes. A transcriptional co-activator PGC-1 and its related family member PRC have previously been shown to interact with NRF-1 and co-activate NRF-1. We show here that NRF-1 can also directly interact with poly(ADP-ribose) polymerase 1 (PARP-1) and co-purify the PARP-1·DNA-PK·Ku80·Ku70·topoisomerase IIβ-containing protein complex. Our in vitro binding experiments show that DNA-binding/dimerization domain of NRF-1 and the N-terminal half of PARP-1, which contains two Zinc fingers and the auto-modification domain, are responsible for the interaction, and that this interaction occurs with or without PARP-1 poly(ADP-ribosyl)ation (PARylation). DNA-bound NRF-1 can form a complex with PARP-1, suggesting that NRF-1 can recruit the PARP-1·DNA-PK·Ku80·Ku70·topoisomerase IIβ-containing protein complex to the promoter. PARP-1 can also PARylate the DNA-binding domain of NRF-1 and negatively regulate NRF-1·PARP-1 interaction. Transient transfection and chromatin immunoprecipitation experiments suggest that PARP-1 plays a role during transcriptional activation by NRF-1. Our finding identifies a new aspect of transcriptional regulation used by NRF-1. PMID:19181665

  3. Orphan nuclear receptor Errγ induces C-reactive protein gene expression through induction of ER-bound Bzip transmembrane transcription factor CREBH.

    PubMed

    Misra, Jagannath; Chanda, Dipanjan; Kim, Don-Kyu; Cho, Seung-Rye; Koo, Seung-Hoi; Lee, Chul-Ho; Back, Sung Hoon; Choi, Hueng-Sik

    2014-01-01

    The orphan nuclear receptor estrogen-related receptor-γ (ERRγ) is a constitutively active transcription factor regulating genes involved in several important cellular processes, including hepatic glucose metabolism, alcohol metabolism, and the endoplasmic reticulum (ER) stress response. cAMP responsive element-binding protein H (CREBH) is an ER-bound bZIP family transcription factor that is activated upon ER stress and regulates genes encoding acute-phase proteins whose expression is increased in response to inflammation. Here, we report that ERRγ directly regulates CREBH gene expression in response to ER stress. ERRγ bound to the ERRγ response element (ERRE) in the CREBH promoter. Overexpression of ERRγ by adenovirus significantly increased expression of CREBH as well as C-reactive protein (CRP), whereas either knockdown of ERRγ or inhibition of ERRγ by ERRγ specific inverse agonist, GSK5182, substantially inhibited ER stress-mediated induction of CREBH and CRP. The transcriptional coactivator PGC1α was required for ERRγ mediated induction of the CREBH gene as demonstrated by the chromatin immunoprecipitation (ChIP) assay showing binding of both ERRγ and PGC1α on the CREBH promoter. The ChIP assay also revealed that histone H3 and H4 acetylation occurred at the ERRγ and PGC1α binding site. Moreover, chronic alcoholic hepatosteatosis, as well as the diabetic obese condition significantly increased CRP gene expression, and this increase was significantly attenuated by GSK5182 treatment. We suggest that orphan nuclear receptor ERRγ directly regulates the ER-bound transcription factor CREBH in response to ER stress and other metabolic conditions. PMID:24466039

  4. Orphan Nuclear Receptor Errγ Induces C-Reactive Protein Gene Expression through Induction of ER-Bound Bzip Transmembrane Transcription Factor CREBH

    PubMed Central

    Misra, Jagannath; Chanda, Dipanjan; Kim, Don-Kyu; Cho, Seung-Rye; Koo, Seung-Hoi; Lee, Chul-Ho; Back, Sung Hoon; Choi, Hueng-Sik

    2014-01-01

    The orphan nuclear receptor estrogen-related receptor-γ (ERRγ) is a constitutively active transcription factor regulating genes involved in several important cellular processes, including hepatic glucose metabolism, alcohol metabolism, and the endoplasmic reticulum (ER) stress response. cAMP responsive element-binding protein H (CREBH) is an ER-bound bZIP family transcription factor that is activated upon ER stress and regulates genes encoding acute-phase proteins whose expression is increased in response to inflammation. Here, we report that ERRγ directly regulates CREBH gene expression in response to ER stress. ERRγ bound to the ERRγ response element (ERRE) in the CREBH promoter. Overexpression of ERRγ by adenovirus significantly increased expression of CREBH as well as C-reactive protein (CRP), whereas either knockdown of ERRγ or inhibition of ERRγ by ERRγ specific inverse agonist, GSK5182, substantially inhibited ER stress-mediated induction of CREBH and CRP. The transcriptional coactivator PGC1α was required for ERRγ mediated induction of the CREBH gene as demonstrated by the chromatin immunoprecipitation (ChIP) assay showing binding of both ERRγ and PGC1α on the CREBH promoter. The ChIP assay also revealed that histone H3 and H4 acetylation occurred at the ERRγ and PGC1α binding site. Moreover, chronic alcoholic hepatosteatosis, as well as the diabetic obese condition significantly increased CRP gene expression, and this increase was significantly attenuated by GSK5182 treatment. We suggest that orphan nuclear receptor ERRγ directly regulates the ER-bound transcription factor CREBH in response to ER stress and other metabolic conditions. PMID:24466039

  5. Src Subfamily Kinases Regulate Nuclear Export and Degradation of Transcription Factor Nrf2 to Switch Off Nrf2-mediated Antioxidant Activation of Cytoprotective Gene Expression*

    PubMed Central

    Niture, Suryakant K.; Jain, Abhinav K.; Shelton, Phillip M.; Jaiswal, Anil K.

