The Chromatin Remodeler SPLAYED Regulates Specific Stress Signaling Pathways

PubMed Central

Walley, Justin W.; Rowe, Heather C.; Xiao, Yanmei; Chehab, E. Wassim; Kliebenstein, Daniel J.; Wagner, Doris; Dehesh, Katayoon

2008-01-01

Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD) is required for the expression of selected genes downstream of the jasmonate (JA) and ethylene (ET) signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks. PMID:19079584

Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog-Dependent Medulloblastoma Initiation and Maintenance

DTIC Science & Technology

2014-10-01

Remodeling Factor in Sonic Hedgehog -Dependent Medulloblastoma Initiation and Maintenance PRINCIPAL INVESTIGATOR: Xuanming Shi CONTRACTING...Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog -Dependent 5b. GRANT NUMBER W81XWH-12-1-0527 Medulloblastoma Initiation and Maintenance...medulloblastoma. 15. SUBJECT TERMS Medulloblastoma, Sonic Hedgehog , Chromatin remodeling, BAF complex, Brg1, mouse model of shh-subtype medulloblastoma

De Novo Mutations in CHD4, an ATP-Dependent Chromatin Remodeler Gene, Cause an Intellectual Disability Syndrome with Distinctive Dysmorphisms.

PubMed

Weiss, Karin; Terhal, Paulien A; Cohen, Lior; Bruccoleri, Michael; Irving, Melita; Martinez, Ariel F; Rosenfeld, Jill A; Machol, Keren; Yang, Yaping; Liu, Pengfei; Walkiewicz, Magdalena; Beuten, Joke; Gomez-Ospina, Natalia; Haude, Katrina; Fong, Chin-To; Enns, Gregory M; Bernstein, Jonathan A; Fan, Judith; Gotway, Garrett; Ghorbani, Mohammad; van Gassen, Koen; Monroe, Glen R; van Haaften, Gijs; Basel-Vanagaite, Lina; Yang, Xiang-Jiao; Campeau, Philippe M; Muenke, Maximilian

2016-10-06

Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler involved in epigenetic regulation of gene transcription, DNA repair, and cell cycle progression. Also known as Mi2β, CHD4 is an integral subunit of a well-characterized histone deacetylase complex. Here we report five individuals with de novo missense substitutions in CHD4 identified through whole-exome sequencing and web-based gene matching. These individuals have overlapping phenotypes including developmental delay, intellectual disability, hearing loss, macrocephaly, distinct facial dysmorphisms, palatal abnormalities, ventriculomegaly, and hypogonadism as well as additional findings such as bone fusions. The variants, c.3380G>A (p.Arg1127Gln), c.3443G>T (p.Trp1148Leu), c.3518G>T (p.Arg1173Leu), and c.3008G>A, (p.Gly1003Asp) (GenBank: NM_001273.3), affect evolutionarily highly conserved residues and are predicted to be deleterious. Previous studies in yeast showed the equivalent Arg1127 and Trp1148 residues to be crucial for SNF2 function. Furthermore, mutations in the same positions were reported in malignant tumors, and a de novo missense substitution in an equivalent arginine residue in the C-terminal helicase domain of SMARCA4 is associated with Coffin Siris syndrome. Cell-based studies of the p.Arg1127Gln and p.Arg1173Leu mutants demonstrate normal localization to the nucleus and HDAC1 interaction. Based on these findings, the mutations potentially alter the complex activity but not its formation. This report provides evidence for the role of CHD4 in human development and expands an increasingly recognized group of Mendelian disorders involving chromatin remodeling and modification. Published by Elsevier Inc.

Chd1 remodelers maintain open chromatin and regulate the epigenetics of differentiation

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

Persson, Jenna; Ekwall, Karl, E-mail: karl.ekwall@ki.se; School of Life Sciences, University College Sodertorn, NOVUM, Huddinge

Eukaryotic DNA is packaged around octamers of histone proteins into nucleosomes, the basic unit of chromatin. In addition to enabling meters of DNA to fit within the confines of a nucleus, the structure of chromatin has functional implications for cell identity. Covalent chemical modifications to the DNA and to histones, histone variants, ATP-dependent chromatin remodelers, small noncoding RNAs and the level of chromatin compaction all contribute to chromosomal structure and to the activity or silencing of genes. These chromatin-level alterations are defined as epigenetic when they are heritable from mother to daughter cell. The great diversity of epigenomes that canmore » arise from a single genome permits a single, totipotent cell to generate the hundreds of distinct cell types found in humans. Two recent studies in mouse and in fly have highlighted the importance of Chd1 chromatin remodelers for maintaining an open, active chromatin state. Based on evidence from fission yeast as a model system, we speculate that Chd1 remodelers are involved in the disassembly of nucleosomes at promoter regions, thus promoting active transcription and open chromatin. It is likely that these nucleosomes are specifically marked for disassembly by the histone variant H2A.Z.« less

Drosophila transcription factor Tramtrack69 binds MEP1 to recruit the chromatin remodeler NuRD.

PubMed

Reddy, B Ashok; Bajpe, Prashanth Kumar; Bassett, Andrew; Moshkin, Yuri M; Kozhevnikova, Elena; Bezstarosti, Karel; Demmers, Jeroen A A; Travers, Andrew A; Verrijzer, C Peter

2010-11-01

ATP-dependent chromatin-remodeling complexes (remodelers) are essential regulators of chromatin structure and gene transcription. How remodelers can act in a gene-selective manner has remained enigmatic. A yeast two-hybrid screen for proteins binding the Drosophila transcription factor Tramtrack69 (TTK69) identified MEP1. Proteomic characterization revealed that MEP1 is a tightly associated subunit of the NuRD remodeler, harboring the Mi2 enzymatic core ATPase. In addition, we identified the fly homolog of human Deleted in oral cancer 1 (DOC1), also known as CDK2-associated protein 1 (CDK2AP1), as a bona fide NuRD subunit. Biochemical and genetic assays supported the functional association between MEP1, Mi2, and TTK69. Genomewide expression analysis established that TTK69, MEP1, and Mi2 cooperate closely to control transcription. The TTK69 transcriptome profile correlates poorly with remodelers other than NuRD, emphasizing the selectivity of remodeler action. On the genes examined, TTK69 is able to bind chromatin in the absence of NuRD, but targeting of NuRD is dependent on TTK69. Thus, there appears to be a hierarchical relationship in which transcription factor binding precedes remodeler recruitment.

Chromatin modification and remodelling: a regulatory landscape for the control of Arabidopsis defence responses upon pathogen attack.

PubMed

Berr, Alexandre; Ménard, Rozenn; Heitz, Thierry; Shen, Wen-Hui

2012-06-01

Due to their sessile lifestyle, plants have to cope with an ever-changing environment and to defend themselves against a multitude of biotic aggressors that compromise their development and reproduction. Responses to various biotic stresses largely depend on the plant's capacity to modulate rapidly and specifically its transcriptome. In a stress type-dependent manner, external signals are translocated into the nucleus to activate transcription factors, resulting in the increased expression of particular sets of defence-related genes. Among mechanisms of transcriptional regulation, chromatin remodelling accomplished through the activity of histone-modifying enzymes and ATP-dependent chromatin-remodelling complexes is emerging as a key process in the orchestration of plant biotic stress responses. In this review, we summarize and discuss roles that chromatin-remodelling mechanisms may play in regulating Arabidopsis defence responses. © 2012 Blackwell Publishing Ltd.

Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog-Dependent Medulloblastoma Initiation and Maintenance

DTIC Science & Technology

2013-10-01

Remodeling Factor in Sonic Hedgehog -Dependent Medulloblastoma Initiation and Maintenance PRINCIPAL INVESTIGATOR: Xuanming Shi CONTRACTING...5a. CONTRACT NUMBER W81XWH-12-1-0527 Function of Brg1 Chromatin Remodeling Factor in Sonic Hedgehog -Dependent 5b. GRANT NUMBER W81XWH-12-1...drug development and therapy of pediatric brain tumor and other Shh- dependent tumors. 15. SUBJECT TERMS Medulloblastoma, Sonic Hedgehog , Chromatin

The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies.

PubMed

Xue, Yutong; Gibbons, Richard; Yan, Zhijiang; Yang, Dafeng; McDowell, Tarra L; Sechi, Salvatore; Qin, Jun; Zhou, Sharleen; Higgs, Doug; Wang, Weidong

2003-09-16

ATRX syndrome is characterized by X-linked mental retardation associated with alpha-thalassemia. The gene mutated in this disease, ATRX, encodes a plant homeodomain-like finger and a SWI2/SNF2-like ATPase motif, both of which are often found in chromatin-remodeling enzymes, but ATRX has not been characterized biochemically. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. The following evidence supports that ATRX and Daxx are components of an ATP-dependent chromatin-remodeling complex: (i) Daxx and ATRX can be coimmunoisolated by antibodies specific for each protein; (ii) a proportion of Daxx cofractionates with ATRX as a complex of 1 MDa by gel-filtration analysis; (iii) in extract from cells of a patient with ATRX syndrome, the level of the Daxx-ATRX complex is correspondingly reduced; (iv) a proportion of ATRX and Daxx colocalize in promyelocytic leukemia nuclear bodies, with which Daxx had previously been located; and (v) the ATRX complex displays ATP-dependent activities that resemble those of other chromatin-remodeling complexes, including triple-helix DNA displacement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI/SNF or NURD complexes, the ATRX complex does not randomize DNA phasing of the mononucleosomes, suggesting that it may remodel chromatin differently. Taken together, the results suggest that ATRX functions in conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATRX syndrome may result from inappropriate expression of genes controlled by this complex.

ATM-dependent pathways of chromatin remodelling and oxidative DNA damage responses.

PubMed

Berger, N Daniel; Stanley, Fintan K T; Moore, Shaun; Goodarzi, Aaron A

2017-10-05

Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase with a master regulatory function in the DNA damage response. In this role, ATM commands a complex biochemical network that signals the presence of oxidative DNA damage, including the dangerous DNA double-strand break, and facilitates subsequent repair. Here, we review the current state of knowledge regarding ATM-dependent chromatin remodelling and epigenomic alterations that are required to maintain genomic integrity in the presence of DNA double-strand breaks and/or oxidative stress. We will focus particularly on the roles of ATM in adjusting nucleosome spacing at sites of unresolved DNA double-strand breaks within complex chromatin environments, and the impact of ATM on preserving the health of cells within the mammalian central nervous system.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

A Nucleotide-Driven Switch Regulates Flanking DNA Length Sensing by a Dimeric Chromatin Remodeler

PubMed Central

Leonard, John D.; Narlikar, Geeta J.

2015-01-01

SUMMARY The ATP-dependent chromatin assembly factor (ACF) is a dimeric motor that spaces nucleosomes to promote formation of silent chromatin. Two copies of its ATPase subunit SNF2h bind opposite sides of a nucleosome, but how these protomers avoid competition is unknown. SNF2h senses the length of DNA flanking a nucleosome via its HAND-SANT-SLIDE (HSS) domain, yet it is unclear how this interaction enhances remodeling. Using covalently connected SNF2h dimers we show that dimerization accelerates remodeling and that the HSS contributes to communication between protomers. We further identify a nucleotide-dependent conformational change in SNF2h. In one conformation the HSS binds flanking DNA, and in another conformation the HSS engages the nucleosome core. Based on these results, we propose a model in which DNA length sensing and translocation are performed by two distinct conformational states of SNF2h. Such separation of function suggests that these activities could be independently regulated to affect remodeling outcomes. PMID:25684208

Drosophila Transcription Factor Tramtrack69 Binds MEP1 To Recruit the Chromatin Remodeler NuRD ▿ †

PubMed Central

Reddy, B. Ashok; Bajpe, Prashanth Kumar; Bassett, Andrew; Moshkin, Yuri M.; Kozhevnikova, Elena; Bezstarosti, Karel; Demmers, Jeroen A. A.; Travers, Andrew A.; Verrijzer, C. Peter

2010-01-01

ATP-dependent chromatin-remodeling complexes (remodelers) are essential regulators of chromatin structure and gene transcription. How remodelers can act in a gene-selective manner has remained enigmatic. A yeast two-hybrid screen for proteins binding the Drosophila transcription factor Tramtrack69 (TTK69) identified MEP1. Proteomic characterization revealed that MEP1 is a tightly associated subunit of the NuRD remodeler, harboring the Mi2 enzymatic core ATPase. In addition, we identified the fly homolog of human Deleted in oral cancer 1 (DOC1), also known as CDK2-associated protein 1 (CDK2AP1), as a bona fide NuRD subunit. Biochemical and genetic assays supported the functional association between MEP1, Mi2, and TTK69. Genomewide expression analysis established that TTK69, MEP1, and Mi2 cooperate closely to control transcription. The TTK69 transcriptome profile correlates poorly with remodelers other than NuRD, emphasizing the selectivity of remodeler action. On the genes examined, TTK69 is able to bind chromatin in the absence of NuRD, but targeting of NuRD is dependent on TTK69. Thus, there appears to be a hierarchical relationship in which transcription factor binding precedes remodeler recruitment. PMID:20733004

Assembly of the Arp5 (Actin-related Protein) Subunit Involved in Distinct INO80 Chromatin Remodeling Activities*

PubMed Central

Yao, Wei; Beckwith, Sean L.; Zheng, Tina; Young, Thomas; Dinh, Van T.; Ranjan, Anand; Morrison, Ashby J.

2015-01-01

ATP-dependent chromatin remodeling, which repositions and restructures nucleosomes, is essential to all DNA-templated processes. The INO80 chromatin remodeling complex is an evolutionarily conserved complex involved in diverse cellular processes, including transcription, DNA repair, and replication. The functional diversity of the INO80 complex can, in part, be attributed to specialized activities of distinct subunits that compose the complex. Furthermore, structural analyses have identified biochemically discrete subunit modules that assemble along the Ino80 ATPase scaffold. Of particular interest is the Saccharomyces cerevisiae Arp5-Ies6 module located proximal to the Ino80 ATPase and the Rvb1-Rvb2 helicase module needed for INO80-mediated in vitro activity. In this study we demonstrate that the previously uncharacterized Ies2 subunit is required for Arp5-Ies6 association with the catalytic components of the INO80 complex. In addition, Arp5-Ies6 module assembly with the INO80 complex is dependent on distinct conserved domains within Arp5, Ies6, and Ino80, including the spacer region within the Ino80 ATPase domain. Arp5-Ies6 interacts with chromatin via assembly with the INO80 complex, as IES2 and INO80 deletion results in loss of Arp5-Ies6 chromatin association. Interestingly, ectopic addition of the wild-type Arp5-Ies6 module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. However, the addition of mutant Arp5 lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding. Collectively, these results define the requirements of Arp5-Ies6 assembly, which are needed to couple ATP hydrolysis to productive nucleosome movement. PMID:26306040

Acetylation-Dependent Chromatin Reorganization by BRDT, a Testis-Specific Bromodomain-Containing Protein

PubMed Central

Pivot-Pajot, Christophe; Caron, Cécile; Govin, Jérôme; Vion, Alexandre; Rousseaux, Sophie; Khochbin, Saadi

2003-01-01

The association between histone acetylation and replacement observed during spermatogenesis prompted us to consider the testis as a source for potential factors capable of remodelling acetylated chromatin. A systematic search of data banks for open reading frames encoding testis-specific bromodomain-containing proteins focused our attention on BRDT, a testis-specific protein of unknown function containing two bromodomains. BRDT specifically binds hyperacetylated histone H4 tail depending on the integrity of both bromodomains. Moreover, in somatic cells, the ectopic expression of BRDT triggered a dramatic reorganization of the chromatin only after induction of histone hyperacetylation by trichostatin A (TSA). We then defined critical domains of BRDT involved in its activity. Both bromodomains of BRDT, as well as flanking regions, were found indispensable for its histone acetylation-dependent remodelling activity. Interestingly, we also observed that recombinant BRDT was capable of inducing reorganization of the chromatin of isolated nuclei in vitro only when the nuclei were from TSA-treated cells. This assay also allowed us to show that the action of BRDT was ATP independent, suggesting a structural role for the protein in the remodelling of acetylated chromatin. This is the first demonstration of a large-scale reorganization of acetylated chromatin induced by a specific factor. PMID:12861021

Epigenomic regulation of oncogenesis by chromatin remodeling.

PubMed

Kumar, R; Li, D-Q; Müller, S; Knapp, S

2016-08-25

Disruption of the intricate gene expression program represents one of major driving factors for the development, progression and maintenance of human cancer, and is often associated with acquired therapeutic resistance. At the molecular level, cancerous phenotypes are the outcome of cellular functions of critical genes, regulatory interactions of histones and chromatin remodeling complexes in response to dynamic and persistent upstream signals. A large body of genetic and biochemical evidence suggests that the chromatin remodelers integrate the extracellular and cytoplasmic signals to control gene activity. Consequently, widespread dysregulation of chromatin remodelers and the resulting inappropriate expression of regulatory genes, together, lead to oncogenesis. We summarize the recent developments and current state of the dysregulation of the chromatin remodeling components as the driving mechanism underlying the growth and progression of human tumors. Because chromatin remodelers, modifying enzymes and protein-protein interactions participate in interpreting the epigenetic code, selective chromatin remodelers and bromodomains have emerged as new frontiers for pharmacological intervention to develop future anti-cancer strategies to be used either as single-agent or in combination therapies with chemotherapeutics or radiotherapy.

The Core Subunit of A Chromatin-Remodeling Complex, ZmCHB101, Plays Essential Roles in Maize Growth and Development.

PubMed

Yu, Xiaoming; Jiang, Lili; Wu, Rui; Meng, Xinchao; Zhang, Ai; Li, Ning; Xia, Qiong; Qi, Xin; Pang, Jinsong; Xu, Zheng-Yi; Liu, Bao

2016-12-05

ATP-dependent chromatin remodeling complexes play essential roles in the regulation of diverse biological processes by formulating a DNA template that is accessible to the general transcription apparatus. Although the function of chromatin remodelers in plant development has been studied in A. thaliana, how it affects growth and development of major crops (e.g., maize) remains uninvestigated. Combining genetic, genomic and bioinformatic analyses, we show here that the maize core subunit of chromatin remodeling complex, ZmCHB101, plays essential roles in growth and development of maize at both vegetative and reproductive stages. Independent ZmCHB101 RNA interference plant lines displayed abaxially curling leaf phenotype due to increase of bulliform cell numbers, and showed impaired development of tassel and cob. RNA-seq-based transcriptome profiling revealed that ZmCHB101 dictated transcriptional reprogramming of a significant set of genes involved in plant development, photosynthesis, metabolic regulation, stress response and gene expressional regulation. Intriguingly, we found that ZmCHB101 was required for maintaining normal nucleosome density and 45 S rDNA compaction. Our findings suggest that the SWI3 protein, ZmCHB101, plays pivotal roles in maize normal growth and development via regulation of chromatin structure.

The chromatin remodeling factor CHD7 controls cerebellar development by regulating reelin expression

PubMed Central

Whittaker, Danielle E.; Riegman, Kimberley L.H.; Kasah, Sahrunizam; Mohan, Conor; Yu, Tian; Sala, Blanca Pijuan; Hebaishi, Husam; Caruso, Angela; Marques, Ana Claudia; Michetti, Caterina; Smachetti, María Eugenia Sanz; Shah, Apar; Sabbioni, Mara; Kulhanci, Omer; Tee, Wee-Wei; Reinberg, Danny; Scattoni, Maria Luisa; McGonnell, Imelda; Wardle, Fiona C.; Fernandes, Cathy

2017-01-01

The mechanisms underlying the neurodevelopmental deficits associated with CHARGE syndrome, which include cerebellar hypoplasia, developmental delay, coordination problems, and autistic features, have not been identified. CHARGE syndrome has been associated with mutations in the gene encoding the ATP-dependent chromatin remodeler CHD7. CHD7 is expressed in neural stem and progenitor cells, but its role in neurogenesis during brain development remains unknown. Here we have shown that deletion of Chd7 from cerebellar granule cell progenitors (GCps) results in reduced GCp proliferation, cerebellar hypoplasia, developmental delay, and motor deficits in mice. Genome-wide expression profiling revealed downregulated expression of the gene encoding the glycoprotein reelin (Reln) in Chd7-deficient GCps. Recessive RELN mutations have been associated with severe cerebellar hypoplasia in humans. We found molecular and genetic evidence that reductions in Reln expression contribute to GCp proliferative defects and cerebellar hypoplasia in GCp-specific Chd7 mouse mutants. Finally, we showed that CHD7 is necessary for maintaining an open, accessible chromatin state at the Reln locus. Taken together, this study shows that Reln gene expression is regulated by chromatin remodeling, identifies CHD7 as a previously unrecognized upstream regulator of Reln, and provides direct in vivo evidence that a mammalian CHD protein can control brain development by modulating chromatin accessibility in neuronal progenitors. PMID:28165338

Genome-wide overlap in the binding location and function of chromatin-remodeling proteins | Center for Cancer Research

Cancer.gov

A single strand of DNA can stretch several meters. Yet dozens of these strands, which can be one-tenth as thin as a human hair, need to fit into the cell’s nucleus. To pack those strands into such a small space, DNA tightly winds itself around histone proteins, forming nucleosomes that are strung together into complexes called chromatin. Beyond efficiently packaging DNA, chromatin also regulates how and when DNA is used. The condensed coiling of the genome makes it inaccessible to proteins such as RNA polymerases and transcription factors that control the expression of specific genes. For DNA to become accessible local chromatin regions need to be “opened” up. This process is called chromatin remodeling, and involves the ATP-dependent removal, ejection, or restructuring of nucleosomes by large, multiprotein enzymes.

Chromatin remodeller SMARCA4 recruits topoisomerase 1 and suppresses transcription-associated genomic instability.

PubMed

Husain, Afzal; Begum, Nasim A; Taniguchi, Takako; Taniguchi, Hisaaki; Kobayashi, Maki; Honjo, Tasuku

2016-02-04

Topoisomerase 1, an enzyme that relieves superhelical tension, is implicated in transcription-associated mutagenesis and genome instability-associated with neurodegenerative diseases as well as activation-induced cytidine deaminase. From proteomic analysis of TOP1-associated proteins, we identify SMARCA4, an ATP-dependent chromatin remodeller; FACT, a histone chaperone; and H3K4me3, a transcriptionally active chromatin marker. Here we show that SMARCA4 knockdown in a B-cell line decreases TOP1 recruitment to chromatin, and leads to increases in Igh/c-Myc chromosomal translocations, variable and switch region mutations and negative superhelicity, all of which are also observed in response to TOP1 knockdown. In contrast, FACT knockdown inhibits association of TOP1 with H3K4me3, and severely reduces DNA cleavage and Igh/c-Myc translocations, without significant effect on TOP1 recruitment to chromatin. We thus propose that SMARCA4 is involved in the TOP1 recruitment to general chromatin, whereas FACT is required for TOP1 binding to H3K4me3 at non-B DNA containing chromatin for the site-specific cleavage.

The BAF60 Subunit of the SWI/SNF Chromatin-Remodeling Complex Directly Controls the Formation of a Gene Loop at FLOWERING LOCUS C in Arabidopsis[W

PubMed Central

Jégu, Teddy; Latrasse, David; Delarue, Marianne; Hirt, Heribert; Domenichini, Séverine; Ariel, Federico; Crespi, Martin; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

2014-01-01

SWI/SNF complexes mediate ATP-dependent chromatin remodeling to regulate gene expression. Many components of these complexes are evolutionarily conserved, and several subunits of Arabidopsis thaliana SWI/SNF complexes are involved in the control of flowering, a process that depends on the floral repressor FLOWERING LOCUS C (FLC). BAF60 is a SWI/SNF subunit, and in this work, we show that BAF60, via a direct targeting of the floral repressor FLC, induces a change at the high-order chromatin level and represses the photoperiod flowering pathway in Arabidopsis. BAF60 accumulates in the nucleus and controls the formation of the FLC gene loop by modulation of histone density, composition, and posttranslational modification. Physiological analysis of BAF60 RNA interference mutant lines allowed us to propose that this chromatin-remodeling protein creates a repressive chromatin configuration at the FLC locus. PMID:24510722

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

PubMed Central

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

2006-01-01

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

Chromatin Remodeling BAF (SWI/SNF) Complexes in Neural Development and Disorders

PubMed Central

Sokpor, Godwin; Xie, Yuanbin; Rosenbusch, Joachim; Tuoc, Tran

2017-01-01

The ATP-dependent BRG1/BRM associated factor (BAF) chromatin remodeling complexes are crucial in regulating gene expression by controlling chromatin dynamics. Over the last decade, it has become increasingly clear that during neural development in mammals, distinct ontogenetic stage-specific BAF complexes derived from combinatorial assembly of their subunits are formed in neural progenitors and post-mitotic neural cells. Proper functioning of the BAF complexes plays critical roles in neural development, including the establishment and maintenance of neural fates and functionality. Indeed, recent human exome sequencing and genome-wide association studies have revealed that mutations in BAF complex subunits are linked to neurodevelopmental disorders such as Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, Kleefstra's syndrome spectrum, Hirschsprung's disease, autism spectrum disorder, and schizophrenia. In this review, we focus on the latest insights into the functions of BAF complexes during neural development and the plausible mechanistic basis of how mutations in known BAF subunits are associated with certain neurodevelopmental disorders. PMID:28824374

Chromatin Remodeling BAF (SWI/SNF) Complexes in Neural Development and Disorders.

PubMed

Sokpor, Godwin; Xie, Yuanbin; Rosenbusch, Joachim; Tuoc, Tran

2017-01-01

The ATP-dependent BRG1/BRM associated factor (BAF) chromatin remodeling complexes are crucial in regulating gene expression by controlling chromatin dynamics. Over the last decade, it has become increasingly clear that during neural development in mammals, distinct ontogenetic stage-specific BAF complexes derived from combinatorial assembly of their subunits are formed in neural progenitors and post-mitotic neural cells. Proper functioning of the BAF complexes plays critical roles in neural development, including the establishment and maintenance of neural fates and functionality. Indeed, recent human exome sequencing and genome-wide association studies have revealed that mutations in BAF complex subunits are linked to neurodevelopmental disorders such as Coffin-Siris syndrome, Nicolaides-Baraitser syndrome, Kleefstra's syndrome spectrum, Hirschsprung's disease, autism spectrum disorder, and schizophrenia. In this review, we focus on the latest insights into the functions of BAF complexes during neural development and the plausible mechanistic basis of how mutations in known BAF subunits are associated with certain neurodevelopmental disorders.

The yeast Fun30 and human SMARCAD1 chromatin remodelers promote DNA end resection

PubMed Central

Costelloe, Thomas; Louge, Raphaël; Tomimatsu, Nozomi; Mukherjee, Bipasha; Martini, Emmanuelle; Khadaroo, Basheer; Dubois, Kenny; Wiegant, Wouter W.; Thierry, Agnès; Burma, Sandeep; van Attikum, Haico; Llorente, Bertrand

2012-01-01

Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5′-3′ degradation of DSB ends that yields 3′ single-stranded DNA (ssDNA) required for loading of checkpoint and recombination proteins. The Mre11-Rad50-Xrs2/NBS1 complex and Sae2/CtIP initiate end resection while long-range resection depends on the exonuclease Exo1 or the helicase-topoisomerase complex Sgs1-Top3-Rmi1 with the endonuclease Dna21-6. DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood7. Here, we show that the yeast S. cerevisiae Fun30 protein and its human counterpart SMARCAD18, two poorly characterized ATP-dependent chromatin remodelers of the Snf2 ATPase family, are novel factors that are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates repair of camptothecin (CPT)-induced DNA lesions, and it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs and the kinetics of recruitment is similar to that of Exo1. Loss of SMARCAD1 impairs end resection, recombinational DNA repair and renders cells hypersensitive to DNA damage resulting from CPT or PARP inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodelers in controlling end resection, homologous recombination and genome stability in the context of chromatin. PMID:22960744

Effects of nucleosome stability on remodeler-catalyzed repositioning

NASA Astrophysics Data System (ADS)

Morgan, Aaron M.; LeGresley, Sarah E.; Briggs, Koan; Al-Ani, Gada; Fischer, Christopher J.

2018-03-01

Chromatin remodelers are molecular motors that play essential roles in the regulation of nucleosome positioning and chromatin accessibility. These machines couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of manipulating chromatin structure through processes that are not completely understood. Here we present a quantitative analysis of nucleosome repositioning by the imitation switch (ISWI) chromatin remodeler and demonstrate that nucleosome stability significantly impacts the observed activity. We show how DNA damage induced changes in the affinity of DNA wrapping within the nucleosome can affect ISWI repositioning activity and demonstrate how assay-dependent limitations can bias studies of nucleosome repositioning. Together, these results also suggest that some of the diversity seen in chromatin remodeler activity can be attributed to the variations in the thermodynamics of interactions between the remodeler, the histones, and the DNA, rather than reflect inherent properties of the remodeler itself.

Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

PubMed

Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

2015-04-28

A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

Structure and Dynamic Properties of a Glucocorticoid Receptor-Induced Chromatin Transition

PubMed Central

Fletcher, Terace M.; Ryu, Byung-Woo; Baumann, Christopher T.; Warren, Barbour S.; Fragoso, Gilberto; John, Sam; Hager, Gordon L.

2000-01-01

Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633–3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a “hit-and-run” mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262–1264, 2000). PMID:10938123

Chromatin associated mechanisms in base excision repair - nucleosome remodeling and DNA transcription, two key players.

PubMed

Menoni, Hervé; Di Mascio, Paolo; Cadet, Jean; Dimitrov, Stefan; Angelov, Dimitar

2017-06-01

Genomic DNA is prone to a large number of insults by a myriad of endogenous and exogenous agents. The base excision repair (BER) is the major mechanism used by cells for the removal of various DNA lesions spontaneously or environmentally induced and the maintenance of genome integrity. The presence of persistent DNA damage is not compatible with life, since abrogation of BER leads to early embryonic lethality in mice. There are several lines of evidences showing existence of a link between deficient BER, cancer proneness and ageing, thus illustrating the importance of this DNA repair pathway in human health. Although the enzymology of BER mechanisms has been largely elucidated using chemically defined DNA damage substrates and purified proteins, the complex interplay of BER with another vital process like transcription or when DNA is in its natural state (i.e. wrapped in nucleosome and assembled in chromatin fiber is largely unexplored. Cells use chromatin remodeling factors to overcome the general repression associated with the nucleosomal organization. It is broadly accepted that energy-dependent nucleosome remodeling factors disrupt histones-DNA interactions at the expense of ATP hydrolysis to favor transcription as well as DNA repair. Importantly, unlike transcription, BER is not part of a regulated developmental process but represents a maintenance system that should be efficient anytime and anywhere in the genome. In this review we will discuss how BER can deal with chromatin organization to maintain genetic information. Emphasis will be placed on the following challenging question: how BER is initiated within chromatin? Copyright © 2017 Elsevier Inc. All rights reserved.

Chromatin remodelling: the industrial revolution of DNA around histones.

PubMed

Saha, Anjanabha; Wittmeyer, Jacqueline; Cairns, Bradley R

2006-06-01

Chromatin remodellers are specialized multi-protein machines that enable access to nucleosomal DNA by altering the structure, composition and positioning of nucleosomes. All remodellers have a catalytic ATPase subunit that is similar to known DNA-translocating motor proteins, suggesting DNA translocation as a unifying aspect of their mechanism. Here, we explore the diversity and specialization of chromatin remodellers, discuss how nucleosome modifications regulate remodeller activity and consider a model for the exposure of nucleosomal DNA that involves the use of directional DNA translocation to pump 'DNA waves' around the nucleosome.

Quantifying transient binding of ISWI chromatin remodelers in living cells by pixel-wise photobleaching profile evolution analysis.

PubMed

Erdel, Fabian; Rippe, Karsten

2012-11-20

Interactions between nuclear proteins and chromatin frequently occur on the time scale of seconds and below. These transient binding events are important for the fast identification of target sites as concluded from our previous analysis of the human chromatin remodelers Snf2H and Snf2L from the imitation switch (ISWI) family. Both ATP-driven molecular motor proteins are able to translocate nucleosomes along the DNA and appear to exert this activity only on a small number of nucleosomes to which they bind more tightly. For mechanistic studies, one needs to distinguish such translocation reactions or other long-lived interactions associated with conformational changes and/or ATP hydrolysis from nonproductive chromatin sampling during target search. These processes can be separated by measuring the duration of nucleosome binding with subsecond time resolution. To reach this goal, we have developed a fluorescence bleaching technique termed pixel-wise photobleaching profile evolution analysis (3PEA). It exploits the inherent time structure of confocal microscopy images and yields millisecond resolution. 3PEA represents a generally applicable approach to quantitate transient chromatin interactions in the 2- to 500-ms time regime within only ∼1 s needed for a measurement. The green autofluorescent protein (GFP)-tagged Snf2H and Snf2L and the inactive Snf2L+13 splice variant were studied by 3PEA in comparison to the isolated GFP or red autofluorescent protein and a GFP pentamer. Our results reveal that the residence time for transient chromatin binding of Snf2H and Snf2L is <2 ms, and strongly support the view that ISWI-type remodelers are only rarely active in unperturbed cells during G1 phase.

TDP-43 Promotes Neurodegeneration by Impairing Chromatin Remodeling.

PubMed

Berson, Amit; Sartoris, Ashley; Nativio, Raffaella; Van Deerlin, Vivianna; Toledo, Jon B; Porta, Sílvia; Liu, Shichong; Chung, Chia-Yu; Garcia, Benjamin A; Lee, Virginia M-Y; Trojanowski, John Q; Johnson, F Brad; Berger, Shelley L; Bonini, Nancy M

2017-12-04

Regulation of chromatin structure is critical for brain development and function. However, the involvement of chromatin dynamics in neurodegeneration is less well understood. Here we find, launching from Drosophila models of amyotrophic lateral sclerosis and frontotemporal dementia, that TDP-43 impairs the induction of multiple key stress genes required to protect from disease by reducing the recruitment of the chromatin remodeler Chd1 to chromatin. Chd1 depletion robustly enhances TDP-43-mediated neurodegeneration and promotes the formation of stress granules. Conversely, upregulation of Chd1 restores nucleosomal dynamics, promotes normal induction of protective stress genes, and rescues stress sensitivity of TDP-43-expressing animals. TDP-43-mediated impairments are conserved in mammalian cells, and, importantly, the human ortholog CHD2 physically interacts with TDP-43 and is strikingly reduced in level in temporal cortex of human patient tissue. These findings indicate that TDP-43-mediated neurodegeneration causes impaired chromatin dynamics that prevents appropriate expression of protective genes through compromised function of the chromatin remodeler Chd1/CHD2. Enhancing chromatin dynamics may be a treatment approach to amyotrophic lateral scleorosis (ALS)/frontotemporal dementia (FTD). Copyright © 2017 Elsevier Ltd. All rights reserved.

Dual role of Brg chromatin remodeling factor in Sonic hedgehog signaling during neural development.

PubMed

Zhan, Xiaoming; Shi, Xuanming; Zhang, Zilai; Chen, Yu; Wu, Jiang I

2011-08-02

Sonic hedgehog (Shh) signaling plays diverse roles during animal development and adult tissue homeostasis through differential regulation of Gli family transcription factors. Dysregulated Shh signaling activities have been linked to birth defects and tumorigenesis. Here we report that Brg, an ATP-dependent chromatin remodeling factor, has dual functions in regulating Shh target gene expression. Using a Brg conditional deletion in Shh-responding neural progenitors and fibroblasts, we demonstrate that Brg is required both for repression of the basal expression and for the activation of signal-induced transcription of Shh target genes. In developing telencephalons deficient for Brg, Shh target genes were derepressed, whereas Brg-deleted cerebellar granule neuron precursors failed to respond to Shh to increase their proliferation. The repressor function of Brg was mediated through Gli3 and both the repressor and activator functions of Brg appeared to be independent of its ATPase activity. Furthermore, Brg facilitates Gli coactivator histone deacetylase (HDAC) binding to the regulatory regions of Shh target genes, providing a possible mechanism for its positive role in Shh signaling. Our results thus reveal that a complex chromatin regulation mechanism underlies the precise transcription outcomes of Shh signaling and its diverse roles during development.

Next-generation sequencing of translocation renal cell carcinoma reveals novel RNA splicing partners and frequent mutations of chromatin-remodeling genes.

PubMed

Malouf, Gabriel G; Su, Xiaoping; Yao, Hui; Gao, Jianjun; Xiong, Liangwen; He, Qiuming; Compérat, Eva; Couturier, Jérôme; Molinié, Vincent; Escudier, Bernard; Camparo, Philippe; Doss, Denaha J; Thompson, Erika J; Khayat, David; Wood, Christopher G; Yu, Willie; Teh, Bin T; Weinstein, John; Tannir, Nizar M

2014-08-01

MITF/TFE translocation renal cell carcinoma (TRCC) is a rare subtype of kidney cancer. Its incidence and the genome-wide characterization of its genetic origin have not been fully elucidated. We performed RNA and exome sequencing on an exploratory set of TRCC (n = 7), and validated our findings using The Cancer Genome Atlas (TCGA) clear-cell RCC (ccRCC) dataset (n = 460). Using the TCGA dataset, we identified seven TRCC (1.5%) cases and determined their genomic profile. We discovered three novel partners of MITF/TFE (LUC7L3, KHSRP, and KHDRBS2) that are involved in RNA splicing. TRCC displayed a unique gene expression signature as compared with other RCC types, and showed activation of MITF, the transforming growth factor β1 and the PI3K complex targets. Genes differentially spliced between TRCC and other RCC types were enriched for MITF and ID2 targets. Exome sequencing of TRCC revealed a distinct mutational spectrum as compared with ccRCC, with frequent mutations in chromatin-remodeling genes (six of eight cases, three of which were from the TCGA). In two cases, we identified mutations in INO80D, an ATP-dependent chromatin-remodeling gene, previously shown to control the amplitude of the S phase. Knockdown of INO80D decreased cell proliferation in a novel cell line bearing LUC7L3-TFE3 translocation. This genome-wide study defines the incidence of TRCC within a ccRCC-directed project and expands the genomic spectrum of TRCC by identifying novel MITF/TFE partners involved in RNA splicing and frequent mutations in chromatin-remodeling genes. ©2014 American Association for Cancer Research.

Actin-related proteins regulate the RSC chromatin remodeler by weakening intramolecular interactions of the Sth1 ATPase.

PubMed

Turegun, Bengi; Baker, Richard W; Leschziner, Andres E; Dominguez, Roberto

2018-01-01

The catalytic subunits of SWI/SNF-family and INO80-family chromatin remodelers bind actin and actin-related proteins (Arps) through an N-terminal helicase/SANT-associated (HSA) domain. Between the HSA and ATPase domains lies a conserved post-HSA (pHSA) domain. The HSA domain of Sth1, the catalytic subunit of the yeast SWI/SNF-family remodeler RSC, recruits the Rtt102-Arp7/9 heterotrimer. Rtt102-Arp7/9 regulates RSC function, but the mechanism is unclear. We show that the pHSA domain interacts directly with another conserved region of the catalytic subunit, protrusion-1. Rtt102-Arp7/9 binding to the HSA domain weakens this interaction and promotes the formation of stable, monodisperse complexes with DNA and nucleosomes. A crystal structure of Rtt102-Arp7/9 shows that ATP binds to Arp7 but not Arp9. However, Arp7 does not hydrolyze ATP. Together, the results suggest that Rtt102 and ATP stabilize a conformation of Arp7/9 that potentiates binding to the HSA domain, which releases intramolecular interactions within Sth1 and controls DNA and nucleosome binding.

Chromatin-Remodeling-Factor ARID1B Represses Wnt/β-Catenin Signaling

PubMed Central

Vasileiou, Georgia; Ekici, Arif B.; Uebe, Steffen; Zweier, Christiane; Hoyer, Juliane; Engels, Hartmut; Behrens, Jürgen; Reis, André; Hadjihannas, Michel V.

2015-01-01

The link of chromatin remodeling to both neurodevelopment and cancer has recently been highlighted by the identification of mutations affecting BAF chromatin-remodeling components, such as ARID1B, in individuals with intellectual disability and cancer. However, the underlying molecular mechanism(s) remains unknown. Here, we show that ARID1B is a repressor of Wnt/β-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/β-catenin target genes are upregulated. Using cellular models of low and high Wnt/β-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/β-catenin target genes and Wnt/β-catenin-dependent transcriptional reporters in a β-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/β-catenin signaling downstream of the β-catenin destruction complex. Both endogenous and exogenous ARID1B associate with β-catenin and repress Wnt/β-catenin-mediated transcription through the BAF core subunit BRG1. Accordingly, mutations in ARID1B leading to partial or complete deletion of its BRG1-binding domain, as is often observed in intellectual disability and cancers, compromise association with β-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/β-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through β-catenin. The data suggest that aberrations in chromatin-remodeling factors, such as ARID1B, might contribute to neurodevelopmental abnormalities and cancer through deregulation of developmental and oncogenic pathways, such as the Wnt/β-catenin signaling pathway. PMID:26340334

Global regulation of H2A.Z localization by the INO80 chromatin remodeling enzyme is essential for genome integrity

PubMed Central

Papamichos-Chronakis, Manolis; Watanabe, Shinya; Rando, Oliver J.; Peterson, Craig L.

2010-01-01

Summary INO80 is an evolutionarily conserved, ATP-dependent chromatin remodeling enzyme that plays roles in transcription, DNA repair, and replication. Here, we show that yeast INO80 facilitates these diverse processes at least in part by controlling genome-wide distribution of the histone variant H2A.Z. In the absence of INO80, H2A.Z nucleosomes are mis-localized, and H2A.Z levels at promoters show reduced responsiveness to transcriptional changes, suggesting that INO80 controls H2A.Z dynamics. Additionally, we demonstrate that INO80 has a novel histone exchange activity in which the enzyme can replace nucleosomal H2A.Z/H2B with free H2A/H2B dimers. Genetic interactions between ino80 and htz1 support a model in which INO80 catalyzes the removal of unacetylated H2A.Z from chromatin as a novel mechanism to promote genome stability. PMID:21241891

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

PubMed Central

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

2013-01-01

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

Chromatin-Remodeling-Factor ARID1B Represses Wnt/β-Catenin Signaling.

PubMed

Vasileiou, Georgia; Ekici, Arif B; Uebe, Steffen; Zweier, Christiane; Hoyer, Juliane; Engels, Hartmut; Behrens, Jürgen; Reis, André; Hadjihannas, Michel V

2015-09-03

The link of chromatin remodeling to both neurodevelopment and cancer has recently been highlighted by the identification of mutations affecting BAF chromatin-remodeling components, such as ARID1B, in individuals with intellectual disability and cancer. However, the underlying molecular mechanism(s) remains unknown. Here, we show that ARID1B is a repressor of Wnt/β-catenin signaling. Through whole-transcriptome analysis, we find that in individuals with intellectual disability and ARID1B loss-of-function mutations, Wnt/β-catenin target genes are upregulated. Using cellular models of low and high Wnt/β-catenin activity, we demonstrate that knockdown of ARID1B activates Wnt/β-catenin target genes and Wnt/β-catenin-dependent transcriptional reporters in a β-catenin-dependent manner. Reciprocally, forced expression of ARID1B inhibits Wnt/β-catenin signaling downstream of the β-catenin destruction complex. Both endogenous and exogenous ARID1B associate with β-catenin and repress Wnt/β-catenin-mediated transcription through the BAF core subunit BRG1. Accordingly, mutations in ARID1B leading to partial or complete deletion of its BRG1-binding domain, as is often observed in intellectual disability and cancers, compromise association with β-catenin, and the resultant ARID1B mutant proteins fail to suppress Wnt/β-catenin signaling. Finally, knockdown of ARID1B in mouse neuroblastoma cells leads to neurite outgrowth through β-catenin. The data suggest that aberrations in chromatin-remodeling factors, such as ARID1B, might contribute to neurodevelopmental abnormalities and cancer through deregulation of developmental and oncogenic pathways, such as the Wnt/β-catenin signaling pathway. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Circadian expression profiles of chromatin remodeling factor genes in Arabidopsis.

PubMed

Lee, Hong Gil; Lee, Kyounghee; Jang, Kiyoung; Seo, Pil Joon

2015-01-01

The circadian clock is a biological time keeper mechanism that regulates biological rhythms to a period of approximately 24 h. The circadian clock enables organisms to anticipate environmental cycles and coordinates internal cellular physiology with external environmental cues. In plants, correct matching of the clock with the environment confers fitness advantages to plant survival and reproduction. Therefore, circadian clock components are regulated at multiple layers to fine-tune the circadian oscillation. Epigenetic regulation provides an additional layer of circadian control. However, little is known about which chromatin remodeling factors are responsible for circadian control. In this work, we analyzed circadian expression of 109 chromatin remodeling factor genes and identified 17 genes that display circadian oscillation. In addition, we also found that a candidate interacts with a core clock component, supporting that clock activity is regulated in part by chromatin modification. As an initial attempt to elucidate the relationship between chromatin modification and circadian oscillation, we identified novel regulatory candidates that provide a platform for future investigations of chromatin regulation of the circadian clock.

Interplay between chromatin modulators and histone acetylation regulates the formation of accessible chromatin in the upstream regulatory region of fission yeast fbp1.

PubMed

Adachi, Akira; Senmatsu, Satoshi; Asada, Ryuta; Abe, Takuya; Hoffman, Charles S; Ohta, Kunihiro; Hirota, Kouji

2018-05-03

Numerous noncoding RNA transcripts are detected in eukaryotic cells. Noncoding RNAs transcribed across gene promoters are involved in the regulation of mRNA transcription via chromatin modulation. This function of noncoding RNA transcription was first demonstrated for the fission yeast fbp1 gene, where a cascade of noncoding RNA transcription events induces chromatin remodeling to facilitate transcription factor binding. We recently demonstrated that the noncoding RNAs from the fbp1 upstream region facilitate binding of the transcription activator Atf1 and thereby promote histone acetylation. Histone acetylation by histone acetyl transferases (HATs) and ATP-dependent chromatin remodelers (ADCRs) are implicated in chromatin remodeling, but the interplay between HATs and ADCRs in this process has not been fully elucidated. Here, we examine the roles played by two distinct ADCRs, Snf22 and Hrp3, and by the HAT Gcn5 in the transcriptional activation of fbp1. Snf22 and Hrp3 redundantly promote disassembly of chromatin in the fbp1 upstream region. Gcn5 critically contributes to nucleosome eviction in the absence of either Snf22 or Hrp3, presumably by recruiting Hrp3 in snf22∆ cells and Snf22 in hrp3∆ cells. Conversely, Gcn5-dependent histone H3 acetylation is impaired in snf22∆/hrp3∆ cells, suggesting that both redundant ADCRs induce recruitment of Gcn5 to the chromatin array in the fbp1 upstream region. These results reveal a previously unappreciated interplay between ADCRs and histone acetylation in which histone acetylation facilitates recruitment of ADCRs, while ADCRs are required for histone acetylation.

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

PubMed

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

2018-05-09

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

A chromatin remodelling complex that loads cohesin onto human chromosomes

NASA Astrophysics Data System (ADS)

Hakimi, Mohamed-Ali; Bochar, Daniel A.; Schmiesing, John A.; Dong, Yuanshu; Barak, Orr G.; Speicher, David W.; Yokomori, Kyoko; Shiekhattar, Ramin

2002-08-01

Nucleosomal DNA is arranged in a higher-order structure that presents a barrier to most cellular processes involving protein DNA interactions. The cellular machinery involved in sister chromatid cohesion, the cohesin complex, also requires access to the nucleosomal DNA to perform its function in chromosome segregation. The machineries that provide this accessibility are termed chromatin remodelling factors. Here, we report the isolation of a human ISWI (SNF2h)-containing chromatin remodelling complex that encompasses components of the cohesin and NuRD complexes. We show that the hRAD21 subunit of the cohesin complex directly interacts with the ATPase subunit SNF2h. Mapping of hRAD21, SNF2h and Mi2 binding sites by chromatin immunoprecipitation experiments reveals the specific association of these three proteins with human DNA elements containing Alu sequences. We find a correlation between modification of histone tails and association of the SNF2h/cohesin complex with chromatin. Moreover, we show that the association of the cohesin complex with chromatin can be regulated by the state of DNA methylation. Finally, we present evidence pointing to a role for the ATPase activity of SNF2h in the loading of hRAD21 on chromatin.

Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells

PubMed Central

Kondo, Toru; Raff, Martin

2004-01-01

We showed previously that purified rat oligodendrocyte precursor cells (OPCs) can be induced by extracellular signals to convert to multipotent neural stem-like cells (NSLCs), which can then generate both neurons and glial cells. Because the conversion of precursor cells to stem-like cells is of both intellectual and practical interest, it is important to understand its molecular basis. We show here that the conversion of OPCs to NSLCs depends on the reactivation of the sox2 gene, which in turn depends on the recruitment of the tumor suppressor protein Brca1 and the chromatin-remodeling protein Brahma (Brm) to an enhancer in the sox2 promoter. Moreover, we show that the conversion is associated with the modification of Lys 4 and Lys 9 of histone H3 at the same enhancer. Our findings suggest that the conversion of OPCs to NSLCs depends on progressive chromatin remodeling, mediated in part by Brca1 and Brm. PMID:15574597

The Chd1 Chromatin Remodeler Shifts Nucleosomal DNA Bidirectionally as a Monomer

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

Qiu, Yupeng; Levendosky, Robert F.; Chakravarthy, Srinivas

Chromatin remodelers catalyze dynamic packaging of the genome by carrying out nucleosome assembly/disassembly, histone exchange, and nucleosome repositioning. Remodeling results in evenly spaced nucleosomes, which requires probing both sides of the nucleosome, yet the way remodelers organize sliding activity to achieve this task is not understood. Here, we show that the monomeric Chd1 remodeler shifts DNA back and forth by dynamically alternating between different segments of the nucleosome. During sliding, Chd1 generates unstable remodeling intermediates that spontaneously relax to a pre-remodeled position. We demonstrate that nucleosome sliding is tightly controlled by two regulatory domains: the DNA-binding domain, which interferes withmore » sliding when its range is limited by a truncated linking segment, and the chromodomains, which play a key role in substrate discrimination. We propose that active interplay of the ATPase motor with the regulatory domains may promote dynamic nucleosome structures uniquely suited for histone exchange and chromatin reorganization during transcription.« less

Drosophila Brahma complex remodels nucleosome organizations in multiple aspects.