    2011-01-01

    Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3β acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis. PMID:21690096

  6. Polymorphisms at positions -22 and -348 in the promoter of the BAT1 gene affect transcription and the binding of nuclear factors.

    PubMed

    Price, Patricia; Wong, Agnes M-L; Williamson, David; Voon, Dominic; Baltic, Svetlana; Allcock, Richard J N; Boodhoo, Alvin; Christiansen, Frank T

    2004-05-01

    BAT1 (D6S81E, UAP56) lies in the central MHC between TNF and HLA-B, a region containing genes that affect susceptibility to immunopathologic disorders. BAT1 protein may be directly responsible for the genetic association, as antisense studies show it can down-regulate inflammatory cytokines. Here we investigate polymorphisms at positions -22 and -348 relative to the BAT1 transcription start site. DNA samples from healthy donors were used to confirm haplotypic associations with the type 1 diabetes-susceptible 8.1 ancestral haplotype (AH; HLA-A1,B8,BAT1-22*C,BAT1-348*C,DR3 ) and the diabetes-resistant 7.1 AH (HLA-A3,B7,BAT1-22*G,BAT1-348*T,DR15). Alleles carried at BAT1-22 and -348 were in linkage disequilibrium. Electrophoretic mobility shift assays using nuclear proteins from T-cells (Jurkat and HT2), monocytes (THP1, U937) and epithelial cells (HeLa and MDA468) demonstrated DNA : protein complexes binding oligonucleotides spanning positions -22 and -348 on the 7.1 AH only. Competition assays, supershifts and molecular weight determinations suggest the complexes include the transcription factors YY1 (at -348) and Oct1 (at -22). Promoter activity was demonstrated using 520 bp and 336 bp fragments cloned from immediately upstream of the transcription start site and carrying all combinations of -22 and -348 alleles, suggesting an unidentified non-polymorphic sequence within 336 bp of the start site drives transcription. The 520 bp fragment of the BAT1 promoter cloned from the 8.1 AH was slightly less efficient than the equivalent from the 7.1 AH, whilst the reverse was observed with 336 bp fragments. This suggests BAT1 transcription on the 7.1 AH is modified by interactions involving DNA flanking positions -22 and -348. PMID:15028669

  7. Primary oocyte transcriptional activation of aqp1ab by the nuclear progestin receptor determines the pelagic egg phenotype of marine teleosts.

    PubMed

    Zapater, Cinta; Chauvigné, François; Tingaud-Sequeira, Angèle; Finn, Roderick Nigel; Cerdà, Joan

    2013-05-15

    In marine teleosts, the aqp1ab water channel plays a vital role in the development of the pelagic egg phenotype. However, the developmental control of aqp1ab activation during oogenesis remains to be established. Here, we report the isolation of the 5'-flanking region of the teleost gilthead seabream aqp1ab gene, in which we identify conserved cis-regulatory elements for the binding of the nuclear progestin receptor (Pgr) and members of the Sox family of transcription factors. Subcellular localization studies indicated that the Pgr, as well as sox3 and -8b transcripts, are co-expressed in seabream oogonia, whereas in meiosis-arrested primary growth (pre-vitellogenic) oocytes, when aqp1ab mRNA and protein are first synthesized, the Pgr appears to be completely translocated from the ooplasm into the nucleus. By contrast, sox9b is highly expressed in more advanced oocytes, coinciding with a strong depletion of aqp1ab transcripts in the oocyte. Functional characterization of wild-type and mutated aqp1ab promoter constructs, using mammalian cells and Xenopus laevis oocytes, demonstrated that aqp1ab transcription is initiated by the Pgr, which is activated by the progestin 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P), the natural ligand of the seabream Pgr. In vitro incubation of seabream primary ovarian explants with the follicle-stimulating hormone or 17,20β-P confirmed that progestin-activated Pgr enhanced Aqp1ab synthesis via the aqp1ab promoter. However, transactivation assays in heterologous systems showed that Sox transcription factors can potentially modulate this mechanism. These data uncover the existence of an endocrine pathway involved in the early activation of a water channel necessary for egg formation in marine teleosts. PMID:23499660

  8. Human THAP7 is a chromatin-associated, histone tail-binding protein that represses transcription via recruitment of HDAC3 and nuclear hormone receptor corepressor.

    PubMed

    Macfarlan, Todd; Kutney, Sara; Altman, Brian; Montross, Rebecca; Yu, Jiujiu; Chakravarti, Debabrata

    2005-02-25

    The identities of signal transducer proteins that integrate histone hypoacetylation and transcriptional repression are largely unknown. Here we demonstrate that THAP7, an uncharacterized member of the recently identified THAP (Thanatos-associated protein) family of proteins, is ubiquitously expressed, associates with chromatin, and represses transcription. THAP7 binds preferentially to hypoacetylated (un-, mono-, and diacetylated) histone H4 tails in vitro via its C-terminal 77 amino acids. Deletion of this domain, or treatment of cells with the histone deacetylase inhibitor TSA, which leads to histone hyperacetylation, partially disrupts THAP7/chromatin association in living cells. THAP7 coimmunoprecipitates with histone deacetylase 3 (HDAC3) and the nuclear hormone receptor corepressor (NCoR) and represses transcription as a Gal4 fusion protein. Chromatin immunoprecipitation assays demonstrate that these corepressors are recruited to promoters in a THAP7 dependent manner and promote histone H3 hypoacetylation. The conserved THAP domain is a key determinant for full HDAC3 association in vitro, and both the THAP domain and the histone interaction domain are important for the repressive properties of THAP7. Full repression mediated by THAP7 is also dependent on NCoR expression. We hypothesize that THAP7 is a dual function repressor protein that actively targets deacetylation of histone H3 necessary to establish transcriptional repression and functions as a signal transducer of the repressive mark of hypoacetylated histone H4. This is the first demonstration of the transcriptional regulatory properties of a human THAP domain protein, and a critical identification of a potential transducer of the repressive signal of hypoacetylated histone H4 in higher eukaryotes. PMID:15561719

  9. Hepatocyte Nuclear Factor 4α (HNF4α) Is a Transcription Factor of Vertebrate Fatty Acyl Desaturase Gene as Identified in Marine Teleost Siganus canaliculatus

    PubMed Central

    Dong, Yewei; Wang, Shuqi; Chen, Junliang; Zhang, Qinghao; Liu, Yang; You, Cuihong; Monroig, Óscar; Tocher, Douglas R.; Li, Yuanyou

    2016-01-01

    Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis. PMID:27472219

  10. Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element.

    PubMed

    Lim, Chun Shen; Brown, Chris M

    2016-09-01

    Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel. PMID:27031749

  11. Rho-kinase signaling controls nucleocytoplasmic shuttling of class IIa Histone Deacetylase (HDAC7) and transcriptional activation of orphan nuclear receptor NR4A1

    SciTech Connect

    Compagnucci, Claudia; Barresi, Sabina; Petrini, Stefania; Bertini, Enrico; Zanni, Ginevra

    2015-04-03

    Rho-kinase (ROCK) has been well documented to play a key role in RhoA-induced actin remodeling. ROCK activation results in myosin light chain (MLC) phosphorylation either by direct action on MLC kinase (MLCK) or by inhibition of MLC phosphatase (MLCP), modulating actin–myosin contraction. We found that inhibition of the ROCK pathway in induced pluripotent stem cells, leads to nuclear export of HDAC7 and transcriptional activation of the orphan nuclear receptor NR4A1 while in cells with constitutive ROCK hyperactivity due to loss of function of the RhoGTPase activating protein Oligophrenin-1 (OPHN1), the orphan nuclear receptor NR4A1 is downregulated. Our study identify a new target of ROCK signaling via myosin phosphatase subunit (MYPT1) and Histone Deacetylase (HDAC7) at the nuclear level and provide new insights in the cellular functions of ROCK. - Highlights: • ROCK regulates nucleocytoplasmic shuttling of HDAC7 via phosphorylation of MYPT1. • Nuclear export of HDAC7 and upregulation of NR4A1 occurs with low ROCK activity. • High levels of ROCK activity due to OPHN1 loss of function downregulate NR4A1.

  12. Alu sequences transcription in X. laevis oocytes: nuclear-cytoplasmic partitioning and evidence for 3' end processing reactions.

    PubMed Central

    Perlino, E; Paonessa, G; Ciliberto, G

    1985-01-01

    A large Alu-family cluster in the 5' flanking region of a human alpha 1-acid glycoprotein gene has been identified and sequenced. Individual members microinjected into X. laevis oocytes are transcribed only when canonical box A and B components of the split pol III promoter are present. Alu transcripts accumulate in the nucleus. An unusually short Alu transcript, able to assume a stable secondary structure, undergoes a 3' end processing reaction similar to the one required for tRNA 3' end maturation. Images PMID:4080545

  13. AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

    SciTech Connect

    Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

    2013-10-15

    The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.

  14. Nuclear respiratory factor 1 controls myocyte enhancer factor 2A transcription to provide a mechanism for coordinate expression of respiratory chain subunits.