PubMed

Shi, Jiejun; Zheng, Meizhu; Ye, Youqiong; Li, Min; Chen, Xiaolong; Hu, Xinjie; Sun, Jin; Zhang, Xiaobai; Jiang, Cizhong

2014-09-01

ATP-dependent chromatin remodeling complexes regulate nucleosome organizations. In Drosophila, gene Brm encodes the core Brahma complex, the ATPase subunit of SWI/SNF class of chromatin remodelers. Its role in modulating the nucleosome landscape in vivo is unclear. In this study, we knocked down Brm in Drosophila third instar larvae to explore the changes in nucleosome profiles and global gene transcription. The results show that Brm knockdown leads to nucleosome occupancy changes throughout the entire genome with a bias in occupancy decrease. In contrast, the knockdown has limited impacts on nucleosome position shift. The knockdown also alters another important physical property of nucleosome positioning, fuzziness. Nucleosome position shift, gain or loss and fuzziness changes are all enriched in promoter regions. Nucleosome arrays around the 5' ends of genes are reorganized in five patterns as a result of Brm knockdown. Intriguingly, the concomitant changes in the genes adjacent to the Brahma-dependent remodeling regions have important roles in development and morphogenesis. Further analyses reveal abundance of AT-rich motifs for transcription factors in the remodeling regions. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Epigenetic regulation and chromatin remodeling in learning and memory.

PubMed

Kim, Somi; Kaang, Bong-Kiun

2017-01-13

Understanding the underlying mechanisms of memory formation and maintenance has been a major goal in the field of neuroscience. Memory formation and maintenance are tightly controlled complex processes. Among the various processes occurring at different levels, gene expression regulation is especially crucial for proper memory processing, as some genes need to be activated while some genes must be suppressed. Epigenetic regulation of the genome involves processes such as DNA methylation and histone post-translational modifications. These processes edit genomic properties or the interactions between the genome and histone cores. They then induce structural changes in the chromatin and lead to transcriptional changes of different genes. Recent studies have focused on the concept of chromatin remodeling, which consists of 3D structural changes in chromatin in relation to gene regulation, and is an important process in learning and memory. In this review, we will introduce three major epigenetic processes involved in memory regulation: DNA methylation, histone methylation and histone acetylation. We will also discuss general mechanisms of long-term memory storage and relate the epigenetic control of learning and memory to chromatin remodeling. Finally, we will discuss how epigenetic mechanisms can contribute to the pathologies of neurological disorders and cause memory-related symptoms.

The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities.

PubMed

Zhang, Y; LeRoy, G; Seelig, H P; Lane, W S; Reinberg, D

1998-10-16

Histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. ATP-dependent nucleosome remodeling, mediated by different multisubunit complexes, was thought to be involved only in transcription activation. Here we report the isolation of a protein complex that contains both histone deacetylation and ATP-dependent nucleosome remodeling activities. The complex contains the histone deacetylases HDAC1/2, histone-binding proteins, the dermatomyositis-specific autoantigen Mi2beta, a polypeptide related to the metastasis-associated protein 1, and a novel polypeptide of 32 kDa. Patients with dermatomyositis have a high rate of malignancy. The finding that Mi2beta exists in a complex containing histone deacetylase and nucleosome remodeling activities suggests a role for chromatin reorganization in cancer metastasis.

Combinatorial Control of Light Induced Chromatin Remodeling and Gene Activation in Neurospora

PubMed Central

Sancar, Cigdem; Ha, Nati; Yilmaz, Rüstem; Tesorero, Rafael; Fisher, Tamas; Brunner, Michael; Sancar, Gencer

2015-01-01

Light is an important environmental cue that affects physiology and development of Neurospora crassa. The light-sensing transcription factor (TF) WCC, which consists of the GATA-family TFs WC1 and WC2, is required for light-dependent transcription. SUB1, another GATA-family TF, is not a photoreceptor but has also been implicated in light-inducible gene expression. To assess regulation and organization of the network of light-inducible genes, we analyzed the roles of WCC and SUB1 in light-induced transcription and nucleosome remodeling. We show that SUB1 co-regulates a fraction of light-inducible genes together with the WCC. WCC induces nucleosome eviction at its binding sites. Chromatin remodeling is facilitated by SUB1 but SUB1 cannot activate light-inducible genes in the absence of WCC. We identified FF7, a TF with a putative O-acetyl transferase domain, as an interaction partner of SUB1 and show their cooperation in regulation of a fraction of light-inducible and a much larger number of non light-inducible genes. Our data suggest that WCC acts as a general switch for light-induced chromatin remodeling and gene expression. SUB1 and FF7 synergistically determine the extent of light-induction of target genes in common with WCC but have in addition a role in transcription regulation beyond light-induced gene expression. PMID:25822411

Chromatin hydrodynamics.

PubMed

Bruinsma, Robijn; Grosberg, Alexander Y; Rabin, Yitzhak; Zidovska, Alexandra

2014-05-06

Following recent observations of large scale correlated motion of chromatin inside the nuclei of live differentiated cells, we present a hydrodynamic theory-the two-fluid model-in which the content of a nucleus is described as a chromatin solution with the nucleoplasm playing the role of the solvent and the chromatin fiber that of a solute. This system is subject to both passive thermal fluctuations and active scalar and vector events that are associated with free energy consumption, such as ATP hydrolysis. Scalar events drive the longitudinal viscoelastic modes (where the chromatin fiber moves relative to the solvent) while vector events generate the transverse modes (where the chromatin fiber moves together with the solvent). Using linear response methods, we derive explicit expressions for the response functions that connect the chromatin density and velocity correlation functions to the corresponding correlation functions of the active sources and the complex viscoelastic moduli of the chromatin solution. We then derive general expressions for the flow spectral density of the chromatin velocity field. We use the theory to analyze experimental results recently obtained by one of the present authors and her co-workers. We find that the time dependence of the experimental data for both native and ATP-depleted chromatin can be well-fitted using a simple model-the Maxwell fluid-for the complex modulus, although there is some discrepancy in terms of the wavevector dependence. Thermal fluctuations of ATP-depleted cells are predominantly longitudinal. ATP-active cells exhibit intense transverse long wavelength velocity fluctuations driven by force dipoles. Fluctuations with wavenumbers larger than a few inverse microns are dominated by concentration fluctuations with the same spectrum as thermal fluctuations but with increased intensity. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Chromatin Hydrodynamics

PubMed Central

Bruinsma, Robijn; Grosberg, Alexander Y.; Rabin, Yitzhak; Zidovska, Alexandra

2014-01-01

Following recent observations of large scale correlated motion of chromatin inside the nuclei of live differentiated cells, we present a hydrodynamic theory—the two-fluid model—in which the content of a nucleus is described as a chromatin solution with the nucleoplasm playing the role of the solvent and the chromatin fiber that of a solute. This system is subject to both passive thermal fluctuations and active scalar and vector events that are associated with free energy consumption, such as ATP hydrolysis. Scalar events drive the longitudinal viscoelastic modes (where the chromatin fiber moves relative to the solvent) while vector events generate the transverse modes (where the chromatin fiber moves together with the solvent). Using linear response methods, we derive explicit expressions for the response functions that connect the chromatin density and velocity correlation functions to the corresponding correlation functions of the active sources and the complex viscoelastic moduli of the chromatin solution. We then derive general expressions for the flow spectral density of the chromatin velocity field. We use the theory to analyze experimental results recently obtained by one of the present authors and her co-workers. We find that the time dependence of the experimental data for both native and ATP-depleted chromatin can be well-fitted using a simple model—the Maxwell fluid—for the complex modulus, although there is some discrepancy in terms of the wavevector dependence. Thermal fluctuations of ATP-depleted cells are predominantly longitudinal. ATP-active cells exhibit intense transverse long wavelength velocity fluctuations driven by force dipoles. Fluctuations with wavenumbers larger than a few inverse microns are dominated by concentration fluctuations with the same spectrum as thermal fluctuations but with increased intensity. PMID:24806919

Different CHD chromatin remodelers are required for expression of distinct gene sets and specific stages during development of Dictyostelium discoideum

PubMed Central

Platt, James L.; Rogers, Benjamin J.; Rogers, Kelley C.; Harwood, Adrian J.; Kimmel, Alan R.

2013-01-01

Control of chromatin structure is crucial for multicellular development and regulation of cell differentiation. The CHD (chromodomain-helicase-DNA binding) protein family is one of the major ATP-dependent, chromatin remodeling factors that regulate nucleosome positioning and access of transcription factors and RNA polymerase to the eukaryotic genome. There are three mammalian CHD subfamilies and their impaired functions are associated with several human diseases. Here, we identify three CHD orthologs (ChdA, ChdB and ChdC) in Dictyostelium discoideum. These CHDs are expressed throughout development, but with unique patterns. Null mutants lacking each CHD have distinct phenotypes that reflect their expression patterns and suggest functional specificity. Accordingly, using genome-wide (RNA-seq) transcriptome profiling for each null strain, we show that the different CHDs regulate distinct gene sets during both growth and development. ChdC is an apparent ortholog of the mammalian Class III CHD group that is associated with the human CHARGE syndrome, and GO analyses of aberrant gene expression in chdC nulls suggest defects in both cell-autonomous and non-autonomous signaling, which have been confirmed through analyses of chdC nulls developed in pure populations or with low levels of wild-type cells. This study provides novel insight into the broad function of CHDs in the regulation development and disease, through chromatin-mediated changes in directed gene expression. PMID:24301467

A SWI/SNF Chromatin Remodelling Protein Controls Cytokinin Production through the Regulation of Chromatin Architecture

PubMed Central

Jégu, Teddy; Domenichini, Séverine; Blein, Thomas; Ariel, Federico; Christ, Aurélie; Kim, Soon-Kap; Crespi, Martin; Boutet-Mercey, Stéphanie; Mouille, Grégory; Bourge, Mickaël; Hirt, Heribert; Bergounioux, Catherine; Raynaud, Cécile; Benhamed, Moussa

2015-01-01

Chromatin architecture determines transcriptional accessibility to DNA and consequently gene expression levels in response to developmental and environmental stimuli. Recently, chromatin remodelers such as SWI/SNF complexes have been recognized as key regulators of chromatin architecture. To gain insight into the function of these complexes during root development, we have analyzed Arabidopsis knock-down lines for one sub-unit of SWI/SNF complexes: BAF60. Here, we show that BAF60 is a positive regulator of root development and cell cycle progression in the root meristem via its ability to down-regulate cytokinin production. By opposing both the deposition of active histone marks and the formation of a chromatin regulatory loop, BAF60 negatively regulates two crucial target genes for cytokinin biosynthesis (IPT3 and IPT7) and one cell cycle inhibitor (KRP7). Our results demonstrate that SWI/SNF complexes containing BAF60 are key factors governing the equilibrium between formation and dissociation of a chromatin loop controlling phytohormone production and cell cycle progression. PMID:26457678

Constraint of gene expression by the chromatin remodelling protein CHD4 facilitates lineage specification

PubMed Central

O'Shaughnessy-Kirwan, Aoife; Signolet, Jason; Costello, Ita; Gharbi, Sarah; Hendrich, Brian

2015-01-01

Chromatin remodelling proteins are essential for different aspects of metazoan biology, yet functional details of why these proteins are important are lacking. Although it is possible to describe the biochemistry of how they remodel chromatin, their chromatin-binding profiles in cell lines, and gene expression changes upon loss of a given protein, in very few cases can this easily translate into an understanding of how the function of that protein actually influences a developmental process. Here, we investigate how the chromatin remodelling protein CHD4 facilitates the first lineage decision in mammalian embryogenesis. Embryos lacking CHD4 can form a morphologically normal early blastocyst, but are unable to successfully complete the first lineage decision and form functional trophectoderm (TE). In the absence of a functional TE, Chd4 mutant blastocysts do not implant and are hence not viable. By measuring transcript levels in single cells from early embryos, we show that CHD4 influences the frequency at which unspecified cells in preimplantation stage embryos express lineage markers prior to the execution of this first lineage decision. In the absence of CHD4, this frequency is increased in 16-cell embryos, and by the blastocyst stage cells fail to properly adopt a TE gene expression programme. We propose that CHD4 allows cells to undertake lineage commitment in vivo by modulating the frequency with which lineage-specification genes are expressed. This provides novel insight into both how lineage decisions are made in mammalian cells, and how a chromatin remodelling protein functions to facilitate lineage commitment. PMID:26116663

SWI/SNF regulates half of its targets without the need of ATP-driven nucleosome remodeling by Brahma.

PubMed

Jordán-Pla, Antonio; Yu, Simei; Waldholm, Johan; Källman, Thomas; Östlund Farrants, Ann-Kristin; Visa, Neus

2018-05-18

Brahma (BRM) is the only catalytic subunit of the SWI/SNF chromatin-remodeling complex of Drosophila melanogaster. The function of SWI/SNF in transcription has long been attributed to its ability to remodel nucleosomes, which requires the ATPase activity of BRM. However, recent studies have provided evidence for a non-catalytic function of BRM in the transcriptional regulation of a few specific genes. Here we have used RNA-seq and ChIP-seq to identify the BRM target genes in S2 cells, and we have used a catalytically inactive BRM mutant (K804R) that is unable to hydrolyze ATP to investigate the magnitude of the non-catalytic function of BRM in transcription regulation. We show that 49% of the BRM target genes in S2 cells are regulated through mechanisms that do not require BRM to have an ATPase activity. We also show that the catalytic and non-catalytic mechanisms of SWI/SNF regulation operate on two subsets of genes that differ in promoter architecture and are linked to different biological processes. This study shows that the non-catalytic role of SWI/SNF in transcription regulation is far more prevalent than previously anticipated and that the genes that are regulated by SWI/SNF through ATPase-dependent and ATPase-independent mechanisms have specialized roles in different cellular and developmental processes.

The Chromatin Remodeler Isw1 Prevents CAG Repeat Expansions During Transcription in Saccharomyces cerevisiae

PubMed Central

Koch, Melissa R.; House, Nealia C. M.; Cosetta, Casey M.; Jong, Robyn M.; Salomon, Christelle G.; Joyce, Cailin E.; Philips, Elliot A.; Su, Xiaofeng A.; Freudenreich, Catherine H.

2018-01-01

CAG/CTG trinucleotide repeats are unstable sequences that are difficult to replicate, repair, and transcribe due to their structure-forming nature. CAG repeats strongly position nucleosomes; however, little is known about the chromatin remodeling needed to prevent repeat instability. In a Saccharomyces cerevisiae model system with CAG repeats carried on a YAC, we discovered that the chromatin remodeler Isw1 is required to prevent CAG repeat expansions during transcription. CAG repeat expansions in the absence of Isw1 were dependent on both transcription-coupled repair (TCR) and base-excision repair (BER). Furthermore, isw1∆ mutants are sensitive to methyl methanesulfonate (MMS) and exhibit synergistic MMS sensitivity when combined with BER or TCR pathway mutants. We conclude that CAG expansions in the isw1∆ mutant occur during a transcription-coupled excision repair process that involves both TCR and BER pathways. We observed increased RNA polymerase II (RNAPII) occupancy at the CAG repeat when transcription of the repeat was induced, but RNAPII binding did not change in isw1∆ mutants, ruling out a role for Isw1 remodeling in RNAPII progression. However, nucleosome occupancy over a transcribed CAG tract was altered in isw1∆ mutants. Based on the known role of Isw1 in the reestablishment of nucleosomal spacing after transcription, we suggest that a defect in this function allows DNA structures to form within repetitive DNA tracts, resulting in inappropriate excision repair and repeat-length changes. These results establish a new function for Isw1 in directly maintaining the chromatin structure at the CAG repeat, thereby limiting expansions that can occur during transcription-coupled excision repair. PMID:29305386

The Chromatin Remodeler BPTF Activates a Stemness Gene-Expression Program Essential for the Maintenance of Adult Hematopoietic Stem Cells.

PubMed

Xu, Bowen; Cai, Ling; Butler, Jason M; Chen, Dongliang; Lu, Xiongdong; Allison, David F; Lu, Rui; Rafii, Shahin; Parker, Joel S; Zheng, Deyou; Wang, Gang Greg

2018-03-13

Self-renewal and differentiation of adult stem cells are tightly regulated partly through configuration of chromatin structure by chromatin remodelers. Using knockout mice, we here demonstrate that bromodomain PHD finger transcription factor (BPTF), a component of the nucleosome remodeling factor (NURF) chromatin-remodeling complex, is essential for maintaining the population size of hematopoietic stem/progenitor cells (HSPCs), including long-term hematopoietic stem cells (HSCs). Bptf-deficient HSCs are defective in reconstituted hematopoiesis, and hematopoietic-specific knockout of Bptf caused profound defects including bone marrow failure and anemia. Genome-wide transcriptome profiling revealed that BPTF loss caused downregulation of HSC-specific gene-expression programs, which contain several master transcription factors (Meis1, Pbx1, Mn1, and Lmo2) required for HSC maintenance and self-renewal. Furthermore, we show that BPTF potentiates the chromatin accessibility of key HSC "stemness" genes. These results demonstrate an essential requirement of the chromatin remodeler BPTF and NURF for activation of "stemness" gene-expression programs and proper function of adult HSCs. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Functional Coupling between HIV-1 Integrase and the SWI/SNF Chromatin Remodeling Complex for Efficient in vitro Integration into Stable Nucleosomes

PubMed Central

Lesbats, Paul; Botbol, Yair; Chevereau, Guillaume; Vaillant, Cédric; Calmels, Christina; Arneodo, Alain; Andreola, Marie-Line; Lavigne, Marc; Parissi, Vincent

2011-01-01

Establishment of stable HIV-1 infection requires the efficient integration of the retroviral genome into the host DNA. The molecular mechanism underlying the control of this process by the chromatin structure has not yet been elucidated. We show here that stably associated nucleosomes strongly inhibit in vitro two viral-end integration by decreasing the accessibility of DNA to integrase. Remodeling of the chromatinized template by the SWI/SNF complex, whose INI1 major component interacts with IN, restores and redirects the full-site integration into the stable nucleosome region. These effects are not observed after remodeling by other human remodeling factors such as SNF2H or BRG1 lacking the integrase binding protein INI1. This suggests that the restoration process depends on the direct interaction between IN and the whole SWI/SNF complex, supporting a functional coupling between the remodeling and integration complexes. Furthermore, in silico comparison between more than 40,000 non-redundant cellular integration sites selected from literature and nucleosome occupancy predictions also supports that HIV-1 integration is promoted in the genomic region of weaker intrinsic nucleosome density in the infected cell. Our data indicate that some chromatin structures can be refractory for integration and that coupling between nucleosome remodeling and HIV-1 integration is required to overcome this natural barrier. PMID:21347347

Fission yeast Tup1-like repressors repress chromatin remodeling at the fbp1+ promoter and the ade6-M26 recombination hotspot.

PubMed Central

Hirota, Kouji; Hoffman, Charles S; Shibata, Takehiko; Ohta, Kunihiro

2003-01-01

Chromatin remodeling plays crucial roles in the regulation of gene expression and recombination. Transcription of the fission yeast fbp1(+) gene and recombination at the meiotic recombination hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ATF-type transcription factor Atf1*Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1(+) transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1(+) promoter and the M26 hotspot. We found that the chromatin structure around two regulatory elements for fbp1(+) was remodeled under derepressed conditions in concert with the robust activation of fbp1(+) transcription. Strains with tup11delta tup12delta double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingly, deletion of rst2(+), encoding a transcription factor controlled by the cAMP-dependent kinase, alleviated the tup11delta tup12delta defects in chromatin regulation but not in transcription repression. The chromatin at the M26 site in mitotic cultures of a tup11delta tup12delta mutant resembled that of wild-type meiotic cells. These observations suggest that these fission yeast Tup1-like corepressors repress chromatin remodeling at CRE-related sequences and that Rst2 antagonizes this function. PMID:14573465

A systematic analysis of factors localized to damaged chromatin reveals PARP-dependent recruitment of transcription factors

PubMed Central

Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C.; Westbrook, Thomas F.; Harper, J. Wade; Elledge, Stephen J.

2015-01-01

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify new DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the ALS candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a PARP-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors and >70% of randomly tested transcription factors localized to sites of DNA damage and approximately 90% were PARP-dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding domain-dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP-dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. PMID:26004182

Extracellular ATP decreases trophoblast invasion, spiral artery remodeling and immune cells in the mesometrial triangle in pregnant rats.

PubMed

Spaans, F; Melgert, B N; Chiang, C; Borghuis, T; Klok, P A; de Vos, P; van Goor, H; Bakker, W W; Faas, M M

2014-08-01

Preeclampsia is characterized by deficient trophoblast invasion and spiral artery remodeling, a process governed by inflammatory cells. High levels of the danger signal extracellular adenosine triphosphate (ATP) have been found in women with preeclampsia and infusion of ATP in pregnant rats induced preeclampsia-like symptoms such as albuminuria and placental ischemia. We hypothesized that ATP inhibits trophoblast invasion and spiral artery remodeling and affects macrophages and natural killer (NK) cells present in the rat mesometrial triangle. Pregnant rats were infused with ATP or saline (control) on day 14 of pregnancy. Rats were sacrificed on day 15, 17 or 20 of pregnancy and placentas with mesometrial triangle were collected. Sections were stained for trophoblast cells, α-smooth muscle actin (spiral artery remodeling), NK cells and various macrophage populations. Expression of various cytokines in the mesometrial triangle was analyzed using real-time RT-PCR. ATP infusion decreased interstitial trophoblast invasion on day 17 and spiral artery remodeling on day 17 and 20, increased activated tartrate resistant acid phosphatase (TRAP)-positive macrophages on day 15, decreased NK cells on day 17 and 20, and decreased inducible nitric oxide synthase (iNOS)-positive and CD206-positive macrophages and TNF-α and IL-33 expression at the end of pregnancy (day 20). Interstitial trophoblast invasion and spiral artery remodeling in the rat mesometrial triangle were decreased by infusion of ATP. These ATP-induced modifications were preceded by an increase in activated TRAP-positive macrophages and coincided with NK cell numbers, suggesting that they are involved. Trophoblast invasion and spiral artery remodeling may be inhibited by ATP-induced activated macrophages and decreased NK cells in the mesometrial triangle in rat pregnancy. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors.

PubMed

Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C; Westbrook, Thomas F; Harper, J Wade; Elledge, Stephen J

2015-06-09

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

The histone chaperone TAF-I/SET/INHAT is required for transcription in vitro of chromatin templates.

PubMed

Gamble, Matthew J; Erdjument-Bromage, Hediye; Tempst, Paul; Freedman, Leonard P; Fisher, Robert P

2005-01-01

To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D3 receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.

Chromatin remodeling regulates catalase expression during cancer cells adaptation to chronic oxidative stress.

PubMed

Glorieux, Christophe; Sandoval, Juan Marcelo; Fattaccioli, Antoine; Dejeans, Nicolas; Garbe, James C; Dieu, Marc; Verrax, Julien; Renard, Patricia; Huang, Peng; Calderon, Pedro Buc

2016-10-01

Regulation of ROS metabolism plays a major role in cellular adaptation to oxidative stress in cancer cells, but the molecular mechanism that regulates catalase, a key antioxidant enzyme responsible for conversion of hydrogen peroxide to water and oxygen, remains to be elucidated. Therefore, we investigated the transcriptional regulatory mechanism controlling catalase expression in three human mammary cell lines: the normal mammary epithelial 250MK primary cells, the breast adenocarcinoma MCF-7 cells and an experimental model of MCF-7 cells resistant against oxidative stress resulting from chronic exposure to H 2 O 2 (Resox), in which catalase was overexpressed. Here we identify a novel promoter region responsible for the regulation of catalase expression at -1518/-1226 locus and the key molecules that interact with this promoter and affect catalase transcription. We show that the AP-1 family member JunB and retinoic acid receptor alpha (RARα) mediate catalase transcriptional activation and repression, respectively, by controlling chromatin remodeling through a histone deacetylases-dependent mechanism. This regulatory mechanism plays an important role in redox adaptation to chronic exposure to H 2 O 2 in breast cancer cells. Our study suggests that cancer adaptation to oxidative stress may be regulated by transcriptional factors through chromatin remodeling, and reveals a potential new mechanism to target cancer cells. Copyright © 2016 Elsevier Inc. All rights reserved.