    PubMed

    Ramachandran, Bindu; Yu, Gengsheng; Gulick, Tod

    2008-05-01

    Nuclear respiratory factors NRF1 and NRF2 regulate the expression of nuclear genes encoding heme biosynthetic enzymes, proteins required for mitochondrial genome transcription and protein import, and numerous respiratory chain subunits. NRFs thereby coordinate the expression of nuclear and mitochondrial genes relevant to mitochondrial biogenesis and respiration. Only two of the nuclear-encoded respiratory chain subunits have evolutionarily conserved tissue-specific forms: the cytochrome c oxidase (COX) subunits VIa and VIIa heart/muscle (H) and ubiquitous (L) isoforms. We used genome comparisons to conclude that the promoter regions of COX6A(H) and COX7A(H) lack NRF sites but have conserved myocyte enhancer factor 2 (MEF2) elements. We show that MEF2A mRNA is induced with forced expression of NRF1 and that the MEF2A 5'-regulatory region contains an evolutionarily conserved canonical element that binds endogenous NRF1 in chromatin immunoprecipitation (ChIP) assays. NRF1 regulates MEF2A promoter-reporters according to overexpression, RNA interference underexpression, and promoter element mutation studies. As there are four mammalian MEF2 isotypes, we used an isoform-specific antibody in ChIP to confirm MEF2A binding to the COX6A(H) promoter. These findings support a role for MEF2A as an intermediary in coordinating respiratory chain subunit expression in heart and muscle through a NRF1 --> MEF2A --> COX(H) transcriptional cascade. MEF2A also bound the MEF2A and PPARGC1A promoters in ChIP, placing it within a feedback loop with PGC1alpha in controlling NRF1 activity. Interruption of this cascade and loop may account for striated muscle mitochondrial defects in mef2a null mice. Our findings also account for the previously described indirect regulation by NRF1 of other MEF2 targets in muscle such as GLUT4. PMID:18222924

  15. Nuclear Respiratory Factor 1 Controls Myocyte Enhancer Factor 2A Transcription to Provide a Mechanism for Coordinate Expression of Respiratory Chain Subunits*S⃞

    PubMed Central

    Ramachandran, Bindu; Yu, Gengsheng; Gulick, Tod

    2008-01-01

    Nuclear respiratory factors NRF1 and NRF2 regulate the expression of nuclear genes encoding heme biosynthetic enzymes, proteins required for mitochondrial genome transcription and protein import, and numerous respiratory chain subunits. NRFs thereby coordinate the expression of nuclear and mitochondrial genes relevant to mitochondrial biogenesis and respiration. Only two of the nuclear-encoded respiratory chain subunits have evolutionarily conserved tissue-specific forms: the cytochrome c oxidase (COX) subunits VIa and VIIa heart/muscle (H) and ubiquitous (L) isoforms. We used genome comparisons to conclude that the promoter regions of COX6AH and COX7AH lack NRF sites but have conserved myocyte enhancer factor 2 (MEF2) elements. We show that MEF2A mRNA is induced with forced expression of NRF1 and that the MEF2A 5′-regulatory region contains an evolutionarily conserved canonical element that binds endogenous NRF1 in chromatin immunoprecipitation (ChIP) assays. NRF1 regulates MEF2A promoter-reporters according to overexpression, RNA interference underexpression, and promoter element mutation studies. As there are four mammalian MEF2 isotypes, we used an isoform-specific antibody in ChIP to confirm MEF2A binding to the COX6AH promoter. These findings support a role for MEF2A as an intermediary in coordinating respiratory chain subunit expression in heart and muscle through a NRF1 → MEF2A → COXH transcriptional cascade. MEF2A also bound the MEF2A and PPARGC1A promoters in ChIP, placing it within a feedback loop with PGC1α in controlling NRF1 activity. Interruption of this cascade and loop may account for striated muscle mitochondrial defects in mef2a null mice. Our findings also account for the previously described indirect regulation by NRF1 of other MEF2 targets in muscle such as GLUT4. PMID:18222924

  16. IL6 Inhibits HBV Transcription by Targeting the Epigenetic Control of the Nuclear cccDNA Minichromosome

    PubMed Central

    Palumbo, Gianna Aurora; Scisciani, Cecilia; Pediconi, Natalia; Lupacchini, Leonardo; Alfalate, Dulce; Guerrieri, Francesca; Calvo, Ludovica; Salerno, Debora; Di Cocco, Silvia; Levrero, Massimo; Belloni, Laura

    2015-01-01

    The HBV covalently closed circular DNA (cccDNA) is organized as a mini-chromosome in the nuclei of infected hepatocytes by histone and non-histone proteins. Transcription from the cccDNA of the RNA replicative intermediate termed pre-genome (pgRNA), is the critical step for genome amplification and ultimately determines the rate of HBV replication. Multiple evidences suggest that cccDNA epigenetic modifications, such as histone modifications and DNA methylation, participate in regulating the transcriptional activity of the HBV cccDNA. Inflammatory cytokines (TNFα, LTβ) and the pleiotropic cytokine interleukin-6 (IL6) inhibit hepatitis B virus (HBV) replication and transcription. Here we show, in HepG2 cells transfected with linear HBV monomers and HBV-infected NTCP-HepG2 cells, that IL6 treatment leads to a reduction of cccDNA-bound histone acetylation paralleled by a rapid decrease in 3.5kb/pgRNA and subgenomic HBV RNAs transcription without affecting cccDNA chromatinization or cccDNA levels. IL6 repressive effect on HBV replication is mediated by a loss of HNF1α and HNF4α binding to the cccDNA and a redistribution of STAT3 binding from the cccDNA to IL6 cellular target genes. PMID:26580974

  17. Adaptive and Specialised Transcriptional Responses to Xenobiotic Stress in Caenorhabditis elegans Are Regulated by Nuclear Hormone Receptors

    PubMed Central

    Jones, Laura M.; Rayson, Samantha J.; Flemming, Anthony J.; Urwin, Peter E.

    2013-01-01

    Characterisation of the pathways by which xenobiotics are metabolised and excreted in both target and non-target organisms is crucial for the rational design of effective and specific novel bioactive molecules. Consequently, we have investigated the induced responses of the model nematode Caenorhabditis elegans to a variety of xenobiotics which represent a range of putative modes of action. The majority of genes that were specifically induced in preliminary microarray analyses encoded enzymes from Phase I and II metabolism, including cytochrome P450s, short chain dehydrogenases, UDP-glucuronosyl transferases and glutathione transferases. Changes in gene expression were confirmed by quantitative PCR and GFP induction in reporter strains driven by promoters for transcription of twelve induced enzymes was investigated. The particular complement of metabolic genes induced was found to be highly contingent on the xenobiotic applied. The known regulators of responses to applied chemicals ahr-1, hif-1, mdt-15 and nhr-8 were not required for any of these inducible responses and skn-1 regulated GFP expression from only two of the promoters. Reporter strains were used in conjunction with systematic RNAi screens to identify transcription factors which drive expression of these genes under xenobiotic exposure. These transcription factors appeared to regulate specific xenobiotic responses and have no reported phenotypes under standard conditions. Focussing on nhr-176 we demonstrate the role of this transcription factor in mediating the resistance to thiabendazole. PMID:23922869

  18. Dissecting nuclear Wingless signalling: recruitment of the transcriptional co-activator Pygopus by a chain of adaptor proteins.

    PubMed

    Städeli, Reto; Basler, Konrad

    2005-11-01

    Members of the Wingless (Wg)/Wnt family of secreted glycoproteins control cell fate during embryonic development and adult homeostasis. Wnt signals regulate the expression of target genes by activating a conserved signal transduction pathway. Upon receptor activation, the signal is transmitted intracellularly by stabilization of Armadillo (Arm)/beta-catenin. Arm/beta-catenin translocates to the nucleus, interacts with DNA-binding factors of the Pangolin (Pan)/TCF/LEF class and activates transcription of target genes in cooperation with the recently identified proteins Legless/BCL9 (Lgs) and Pygopus (Pygo). Here, we analyse the mode of action of Pan, Arm, Lgs, and Pygo in Drosophila cultured cells. We provide evidence that together these four proteins form a 'chain of adaptors' linking the NH2-terminal homology domain (NHD) of Pygo to the DNA-binding domain of Pan. We show that the NHD has potent transcriptional activation capacity, which differs from that of acidic activator domains and depends on a conserved NPF tripeptide. A single point mutation within this NPF motif abolishes the transcriptional activity of the Pygo NHD in vitro and strongly reduces Wg signalling in vivo. Together, our results suggest that the transcriptional output of Wg pathway activity largely relies on a 'chain of adaptors' design to direct the Pygo NHD to Wg target promoters in an Arm-dependent manner. PMID:16169192

  19. Nucleotide sequence and transcriptional analysis of the HindIII P region of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus.

    PubMed

    Carstens, E B; Lu, A

    1990-12-01

    DNA sequence analysis of the HindIII P region of a temperature-sensitive mutant of Autographa californica nuclear polyhedrosis virus confirmed the specific amplification of 1.4 kb of viral DNA from this region of the genome. The sequenced region included an open reading frame, translated in a counterclockwise direction, which would potentially encode a 74K protein. The amplified DNA was contained within this open reading frame, resulting in in-frame amplifications of a domain within the protein. Transcription studies revealed the presence of a ladder of viral RNA species corresponding to a 2.5 kb transcript carrying tandem repeats of about 1.4 kb. This indicated that the duplicated DNA was transcribed in the same orientation as the p10 gene. We predict that transcripts synthesized from the opposite DNA strand also consist of a ladder of related mRNAs which would be translated to produce a family of p74 proteins with multiple internal domains. PMID:2273394

  20. Transcriptional Regulation of the Intestinal Nuclear Bile Acid Farnesoid X Receptor (FXR) by the caudal-related Homeobox 2 (CDX2)*

    PubMed Central

    Modica, Salvatore; Cariello, Marica; Morgano, Annalisa; Gross, Isabelle; Vegliante, Maria Carmela; Murzilli, Stefania; Salvatore, Lorena; Freund, Jean-Noel; Sabbà, Carlo; Moschetta, Antonio

    2014-01-01

    Farnesoid X receptor (FXR, NR1H4) is a bile acid-activated transcription factor that belongs to the nuclear receptor superfamily. It is highly expressed in the enterohepatic system, where it senses bile acid levels to consequently reduce their synthesis while inducing their detoxification. Bile acids are intestinal tumor promoters and their concentrations have to be tightly regulated. Indeed, reduced expression of FXR in the intestine increases colorectal cancer susceptibility in mice, whereas its activation can promote apoptosis in genetically modified cells. Notably, despite the broad knowledge of the FXR enterohepatic transcriptional activity, the molecular mechanisms regulating FXR expression in the intestine are still unknown. Herein, by combining both gain and loss of function approaches and FXR promoter activity studies, we identified caudal-related homeobox 2 (CDX2) transcription factor as a positive regulator of FXR expression in the enterocytes. Our results provide a putative novel tool for modulating FXR expression against bile acid-related colorectal cancer progression. PMID:25138215