The Chromatin Remodeling Factor SMARCB1 Forms a Complex with Human Cytomegalovirus Proteins UL114 and UL44

PubMed Central

Ranneberg-Nilsen, Toril; Rollag, Halvor; Slettebakk, Ragnhild; Backe, Paul Hoff; Olsen, Øyvind; Luna, Luisa; Bjørås, Magnar

2012-01-01

Background Human cytomegalovirus (HCMV) uracil DNA glycosylase, UL114, is required for efficient viral DNA replication. Presumably, UL114 functions as a structural partner to other factors of the DNA-replication machinery and not as a DNA repair protein. UL114 binds UL44 (HCMV processivity factor) and UL54 (HCMV-DNA-polymerase). In the present study we have searched for cellular partners of UL114. Methodology/Principal Findings In a yeast two-hybrid screen SMARCB1, a factor of the SWI/SNF chromatin remodeling complex, was found to be an interacting partner of UL114. This interaction was confirmed in vitro by co-immunoprecipitation and pull-down. Immunofluorescence microscopy revealed that SMARCB1 along with BRG-1, BAF170 and BAF155, which are the core SWI/SNF components required for efficient chromatin remodeling, were present in virus replication foci 24–48 hours post infection (hpi). Furthermore a direct interaction was also demonstrated for SMARCB1 and UL44. Conclusions/Significance The core SWI/SNF factors required for efficient chromatin remodeling are present in the HCMV replication foci throughout infection. The proteins UL44 and UL114 interact with SMARCB1 and may participate in the recruitment of the SWI/SNF complex to the chromatinized virus DNA. Thus, the presence of the SWI/SNF chromatin remodeling complex in replication foci and its association with UL114 and with UL44 might imply its involvement in different DNA transactions. PMID:22479537

Concerted action of the PHD, chromo and motor domains regulates the human chromatin remodelling ATPase CHD4.

PubMed

Morra, Rosa; Lee, Benjamin M; Shaw, Heather; Tuma, Roman; Mancini, Erika J

2012-07-30

CHD4, the core subunit of the Nucleosome Remodelling and Deacetylase (NuRD) complex, is a chromatin remodelling ATPase that, in addition to a helicase domain, harbors tandem plant homeo finger and chromo domains. By using a panel of domain constructs we dissect their roles and demonstrate that DNA binding, histone binding and ATPase activities are allosterically regulated. Molecular shape reconstruction from small-angle X-ray scattering reveals extensive domain-domain interactions, which provide a structural explanation for the regulation of CHD4 activities by intramolecular domain communication. Our results demonstrate functional interdependency between domains within a chromatin remodeller. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mutation of Neuron-Specific Chromatin Remodeling Subunit BAF53b: Rescue of Plasticity and Memory by Manipulating Actin Remodeling

ERIC Educational Resources Information Center

Ciernia, Annie Vogel; Kramár, Enikö A.; Matheos, Dina P.; Havekes, Robbert; Hemstedt, Thekla J.; Magnan, Christophe N.; Sakata, Keith; Tran, Ashley; Azzawi, Soraya; Lopez, Alberto; Dang, Richard; Wang, Weisheng; Trieu, Brian; Tong, Joyce; Barrett, Ruth M.; Post, Rebecca J.; Baldi, Pierre; Abel, Ted; Lynch, Gary; Wood, Marcelo A.

2017-01-01

Recent human exome-sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several intellectual disabilities and cognitive disorders, including autism. However, it remains unclear how mutations in BAF complexes result in impaired cognitive function. Post-mitotic…

A Network of Chromatin Factors Is Regulating the Transition to Postembryonic Development in Caenorhabditis elegans

PubMed Central

Erdelyi, Peter; Wang, Xing; Suleski, Marina; Wicky, Chantal

2016-01-01

Mi2 proteins are evolutionarily conserved, ATP-dependent chromatin remodelers of the CHD family that play key roles in stem cell differentiation and reprogramming. In Caenorhabditis elegans, the let-418 gene encodes one of the two Mi2 homologs, which is part of at least two chromatin complexes, namely the Nucleosome Remodeling and histone Deacetylase (NuRD) complex and the MEC complex, and functions in larval development, vulval morphogenesis, lifespan regulation, and cell fate determination. To explore the mechanisms involved in the action of LET-418/Mi2, we performed a genome-wide RNA interference (RNAi) screen for suppressors of early larval arrest associated with let-418 mutations. We identified 29 suppressor genes, of which 24 encode chromatin regulators, mostly orthologs of proteins present in transcriptional activator complexes. The remaining five genes vary broadly in their predicted functions. All suppressor genes could suppress multiple aspects of the let-418 phenotype, including developmental arrest and ectopic expression of germline genes in the soma. Analysis of available transcriptomic data and quantitative PCR revealed that LET-418 and the suppressors of early larval arrest are regulating common target genes. These suppressors might represent direct competitors of LET-418 complexes for chromatin regulation of crucial genes involved in the transition to postembryonic development. PMID:28007841

A Network of Chromatin Factors Is Regulating the Transition to Postembryonic Development in Caenorhabditis elegans.

PubMed

Erdelyi, Peter; Wang, Xing; Suleski, Marina; Wicky, Chantal

2017-02-09

Mi2 proteins are evolutionarily conserved, ATP-dependent chromatin remodelers of the CHD family that play key roles in stem cell differentiation and reprogramming. In Caenorhabditis elegans , the let-418 gene encodes one of the two Mi2 homologs, which is part of at least two chromatin complexes, namely the Nucleosome Remodeling and histone Deacetylase (NuRD) complex and the MEC complex, and functions in larval development, vulval morphogenesis, lifespan regulation, and cell fate determination. To explore the mechanisms involved in the action of LET-418/Mi2, we performed a genome-wide RNA interference (RNAi) screen for suppressors of early larval arrest associated with let-418 mutations. We identified 29 suppressor genes, of which 24 encode chromatin regulators, mostly orthologs of proteins present in transcriptional activator complexes. The remaining five genes vary broadly in their predicted functions. All suppressor genes could suppress multiple aspects of the let-418 phenotype, including developmental arrest and ectopic expression of germline genes in the soma. Analysis of available transcriptomic data and quantitative PCR revealed that LET-418 and the suppressors of early larval arrest are regulating common target genes. These suppressors might represent direct competitors of LET-418 complexes for chromatin regulation of crucial genes involved in the transition to postembryonic development. Copyright © 2017 Erdelyi et al.

In vivo transcriptional profile analysis reveals RNA splicing and chromatin remodeling as prominent processes for adult neurogenesis.

PubMed

Lim, Daniel A; Suárez-Fariñas, Mayte; Naef, Felix; Hacker, Coleen R; Menn, Benedicte; Takebayashi, Hirohide; Magnasco, Marcelo; Patil, Nila; Alvarez-Buylla, Arturo

2006-01-01

Neural stem cells and neurogenesis persist in the adult mammalian brain subventricular zone (SVZ). Cells born in the rodent SVZ migrate to the olfactory bulb (Ob) where they differentiate into interneurons. To determine the gene expression and functional profile of SVZ neurogenesis, we performed three complementary sets of transcriptional analysis experiments using Affymetrix GeneChips: (1) comparison of adult mouse SVZ and Ob gene expression profiles with those of the striatum, cerebral cortex, and hippocampus; (2) profiling of SVZ stem cells and ependyma isolated by fluorescent-activated cell sorting (FACS); and (3) analysis of gene expression changes during in vivo SVZ regeneration after anti-mitotic treatment. Gene Ontology (GO) analysis of data from these three separate approaches showed that in adult SVZ neurogenesis, RNA splicing and chromatin remodeling are biological processes as statistically significant as cell proliferation, transcription, and neurogenesis. In non-neurogenic brain regions, RNA splicing and chromatin remodeling were not prominent processes. Fourteen mRNA splicing factors including Sf3b1, Sfrs2, Lsm4, and Khdrbs1/Sam68 were detected along with 9 chromatin remodeling genes including Mll, Bmi1, Smarcad1, Baf53a, and Hat1. We validated the transcriptional profile data with Northern blot analysis and in situ hybridization. The data greatly expand the catalogue of cell cycle components, transcription factors, and migration genes for adult SVZ neurogenesis and reveal RNA splicing and chromatin remodeling as prominent biological processes for these germinal cells.

Chromatin remodelling and autocrine TNFα are required for optimal interleukin-6 expression in activated human neutrophils.

PubMed

Zimmermann, Maili; Aguilera, Francisco Bianchetto; Castellucci, Monica; Rossato, Marzia; Costa, Sara; Lunardi, Claudio; Ostuni, Renato; Girolomoni, Giampiero; Natoli, Gioacchino; Bazzoni, Flavia; Tamassia, Nicola; Cassatella, Marco A

2015-01-23

Controversy currently exists about the ability of human neutrophils to produce IL-6. Here, we show that the chromatin organization of the IL-6 genomic locus in human neutrophils is constitutively kept in an inactive configuration. However, we also show that upon exposure to stimuli that trigger chromatin remodelling at the IL-6 locus, such as ligands for TLR8 or, less efficiently, TLR4, highly purified neutrophils express and secrete IL-6. In TLR8-activated neutrophils, but not monocytes, IL-6 expression is preceded by the induction of a latent enhancer located 14 kb upstream of the IL-6 transcriptional start site. In addition, IL-6 induction is potentiated by endogenous TNFα, which prolongs the synthesis of the IκBζ co-activator and sustains C/EBPβ recruitment and histone acetylation at IL-6 regulatory regions. Altogether, these data clarify controversial literature on the ability of human neutrophils to generate IL-6 and uncover chromatin-dependent layers of regulation of IL-6 in these cells.

Chromatin Remodeling Function of BRCA1 and Its Implication in Regulation of DNA Replication

DTIC Science & Technology

2001-09-01

Remodeling Function of BRCAI and its Implication in Regulation of DNA Replication PRINCIPAL INVESTIGATOR: Rong Li, Ph.D. CONTRACTING ORGANIZATION: University...of BRCAI and its Implication DAMD17-99-1-9572 in Regulation of DNA Replication 6. AUTHOR(S) Rong Li, Ph.D. 7. PERFORMING ORGANIZATION NAME(S) AND...1-mediated nuclear functions. 14. SUBJECT TERMS 15. NUMBER OF PAGES Breast Cancer, DNA replication , chromatin remodeling, transcription, 19 cell cycle

Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin)*

PubMed Central

Duan, Ming-Rui; Smerdon, Michael J.

2014-01-01

Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage. PMID:24515106

Analysis of DNA replication associated chromatin decondensation: in vivo assay for understanding chromatin remodeling mechanisms of selected proteins.

PubMed

Borysov, Sergiy; Bryant, Victoria L; Alexandrow, Mark G

2015-01-01

mechanisms underlying DNA replication associated chromatin accessibility, this unique and powerful experimental system has the propensity to be a valuable tool for understanding chromatin remodeling mechanisms orchestrated by other cellular processes such as DNA repair, recombination, mitotic chromosome condensation, or other chromosome dynamics involving chromatin alterations and accessibility.

SWI/SNF Chromatin-remodeling Factors: Multiscale Analyses and Diverse Functions*

PubMed Central

Euskirchen, Ghia; Auerbach, Raymond K.; Snyder, Michael

2012-01-01

Chromatin-remodeling enzymes play essential roles in many biological processes, including gene expression, DNA replication and repair, and cell division. Although one such complex, SWI/SNF, has been extensively studied, new discoveries are still being made. Here, we review SWI/SNF biochemistry; highlight recent genomic and proteomic advances; and address the role of SWI/SNF in human diseases, including cancer and viral infections. These studies have greatly increased our understanding of complex nuclear processes. PMID:22952240

The SWI2/SNF2 Chromatin-Remodeling ATPase BRAHMA Regulates Chlorophyll Biosynthesis in Arabidopsis.

PubMed

Zhang, Dong; Li, Yuhong; Zhang, Xinyu; Zha, Ping; Lin, Rongcheng

2017-01-09

Chlorophyll biosynthesis is critical for chloroplast development and photosynthesis in plants. Although reactions in the chlorophyll biosynthetic pathway have been largely known, little is known about the regulatory mechanisms of this pathway. In this study, we found that the dark-grown knockout and knockdown mutants as well as RNA-interference transgenic seedlings of BRAHMA (BRM), which encodes an SWI2/SNF2 chromatin-remodeling ATPase, had higher greening rates, accumulated less protochlorophyllide, and produced less reactive oxygen species than Arabidopsis wild-type plants did upon light exposure. The expression of NADPH:protochlorophyllide oxidoreductase A (PORA), PORB, and PORC, which catalyze a key step in chlorophyll biosynthesis, was increased in the brm mutants. We found that BRM physically interacted with the bHLH transcription factor PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) through its N-terminal domains. Furthermore, we demonstrated that BRM was directly recruited to the cis-regulatory regions of PORC, but not of PORA and PORB, at least partially in a PIF1-dependent manner and the level of histone H3 lysine 4 tri-methylation (H3K4me3) at PORC loci was increased in the brm mutant. Taken together, our data indicate that the chromatin-remodeling enzyme BRM modulates PORC expression through interacting with PIF1, providing a novel regulatory mechanism by which plants fine-tune chlorophyll biosynthesis during the transition from heterotrophic to autotrophic growth. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure

PubMed Central

Rosa-Garrido, Manuel; Chapski, Douglas J.; Schmitt, Anthony D.; Kimball, Todd H.; Karbassi, Elaheh; Monte, Emma; Balderas, Enrique; Pellegrini, Matteo; Shih, Tsai-Ting; Soehalim, Elizabeth; Liem, David; Ping, Peipei; Galjart, Niels J.; Ren, Shuxun; Wang, Yibin; Ren, Bing

2017-01-01

Background: Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. Methods: To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload–induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Results: Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. Conclusions: These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy. PMID

High-Resolution Mapping of Chromatin Conformation in Cardiac Myocytes Reveals Structural Remodeling of the Epigenome in Heart Failure.

PubMed

Rosa-Garrido, Manuel; Chapski, Douglas J; Schmitt, Anthony D; Kimball, Todd H; Karbassi, Elaheh; Monte, Emma; Balderas, Enrique; Pellegrini, Matteo; Shih, Tsai-Ting; Soehalim, Elizabeth; Liem, David; Ping, Peipei; Galjart, Niels J; Ren, Shuxun; Wang, Yibin; Ren, Bing; Vondriska, Thomas M

2017-10-24

Cardiovascular disease is associated with epigenomic changes in the heart; however, the endogenous structure of cardiac myocyte chromatin has never been determined. To investigate the mechanisms of epigenomic function in the heart, genome-wide chromatin conformation capture (Hi-C) and DNA sequencing were performed in adult cardiac myocytes following development of pressure overload-induced hypertrophy. Mice with cardiac-specific deletion of CTCF (a ubiquitous chromatin structural protein) were generated to explore the role of this protein in chromatin structure and cardiac phenotype. Transcriptome analyses by RNA-seq were conducted as a functional readout of the epigenomic structural changes. Depletion of CTCF was sufficient to induce heart failure in mice, and human patients with heart failure receiving mechanical unloading via left ventricular assist devices show increased CTCF abundance. Chromatin structural analyses revealed interactions within the cardiac myocyte genome at 5-kb resolution, enabling examination of intra- and interchromosomal events, and providing a resource for future cardiac epigenomic investigations. Pressure overload or CTCF depletion selectively altered boundary strength between topologically associating domains and A/B compartmentalization, measurements of genome accessibility. Heart failure involved decreased stability of chromatin interactions around disease-causing genes. In addition, pressure overload or CTCF depletion remodeled long-range interactions of cardiac enhancers, resulting in a significant decrease in local chromatin interactions around these functional elements. These findings provide a high-resolution chromatin architecture resource for cardiac epigenomic investigations and demonstrate that global structural remodeling of chromatin underpins heart failure. The newly identified principles of endogenous chromatin structure have key implications for epigenetic therapy. © 2017 The Authors.

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

PubMed Central

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

2008-01-01

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

Essential Role of Chromatin Remodeling Protein Bptf in Early Mouse Embryos and Embryonic Stem Cells

PubMed Central

Landry, Joseph; Sharov, Alexei A.; Piao, Yulan; Sharova, Lioudmila V.; Xiao, Hua; Southon, Eileen; Matta, Jennifer; Tessarollo, Lino; Zhang, Ying E.; Ko, Minoru S. H.; Kuehn, Michael R.; Yamaguchi, Terry P.; Wu, Carl

2008-01-01

We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf−/− embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf−/− embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo. PMID:18974875

ATP binding cassette G1-dependent cholesterol efflux during inflammation.

PubMed

de Beer, Maria C; Ji, Ailing; Jahangiri, Anisa; Vaughan, Ashley M; de Beer, Frederick C; van der Westhuyzen, Deneys R; Webb, Nancy R

2011-02-01

ATP binding cassette transporter G1 (ABCG1) mediates the transport of cellular cholesterol to HDL, and it plays a key role in maintaining macrophage cholesterol homeostasis. During inflammation, HDL undergoes substantial remodeling, acquiring lipid changes and serum amyloid A (SAA) as a major apolipoprotein. In the current study, we investigated whether remodeling of HDL that occurs during acute inflammation impacts ABCG1-dependent efflux. Our data indicate that lipid free SAA acts similarly to apolipoprotein A-I (apoA-I) in mediating sequential efflux from ABCA1 and ABCG1. Compared with normal mouse HDL, acute phase (AP) mouse HDL containing SAA exhibited a modest but significant 17% increase in ABCG1-dependent efflux. Interestingly, AP HDL isolated from mice lacking SAA (SAAKO mice) was even more effective in promoting ABCG1 efflux. Hydrolysis with Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) significantly reduced the ability of AP HDL from SAAKO mice to serve as a substrate for ABCG1-mediated cholesterol transfer, indicating that phospholipid (PL) enrichment, and not the presence of SAA, is responsible for alterations in efflux. AP human HDL, which is not PL-enriched, was somewhat less effective in mediating ABCG1-dependent efflux compared with normal human HDL. Our data indicate that inflammatory remodeling of HDL impacts ABCG1-dependent efflux independent of SAA.

NPM1/B23: A Multifunctional Chaperone in Ribosome Biogenesis and Chromatin Remodeling

PubMed Central

Lindström, Mikael S.

2011-01-01

At a first glance, ribosome biogenesis and chromatin remodeling are quite different processes, but they share a common problem involving interactions between charged nucleic acids and small basic proteins that may result in unwanted intracellular aggregations. The multifunctional nuclear acidic chaperone NPM1 (B23/nucleophosmin) is active in several stages of ribosome biogenesis, chromatin remodeling, and mitosis as well as in DNA repair, replication and transcription. In addition, NPM1 plays an important role in the Myc-ARF-p53 pathway as well as in SUMO regulation. However, the relative importance of NPM1 in these processes remains unclear. Provided herein is an update on the expanding list of the diverse activities and interacting partners of NPM1. Mechanisms of NPM1 nuclear export functions of NPM1 in the nucleolus and at the mitotic spindle are discussed in relation to tumor development. It is argued that the suggested function of NPM1 as a histone chaperone could explain several, but not all, of the effects observed in cells following changes in NPM1 expression. A future challenge is to understand how NPM1 is activated, recruited, and controlled to carry out its functions. PMID:21152184

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures

PubMed Central

Naughton, Catherine; Avlonitis, Nicolaos; Corless, Samuel; Prendergast, James G.; Mati, Ioulia K.; Eijk, Paul P.; Cockroft, Scott L.; Bradley, Mark; Ylstra, Bauke; Gilbert, Nick

2013-01-01

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes. PMID:23416946

Reduction in Histone H3 Acetylation and Chromatin Remodeling in Corneas of Alloxan-Induced Diabetic Rats.

PubMed

Herencia-Bueno, Karina E; Aldrovani, Marcela; Crivelaro, Roberta M; Thiesen, Roberto; Barros-Sobrinho, Alexandre A F; Claros-Chacaltana, Flor D Y; Padua, Ivan R M; Santos, Daniela M; Laus, José L

2018-05-01

To evaluate acetylation of histone H3, chromatin remodeling, nuclear size and shape, DNA ploidy, and distribution of nucleolus organizing regions (NORs) in corneal epithelial and stromal cells of diabetic and nondiabetic rats. Diabetes was induced by a single intraperitoneal injection of alloxan. All diabetic rats (n = 20) included in the study had 4 weeks of moderate-to-severe hyperglycemia (plasma glucose levels >400 mg/dL). Acetylated histone H3 levels were quantified in corneal tissue using a colorimetric assay. Chromatin remodeling, nuclear sizes (area/perimeter) and shapes (circularity), and DNA ploidies were evaluated from Feulgen-stained tissue sections using video image analysis. Distributions of NORs were studied in tissue sections impregnated with silver ions. Ophthalmic clinical parameters, including corneal sensitivity, were investigated. Twenty nondiabetic rats were used as controls. Acetylation of histone H3 was reduced in the corneas of the diabetic rats. Nuclei in corneal epithelial cells of diabetic rats compacted chromatin, increased in size, modified their shapes, and elevated DNA ploidy. The only nuclear change observed in the corneal stromal cells of diabetic rats was chromatin decompaction. The size of the silver-stained NOR did not differ between the study samples. The corneal sensitivity in diabetic rats was 51.8% lower than that in nondiabetic rats. The results of this study show that alloxan-induced diabetes altered the histone H3 acetylation pattern and compromised the chromatin supraorganization in corneal tissue/cells. Continued research is needed to understand the clinical and morphofunctional significance of changes in corneal cell nuclei of diabetic individuals.

Actin Family Proteins in the Human INO80 Chromatin Remodeling Complex Exhibit Functional Roles in the Induction of Heme Oxygenase-1 with Hemin.

PubMed

Takahashi, Yuichiro; Murakami, Hirokazu; Akiyama, Yusuke; Katoh, Yasutake; Oma, Yukako; Nishijima, Hitoshi; Shibahara, Kei-Ichi; Igarashi, Kazuhiko; Harata, Masahiko

2017-01-01

Nuclear actin family proteins, comprising of actin and actin-related proteins (Arps), are essential functional components of the multiple chromatin remodeling complexes. The INO80 chromatin remodeling complex, which is evolutionarily conserved and has roles in transcription, DNA replication and repair, consists of actin and actin-related proteins Arp4, Arp5, and Arp8. We generated Arp5 knockout (KO) and Arp8 KO cells from the human Nalm-6 pre-B cell line and used these KO cells to examine the roles of Arp5 and Arp8 in the transcriptional regulation mediated by the INO80 complex. In both of Arp5 KO and Arp8 KO cells, the oxidative stress-induced expression of HMOX1 gene, encoding for heme oxygenase-1 (HO-1), was significantly impaired. Consistent with these observations, chromatin immunoprecipitation (ChIP) assay revealed that oxidative stress caused an increase in the binding of the INO80 complex to the regulatory sites of HMOX1 in wild-type cells. The binding of INO80 complex to chromatin was reduced in Arp8 KO cells compared to that in the wild-type cells. On the other hand, the binding of INO80 complex to chromatin in Arp5 KO cells was similar to that in the wild-type cells even under the oxidative stress condition. However, both remodeling of chromatin at the HMOX1 regulatory sites and binding of a transcriptional activator to these sites were impaired in Arp5 KO cells, indicating that Arp5 is required for the activation of the INO80 complex. Collectively, these results suggested that these nuclear Arps play indispensable roles in the function of the INO80 chromatin remodeling complex.

Chromatin remodeling in stem cell maintenance in Arabidopsis thaliana.

PubMed

Shen, Wen-Hui; Xu, Lin

2009-07-01

Pluripotent stem cells are able to both self-renew and generate undifferentiated cells for the formation of new tissues and organs. In higher plants, stem cells found in the shoot apical meristem (SAM) and the root apical meristem (RAM) are origins of organogenesis occurring post-embryonically. It is important to understand how the regulation of stem cell fate is coordinated to enable the meristem to constantly generate different types of lateral organs. Much knowledge has accumulated on specific transcription factors controlling SAM and RAM activity. Here, we review recent evidences for a role of chromatin remodeling in the maintenance of stable expression states of transcription factor genes and the control of stem cell activity in Arabidopsis.

Micron-scale coherence in interphase chromatin dynamics

PubMed Central

Zidovska, Alexandra; Weitz, David A.; Mitchison, Timothy J.