  1. The nuclear localization of the Arabidopsis transcription factor TIP is blocked by its interaction with the coat protein of Turnip crinkle virus

    SciTech Connect

    Ren Tao; Qu Feng; Morris, T. Jack . E-mail: jmorris@unlnotes.unl.edu

    2005-01-20

    We have previously reported that TIP, an Arabidopsis protein, interacts with the coat protein (CP) of Turnip crinkle virus (TCV) in yeast cells and that this interaction correlated with the resistance response in the TCV-resistant Arabidopsis ecotype Dijon-17. TIP was also able to activate transcription of reporter genes in yeast cells, suggesting that it is likely a transcription factor. We have now verified the physical interaction between TIP and TCV CP in vitro and showed that CP mutants unable to interact with TIP in yeast cells bind TIP with much lower affinity in vitro. Secondly, we have performed gel shift experiments demonstrating that TIP does not bind to DNA in a sequence-specific manner. The subcellular localization of TIP was also investigated by transiently expressing green fluorescence protein (GFP)-tagged TIP in Nicotiana benthamiana plant cells, which showed that GFP-tagged TIP localizes primarily to nuclei. Significantly, co-expression of TCVCP and GFP-TIP prevented the nuclear localization of TIP. Together, these results suggest that TIP might be a transcription factor involved in regulating the defense response of Arabidopsis to TCV and that its normal role is compromised by interaction with the invading viral CP.

  2. Transcriptional Regulation of X-Box-binding Protein One (XBP1) by Hepatocyte Nuclear Factor 4α (HNF4Α) Is Vital to Beta-cell Function.

    PubMed

    Moore, Benjamin D; Jin, Ramon U; Lo, Heiyong; Jung, Min; Wang, Haiyan; Battle, Michele A; Wollheim, Claes B; Urano, Fumihiko; Mills, Jason C

    2016-03-18

    The transcription factor, X-box-binding protein-1 (XBP1), controls the development and maintenance of the endoplasmic reticulum (ER) in multiple secretory cell lineages. We show here that Hepatocyte Nuclear Factor 4α (HNF4α) directly induces XBP1 expression. Mutations in HNF4α cause Mature-Onset Diabetes of the Young I (MODYI), a subset of diabetes characterized by diminished GSIS. In mouse models, cell lines, and ex vivo islets, using dominant negative and human- disease-allele point mutants or knock-out and knockdown models, we show that disruption of HNF4α caused decreased expression of XBP1 and reduced cellular ER networks. GSIS depends on ER Ca(2+) signaling; we show that diminished XBP1 and/or HNF4α in β-cells led to impaired ER Ca(2+) homeostasis. Restoring XBP1 expression was sufficient to completely rescue GSIS in HNF4α-deficient β-cells. Our findings uncover a transcriptional relationship between HNF4α and Xbp1 with potentially broader implications about MODYI and the importance of transcription factor signaling in the regulation of secretion. PMID:26792861

  3. Exo70 is transcriptionally up-regulated by hepatic nuclear factor 4α and contributes to cell cycle control in hepatoma cells

    PubMed Central

    Zhao, Yujie; Hou, Jihuan; Mi, Panying; Mao, Liyuan; Xu, Liang; Zhang, Youyu; Xiao, Li; Cao, Hanwei; Zhang, Wenqing; Zhang, Bing; Song, Gang; Hu, Tianhui; Zhan, Yan-yan

    2016-01-01

    Exo70, a member of the exocyst complex, is involved in cell exocytosis, migration, invasion and autophagy. However, the expression regulation and function of Exo70 in hepatocellular carcinoma are still poorly understood. In this study, we found Exo70 expression in human hepatoma cells was greatly reduced after knocking down hepatic nuclear factor 4α (HNF4α), the most important and abundant transcription factor in liver. This regulation occurred at the transcriptional level but not post-translational level. HNF4α transactivated Exo70 promoter through directly binding to the HNF4α-response element in this promoter. Cell cycle analysis further revealed that down-regulation of HNF4α and Exo70 was essential to berberine-stimulated G2/M cell cycle arrest in hepatoma cells. Moreover, knocking down either Exo70 or HNF4α induced G2/M phase arrest of hepatoma cells. Exo70 acted downstream of HNF4α to stimulate G2/M transition via increasing Cdc2 expression. Together, our results identify Exo70 as a novel transcriptional target of HNF4α to promote cell cycle progression in hepatoma, thus provide a basis for the development of therapeutic strategies for hepatocellular carcinoma. PMID:26848864

  4. The restricted promoter activity of the liver transcription factor hepatocyte nuclear factor 3 beta involves a cell-specific factor and positive autoactivation.

    PubMed Central

    Pani, L; Quian, X B; Clevidence, D; Costa, R H

    1992-01-01

    The transcription factor hepatocyte nuclear factor 3 (HNF-3) is involved in the coordinate expression of several liver genes. HNF-3 DNA binding activity is composed of three different liver proteins which recognize the same DNA site. The HNF-3 proteins (designated alpha, beta, and gamma) possess homology in the DNA binding domain and in several additional regions. To understand the cell-type-specific expression of HNF-3 beta, we have defined the regulatory sequences that elicit hepatoma-specific expression. Promoter activity requires -134 bp of HNF-3 beta proximal sequences and binds four nuclear proteins, including two ubiquitous factors. One of these promoter sites interacts with a novel cell-specific factor, LF-H3 beta, whose binding activity correlates with the HNF-3 beta tissue expression pattern. Furthermore, there is a binding site for the HNF-3 protein within its own promoter, suggesting that an autoactivation mechanism is involved in the establishment of HNF-3 beta expression. We propose that both the LF-H3 beta and HNF-3 sites play an important role in the cell-type-specific expression of the HNF-3 beta transcription factor. Images PMID:1732730

  5. Enhanced Cardiac Akt/Protein Kinase B Signaling Contributes to Pathological Cardiac Hypertrophy in Part by Impairing Mitochondrial Function via Transcriptional Repression of Mitochondrion-Targeted Nuclear Genes

    PubMed Central

    Wende, Adam R.; O'Neill, Brian T.; Bugger, Heiko; Riehle, Christian; Tuinei, Joseph; Buchanan, Jonathan; Tsushima, Kensuke; Wang, Li; Caro, Pilar; Guo, Aili; Sloan, Crystal; Kim, Bum Jun; Wang, Xiaohui; Pereira, Renata O.; McCrory, Mark A.; Nye, Brenna G.; Benavides, Gloria A.; Darley-Usmar, Victor M.; Shioi, Tetsuo; Weimer, Bart C.

    2014-01-01

    Sustained Akt activation induces cardiac hypertrophy (LVH), which may lead to heart failure. This study tested the hypothesis that Akt activation contributes to mitochondrial dysfunction in pathological LVH. Akt activation induced LVH and progressive repression of mitochondrial fatty acid oxidation (FAO) pathways. Preventing LVH by inhibiting mTOR failed to prevent the decline in mitochondrial function, but glucose utilization was maintained. Akt activation represses expression of mitochondrial regulatory, FAO, and oxidative phosphorylation genes in vivo that correlate with the duration of Akt activation in part by reducing FOXO-mediated transcriptional activation of mitochondrion-targeted nuclear genes in concert with reduced signaling via peroxisome proliferator-activated receptor α (PPARα)/PGC-1α and other transcriptional regulators. In cultured myocytes, Akt activation disrupted mitochondrial bioenergetics, which could be partially reversed by maintaining nuclear FOXO but not by increasing PGC-1α. Thus, although short-term Akt activation may be cardioprotective during ischemia by reducing mitochondrial metabolism and increasing glycolysis, long-term Akt activation in the adult heart contributes to pathological LVH in part by reducing mitochondrial oxidative capacity. PMID:25535334

  6. Death-domain associated protein-6 (DAXX) mediated apoptosis in hantavirus infection is counter-balanced by activation of interferon-stimulated nuclear transcription factors

    SciTech Connect

    Khaiboullina, Svetlana F.; Morzunov, Sergey P.; Boichuk, Sergei V.; Palotás, András; Jeor, Stephen St.; Lombardi, Vincent C.; Rizvanov, Albert A.

    2013-09-01

    Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20 kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirus triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.

  7. Down-regulation of the zinc-finger homeobox protein TSHZ2 releases GLI1 from the nuclear repressor complex to restore its transcriptional activity during mammary tumorigenesis

    PubMed Central

    Riku, Miho; Inaguma, Shingo; Ito, Hideaki; Tsunoda, Takumi; Ikeda, Hiroshi; Kasai, Kenji

    2016-01-01

    Although breast cancer is one of the most common malignancies, the molecular mechanisms underlying its development and progression are not fully understood. To identify key molecules involved, we screened publicly available microarray datasets for genes differentially expressed between breast cancers and normal mammary glands. We found that three of the genes predicted in this analysis were differentially expressed among human mammary tissues and cell lines. Of these genes, we focused on the role of the zinc-finger homeobox protein TSHZ2, which is down-regulated in breast cancer cells. We found that TSHZ2 is a nuclear protein harboring a bipartite nuclear localization signal, and we confirmed its function as a C-terminal binding protein (CtBP)-dependent transcriptional repressor. Through comprehensive screening, we identified TSHZ2-suppressing genes such as AEBP1 and CXCR4, which are conversely up-regulated by GLI1, the downstream transcription factor of Hedgehog signaling. We found that GLI1 forms a ternary complex with CtBP2 in the presence of TSHZ2 and that the transcriptional activity of GLI1 is suppressed by TSHZ2 in a CtBP-dependent manner. Indeed, knockdown of TSHZ2 increases the expression of AEBP1 and CXCR4 in TSHZ2-expressing immortalized mammary duct epithelium. Concordantly, immunohistochemical staining of mammary glands revealed that normal duct cells expresses GLI1 in the nucleus along with TSHZ2 and CtBP2, whereas invasive ductal carcinoma cells, which does not express TSHZ2, show the increase in the expression of AEBP1 and CXCR4 and in the cytoplasmic localization of GLI1. Thus, we propose that down-regulation of TSHZ2 is crucial for mammary tumorigenesis via the activation of GLI1. PMID:26744317

  8. Down-regulation of the zinc-finger homeobox protein TSHZ2 releases GLI1 from the nuclear repressor complex to restore its transcriptional activity during mammary tumorigenesis.