2013-01-01

Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood, especially at large length scales. We developed an approach, displacement correlation spectroscopy based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. This method revealed that chromatin movement was coherent across large regions (4–5 µm) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP dependent and unidirectional for several seconds, perhaps accounting for ATP-dependent directed movement of single genes. Perturbation of major nuclear ATPases such as DNA polymerase, RNA polymerase II, and topoisomerase II eliminated micron-scale coherence, while causing rapid, local movement to increase; i.e., local motions accelerated but became uncoupled from their neighbors. We observe similar trends in chromatin dynamics upon inducing a direct DNA damage; thus we hypothesize that this may be due to DNA damage responses that physically relax chromatin and block long-distance communication of forces. PMID:24019504

Chromatin remodeling enzyme Brg1 is required for mouse lens fiber cell terminal differentiation and its denucleation

PubMed Central

2010-01-01

Background Brahma-related gene 1 (Brg1, also known as Smarca4 and Snf2β) encodes an adenosine-5'-triphosphate (ATP)-dependent catalytical subunit of the (switch/sucrose nonfermentable) (SWI/SNF) chromatin remodeling complexes. SWI/SNF complexes are recruited to chromatin through multiple mechanisms, including specific DNA-binding factors (for example, heat shock transcription factor 4 (Hsf4) and paired box gene 6 (Pax6)), chromatin structural proteins (for example, high-mobility group A1 (HMGA1)) and/or acetylated core histones. Previous studies have shown that a single amino acid substitution (K798R) in the Brg1 ATPase domain acts via a dominant-negative (dn) mechanism. Genetic studies have demonstrated that Brg1 is an essential gene for early (that is, prior implantation) mouse embryonic development. Brg1 also controls neural stem cell maintenance, terminal differentiation of multiple cell lineages and organs including the T-cells, glial cells and limbs. Results To examine the roles of Brg1 in mouse lens development, a dnBrg1 transgenic construct was expressed using the lens-specific αA-crystallin promoter in postmitotic lens fiber cells. Morphological studies revealed abnormal lens fiber cell differentiation in transgenic lenses resulting in cataract. Electron microscopic studies showed abnormal lens suture formation and incomplete karyolysis (that is, denucleation) of lens fiber cells. To identify genes regulated by Brg1, RNA expression profiling was performed in embryonic day 15.5 (E15.5) wild-type and dnBrg1 transgenic lenses. In addition, comparisons between differentially expressed genes in dnBrg1 transgenic, Pax6 heterozygous and Hsf4 homozygous lenses identified multiple genes coregulated by Brg1, Hsf4 and Pax6. DNase IIβ, a key enzyme required for lens fiber cell denucleation, was found to be downregulated in each of the Pax6, Brg1 and Hsf4 model systems. Lens-specific deletion of Brg1 using conditional gene targeting demonstrated that Brg1 was

HTLV-1 Tax Mediated Downregulation of miRNAs Associated with Chromatin Remodeling Factors in T Cells with Stably Integrated Viral Promoter

PubMed Central

Rahman, Saifur; Quann, Kevin; Pandya, Devanshi; Singh, Shruti; Khan, Zafar K.; Jain, Pooja

2012-01-01

RNA interference (RNAi) is a natural cellular mechanism to silence gene expression and is predominantly mediated by microRNAs (miRNAs) that target messenger RNA. Viruses can manipulate the cellular processes necessary for their replication by targeting the host RNAi machinery. This study explores the effect of human T-cell leukemia virus type 1 (HTLV-1) transactivating protein Tax on the RNAi pathway in the context of a chromosomally integrated viral long terminal repeat (LTR) using a CD4+ T-cell line, Jurkat. Transcription factor profiling of the HTLV-1 LTR stably integrated T-cell clone transfected with Tax demonstrates increased activation of substrates and factors associated with chromatin remodeling complexes. Using a miRNA microarray and bioinformatics experimental approach, Tax was also shown to downregulate the expression of miRNAs associated with the translational regulation of factors required for chromatin remodeling. These observations were validated with selected miRNAs and an HTLV-1 infected T cells line, MT-2. miR-149 and miR-873 were found to be capable of directly targeting p300 and p/CAF, chromatin remodeling factors known to play critical role in HTLV-1 pathogenesis. Overall, these results are first in line establishing HTLV-1/Tax-miRNA-chromatin concept and open new avenues toward understanding retroviral latency and/or replication in a given cell type. PMID:22496815

Dysregulation of chromatin remodelling complexes in amyotrophic lateral sclerosis.

PubMed

Tibshirani, Michael; Zhao, Beibei; Gentil, Benoit J; Minotti, Sandra; Marques, Christine; Keith, Julia; Rogaeva, Ekaterina; Zinman, Lorne; Rouaux, Caroline; Robertson, Janice; Durham, Heather D

2017-11-01

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease with paralysis resulting from dysfunction and loss of motor neurons. A common neuropathological finding is attrition of motor neuron dendrites, which make central connections vital to motor control. The chromatin remodelling complex, neuronal Brahma-related gene 1 (Brg1)-associated factor complex (nBAF), is critical for neuronal differentiation, dendritic extension and synaptic function. We have identified loss of the crucial nBAF subunits Brg1, Brg1-associated factor 53b and calcium responsive transactivator in cultured motor neurons expressing FUS or TAR-DNA Binding Protein 43 (TDP-43) mutants linked to familial ALS. When plasmids encoding wild-type or mutant human FUS or TDP-43 were expressed in motor neurons of dissociated spinal cord cultures prepared from E13 mice, mutant proteins in particular accumulated in the cytoplasm. Immunolabelling of nBAF subunits was reduced in proportion to loss of nuclear FUS or TDP-43 and depletion of Brg1 was associated with nuclear retention of Brg1 mRNA. Dendritic attrition (loss of intermediate and terminal dendritic branches) occurred in motor neurons expressing mutant, but not wild-type, FUS or TDP-43. This attrition was delayed by ectopic over-expression of Brg1 and was reproduced by inhibiting Brg1 activity either through genetic manipulation or treatment with the chemical inhibitor, (E)-1-(2-Hydroxyphenyl)-3-((1R, 4R)-5-(pyridin-2-yl)-2, 5-diazabicyclo[2.2.1]heptan-2-yl)prop-2-en-1-one, demonstrating the importance of Brg1 to maintenance of dendritic architecture. Loss of nBAF subunits was also documented in spinal motor neurons in autopsy tissue from familial amyotrophic sclerosis (chromosome 9 open reading frame 72 with G4C2 nucleotide expansion) and from sporadic cases with no identified mutation, pointing to dysfunction of nBAF chromatin remodelling in multiple forms of ALS. © The Author 2017. Published by Oxford University Press. All rights reserved

A comprehensive molecular study on Coffin-Siris and Nicolaides-Baraitser syndromes identifies a broad molecular and clinical spectrum converging on altered chromatin remodeling.

PubMed

Wieczorek, Dagmar; Bögershausen, Nina; Beleggia, Filippo; Steiner-Haldenstätt, Sabine; Pohl, Esther; Li, Yun; Milz, Esther; Martin, Marcel; Thiele, Holger; Altmüller, Janine; Alanay, Yasemin; Kayserili, Hülya; Klein-Hitpass, Ludger; Böhringer, Stefan; Wollstein, Andreas; Albrecht, Beate; Boduroglu, Koray; Caliebe, Almuth; Chrzanowska, Krystyna; Cogulu, Ozgur; Cristofoli, Francesca; Czeschik, Johanna Christina; Devriendt, Koenraad; Dotti, Maria Teresa; Elcioglu, Nursel; Gener, Blanca; Goecke, Timm O; Krajewska-Walasek, Malgorzata; Guillén-Navarro, Encarnación; Hayek, Joussef; Houge, Gunnar; Kilic, Esra; Simsek-Kiper, Pelin Özlem; López-González, Vanesa; Kuechler, Alma; Lyonnet, Stanislas; Mari, Francesca; Marozza, Annabella; Mathieu Dramard, Michèle; Mikat, Barbara; Morin, Gilles; Morice-Picard, Fanny; Ozkinay, Ferda; Rauch, Anita; Renieri, Alessandra; Tinschert, Sigrid; Utine, G Eda; Vilain, Catheline; Vivarelli, Rossella; Zweier, Christiane; Nürnberg, Peter; Rahmann, Sven; Vermeesch, Joris; Lüdecke, Hermann-Josef; Zeschnigk, Michael; Wollnik, Bernd

2013-12-20

Chromatin remodeling complexes are known to modify chemical marks on histones or to induce conformational changes in the chromatin in order to regulate transcription. De novo dominant mutations in different members of the SWI/SNF chromatin remodeling complex have recently been described in individuals with Coffin-Siris (CSS) and Nicolaides-Baraitser (NCBRS) syndromes. Using a combination of whole-exome sequencing, NGS-based sequencing of 23 SWI/SNF complex genes, and molecular karyotyping in 46 previously undescribed individuals with CSS and NCBRS, we identified a de novo 1-bp deletion (c.677delG, p.Gly226Glufs*53) and a de novo missense mutation (c.914G>T, p.Cys305Phe) in PHF6 in two individuals diagnosed with CSS. PHF6 interacts with the nucleosome remodeling and deacetylation (NuRD) complex implicating dysfunction of a second chromatin remodeling complex in the pathogenesis of CSS-like phenotypes. Altogether, we identified mutations in 60% of the studied individuals (28/46), located in the genes ARID1A, ARID1B, SMARCB1, SMARCE1, SMARCA2, and PHF6. We show that mutations in ARID1B are the main cause of CSS, accounting for 76% of identified mutations. ARID1B and SMARCB1 mutations were also found in individuals with the initial diagnosis of NCBRS. These individuals apparently belong to a small subset who display an intermediate CSS/NCBRS phenotype. Our proposed genotype-phenotype correlations are important for molecular screening strategies.

CHD5, a brain-specific paralog of Mi2 chromatin remodeling enzymes, regulates expression of neuronal genes.

PubMed

Potts, Rebecca Casaday; Zhang, Peisu; Wurster, Andrea L; Precht, Patricia; Mughal, Mohamed R; Wood, William H; Zhang, Yonqing; Becker, Kevin G; Mattson, Mark P; Pazin, Michael J

2011-01-01

CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease.

CHD5, a Brain-Specific Paralog of Mi2 Chromatin Remodeling Enzymes, Regulates Expression of Neuronal Genes

PubMed Central

Potts, Rebecca Casaday; Zhang, Peisu; Wurster, Andrea L.; Precht, Patricia; Mughal, Mohamed R.; Wood, William H.; Zhang, Yonqing; Becker, Kevin G.; Mattson, Mark P.; Pazin, Michael J.

2011-01-01

CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease. PMID:21931736

The Seed Repair Response during Germination: Disclosing Correlations between DNA Repair, Antioxidant Response, and Chromatin Remodeling in Medicago truncatula

PubMed Central

Pagano, Andrea; Araújo, Susana de Sousa; Macovei, Anca; Leonetti, Paola; Balestrazzi, Alma

2017-01-01

FAMILY) and MtADA2A (TRANSCRIPTIONAL ADAPTOR) showed tissue- and dose-dependent fluctuations in transcript levels. PCA (Principal Component Analysis) and correlation analyses suggest for a new putative link between DNA repair and chromatin remodeling that involves MtOGG1 and MtTRRAP genes, in the context of seed germination. Interesting correlations also connect DNA repair and chromatin remodeling with antioxidant players and proliferation markers. PMID:29184569

Stress and the Emerging Roles of Chromatin Remodeling in Signal Integration and Stable Transmission of Reversible Phenotypes

PubMed Central

Weaver, Ian C. G.; Korgan, Austin C.; Lee, Kristen; Wheeler, Ryan V.; Hundert, Amos S.; Goguen, Donna

2017-01-01

The influence of early life experience and degree of parental-infant attachment on emotional development in children and adolescents has been comprehensively studied. Structural and mechanistic insight into the biological foundation and maintenance of mammalian defensive systems (metabolic, immune, nervous and behavioral) is slowly advancing through the emerging field of developmental molecular (epi)genetics. Initial evidence revealed that differential nurture early in life generates stable differences in offspring hypothalamic-pituitary-adrenal (HPA) regulation, in part, through chromatin remodeling and changes in DNA methylation of specific genes expressed in the brain, revealing physical, biochemical and molecular paths for the epidemiological concept of gene-environment interactions. Herein, a primary molecular mechanism underpinning the early developmental programming and lifelong maintenance of defensive (emotional) responses in the offspring is the alteration of chromatin domains of specific genomic regions from a condensed state (heterochromatin) to a transcriptionally accessible state (euchromatin). Conversely, DNA methylation promotes the formation of heterochromatin, which is essential for gene silencing, genomic integrity and chromosome segregation. Therefore, inter-individual differences in chromatin modifications and DNA methylation marks hold great potential for assessing the impact of both early life experience and effectiveness of intervention programs—from guided psychosocial strategies focused on changing behavior to pharmacological treatments that target chromatin remodeling and DNA methylation enzymes to dietary approaches that alter cellular pools of metabolic intermediates and methyl donors to affect nutrient bioavailability and metabolism. In this review article, we discuss the potential molecular mechanism(s) of gene regulation associated with chromatin modeling and programming of endocrine (e.g., HPA and metabolic or cardiovascular) and

dDYRK2 and Minibrain interact with the chromatin remodelling factors SNR1 and TRX.

PubMed

Kinstrie, Ross; Lochhead, Pamela A; Sibbet, Gary; Morrice, Nick; Cleghon, Vaughn

2006-08-15

The DYRKs (dual specificity tyrosine phosphorylation-regulated kinases) are a conserved family of protein kinases that autophosphorylate a tyrosine residue in their activation loop by an intra-molecular mechanism and phosphorylate exogenous substrates on serine/threonine residues. Little is known about the identity of true substrates for DYRK family members and their binding partners. To address this question, we used full-length dDYRK2 (Drosophila DYRK2) as bait in a yeast two-hybrid screen of a Drosophila embryo cDNA library. Of 14 independent dDYRK2 interacting clones identified, three were derived from the chromatin remodelling factor, SNR1 (Snf5-related 1), and three from the essential chromatin component, TRX (trithorax). The association of dDYRK2 with SNR1 and TRX was confirmed by co-immunoprecipitation studies. Deletion analysis showed that the C-terminus of dDYRK2 modulated the interaction with SNR1 and TRX. DYRK family member MNB (Minibrain) was also found to co-precipitate with SNR1 and TRX, associations that did not require the C-terminus of the molecule. dDYRK2 and MNB were also found to phosphorylate SNR1 at Thr102 in vitro and in vivo. This phosphorylation required the highly conserved DH-box (DYRK homology box) of dDYRK2, whereas the DH-box was not essential for phosphorylation by MNB. This is the first instance of phosphorylation of SNR1 or any of its homologues and implicates the DYRK family of kinases with a role in chromatin remodelling.

The structure of the core NuRD repression complex provides insights into its interaction with chromatin

PubMed Central

Millard, Christopher J; Varma, Niranjan; Saleh, Almutasem; Morris, Kyle; Watson, Peter J; Bottrill, Andrew R; Fairall, Louise; Smith, Corinne J; Schwabe, John WR

2016-01-01

The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin. DOI: http://dx.doi.org/10.7554/eLife.13941.001 PMID:27098840

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

PubMed Central

Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

2016-01-01

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

Compact tomato seedlings and plants upon overexpression of a tomato chromatin remodelling ATPase gene.

PubMed

Folta, Adam; Bargsten, Joachim W; Bisseling, Ton; Nap, Jan-Peter; Mlynarova, Ludmila

2016-02-01

Control of plant growth is an important aspect of crop productivity and yield in agriculture. Overexpression of the AtCHR12/23 genes in Arabidopsis thaliana reduced growth habit without other morphological changes. These two genes encode Snf2 chromatin remodelling ATPases. Here, we translate this approach to the horticultural crop tomato (Solanum lycopersicum). We identified and cloned the single tomato ortholog of the two Arabidopsis Snf2 genes, designated SlCHR1. Transgenic tomato plants (cv. Micro-Tom) that constitutively overexpress the coding sequence of SlCHR1 show reduced growth in all developmental stages of tomato. This confirms that SlCHR1 combines the functions of both Arabidopsis genes in tomato. Compared to the wild type, the transgenic seedlings of tomato have significantly shorter roots, hypocotyls and reduced cotyledon size. Transgenic plants have a much more compact growth habit with markedly reduced plant height, severely compacted reproductive structures with smaller flowers and smaller fruits. The results indicate that either GMO-based or non-GMO-based approaches to modulate the expression of chromatin remodelling ATPase genes could develop into methods to control plant growth, for example to replace the use of chemical growth retardants. This approach is likely to be applicable and attractive for any crop for which growth habit reduction has added value. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

The chromatin remodeler chd5 is necessary for proper head development during embryogenesis of Danio rerio.

PubMed

Bishop, Brett; Ho, Kwok Ki; Tyler, Kim; Smith, Amanda; Bonilla, Sylvia; Leung, Yuk Fai; Ogas, Joe

2015-08-01

The chromatin remodeler CHD5 plays a critical role in tumor suppression and neurogenesis in mammals. CHD5 contributes to gene expression during neurogenesis, but there is still much to learn regarding how this class of remodelers contributes to differentiation and development. CHD5 remodelers are vertebrate-specific, raising the prospect that CHD5 plays one or more conserved roles in this phylum. Expression of chd5 in adult fish closely mirrors expression of CHD5 in adult mammals. Knockdown of Chd5 during embryogenesis suggests new roles for CHD5 remodelers based on resulting defects in craniofacial development including reduced head and eye size as well as reduced cartilage formation in the head. In addition, knockdown of Chd5 results in altered expression of neural markers in the developing brain and eye as well as a profound defect in differentiation of dopaminergic amacrine cells. Recombinant zebrafish Chd5 protein exhibits nucleosome remodeling activity in vitro, suggesting that it is the loss of this activity that contributes to the observed phenotypes. Our studies indicate that zebrafish is an appropriate model for functional characterization of CHD5 remodelers in vertebrates and highlight the potential of this model for generating novel insights into the role of this vital class of remodelers. Copyright © 2015. Published by Elsevier B.V.

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.

Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells.

PubMed

Laurette, Patrick; Strub, Thomas; Koludrovic, Dana; Keime, Céline; Le Gras, Stéphanie; Seberg, Hannah; Van Otterloo, Eric; Imrichova, Hana; Siddaway, Robert; Aerts, Stein; Cornell, Robert A; Mengus, Gabrielle; Davidson, Irwin

2015-03-24

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.

Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization

PubMed Central

Emelyanov, Alexander V.; Rabbani, Joshua; Mehta, Monika; Vershilova, Elena; Keogh, Michael C.

2014-01-01

Nuclear DNA in the male gamete of sexually reproducing animals is organized as sperm chromatin compacted primarily by sperm-specific protamines. Fertilization leads to sperm chromatin remodeling, during which protamines are expelled and replaced by histones. Despite our increased understanding of the factors that mediate nucleosome assembly in the nascent male pronucleus, the machinery for protamine removal remains largely unknown. Here we identify four Drosophila protamine chaperones that mediate the dissociation of protamine–DNA complexes: NAP-1, NLP, and nucleophosmin are previously characterized histone chaperones, and TAP/p32 has no known function in chromatin metabolism. We show that TAP/p32 is required for the removal of Drosophila protamine B in vitro, whereas NAP-1, NLP, and Nph share roles in the removal of protamine A. Embryos from P32-null females show defective formation of the male pronucleus in vivo. TAP/p32, similar to NAP-1, NLP, and Nph, facilitates nucleosome assembly in vitro and is therefore a histone chaperone. Furthermore, mutants of P32, Nlp, and Nph exhibit synthetic-lethal genetic interactions. In summary, we identified factors mediating protamine removal from DNA and reconstituted in a defined system the process of sperm chromatin remodeling that exchanges protamines for histones to form the nucleosome-based chromatin characteristic of somatic cells. PMID:25228646

The yeast prefoldin-like URI-orthologue Bud27 associates with the RSC nucleosome remodeler and modulates transcription

PubMed Central

Mirón-García, María Carmen; Garrido-Godino, Ana Isabel; Martínez-Fernández, Verónica; Fernández-Pevida, Antonio; Cuevas-Bermúdez, Abel; Martín-Expósito, Manuel; Chávez, Sebastián; de la Cruz, Jesús; Navarro, Francisco

2014-01-01

Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATP-independent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity. PMID:25081216

Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells.

PubMed

Heo, Su-Jin; Han, Woojin M; Szczesny, Spencer E; Cosgrove, Brian D; Elliott, Dawn M; Lee, David A; Duncan, Randall L; Mauck, Robert L

2016-08-23

Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in chromatin condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in chromatin condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced chromatin condensation. With loading, ATP release occurred very rapidly (within the first 10-20 s), whereas changes in chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that chromatin condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

The chromatin remodeling complex Swi/Snf regulates splicing of meiotic transcripts in Saccharomyces cerevisiae

PubMed Central

Douglass, Stephen; Galivanche, Anoop R.

2017-01-01

Abstract Despite its relatively streamlined genome, there are important examples of regulated RNA splicing in Saccharomyces cerevisiae, such as splicing of meiotic transcripts. Like other eukaryotes, S. cerevisiae undergoes a dramatic reprogramming of gene expression during meiosis, including regulated splicing of a number of crucial meiosis-specific RNAs. Splicing of a subset of these is dependent upon the splicing activator Mer1. Here we show a crucial role for the chromatin remodeler Swi/Snf in regulation of splicing of meiotic genes and find that the complex affects meiotic splicing in two ways. First, we show that Swi/Snf regulates nutrient-dependent downregulation of ribosomal protein encoding RNAs, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs (the ribosomal protein genes) to Mer1-regulated transcripts. We also demonstrate that Mer1 expression is dependent on Snf2, its acetylation state and histone H3 lysine 9 acetylation at the MER1 locus. Hence, Snf2 exerts systems level control of meiotic gene expression through two temporally distinct mechanisms, demonstrating that it is a key regulator of meiotic splicing in S. cerevisiae. We also reveal an evolutionarily conserved mechanism whereby the cell redirects its energy from maintaining its translational capacity to the process of meiosis. PMID:28637241

Wongabel rhabdovirus accessory protein U3 targets the SWI/SNF chromatin remodeling complex.

PubMed

Joubert, D Albert; Rodriguez-Andres, Julio; Monaghan, Paul; Cummins, Michelle; McKinstry, William J; Paradkar, Prasad N; Moseley, Gregory W; Walker, Peter J

2015-01-15

Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G, and L), the 13.2-kb negative-sense single-stranded RNA (ssRNA) WONV genome contains five uncharacterized accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3), and a fifth one overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-amino-acid (aa) C-terminal domain of SNF5, a component of the SWI/SNF chromatin remodeling complex. The interaction was confirmed by affinity chromatography, and nuclear colocalization was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are upregulated during infection of mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knockdown results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine gene CSF1. The data suggest that WONV U3 targets the SWI/SNF complex to block the host response to infection. The rhabdoviruses comprise a large family of RNA viruses infecting plants, vertebrates, and invertebrates. In addition to the major structural proteins (N, P, M, G, and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the nucleus and interacts

Wongabel Rhabdovirus Accessory Protein U3 Targets the SWI/SNF Chromatin Remodeling Complex

PubMed Central

Joubert, D. Albert; Rodriguez-Andres, Julio; Monaghan, Paul; Cummins, Michelle; McKinstry, William J.; Paradkar, Prasad N.; Moseley, Gregory W.

2014-01-01

ABSTRACT Wongabel virus (WONV) is an arthropod-borne rhabdovirus that infects birds. It is one of the growing array of rhabdoviruses with complex genomes that encode multiple accessory proteins of unknown function. In addition to the five canonical rhabdovirus structural protein genes (N, P, M, G, and L), the 13.2-kb negative-sense single-stranded RNA (ssRNA) WONV genome contains five uncharacterized accessory genes, one overlapping the N gene (Nx or U4), three located between the P and M genes (U1 to U3), and a fifth one overlapping the G gene (Gx or U5). Here we show that WONV U3 is expressed during infection in insect and mammalian cells and is required for efficient viral replication. A yeast two-hybrid screen against a mosquito cell cDNA library identified that WONV U3 interacts with the 83-amino-acid (aa) C-terminal domain of SNF5, a component of the SWI/SNF chromatin remodeling complex. The interaction was confirmed by affinity chromatography, and nuclear colocalization was established by confocal microscopy. Gene expression studies showed that SNF5 transcripts are upregulated during infection of mosquito cells with WONV, as well as West Nile virus (Flaviviridae) and bovine ephemeral fever virus (Rhabdoviridae), and that SNF5 knockdown results in increased WONV replication. WONV U3 also inhibits SNF5-regulated expression of the cytokine gene CSF1. The data suggest that WONV U3 targets the SWI/SNF complex to block the host response to infection. IMPORTANCE The rhabdoviruses comprise a large family of RNA viruses infecting plants, vertebrates, and invertebrates. In addition to the major structural proteins (N, P, M, G, and L), many rhabdoviruses encode a diverse array of accessory proteins of largely unknown function. Understanding the role of these proteins may reveal much about host-pathogen interactions in infected cells. Here we examine accessory protein U3 of Wongabel virus, an arthropod-borne rhabdovirus that infects birds. We show that U3 enters the

Aging by epigenetics-A consequence of chromatin damage?

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

Sedivy, John M.; Banumathy, Gowrishankar; Adams, Peter D.

Chromatin structure is not fixed. Instead, chromatin is dynamic and is subject to extensive developmental and age-associated remodeling. In some cases, this remodeling appears to counter the aging and age-associated diseases, such as cancer, and extend organismal lifespan. However, stochastic non-deterministic changes in chromatin structure might, over time, also contribute to the break down of nuclear, cell and tissue function, and consequently aging and age-associated diseases.

Regulating the chromatin landscape: structural and mechanistic perspectives.

PubMed

Bartholomew, Blaine

2014-01-01

A large family of chromatin remodelers that noncovalently modify chromatin is crucial in cell development and differentiation. They are often the targets of cancer, neurological disorders, and other human diseases. These complexes alter nucleosome positioning, higher-order chromatin structure, and nuclear organization. They also assemble chromatin, exchange out histone variants, and disassemble chromatin at defined locations. We review aspects of the structural organization of these complexes, the functional properties of their protein domains, and variation between complexes. We also address the mechanistic details of these complexes in mobilizing nucleosomes and altering chromatin structure. A better understanding of these issues will be vital for further analyses of subunits of these chromatin remodelers, which are being identified as targets in human diseases by NGS (next-generation sequencing).

Chromatin Remodeling Factors Isw2 and Ino80 Regulate Checkpoint Activity and Chromatin Structure in S Phase

PubMed Central

Lee, Laura; Rodriguez, Jairo; Tsukiyama, Toshio

2015-01-01

When cells undergo replication stress, proper checkpoint activation and deactivation are critical for genomic stability and cell survival and therefore must be highly regulated. Although mechanisms of checkpoint activation are well studied, mechanisms of checkpoint deactivation are far less understood. Previously, we reported that chromatin remodeling factors Isw2 and Ino80 attenuate the S-phase checkpoint activity in Saccharomyces cerevisiae, especially during recovery from hydroxyurea. In this study, we found that Isw2 and Ino80 have a more pronounced role in attenuating checkpoint activity during late S phase in the presence of methyl methanesulfonate (MMS). We therefore screened for checkpoint factors required for Isw2 and Ino80 checkpoint attenuation in the presence of MMS. Here we demonstrate that Isw2 and Ino80 antagonize checkpoint activators and attenuate checkpoint activity in S phase in MMS either through a currently unknown pathway or through RPA. Unexpectedly, we found that Isw2 and Ino80 increase chromatin accessibility around replicating regions in the presence of MMS through a novel mechanism. Furthermore, through growth assays, we provide additional evidence that Isw2 and Ino80 partially counteract checkpoint activators specifically in the presence of MMS. Based on these results, we propose that Isw2 and Ino80 attenuate S-phase checkpoint activity through a novel mechanism. PMID:25701287

Kinases and chromatin structure

PubMed Central

Miotto, Benoit

2013-01-01

Chromatin structure is regulated by families of proteins that are able to covalently modify the histones and the DNA, as well as to regulate the spacing of nucleosomes along the DNA. Over the years, these chromatin remodeling factors have been proven to be essential to a variety of processes, including gene expression, DNA replication, and chromosome cohesion. The function of these remodeling factors is regulated by a number of chemical and developmental signals and, in turn, changes in the chromatin structure eventually contribute to the response to changes in the cellular environment. Exciting new research findings by the laboratories of Sharon Dent and Steve Jackson indicate, in two different contexts, that changes in the chromatin structure may, in reverse, signal to intracellular signaling pathways to regulate cell fate. The discoveries clearly challenge our traditional view of ‘epigenetics’, and may have important implications in human health. PMID:23917692

Drosophila TAP/p32 is a core histone chaperone that cooperates with NAP-1, NLP, and nucleophosmin in sperm chromatin remodeling during fertilization.

PubMed

Emelyanov, Alexander V; Rabbani, Joshua; Mehta, Monika; Vershilova, Elena; Keogh, Michael C; Fyodorov, Dmitry V

2014-09-15

Nuclear DNA in the male gamete of sexually reproducing animals is organized as sperm chromatin compacted primarily by sperm-specific protamines. Fertilization leads to sperm chromatin remodeling, during which protamines are expelled and replaced by histones. Despite our increased understanding of the factors that mediate nucleosome assembly in the nascent male pronucleus, the machinery for protamine removal remains largely unknown. Here we identify four Drosophila protamine chaperones that mediate the dissociation of protamine-DNA complexes: NAP-1, NLP, and nucleophosmin are previously characterized histone chaperones, and TAP/p32 has no known function in chromatin metabolism. We show that TAP/p32 is required for the removal of Drosophila protamine B in vitro, whereas NAP-1, NLP, and Nph share roles in the removal of protamine A. Embryos from P32-null females show defective formation of the male pronucleus in vivo. TAP/p32, similar to NAP-1, NLP, and Nph, facilitates nucleosome assembly in vitro and is therefore a histone chaperone. Furthermore, mutants of P32, Nlp, and Nph exhibit synthetic-lethal genetic interactions. In summary, we identified factors mediating protamine removal from DNA and reconstituted in a defined system the process of sperm chromatin remodeling that exchanges protamines for histones to form the nucleosome-based chromatin characteristic of somatic cells. © 2014 Emelyanov et al.; Published by Cold Spring Harbor Laboratory Press.

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

Functions of Fun30 Chromatin Remodeler in Regulating Cellular Resistance to Genotoxic Stress

PubMed Central

Bi, Xin; Yu, Qun; Siler, Jasmine; Li, Chong; Khan, Ali

2015-01-01

The Saccharomyces cerevisiae Fun30 chromatin remodeler has recently been shown to facilitate long-range resection of DNA double strand break (DSB) ends, which proceeds homologous recombination (HR). This is believed to underlie the role of Fun30 in promoting cellular resistance to DSB inducing agent camptothecin. We show here that Fun30 also contributes to cellular resistance to genotoxins methyl methanesulfonate (MMS) and hydroxyurea (HU) that can stall the progression of DNA replication. We present evidence implicating DNA end resection in Fun30-dependent MMS-resistance. On the other hand, we show that Fun30 deletion suppresses the MMS- and HU-sensitivity of cells lacking the Rad5/Mms2/Ubc13-dependent error-free DNA damage tolerance mechanism. This suppression is not the result of a reduction in DNA end resection, and is dependent on the key HR component Rad51. We further show that Fun30 negatively regulates the recovery of rad5Δ mutant from MMS induced G2/M arrest. Therefore, Fun30 has two functions in DNA damage repair: one is the promotion of cellular resistance to genotoxic stress by aiding in DNA end resection, and the other is the negative regulation of a Rad51-dependent, DNA end resection-independent mechanism for countering replicative stress. The latter becomes manifest when Rad5 dependent DNA damage tolerance is impaired. In addition, we find that the putative ubiquitin-binding CUE domain of Fun30 serves to restrict the ability of Fun30 to hinder MMS- and HU-tolerance in the absence of Rad5. PMID:25806814

Engagement of Arginine Finger to ATP Triggers Large Conformational Changes in NtrC1 AAA+ ATPase for Remodeling Bacterial RNA Polymerase

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

Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat

The NtrC-like AAA+ ATPases control virulence and other important bacterial activities through delivering mechanical work to {sigma}54-RNA polymerase to activate transcription from {sigma}54-dependent genes. We report the first crystal structure for such an ATPase, NtrC1 of Aquifex aeolicus, in which the catalytic arginine engages the {gamma}-phosphate of ATP. Comparing the new structure with those previously known for apo and ADP-bound states supports a rigid-body displacement model that is consistent with large-scale conformational changes observed by low-resolution methods. First, the arginine finger induces rigid-body roll, extending surface loops above the plane of the ATPase ring to bind {sigma}54. Second, ATP hydrolysismore » permits Pi release and retraction of the arginine with a reversed roll, remodeling {sigma}54-RNAP. This model provides a fresh perspective on how ATPase subunits interact within the ring-ensemble to promote transcription, directing attention to structural changes on the arginine-finger side of an ATP-bound interface.« less

LINE-1 silencing by retinoblastoma proteins is effected through the nucleosomal and remodeling deacetylase multiprotein complex.

PubMed

Montoya-Durango, Diego E; Ramos, Kenneth A; Bojang, Pasano; Ruiz, Lorell; Ramos, Irma N; Ramos, Kenneth S

2016-01-25

Long Interspersed Nuclear Element-1 (L1) is an oncogenic mammalian retroelement silenced early in development via tightly controlled epigenetic mechanisms. We have previously shown that the regulatory region of human and murine L1s interact with retinoblastoma (RB) proteins to effect retroelement silencing. The present studies were conducted to identify the corepressor complex responsible for RB-mediated silencing of L1. Chromatin immunoprecipitation and silencing RNA technology were used to identify the repressor complex that silences L1 in human and murine cells. Components of the Nucleosomal and Remodeling Deacetylase (NuRD) multiprotein complex specifically enriched the L1 5'-untranslated DNA sequence in human and murine cells. Genetic ablation of RB proteins in murine cells destabilized interactions within the NuRD macromolecular complex and mediated nuclear rearrangement of Mi2-β, an ATP-dependent helicase subunit with nucleosome remodeling activity. Depletion of Mi2-β, RbAP46 and HDAC2 reduced the repressor activity of the NuRD complex and reactivated a synthetic L1 reporter in human cells. Epigenetic reactivation of L1 in RB-null cells by DNA damage was markedly enhanced compared to wild type cells. RB proteins stabilize interactions of the NuRD corepressor complex within the L1 promoter to effect L1 silencing. L1 retroelements may serve as a scaffold on which RB builds heterochromatic regions that regulate chromatin function.

SMARCA4 regulates gene expression and higher-order chromatin structure in proliferating mammary epithelial cells

PubMed Central

Barutcu, A. Rasim; Lajoie, Bryan R.; Fritz, Andrew J.; McCord, Rachel P.; Nickerson, Jeffrey A.; van Wijnen, Andre J.; Lian, Jane B.; Stein, Janet L.; Dekker, Job; Stein, Gary S.; Imbalzano, Anthony N.

2016-01-01

The packaging of DNA into chromatin plays an important role in transcriptional regulation and nuclear processes. Brahma-related gene-1 SMARCA4 (also known as BRG1), the essential ATPase subunit of the mammalian SWI/SNF chromatin remodeling complex, uses the energy from ATP hydrolysis to disrupt nucleosomes at target regions. Although the transcriptional role of SMARCA4 at gene promoters is well-studied, less is known about its role in higher-order genome organization. SMARCA4 knockdown in human mammary epithelial MCF-10A cells resulted in 176 up-regulated genes, including many related to lipid and calcium metabolism, and 1292 down-regulated genes, some of which encode extracellular matrix (ECM) components that can exert mechanical forces and affect nuclear structure. ChIP-seq analysis of SMARCA4 localization and SMARCA4-bound super-enhancers demonstrated extensive binding at intergenic regions. Furthermore, Hi-C analysis showed extensive SMARCA4-mediated alterations in higher-order genome organization at multiple resolutions. First, SMARCA4 knockdown resulted in clustering of intra- and inter-subtelomeric regions, demonstrating a novel role for SMARCA4 in telomere organization. SMARCA4 binding was enriched at topologically associating domain (TAD) boundaries, and SMARCA4 knockdown resulted in weakening of TAD boundary strength. Taken together, these findings provide a dynamic view of SMARCA4-dependent changes in higher-order chromatin organization and gene expression, identifying SMARCA4 as a novel component of chromatin organization. PMID:27435934

DDB2 promotes chromatin decondensation at UV-induced DNA damage

PubMed Central

Lindh, Michael; Acs, Klara; Vrouwe, Mischa G.; Pines, Alex; van Attikum, Haico; Mullenders, Leon H.

2012-01-01

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A–RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)–dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]–ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains. PMID:22492724

Transcription coactivator SAYP combines chromatin remodeler Brahma and transcription initiation factor TFIID into a single supercomplex

PubMed Central

Vorobyeva, Nadezhda E.; Soshnikova, Nataliya V.; Nikolenko, Julia V.; Kuzmina, Julia L.; Nabirochkina, Elena N.; Georgieva, Sofia G.; Shidlovskii, Yulii V.

2009-01-01

Transcription activation by RNA polymerase II is a complicated process driven by combined, precisely coordinated action of a wide array of coactivator complexes, which carry out chromatin-directed activities and nucleate the assembly of the preinitiation complex on the promoter. Using various techniques, we have shown the existence of a stable coactivator supercomplex consisting of the chromatin-remodeling factor Brahma (SWI/SNF) and the transcription initiation factor TFIID, named BTFly (Brahma and TFIID in one assembly). The coupling of Brahma and TFIID is mediated by the SAYP factor, whose evolutionarily conserved activation domain SAY can directly bind to both BAP170 subunit of Brahma and TAF5 subunit of TFIID. The integrity of BTFly is crucial for its ability to activate transcription. BTFly is distributed genome-wide and appears to be a means of effective transcription activation. PMID:19541607

Assembly of transcriptionally inactive chromatin in vitro.

PubMed

Shanahan, M M; Kmiec, E B

1989-07-01

We have successfully uncoupled the previously interlocked activities of chromatin assembly and in vitro transcription promoted by the Xenopus oocyte S-150 cell-free extract. Our isolated fraction catalyzes extensive chromatin assembly measured both by changes in DNA topology and Micrococcal nuclease digestions. The assembly of chromatin is slowed by the exogenous addition of ATP. In the absence of exogenously added ATP, the fraction forms a chromatin template that is transcriptionally inert. Addition of small amounts of the HeLa cell extract (S-100) converts these templates into transcriptionally active ones without disrupting the chromatin structure. Our protocol defines a method for the isolation of a fraction from the Xenopus cell free extract that catalyzes the assembly of transcriptionally inactive chromatin. We characterize this reaction and establish conditions for the transcriptional activation of these inactive minichromosomes.

SHORT HYPOCOTYL1 Encodes a SMARCA3-Like Chromatin Remodeling Factor Regulating Elongation1[OPEN

PubMed Central

Bo, Kailiang; Behera, Tusar K.; Pandey, Sudhakar; Wen, Changlong; Wang, Yuhui; Simon, Philipp W.; Li, Yuhong

2016-01-01

In Arabidopsis (Arabidopsis thaliana), the UVR8-mediated signaling pathway is employed to attain UVB protection and acclimation to deal with low-dosage UVB (LDUVB)-induced stresses. Here, we identified SHORT HYPOCOTYL1 (SH1) in cucumber (Cucumis sativus), which regulates LDUVB-dependent hypocotyl elongation by modulating the UVR8 signaling pathway. We showed that hypocotyl elongation in cucumbers carrying the recessive sh1 allele was LDUVB insensitive and that Sh1 encoded a human SMARCA3-like chromatin remodeling factor. The allele frequency and distribution pattern at this locus among natural populations supported the wild cucumber origin of sh1 for local adaptation, which was under selection during domestication. The cultivated cucumber carries predominantly the Sh1 allele; the sh1 allele is nearly fixed in the semiwild Xishuangbanna cucumber, and the wild cucumber population is largely at Hardy-Weinberg equilibrium for the two alleles. The SH1 protein sequence was highly conserved among eukaryotic organisms, but its regulation of hypocotyl elongation in cucumber seems to be a novel function. While Sh1 expression was inhibited by LDUVB, its transcript abundance was highly correlated with hypocotyl elongation rate and the expression level of cell-elongation-related genes. Expression profiling of key regulators in the UVR8 signaling pathway revealed significant differential expression of CsHY5 between two near isogenic lines of Sh1. Sh1 and CsHY5 acted antagonistically at transcriptional level. A working model was proposed in which Sh1 regulates LDUVB-dependent hypocotyl elongation in cucumber through changing the chromatin states and thus the accessibility of CsHY5 in the UVR8 signaling pathway to promoters of LDUVB-responsive genes for hypocotyl elongation. PMID:27559036

ATP Dependence of Na+/H+ Exchange

PubMed Central

Demaurex, Nicolas; Romanek, Robert R.; Orlowski, John; Grinstein, Sergio

1997-01-01

We studied the ATP dependence of NHE-1, the ubiquitous isoform of the Na+/H+ antiporter, using the whole-cell configuration of the patch-clamp technique to apply nucleotides intracellularly while measuring cytosolic pH (pHi) by microfluorimetry. Na+/H+ exchange activity was measured as the Na+-driven pHi recovery from an acid load, which was imposed via the patch pipette. In Chinese hamster ovary (CHO) fibroblasts stably transfected with NHE-1, omission of ATP from the pipette solution inhibited Na+/H+ exchange. Conversely, ATP perfusion restored exchange activity in cells that had been metabolically depleted by 2-deoxy-d-glucose and oligomycin. In cells dialyzed in the presence of ATP, no “run-down” was observed even after extended periods, suggesting that the nucleotide is the only diffusible factor required for optimal NHE-1 activity. Half-maximal activation of the antiporter was obtained at ∼5 mM Mg-ATP. Submillimolar concentrations failed to sustain Na+/H+ exchange even when an ATP regenerating system was included in the pipette solution. High ATP concentrations are also known to be required for the optimal function of other cation exchangers. In the case of the Na/Ca2+ exchanger, this requirement has been attributed to an aminophospholipid translocase, or “flippase.” The involvement of this enzyme in Na+/H+ exchange was examined using fluorescent phosphatidylserine, which is actively translocated by the flippase. ATP depletion decreased the transmembrane uptake of NBD-labeled phosphatidylserine (NBD-PS), indicating that the flippase was inhibited. Diamide, an agent reported to block the flippase, was as potent as ATP depletion in reducing NBD-PS uptake. However, diamide had no effect on Na+/H+ exchange, implying that the effect of ATP is not mediated by changes in lipid distribution across the plasma membrane. K-ATP and ATPγS were as efficient as Mg-ATP in sustaining NHE-1 activity, while AMP-PNP and AMP-PCP only partially substituted for ATP. In

A Brg1 mutation that uncouples ATPase activity from chromatin remodeling reveals an essential role for SWI/SNF-related complexes in β-globin expression and erythroid development

PubMed Central

Bultman, Scott J.; Gebuhr, Thomas C.; Magnuson, Terry

2005-01-01

The Brg1 catalytic subunit of SWI/SNF-related complexes has been implicated in many developmental and physiological processes, but null homozygotes die as blastocysts prior to implantation. To circumvent this early embryonic lethality, we performed an ENU mutagenesis screen and generated a Brg1 hypomorph mutation in the ATPase domain. The mutant Brg1 protein is stable, assembles into SWI/SNF-related complexes, and exhibits normal ATPase activity but is unable to establish DNase I hypersensitivity sites characteristic of open chromatin. Mutant embryos develop normally until midgestation but then exhibit a distinct block in the development of the erythroid lineage, leading to anemia and death. The mutant Brg1 protein is recruited to the β-globin locus, but chromatin remodeling and transcription are perturbed. Histone acetylation and DNA methylation are also affected. To our knowledge, Brg1 is the first chromatin-modifying factor shown to be required for β-globin regulation and erythropoiesis in vivo. Not only does this mutation establish a role for Brg1 during organogenesis, it also demonstrates that ATPase activity can be uncoupled from chromatin remodeling. PMID:16287714

The yeast prefoldin-like URI-orthologue Bud27 associates with the RSC nucleosome remodeler and modulates transcription.

PubMed

Mirón-García, María Carmen; Garrido-Godino, Ana Isabel; Martínez-Fernández, Verónica; Fernández-Pevida, Antonio; Cuevas-Bermúdez, Abel; Martín-Expósito, Manuel; Chávez, Sebastián; de la Cruz, Jesús; Navarro, Francisco

2014-09-01

Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATP-independent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

New Face for Chromatin-Related Mesenchymal Modulator: n-CHD9 Localizes to Nucleoli and Interacts With Ribosomal Genes.

PubMed

Salomon-Kent, Ronit; Marom, Ronit; John, Sam; Dundr, Miroslav; Schiltz, Louis R; Gutierrez, Jose; Workman, Jerry; Benayahu, Dafna; Hager, Gordon L

2015-09-01

Mesenchymal stem cells' differentiation into several lineages is coordinated by a complex of transcription factors and co-regulators which bind to specific gene promoters. The Chromatin-Related Mesenchymal Modulator, CHD9 demonstrated in vitro its ability for remodeling activity to reposition nucleosomes in an ATP-dependent manner. Epigenetically, CHD9 binds with modified H3-(K9me2/3 and K27me3). Previously, we presented a role for CHD9 with RNA Polymerase II (Pol II)-dependent transcription of tissue specific genes. Far less is known about CHD9 function in RNA Polymerase I (Pol I) related transcription of the ribosomal locus that also drives specific cell fate. We here describe a new form, the nucleolar CHD9 (n-CHD9) that is dynamically associated with Pol I, fibrillarin, and upstream binding factor (UBF) in the nucleoli, as shown by imaging and molecular approaches. Inhibitors of transcription disorganized the nucleolar compartment of transcription sites where rDNA is actively transcribed. Collectively, these findings link n-CHD9 with RNA pol I transcription in fibrillar centers. Using chromatin immunoprecipitation (ChIP) and tilling arrays (ChIP- chip), we find an association of n-CHD9 with Pol I related to rRNA biogenesis. Our new findings support the role for CHD9 in chromatin regulation and association with rDNA genes, in addition to its already known function in transcription control of tissue specific genes. © 2015 Wiley Periodicals, Inc.

Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly

PubMed Central

Chereji, Răzvan V.; Bharatula, Vasudha; Elfving, Nils; Blomberg, Jeanette; Larsson, Miriam; Morozov, Alexandre V.; Broach, James R.

2017-01-01

Abstract Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Saccharomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3′-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization. PMID:28575439

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

PubMed Central

Singh, Ajeet Pratap; Archer, Trevor K.

2014-01-01

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

Human ISWI complexes are targeted by SMARCA5 ATPase and SLIDE domains to help resolve lesion-stalled transcription

PubMed Central

Aydin, Özge Z.; Marteijn, Jurgen A.; Ribeiro-Silva, Cristina; Rodríguez López, Aida; Wijgers, Nils; Smeenk, Godelieve; van Attikum, Haico; Poot, Raymond A.; Vermeulen, Wim; Lans, Hannes

2014-01-01

Chromatin compaction of deoxyribonucleic acid (DNA) presents a major challenge to the detection and removal of DNA damage. Helix-distorting DNA lesions that block transcription are specifically repaired by transcription-coupled nucleotide excision repair, which is initiated by binding of the CSB protein to lesion-stalled RNA polymerase II. Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription. Human ISWI isoform SMARCA5/SNF2H and its binding partners ACF1 and WSTF are rapidly recruited to UV-C induced DNA damage to specifically facilitate CSB binding and to promote transcription recovery. SMARCA5 targeting to UV-C damage depends on transcription and histone modifications and requires functional SWI2/SNF2-ATPase and SLIDE domains. After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain. Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA. PMID:24990377

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators

PubMed Central

Polstein, Lauren R.; Perez-Pinera, Pablo; Kocak, D. Dewran; Vockley, Christopher M.; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E.; Reddy, Timothy E.; Gersbach, Charles A.

2015-01-01

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. PMID:26025803

The ''self-stirred'' genome: Bulk and surface dynamics of the chromatin globule

NASA Astrophysics Data System (ADS)

Zidovska, Alexandra

Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood. In interphase, time between two cell divisions, chromatin fills the cell nucleus in its minimally condensed polymeric state. Chromatin serves as substrate to a number of biological processes, e.g. gene expression and DNA replication, which require it to become locally restructured. These are energy-consuming processes giving rise to non-equilibrium dynamics. Chromatin dynamics has been traditionally studied by imaging of fluorescently labeled nuclear proteins and single DNA-sites, thus focusing only on a small number of tracer particles. Recently, we developed an approach, displacement correlation spectroscopy (DCS) based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. DCS revealed that chromatin movement was coherent across large regions (4-5 μm) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP-dependent and unidirectional for several seconds. Following these observations, we developed a hydrodynamic theory of active chromatin dynamics, using the two-fluid model and describing the content of cell nucleus as a chromatin solution, which is subject to both passive thermal fluctuations and active (ATP-consuming) scalar and vector events. In this work we continue in our efforts to elucidate the mechanism and function of the chromatin dynamics in interphase. We investigate the chromatin interactions with the nuclear envelope and compare the surface dynamics of the chromatin globule with its bulk dynamics.

Haploinsufficiency of ARID1B, a Member of the SWI/SNF-A Chromatin-Remodeling Complex, Is a Frequent Cause of Intellectual Disability

PubMed Central

Hoyer, Juliane; Ekici, Arif B.; Endele, Sabine; Popp, Bernt; Zweier, Christiane; Wiesener, Antje; Wohlleber, Eva; Dufke, Andreas; Rossier, Eva; Petsch, Corinna; Zweier, Markus; Göhring, Ina; Zink, Alexander M.; Rappold, Gudrun; Schröck, Evelin; Wieczorek, Dagmar; Riess, Olaf; Engels, Hartmut; Rauch, Anita; Reis, André

2012-01-01

Intellectual disability (ID) is a clinically and genetically heterogeneous common condition that remains etiologically unresolved in the majority of cases. Although several hundred diseased genes have been identified in X-linked, autosomal-recessive, or syndromic types of ID, the establishment of an etiological basis remains a difficult task in unspecific, sporadic cases. Just recently, de novo mutations in SYNGAP1, STXBP1, MEF2C, and GRIN2B were reported as relatively common causes of ID in such individuals. On the basis of a patient with severe ID and a 2.5 Mb microdeletion including ARID1B in chromosomal region 6q25, we performed mutational analysis in 887 unselected patients with unexplained ID. In this cohort, we found eight (0.9%) additional de novo nonsense or frameshift mutations predicted to cause haploinsufficiency. Our findings indicate that haploinsufficiency of ARID1B, a member of the SWI/SNF-A chromatin-remodeling complex, is a common cause of ID, and they add to the growing evidence that chromatin-remodeling defects are an important contributor to neurodevelopmental disorders. PMID:22405089

Active PHO5 chromatin encompasses variable numbers of nucleosomes at individual promoters.