    PubMed

    Riku, Miho; Inaguma, Shingo; Ito, Hideaki; Tsunoda, Takumi; Ikeda, Hiroshi; Kasai, Kenji

    2016-02-01

    Although breast cancer is one of the most common malignancies, the molecular mechanisms underlying its development and progression are not fully understood. To identify key molecules involved, we screened publicly available microarray datasets for genes differentially expressed between breast cancers and normal mammary glands. We found that three of the genes predicted in this analysis were differentially expressed among human mammary tissues and cell lines. Of these genes, we focused on the role of the zinc-finger homeobox protein TSHZ2, which is down-regulated in breast cancer cells. We found that TSHZ2 is a nuclear protein harboring a bipartite nuclear localization signal, and we confirmed its function as a C-terminal binding protein (CtBP)-dependent transcriptional repressor. Through comprehensive screening, we identified TSHZ2-suppressing genes such as AEBP1 and CXCR4, which are conversely up-regulated by GLI1, the downstream transcription factor of Hedgehog signaling. We found that GLI1 forms a ternary complex with CtBP2 in the presence of TSHZ2 and that the transcriptional activity of GLI1 is suppressed by TSHZ2 in a CtBP-dependent manner. Indeed, knockdown of TSHZ2 increases the expression of AEBP1 and CXCR4 in TSHZ2-expressing immortalized mammary duct epithelium. Concordantly, immunohistochemical staining of mammary glands revealed that normal duct cells expresses GLI1 in the nucleus along with TSHZ2 and CtBP2, whereas invasive ductal carcinoma cells, which does not express TSHZ2, show the increase in the expression of AEBP1 and CXCR4 and in the cytoplasmic localization of GLI1. Thus, we propose that down-regulation of TSHZ2 is crucial for mammary tumorigenesis via the activation of GLI1. PMID:26744317

  9. Post-transcriptional regulation of cyclins D1, D3 and G1 and proliferation of human cancer cells depend on IMP-3 nuclear localization.

    PubMed

    Rivera Vargas, T; Boudoukha, S; Simon, A; Souidi, M; Cuvellier, S; Pinna, G; Polesskaya, A

    2014-05-29

    RNA-binding proteins of the IMP family (insulin-like growth factor 2 (IGF2) mRNA-binding proteins 1-3) are important post-transcriptional regulators of gene expression. Multiple studies have linked high expression of IMP proteins, and especially of IMP-3, to an unfavorable prognosis in numerous types of cancer. The specific importance of IMP-3 for cancer transformation remains poorly understood. We here show that all three IMPs can directly bind the mRNAs of cyclins D1, D3 and G1 (CCND1, D3 and G1) in vivo and in vitro, and yet only IMP-3 regulates the expression of these cyclins in a significant manner in six human cancer cell lines of different origins. In the absence of IMP-3, the levels of CCND1, D3 and G1 proteins fall dramatically, and the cells accumulate in the G1 phase of the cell cycle, leading to almost complete proliferation arrest. Our results show that, compared with IMP-1 and IMP-2, IMP-3 is enriched in the nucleus, where it binds the transcripts of CCND1, D3 and G1. The nuclear localization of IMP-3 depends on its protein partner HNRNPM and is indispensable for the post-transcriptional regulation of expression of the cyclins. Cytoplasmic retention of IMP-3 and HNRNPM in human cancer cells leads to significant drop in proliferation. In conclusion, a nuclear IMP-3-HNRNPM complex is important for the efficient synthesis of CCND1, D3 and G1 and for the proliferation of human cancer cells. PMID:23812426

  10. Expression of U1 small nuclear ribonucleoprotein 70K antisense transcript using APETALA3 promoter suppresses the development of sepals and petals.

    PubMed

    Golovkin, Maxim; Reddy, Anireddy S N

    2003-08-01

    U1 small nuclear ribonucleoprotein (snRNP)-70K (U1-70K), a U1 snRNP-specific protein, is involved in the early stages of spliceosome formation. In non-plant systems, it is involved in constitutive and alternative splicing. It has been shown that U1snRNP is dispensable for in vitro splicing of some animal pre-mRNAs, and inactivation of U1-70K in yeast (Saccharomyces cerevisiae) is not lethal. As in yeast and humans (Homo sapiens), plant U1-70K is coded by a single gene. In this study, we blocked the expression of Arabidopsis U1-70K in petals and stamens by expressing U1-70K antisense transcript using the AP3 (APETALA3) promoter specific to these floral organs. Flowers of transgenic Arabidopsis plants expressing U1-70K antisense transcript showed partially developed stamens and petals that are arrested at different stages of development. In some transgenic lines, flowers have rudimentary petals and stamens and are male sterile. The severity of the phenotype is correlated with the level of the antisense transcript. Molecular analysis of transgenic plants has confirmed that the observed phenotype is not due to disruption of whorl-specific homeotic genes, AP3 or PISTILLATA, responsible for petal and stamen development. The AP3 transcript was not detected in transgenic flowers with severe phenotype. Flowers of Arabidopsis plants transformed with a reporter gene driven by the same promoter showed no abnormalities. These results show that U1-70K is necessary for the development of sepals and petals and is an essential gene in plants. PMID:12913145