PubMed

Jessen, Walter J; Hoose, Scott A; Kilgore, Jessica A; Kladde, Michael P

2006-03-01

Transcriptional activation is often associated with chromatin remodeling. However, little is known about the dynamics of remodeling of nucleosome arrays in vivo. Upon induction of Saccharomyces cerevisiae PHO5, a novel kinetic assay of DNA methyltransferase accessibility showed that nucleosomes adjacent to the histone-free upstream activating sequence (UASp1) are disrupted earlier and at higher frequency in the cell population than are those more distal. Individually cloned molecules, each representing the chromatin state of a full promoter from a single cell, revealed multiple promoter classes with either no remodeling or variable numbers of disrupted nucleosomes. Individual promoters in the remodeled fraction were highly enriched for contiguous blocks of disrupted nucleosomes, the majority of which overlapped the UAS region. These results support a probabilistic model in which chromatin remodeling at PHO5 spreads from sites of transactivator association with DNA and attenuates with distance.

Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly.

PubMed

Chereji, Razvan V; Bharatula, Vasudha; Elfving, Nils; Blomberg, Jeanette; Larsson, Miriam; Morozov, Alexandre V; Broach, James R; Björklund, Stefan

2017-09-06

Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Saccharomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Transient Rise in Free Mg2+ Ions Released from ATP-Mg Hydrolysis Contributes to Mitotic Chromosome Condensation.

PubMed

Maeshima, Kazuhiro; Matsuda, Tomoki; Shindo, Yutaka; Imamura, Hiromi; Tamura, Sachiko; Imai, Ryosuke; Kawakami, Syoji; Nagashima, Ryosuke; Soga, Tomoyoshi; Noji, Hiroyuki; Oka, Kotaro; Nagai, Takeharu

2018-02-05

For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1-5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appropriate chromosome shape [6-17], free divalent cations such as Mg 2+ and Ca 2+ , which condense chromatin or chromosomes in vitro [18-28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like "beads on a string" by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracellular free divalent cations, but not total cations [30], especially Mg 2+ , have prevented us from elucidating their function. Here, we developed a Förster resonance energy transfer (FRET)-based Mg 2+ indicator that monitors free Mg 2+ dynamics throughout the cell cycle. By combining this indicator with Ca 2+ [31] and adenosine triphosphate (ATP) [32] indicators, we demonstrate that the levels of free Mg 2+ , but not Ca 2+ , increase during mitosis. The Mg 2+ increase is coupled with a decrease in ATP, which is normally bound to Mg 2+ in the cell [33]. ATP inhibited Mg 2+ -dependent chromatin condensation in vitro. Chelating Mg 2+ induced mitotic cell arrest and chromosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP-bound Mg 2+ is released by ATP hydrolysis and contributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg 2+ -ATP balance. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Environmental-stress-induced Chromatin Regulation and its Heritability

PubMed Central

Fang, Lei; Wuptra, Kenly; Chen, Danqi; Li, Hongjie; Huang, Shau-Ku; Jin, Chunyuan; Yokoyama, Kazunari K

2014-01-01

Chromatin is subject to proofreading and repair mechanisms during the process of DNA replication, as well as repair to maintain genetic and epigenetic information and genome stability. The dynamic structure of chromatin modulates various nuclear processes, including transcription and replication, by altering the accessibility of the DNA to regulatory factors. Structural changes in chromatin are affected by the chemical modification of histone proteins and DNA, remodeling of nucleosomes, incorporation of variant histones, noncoding RNAs, and nonhistone DNA-binding proteins. Phenotypic diversity and fidelity can be balanced by controlling stochastic switching of chromatin structure and dynamics in response to the environmental disruptors and endogenous stresses. The dynamic chromatin remodeling can, therefore, serve as a sensor, through which environmental and/or metabolic agents can alter gene expression, leading to global cellular changes involving multiple interactive networks. Furthermore its recent evidence also suggests that the epigenetic changes are heritable during the development. This review will discuss the environmental sensing system for chromatin regulation and genetic and epigenetic controls from developmental perspectives. PMID:25045581

Cardiac Metabolism in Heart Failure - Implications beyond ATP production

PubMed Central

Doenst, Torsten; Nguyen, T. Dung; Abel, E. Dale

2013-01-01

The heart has a high rate of ATP production and turnover which is required to maintain its continuous mechanical work. Perturbations in ATP generating processes may therefore affect contractile function directly. Characterizing cardiac metabolism in heart failure revealed several metabolic alterations termed metabolic remodeling, ranging from changes in substrate utilization to mitochondrial dysfunction, ultimately resulting in ATP deficiency and impaired contractility. However, ATP depletion is not the only relevant consequence of metabolic remodeling during heart failure. By providing cellular building blocks and signaling molecules, metabolic pathways control essential processes such as cell growth and regeneration. Thus, alterations in cardiac metabolism may also affect the progression to heart failure by mechanisms beyond ATP supply. Our aim is therefore to highlight that metabolic remodeling in heart failure not only results in impaired cardiac energetics, but also induces other processes implicated in the development of heart failure such as structural remodeling and oxidative stress. Accordingly, modulating cardiac metabolism in heart failure may have significant therapeutic relevance that goes beyond the energetic aspect. PMID:23989714

Quantitative Dynamics of Chromatin Remodeling during Germ Cell Specification from Mouse Embryonic Stem Cells.

PubMed

Kurimoto, Kazuki; Yabuta, Yukihiro; Hayashi, Katsuhiko; Ohta, Hiroshi; Kiyonari, Hiroshi; Mitani, Tadahiro; Moritoki, Yoshinobu; Kohri, Kenjiro; Kimura, Hiroshi; Yamamoto, Takuya; Katou, Yuki; Shirahige, Katsuhiko; Saitou, Mitinori

2015-05-07

Germ cell specification is accompanied by epigenetic remodeling, the scale and specificity of which are unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome. Copyright © 2015 Elsevier Inc. All rights reserved.

Plant chromatin warms up in Madrid

PubMed Central

Jarillo, José A; Gaudin, Valerie; Hennig, Lars; Köhler, Claudia; Piñeiro, Manuel

2014-01-01

The 3rd European Workshop on Plant Chromatin (EWPC) was held on August 2013 in Madrid, Spain. A number of different topics on plant chromatin were presented during the meeting, including new factors mediating Polycomb Group protein function in plants, chromatin-mediated reprogramming in plant developmental transitions, the role of histone variants, and newly identified chromatin remodeling factors. The function of interactions between chromatin and transcription factors in the modulation of gene expression, the role of chromatin dynamics in the control of nuclear processes and the influence of environmental factors on chromatin organization were also reported. In this report, we highlight some of the new insights emerging in this growing area of research, presented at the 3rd EWPC. PMID:24504145

The BRG1 chromatin remodeling enzyme links cancer cell metabolism and proliferation

PubMed Central

Wu, Qiong; Madany, Pasil; Dobson, Jason R.; Schnabl, Jake M.; Sharma, Soni; Smith, Tara C.; van Wijnen, Andre J.; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.; Muthuswami, Rohini; Imbalzano, Anthony N.; Nickerson, Jeffrey A.

2016-01-01

Cancer cells reprogram cellular metabolism to meet the demands of growth. Identification of the regulatory machinery that regulates cancer-specific metabolic changes may open new avenues for anti-cancer therapeutics. The epigenetic regulator BRG1 is a catalytic ATPase for some mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is a well-characterized tumor suppressor in some human cancers, but is frequently overexpressed without mutation in other cancers, including breast cancer. Here we demonstrate that BRG1 upregulates de novo lipogenesis and that this is crucial for cancer cell proliferation. Knockdown of BRG1 attenuates lipid synthesis by impairing the transcription of enzymes catalyzing fatty acid and lipid synthesis. Remarkably, exogenous addition of palmitate, the key intermediate in fatty acid synthesis, rescued the cancer cell proliferation defect caused by BRG1 knockdown. Our work suggests that targeting BRG1 to reduce lipid metabolism and, thereby, to reduce proliferation, has promise for epigenetic therapy in triple negative breast cancer. PMID:27223259

ATP-Sensitive K+ Channel Knockout Induces Cardiac Proteome Remodeling Predictive of Heart Disease Susceptibility

PubMed Central

Arrell, D. Kent; Zlatkovic, Jelena; Kane, Garvan C.; Yamada, Satsuki; Terzic, Andre

2010-01-01

Forecasting disease susceptibility requires detection of maladaptive signatures prior to onset of overt symptoms. A case-in-point are cardiac ATP-sensitive K+ (KATP) channelopathies, for which the substrate underlying disease vulnerability remains to be identified. Resolving molecular pathobiology, even for single genetic defects, mandates a systems platform to reliably diagnose disease predisposition. High-throughput proteomic analysis was here integrated with network biology to decode consequences of Kir6.2 KATP channel pore deletion. Differential two-dimensional gel electrophoresis reproducibly resolved > 800 protein species from hearts of asymptomatic wild-type and Kir6.2-knockout counterparts. KATP channel ablation remodeled the cardiac proteome, significantly altering 71 protein spots, from which 102 unique identities were assigned following hybrid linear ion trap quadrupole-Orbitrap tandem mass spectrometry. Ontological annotation stratified the KATP channel-dependent protein cohort into a predominant bioenergetic module (63 resolved identities), with additional focused sets representing signaling molecules (6), oxidoreductases (8), chaperones (6), and proteins involved in catabolism (6), cytostructure (8), and transcription and translation (5). Protein interaction mapping, in conjunction with expression level changes, localized a KATP channel-associated subproteome within a nonstochastic scale-free network. Global assessment of the KATP channel deficient environment verified the primary impact on metabolic pathways and revealed overrepresentation of markers associated with cardiovascular disease. Experimental imposition of graded stress precipitated exaggerated structural and functional myocardial defects in the Kir6.2-knockout, decreasing survivorship and validating the forecast of disease susceptibility. Proteomic cartography thus provides an integral view of molecular remodeling in the heart induced by KATP channel deletion, establishing a systems

Chromatin and Transcription in Yeast

PubMed Central

Rando, Oliver J.; Winston, Fred

2012-01-01

Understanding the mechanisms by which chromatin structure controls eukaryotic transcription has been an intense area of investigation for the past 25 years. Many of the key discoveries that created the foundation for this field came from studies of Saccharomyces cerevisiae, including the discovery of the role of chromatin in transcriptional silencing, as well as the discovery of chromatin-remodeling factors and histone modification activities. Since that time, studies in yeast have continued to contribute in leading ways. This review article summarizes the large body of yeast studies in this field. PMID:22345607

Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release.

PubMed

Sabirov, R Z; Dutta, A K; Okada, Y

2001-09-01

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl(-) channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around +/-25 mV. The whole-cell current was selective for anions and sensitive to Gd(3)+. In on-cell patches, single-channel events appeared with a lag period of approximately 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of approximately 400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC(50) of 12.3 mM and an electric distance (delta) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC(50) of 12.9 mM and delta of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with P(ATP)/P(Cl) of 0.09. The single-channel activity was sensitive to Gd(3)+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.

Dietary polyphenols and chromatin remodeling.

PubMed

Russo, Gian Luigi; Vastolo, Viviana; Ciccarelli, Marco; Albano, Luigi; Macchia, Paolo Emidio; Ungaro, Paola

2017-08-13

Polyphenols are the most abundant phytochemicals in fruits, vegetables, and plant-derived beverages. Recent findings suggest that polyphenols display the ability to reverse adverse epigenetic regulation involved in pathological conditions, such as obesity, metabolic disorder, cardiovascular and neurodegenerative diseases, and various forms of cancer. Epigenetics, defined as heritable changes to the transcriptome, independent from those occurring in the genome, includes DNA methylation, histone modifications, and posttranscriptional gene regulation by noncoding RNAs. Sinergistically and cooperatively, these processes regulate gene expression by changing chromatin organization and DNA accessibility. Such induced epigenetic changes can be inherited during cell division, resulting in permanent maintenance of the acquired phenotype, but they may also occur throughout an individual life-course and may ultimately influence phenotypic outcomes (health and disease risk). In the last decade, a number of studies have shown that nutrients can affect metabolic traits by altering the structure of chromatin and directly regulate both transcription and translational processes. In this context, dietary polyphenol-targeted epigenetics becomes an attractive approach for disease prevention and intervention. Here, we will review how polyphenols, including flavonoids, curcuminoids, and stilbenes, modulate the establishment and maintenance of key epigenetic marks, thereby influencing gene expression and, hence, disease risk and health.

Chromatin dynamics in plants.

PubMed

Fransz, Paul F; de Jong, J Hans

2002-12-01

Recent studies in yeast, animals and plants have provided major breakthroughs in unraveling the molecular mechanism of higher-order gene regulation. In conjunction with the DNA code, proteins that are involved in chromatin remodeling, histone modification and epigenetic imprinting form a large network of interactions that control the nuclear programming of cell identity. New insight into how chromatin conformations are regulated in plants sheds light on the relationships between chromosome function, cell differentiation and developmental patterns.

Chromatin in embryonic stem cell neuronal differentiation.

PubMed

Meshorer, E

2007-03-01

Chromatin, the basic regulatory unit of the eukaryotic genetic material, is controlled by epigenetic mechanisms including histone modifications, histone variants, DNA methylation and chromatin remodeling. Cellular differentiation involves large changes in gene expression concomitant with alterations in genome organization and chromatin structure. Such changes are particularly evident in self-renewing pluripotent embryonic stem cells, which begin, in terms of cell fate, as a tabula rasa, and through the process of differentiation, acquire distinct identities. Here I describe the changes in chromatin that accompany neuronal differentiation, particularly of embryonic stem cells, and discuss how chromatin serves as the master regulator of cellular destiny.

Genome-wide specificity of DNA binding, gene regulation, and chromatin remodeling by TALE- and CRISPR/Cas9-based transcriptional activators.

PubMed

Polstein, Lauren R; Perez-Pinera, Pablo; Kocak, D Dewran; Vockley, Christopher M; Bledsoe, Peggy; Song, Lingyun; Safi, Alexias; Crawford, Gregory E; Reddy, Timothy E; Gersbach, Charles A

2015-08-01

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. © 2015 Polstein et al.; Published by Cold Spring Harbor Laboratory Press.

Molecular cloning and characterization of an SRCAP chromatin remodeling homologue in Toxoplasma gondii.

PubMed

Sullivan, William J; Monroy, M Alexandra; Bohne, Wolfgang; Nallani, Karuna C; Chrivia, John; Yaciuk, Peter; Smith, Charles K; Queener, Sherry F

2003-05-01

We have identified and mapped a gene in Toxoplasma gondii that encodes a homologue of SRCAP (Snf2-related CBP activator protein), a member of the SNF/SWI family of chromatin remodeling factors. The genomic locus (TgSRCAP) is present as a single copy and contains 16 introns. The predicted cDNA contains an open reading frame of 8,775 bp and encodes a protein of 2,924 amino acids. We have identified additional SRCAP-like sequences in Apicomplexa for comparison by screening genomic databases. An analysis of SRCAP homologues between species reveals signature features that may be indicative of SRCAP members. Expression of mRNA encoding TgSRCAP is upregulated when tachyzoite (invasive form) parasites are induced to differentiate into bradyzoites (encysted form) in vitro. Recombinant TgSRCAP protein is functionally equivalent to the human homologue, being capable of increasing transcription mediated by CREB.

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

PubMed

Matysiak, Julien; Lesbats, Paul; Mauro, Eric; Lapaillerie, Delphine; Dupuy, Jean-William; Lopez, Angelica P; Benleulmi, Mohamed Salah; Calmels, Christina; Andreola, Marie-Line; Ruff, Marc; Llano, Manuel; Delelis, Olivier; Lavigne, Marc; Parissi, Vincent

2017-07-28

Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

The Chromatin Remodeler SPLAYED Negatively Regulates SNC1-Mediated Immunity.

PubMed

Johnson, Kaeli C M; Xia, Shitou; Feng, Xiaoqi; Li, Xin

2015-08-01

SNC1 (SUPPRESSOR OF NPR1, CONSTITUTIVE 1) is one of a suite of intracellular Arabidopsis NOD-like receptor (NLR) proteins which, upon activation, result in the induction of defense responses. However, the molecular mechanisms underlying NLR activation and the subsequent provocation of immune responses are only partially characterized. To identify negative regulators of NLR-mediated immunity, a forward genetic screen was undertaken to search for enhancers of the dwarf, autoimmune gain-of-function snc1 mutant. To avoid lethality resulting from severe dwarfism, the screen was conducted using mos4 (modifier of snc1, 4) snc1 plants, which display wild-type-like morphology and resistance. M2 progeny were screened for mutant, snc1-enhancing (muse) mutants displaying a reversion to snc1-like phenotypes. The muse9 mos4 snc1 triple mutant was found to exhibit dwarf morphology, elevated expression of the pPR2-GUS defense marker reporter gene and enhanced resistance to the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. Via map-based cloning and Illumina sequencing, it was determined that the muse9 mutation is in the gene encoding the SWI/SNF chromatin remodeler SYD (SPLAYED), and was thus renamed syd-10. The syd-10 single mutant has no observable alteration from wild-type-like resistance, although the syd-4 T-DNA insertion allele displays enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Transcription of SNC1 is increased in both syd-4 and syd-10. These data suggest that SYD plays a subtle, specific role in the regulation of SNC1 expression and SNC1-mediated immunity. SYD may work with other proteins at the chromatin level to repress SNC1 transcription; such regulation is important for fine-tuning the expression of NLR-encoding genes to prevent unpropitious autoimmunity. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please

Nuclear size as estrogen-responsive chromatin quality parameter of mouse spermatozoa.

PubMed

Cacciola, Giovanna; Chioccarelli, Teresa; Altucci, Lucia; Viggiano, Andrea; Fasano, Silvia; Pierantoni, Riccardo; Cobellis, Gilda

2013-11-01

Recently, we have investigated the endocannabinoid involvement in chromatin remodeling events occurring in male spermatids. Indeed, we have demonstrated that genetic inactivation of the cannabinoid receptor type 1 (Cnr1) negatively influences chromatin remodeling mechanisms, by reducing histone displacement and indices of sperm chromatin quality (chromatin condensation and DNA integrity). Conversely, Cnr1 knock-out (Cnr1(-/-)) male mice, treated with estrogens, replaced histones and rescued chromatin condensation as well as DNA integrity. In the present study, by exploiting Cnr1(+/+), Cnr(+/-) and Cnr1(-/-) epididymal sperm samples, we show that histone retention directly correlates with low values of sperm chromatin quality indices determining sperm nuclear size elongation. Moreover, we demonstrate that estrogens, by promoting histone displacement and chromatin condensation rescue, are able to efficiently reduce the greater nuclear length observed in Cnr1(-/-) sperm. As a consequence of our results, we suggest that nucleus length may be used as a morphological parameter useful to screen out spermatozoa with low chromatin quality. Copyright © 2013 Elsevier Inc. All rights reserved.

Lipid droplet-associated gene expression and chromatin remodelling in LIPASE 5'-upstream region from beginning- to mid-endodormant bud in 'Fuji' apple.

PubMed

Saito, Takanori; Wang, Shanshan; Ohkawa, Katsuya; Ohara, Hitoshi; Ikeura, Hiromi; Ogawa, Yukiharu; Kondo, Satoru

2017-11-01

We found that lipid accumulation in the meristem region and the expression of MdLIP2A, which appears to be regulated by chromatin remodeling, coincided with endodormancy induction in the 'Fuji' apple. In deciduous trees, including apples (Malus × domestica Borkh.), lipid accumulation in the meristem region towards endodormancy induction has been thought to be an important process for the acquisition of cold tolerance. In this study, we conducted histological staining of crude lipids in the meristem region of 'Fuji' apples and found that lipid accumulation coincided with endodormancy induction. Since a major component of lipid bodies (triacylglycerol) is esterified fatty acids, we analysed fatty acid-derived volatile compounds and genes encoding fatty acid-modifying enzymes (MdLOX1A and MdHPL2A); the reduction of lipid breakdown also coincided with endodormancy induction. We then characterised the expression patterns of lipid body-regulatory genes MdOLE1 and MdLIP2A during endodormancy induction and found that the expression of MdLIP2A correlated well with lipid accumulation towards endodormancy induction. Based on these results, we conducted chromatin remodelling studies and localized the cis-element in the 5'-upstream region of MdLIP2A to clarify its regulatory mechanism. Finally, we revealed that chromatin was concentrated - 764 to - 862 bp of the 5'-upstream region of MdLIP2A, which harbours the GARE [gibberellin responsive MYB transcription factor binding site] and CArG [MADS-box transcription factor binding site] motifs-meristem development-related protein-binding sites.

Human cells contain a factor that facilitates the DNA glycosylase-mediated excision of oxidized bases from occluded sites in nucleosomes.

PubMed

Maher, R L; Marsden, C G; Averill, A M; Wallace, S S; Sweasy, J B; Pederson, D S

2017-09-01

Reactive oxygen species generate some 20,000 base lesions per human cell per day. The vast majority of these potentially mutagenic or cytotoxic lesions are subject to base excision repair (BER). Although chromatin remodelers have been shown to enhance the excision of oxidized bases from nucleosomes in vitro, it is not clear that they are recruited to and act at sites of BER in vivo. To test the hypothesis that cells possess factors that enhance BER in chromatin, we assessed the capacity of nuclear extracts from human cells to excise thymine glycol (Tg) lesions from exogenously added, model nucleosomes. The DNA glycosylase NTHL1 in these extracts was able to excise Tg from both naked DNA and sites in nucleosomes that earlier studies had shown to be sterically accessible. However, the same extracts were able to excise lesions from sterically-occluded sites in nucleosomes only after the addition of Mg 2+ /ATP. Gel mobility shift assays indicated that nucleosomes remain largely intact following the Mg 2+ /ATP -dependent excision reaction. Size exclusion chromatography indicated that the NTHL1-stimulating activity has a relatively low molecular weight, close to that of NTHL1 and other BER glycosylases; column fractions that contained the very large chromatin remodeling complexes did not exhibit this same stimulatory activity. These results indicate that cells possess a factor(s) that promotes the initiation of BER in chromatin, but differs from most known chromatin remodeling complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of chromatin in water stress responses in plants

PubMed Central

Han, Soon-Ki; Wagner, Doris

2014-01-01

As sessile organisms, plants are exposed to environmental stresses throughout their life. They have developed survival strategies such as developmental and morphological adaptations, as well as physiological responses, to protect themselves from adverse environments. In addition, stress sensing triggers large-scale transcriptional reprogramming directed at minimizing the deleterious effect of water stress on plant cells. Here, we review recent findings that reveal a role of chromatin in water stress responses. In addition, we discuss data in support of the idea that chromatin remodelling and modifying enzymes may be direct targets of stress signalling pathways. Modulation of chromatin regulator activity by these signaling pathways may be critical in minimizing potential trade-offs between growth and stress responses. Alterations in the chromatin organization and/or in the activity of chromatin remodelling and modifying enzymes may furthermore contribute to stress memory. Mechanistic insight into these phenomena derived from studies in model plant systems should allow future engineering of broadly drought-tolerant crop plants that do not incur unnecessary losses in yield or growth. PMID:24302754

DNA replication through a chromatin environment.

PubMed

Bellush, James M; Whitehouse, Iestyn

2017-10-05

Compaction of the genome into the nuclear space is achieved by wrapping DNA around octameric assemblies of histone proteins to form nucleosomes, the fundamental repeating unit of chromatin. Aside from providing a means by which to fit larger genomes into the cell, chromatinization of DNA is a crucial means by which the cell regulates access to the genome. While the complex role that chromatin plays in gene transcription has been appreciated for a long time, it is now also apparent that crucial aspects of DNA replication are linked to the biology of chromatin. This review will focus on recent advances in our understanding of how the chromatin environment influences key aspects of DNA replication.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'. © 2017 The Author(s).

Mutation of neuron-specific chromatin remodeling subunit BAF53b: rescue of plasticity and memory by manipulating actin remodeling.

PubMed

Vogel Ciernia, Annie; Kramár, Enikö A; Matheos, Dina P; Havekes, Robbert; Hemstedt, Thekla J; Magnan, Christophe N; Sakata, Keith; Tran, Ashley; Azzawi, Soraya; Lopez, Alberto; Dang, Richard; Wang, Weisheng; Trieu, Brian; Tong, Joyce; Barrett, Ruth M; Post, Rebecca J; Baldi, Pierre; Abel, Ted; Lynch, Gary; Wood, Marcelo A

2017-05-01

Recent human exome-sequencing studies have implicated polymorphic Brg1-associated factor (BAF) complexes (mammalian SWI/SNF chromatin remodeling complexes) in several intellectual disabilities and cognitive disorders, including autism. However, it remains unclear how mutations in BAF complexes result in impaired cognitive function. Post-mitotic neurons express a neuron-specific assembly, nBAF, characterized by the neuron-specific subunit BAF53b. Subdomain 2 of BAF53b is essential for the differentiation of neuronal precursor cells into neurons. We generated transgenic mice lacking subdomain 2 of Baf53b (BAF53bΔSB2). Long-term synaptic potentiation (LTP) and long-term memory, both of which are associated with phosphorylation of the actin severing protein cofilin, were assessed in these animals. A phosphorylation mimic of cofilin was stereotaxically delivered into the hippocampus of BAF53bΔSB2 mice in an effort to rescue LTP and memory. BAF53bΔSB2 mutant mice show impairments in phosphorylation of synaptic cofilin, LTP, and memory. Both the synaptic plasticity and memory deficits are rescued by overexpression of a phosphorylation mimetic of cofilin. Baseline physiology and behavior were not affected by the mutation or the experimental treatment. This study suggests a potential link between nBAF function, actin cytoskeletal remodeling at the dendritic spine, and memory formation. This work shows that a targeted manipulation of synaptic function can rescue adult plasticity and memory deficits caused by manipulations of nBAF, and thereby provides potential novel avenues for therapeutic development for multiple intellectual disability disorders. © 2017 Vogel Ciernia et al.; Published by Cold Spring Harbor Laboratory Press.

Relocalization of human chromatin remodeling cofactor TIP48 in mitosis

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

Sigala, Barbara; Edwards, Mina; Puri, Teena

2005-11-01

TIP48 is a highly conserved eukaryotic AAA{sup +} protein which is an essential cofactor for several complexes involved in chromatin acetylation and remodeling, transcriptional and developmental regulation and nucleolar organization and trafficking. We show that TIP48 abundance in HeLa cells did not change during the cell cycle, nor did its distribution in various biochemical fractions. However, we observed distinct changes in the subcellular localization of TIP48 during M phase using immunofluorescence microscopy. Our studies demonstrate that in interphase cells TIP48 was found mainly in the nucleus and exhibited a distinct localization in the nuclear periphery. As the cells entered mitosis,more » TIP48 was excluded from the condensing chromosomes but showed association with the mitotic apparatus. During anaphase, some TIP48 was detected in the centrosome colocalizing with tubulin but the strongest staining appeared in the mitotic equator associated with the midzone central spindle. Accumulation of TIP48 in the midzone and the midbody was observed in late telophase and cytokinesis. This redeployment of TIP48 during anaphase and cytokinesis was independent of microtubule assembly. The relocation of endogenous TIP48 to the midzone/midbody under physiological conditions suggests a novel and distinct function for TIP48 in mitosis and possible involvement in the exit of mitosis.« less

Chromatin versus pathogens: the function of epigenetics in plant immunity

PubMed Central

Ding, Bo; Wang, Guo-Liang

2015-01-01

To defend against pathogens, plants have developed a sophisticated innate immunity that includes effector recognition, signal transduction, and rapid defense responses. Recent evidence has demonstrated that plants utilize the epigenetic control of gene expression to fine-tune their defense when challenged by pathogens. In this review, we highlight the current understanding of the molecular mechanisms of histone modifications (i.e., methylation, acetylation, and ubiquitination) and chromatin remodeling that contribute to plant immunity against pathogens. Functions of key histone-modifying and chromatin remodeling enzymes are discussed. PMID:26388882

Chromatin versus pathogens: the function of epigenetics in plant immunity.

PubMed

Ding, Bo; Wang, Guo-Liang

2015-01-01

To defend against pathogens, plants have developed a sophisticated innate immunity that includes effector recognition, signal transduction, and rapid defense responses. Recent evidence has demonstrated that plants utilize the epigenetic control of gene expression to fine-tune their defense when challenged by pathogens. In this review, we highlight the current understanding of the molecular mechanisms of histone modifications (i.e., methylation, acetylation, and ubiquitination) and chromatin remodeling that contribute to plant immunity against pathogens. Functions of key histone-modifying and chromatin remodeling enzymes are discussed.

Synergistic activation of Arg1 gene by retinoic acid and IL-4 involves chromatin remodeling for transcription initiation and elongation coupling

PubMed Central

Lee, Bomi; Wu, Cheng-Ying; Lin, Yi-Wei; Park, Sung Wook; Wei, Li-Na

2016-01-01

All-trans Retinoic acid (RA) and its derivatives are potent therapeutics for immunological functions including wound repair. However, the molecular mechanism of RA modulation in innate immunity is poorly understood, especially in macrophages. We found that topical application of RA significantly improves wound healing and that RA and IL-4 synergistically activate Arg1, a critical gene for tissue repair, in M2 polarized macrophages. This involves feed forward regulation of Raldh2, a rate-limiting enzyme for RA biosynthesis, and requires Med25 to coordinate RAR, STAT6 and chromatin remodeler, Brg1 to remodel the +1 nucleosome of Arg1 for transcription initiation. By recruiting elongation factor TFIIS, Med25 also facilitates transcriptional initiation-elongation coupling. This study uncovers synergistic activation of Arg1 by RA and IL-4 in M2 macrophages that involves feed forward regulation of RA synthesis and dual functions of Med25 in nucleosome remodeling and transcription initiation-elongation coupling that underlies robust modulatory activity of RA in innate immunity. PMID:27166374

Nucleoporins and chromatin metabolism.

PubMed

Ptak, Christopher; Wozniak, Richard W

2016-06-01

Mounting evidence has implicated a group of proteins termed nucleoporins, or Nups, in various processes that regulate chromatin structure and function. Nups were first recognized as building blocks for nuclear pore complexes, but several members of this group of proteins also reside in the cytoplasm and within the nucleus. Moreover, many are dynamic and move between these various locations. Both at the nuclear envelope, as part of nuclear pore complexes, and within the nucleoplasm, Nups interact with protein complexes that function in gene transcription, chromatin remodeling, DNA repair, and DNA replication. Here, we review recent studies that provide further insight into the molecular details of these interactions and their role in regulating the activity of chromatin modifying factors. Copyright © 2016. Published by Elsevier Ltd.

Histone variant H3.3-mediated chromatin remodeling is essential for paternal genome activation in mouse preimplantation embryos.

PubMed

Kong, Qingran; Banaszynski, Laura A; Geng, Fuqiang; Zhang, Xiaolei; Zhang, Jiaming; Zhang, Heng; O'Neill, Claire L; Yan, Peidong; Liu, Zhonghua; Shido, Koji; Palermo, Gianpiero D; Allis, C David; Rafii, Shahin; Rosenwaks, Zev; Wen, Duancheng

2018-03-09

Derepression of chromatin-mediated transcriptional repression of paternal and maternal genomes is considered the first major step that initiates zygotic gene expression after fertilization. The histone variant H3.3 is present in both male and female gametes and is thought to be important for remodeling the paternal and maternal genomes for activation during both fertilization and embryogenesis. However, the underlying mechanisms remain poorly understood. Using our H3.3B-HA-tagged mouse model, engineered to report H3.3 expression in live animals and to distinguish different sources of H3.3 protein in embryos, we show here that sperm-derived H3.3 (sH3.3) protein is removed from the sperm genome shortly after fertilization and extruded from the zygotes via the second polar bodies (PBII) during embryogenesis. We also found that the maternal H3.3 (mH3.3) protein is incorporated into the paternal genome as early as 2 h postfertilization and is detectable in the paternal genome until the morula stage. Knockdown of maternal H3.3 resulted in compromised embryonic development both of fertilized embryos and of androgenetic haploid embryos. Furthermore, we report that mH3.3 depletion in oocytes impairs both activation of the Oct4 pluripotency marker gene and global de novo transcription from the paternal genome important for early embryonic development. Our results suggest that H3.3-mediated paternal chromatin remodeling is essential for the development of preimplantation embryos and the activation of the paternal genome during embryogenesis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The Catalytic and Non-catalytic Functions of the Brahma Chromatin-Remodeling Protein Collaborate to Fine-Tune Circadian Transcription in Drosophila

PubMed Central

Kwok, Rosanna S.; Li, Ying H.; Lei, Anna J.; Edery, Isaac; Chiu, Joanna C.

2015-01-01

Daily rhythms in gene expression play a critical role in the progression of circadian clocks, and are under regulation by transcription factor binding, histone modifications, RNA polymerase II (RNAPII) recruitment and elongation, and post-transcriptional mechanisms. Although previous studies have shown that clock-controlled genes exhibit rhythmic chromatin modifications, less is known about the functions performed by chromatin remodelers in animal clockwork. Here we have identified the Brahma (Brm) complex as a regulator of the Drosophila clock. In Drosophila, CLOCK (CLK) is the master transcriptional activator driving cyclical gene expression by participating in an auto-inhibitory feedback loop that involves stimulating the expression of the main negative regulators, period (per) and timeless (tim). BRM functions catalytically to increase nucleosome density at the promoters of per and tim, creating an overall restrictive chromatin landscape to limit transcriptional output during the active phase of cycling gene expression. In addition, the non-catalytic function of BRM regulates the level and binding of CLK to target promoters and maintains transient RNAPII stalling at the per promoter, likely by recruiting repressive and pausing factors. By disentangling its catalytic versus non-catalytic functions at the promoters of CLK target genes, we uncovered a multi-leveled mechanism in which BRM fine-tunes circadian transcription. PMID:26132408

ERECTA signaling controls Arabidopsis inflorescence architecture through chromatin-mediated activation of PRE1 expression.

PubMed

Cai, Hanyang; Zhao, Lihua; Wang, Lulu; Zhang, Man; Su, Zhenxia; Cheng, Yan; Zhao, Heming; Qin, Yuan

2017-06-01

Flowering plants display a remarkable diversity in inflorescence architecture, and pedicel length is one of the key contributors to this diversity. In Arabidopsis thaliana, the receptor-like kinase ERECTA (ER) mediated signaling pathway plays important roles in regulating inflorescence architecture by promoting cell proliferation. However, the regulating mechanism remains elusive in the pedicel. Genetic interactions between ERECTA signaling and the chromatin remodeling complex SWR1 in the control of inflorescence architecture were studied. Comparative transcriptome analysis was applied to identify downstream components. Chromatin immunoprecipitation and nucleosome occupancy was further investigated. The results indicated that the chromatin remodeler SWR1 coordinates with ERECTA signaling in regulating inflorescence architecture by activating the expression of PRE1 family genes and promoting pedicel elongation. It was found that SWR1 is required for the incorporation of the H2A.Z histone variant into nucleosomes of the whole PRE1 gene family and the ERECTA controlled expression of PRE1 gene family through regulating nucleosome dynamics. We propose that utilization of a chromatin remodeling complex to regulate gene expression is a common theme in developmental control across kingdoms. These findings shed light on the mechanisms through which chromatin remodelers orchestrate complex transcriptional regulation of gene expression in coordination with a developmental cue. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

A chromatin activity based chemoproteomic approach reveals a transcriptional repressome for gene-specific silencing

PubMed Central

Liu, Cui; Yu, Yanbao; Liu, Feng; Wei, Xin; Wrobel, John A.; Gunawardena, Harsha P.; Zhou, Li; Jin, Jian; Chen, Xian

2015-01-01

Immune cells develop endotoxin tolerance (ET) after prolonged stimulation. ET increases the level of a repression mark H3K9me2 in the transcriptional-silent chromatin specifically associated with pro-inflammatory genes. However, it is not clear what proteins are functionally involved in this process. Here we show that a novel chromatin activity based chemoproteomic (ChaC) approach can dissect the functional chromatin protein complexes that regulate ET-associated inflammation. Using UNC0638 that binds the enzymatically active H3K9-specific methyltransferase G9a/GLP, ChaC reveals that G9a is constitutively active at a G9a-dependent mega-dalton repressome in primary endotoxin-tolerant macrophages. G9a/GLP broadly impacts the ET-specific reprogramming of the histone code landscape, chromatin remodeling, and the activities of select transcription factors. We discover that the G9a-dependent epigenetic environment promotes the transcriptional repression activity of c-Myc for gene-specific co-regulation of chronic inflammation. ChaC may be also applicable to dissect other functional protein complexes in the context of phenotypic chromatin architectures. PMID:25502336

Unexpected Role of the Copper Transporter ATP7A in PDGF-Induced Vascular Smooth Muscle Cell Migration

PubMed Central

Ashino, Takashi; Sudhahar, Varadarajan; Urao, Norifumi; Oshikawa, Jin; Chen, Gin-Fu; Wang, Huan; Huo, Yuqing; Finney, Lydia; Vogt, Stefan; McKinney, Ronald D.; Maryon, Edward B.; Kaplan, Jack H.; Ushio-Fukai, Masuko; Fukai, Tohru

2010-01-01

Rationale Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1, but also by the copper exporter ATP7A (Menke ATPase) whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. Objective To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Methods and Results Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A siRNA or CTR siRNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor pro-lysyl oxidase (Pro-LOX) in lipid raft fraction as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based X-ray fluorescence microscopy at neointimal VSMCs in wire injury model. Conclusions These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis. PMID:20671235

Unexpected role of the copper transporter ATP7A in PDGF-induced vascular smooth muscle cell migration.

PubMed

Ashino, Takashi; Sudhahar, Varadarajan; Urao, Norifumi; Oshikawa, Jin; Chen, Gin-Fu; Wang, Huan; Huo, Yuqing; Finney, Lydia; Vogt, Stefan; McKinney, Ronald D; Maryon, Edward B; Kaplan, Jack H; Ushio-Fukai, Masuko; Fukai, Tohru

2010-09-17

Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.

The RSF1 histone-remodelling factor facilitates DNA double-strand break repair by recruiting centromeric and Fanconi Anaemia proteins.

PubMed

Pessina, Fabio; Lowndes, Noel F

2014-05-01

ATM is a central regulator of the cellular responses to DNA double-strand breaks (DSBs). Here we identify a biochemical interaction between ATM and RSF1 and we characterise the role of RSF1 in this response. The ATM-RSF1 interaction is dependent upon both DSBs and ATM kinase activity. Together with SNF2H/SMARCA5, RSF1 forms the RSF chromatin-remodelling complex. Although RSF1 is specific to the RSF complex, SNF2H/SMARCA5 is a catalytic subunit of several other chromatin-remodelling complexes. Although not required for checkpoint signalling, RSF1 is required for efficient repair of DSBs via both end-joining and homology-directed repair. Specifically, the ATM-dependent recruitment to sites of DSBs of the histone fold proteins CENPS/MHF1 and CENPX/MHF2, previously identified at centromeres, is RSF1-dependent. In turn these proteins recruit and regulate the mono-ubiquitination of the Fanconi Anaemia proteins FANCD2 and FANCI. We propose that by depositing CENPS/MHF1 and CENPX/MHF2, the RSF complex either directly or indirectly contributes to the reorganisation of chromatin around DSBs that is required for efficient DNA repair.

The RSF1 Histone-Remodelling Factor Facilitates DNA Double-Strand Break Repair by Recruiting Centromeric and Fanconi Anaemia Proteins

PubMed Central

Pessina, Fabio; Lowndes, Noel F.

2014-01-01

ATM is a central regulator of the cellular responses to DNA double-strand breaks (DSBs). Here we identify a biochemical interaction between ATM and RSF1 and we characterise the role of RSF1 in this response. The ATM–RSF1 interaction is dependent upon both DSBs and ATM kinase activity. Together with SNF2H/SMARCA5, RSF1 forms the RSF chromatin-remodelling complex. Although RSF1 is specific to the RSF complex, SNF2H/SMARCA5 is a catalytic subunit of several other chromatin-remodelling complexes. Although not required for checkpoint signalling, RSF1 is required for efficient repair of DSBs via both end-joining and homology-directed repair. Specifically, the ATM-dependent recruitment to sites of DSBs of the histone fold proteins CENPS/MHF1 and CENPX/MHF2, previously identified at centromeres, is RSF1-dependent. In turn these proteins recruit and regulate the mono-ubiquitination of the Fanconi Anaemia proteins FANCD2 and FANCI. We propose that by depositing CENPS/MHF1 and CENPX/MHF2, the RSF complex either directly or indirectly contributes to the reorganisation of chromatin around DSBs that is required for efficient DNA repair. PMID:24800743

Dependence of the Linker Histone and Chromatin Condensation on the Nucleosome Environment.

PubMed

Perišić, Ognjen; Schlick, Tamar

2017-08-24

The linker histone (LH), an auxiliary protein that can bind to chromatin and interact with the linker DNA to form stem motifs, is a key element of chromatin compaction. By affecting the chromatin condensation level, it also plays an active role in gene expression. However, the presence and variable concentration of LH in chromatin fibers with different DNA linker lengths indicate that its folding and condensation are highly adaptable and dependent on the immediate nucleosome environment. Recent experimental studies revealed that the behavior of LH in mononucleosomes markedly differs from that in small nucleosome arrays, but the associated mechanism is unknown. Here we report a structural analysis of the behavior of LH in mononucleosomes and oligonucleosomes (2-6 nucleosomes) using mesoscale chromatin simulations. We show that the adapted stem configuration heavily depends on the strength of electrostatic interactions between LH and its parental DNA linkers, and that those interactions tend to be asymmetric in small oligonucleosome systems. Namely, LH in oligonucleosomes dominantly interacts with one DNA linker only, as opposed to mononucleosomes where LH has similar interactions with both linkers and forms a highly stable nucleosome stem. Although we show that the LH condensation depends sensitively on the electrostatic interactions with entering and exiting DNA linkers, other interactions, especially by nonparental cores and nonparental linkers, modulate the structural condensation by softening LH and thus making oligonucleosomes more flexible, in comparison to to mono- and dinucleosomes. We also find that the overall LH/chromatin interactions sensitively depend on the linker length because the linker length determines the maximal nucleosome stem length. For mononucleosomes with DNA linkers shorter than LH, LH condenses fully, while for DNA linkers comparable or longer than LH, the LH extension in mononucleosomes strongly follows the length of DNA linkers

Regulation of chromatin structure in the cardiovascular system.

PubMed

Rosa-Garrido, Manuel; Karbassi, Elaheh; Monte, Emma; Vondriska, Thomas M

2013-01-01

It has been appreciated for some time that cardiovascular disease involves large-scale transcriptional changes in various cell types. What has become increasingly clear only in the past few years, however, is the role of chromatin remodeling in cardiovascular phenotypes in normal physiology, as well as in development and disease. This review summarizes the state of the chromatin field in terms of distinct mechanisms to regulate chromatin structure in vivo, identifying when these modes of regulation have been demonstrated in cardiovascular tissues. We describe areas in which a better understanding of chromatin structure is leading to new insights into the fundamental biology of cardiovascular disease. 

Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte.

PubMed

Yang, Feikun; Baumann, Claudia; Viveiros, Maria M; De La Fuente, Rabindranath

2012-01-01

Histone acetylation regulates higher-order chromatin structure and function and is critical for the control of gene expression. Histone deacetylase inhibitors (HDACi) are currently under investigation as novel cancer therapeutic drugs. Here, we show that female germ cells are extremely susceptible to chromatin changes induced by HDACi. Our results indicate that exposure to trichostatin A (TSA) at nanomolar levels interferes with major chromatin remodeling events in the mammalian oocyte leading to chromosome instability. High resolution analysis of chromatin structure and live-cell imaging revealed a striking euchromatin decondensation associated with histone H4 hyperacetylation following exposure to 15 nM TSA in >90% of pre-ovulatory oocytes. Dynamic changes in large-scale chromatin structure were detected after 2 h of exposure and result in the formation of misaligned chromosomes in >75% (P<0.05) of in vitro matured oocytes showing chromosome lagging as well as abnormal sister chromatid separation at anaphase I. Abnormal axial chromatid condensation during meiosis results in the formation of elongated chromosomes exhibiting hyperacetylation of histone H4 at lysine 5 and lysine 16 at interstitial chromosome segments, but not pericentric heterochromatin, while highly decondensed bivalents exhibit prominent histone H3 phosphorylation at centromeric domains. Notably, no changes were observed in the chromosomal localization of the condensin protein SMC4. These results indicate that HDAC activity is required for proper chromosome condensation in the mammalian oocyte and that HDACi may induce abnormal chromosome segregation by interfering with both chromosome-microtubule interactions, as well as sister chromatid separation. Thus, HDACi, proposed for cancer therapy, may disrupt the epigenetic status of female germ cells, predisposing oocytes to aneuploidy at previously unrecognized low doses.

RSC-dependent constructive and destructive interference between opposing arrays of phased nucleosomes in yeast

PubMed Central

Ganguli, Dwaipayan; Chereji, Răzvan V.; Iben, James R.; Cole, Hope A.

2014-01-01

RSC and SWI/SNF are related ATP-dependent chromatin remodeling machines that move nucleosomes, regulating access to DNA. We addressed their roles in nucleosome phasing relative to transcription start sites in yeast. SWI/SNF has no effect on phasing at the global level. In contrast, RSC depletion results in global nucleosome repositioning: Both upstream and downstream nucleosomal arrays shift toward the nucleosome-depleted region (NDR), with no change in spacing, resulting in a narrower and partly filled NDR. The global picture of RSC-depleted chromatin represents the average of a range of chromatin structures, with most genes showing a shift of the +1 or the −1 nucleosome into the NDR. Using RSC ChIP data reported by others, we show that RSC occupancy is highest on the coding regions of heavily transcribed genes, though not at their NDRs. We propose that RSC has a role in restoring chromatin structure after transcription. Analysis of gene pairs in different orientations demonstrates that phasing patterns reflect competition between phasing signals emanating from neighboring NDRs. These signals may be in phase, resulting in constructive interference and a regular array, or out of phase, resulting in destructive interference and fuzzy positioning. We propose a modified barrier model, in which a stable complex located at the NDR acts as a bidirectional phasing barrier. In RSC-depleted cells, this barrier has a smaller footprint, resulting in narrower NDRs. Thus, RSC plays a critical role in organizing yeast chromatin. PMID:25015381

Preparation and Analysis of Positioned Mononucleosomes

PubMed Central

Kulaeva, Olga; Studitsky, Vasily M.

2016-01-01

Short DNA fragments containing single nucleosomes have been extensively employed as simple model experimental systems for analysis of many intranuclear processes, including binding of proteins to nucleosomes, covalent histone modifications, transcription, DNA repair and ATP-dependent chromatin remodeling. Here we describe several recently developed procedures for obtaining and analysis of mononucleosomes assembled on 200–350-bp DNA fragments. PMID:25827872

The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression.

PubMed

Klochendler-Yeivin, A; Fiette, L; Barra, J; Muchardt, C; Babinet, C; Yaniv, M

2000-12-01

The assembly of eukaryotic DNA into nucleosomes and derived higher order structures constitutes a barrier for transcription, replication and repair. A number of chromatin remodeling complexes, as well as histone acetylation, were shown to facilitate gene activation. To investigate the function of two closely related mammalian SWI/SNF complexes in vivo, we inactivated the murine SNF5/INI1 gene, a common subunit of these two complexes. Mice lacking SNF5 protein stop developing at the peri-implantation stage, showing that the SWI/SNF complex is essential for early development and viability of early embryonic cells. Furthermore, heterozygous mice develop nervous system and soft tissue sarcomas. In these tumors the wild-type allele was lost, providing further evidence that SNF5 functions as a tumor suppressor gene in certain cell types.

The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression

PubMed Central

Klochendler-Yeivin, Agnes; Fiette, Laurence; Barra, Jaqueline; Muchardt, Christian; Babinet, Charles; Yaniv, Moshe

2000-01-01

The assembly of eukaryotic DNA into nucleosomes and derived higher order structures constitutes a barrier for transcription, replication and repair. A number of chromatin remodeling complexes, as well as histone acetylation, were shown to facilitate gene activation. To investigate the function of two closely related mammalian SWI/SNF complexes in vivo, we inactivated the murine SNF5/INI1 gene, a common subunit of these two complexes. Mice lacking SNF5 protein stop developing at the peri-implantation stage, showing that the SWI/SNF complex is essential for early development and viability of early embryonic cells. Furthermore, heterozygous mice develop nervous system and soft tissue sarcomas. In these tumors the wild-type allele was lost, providing further evidence that SNF5 functions as a tumor suppressor gene in certain cell types. PMID:11263494

Skeletal muscle and liver contain a soluble ATP + ubiquitin-dependent proteolytic system.

PubMed Central

Fagan, J M; Waxman, L; Goldberg, A L

1987-01-01

Although protein breakdown in most cells seems to require metabolic energy, it has only been possible to establish a soluble ATP-dependent proteolytic system in extracts of reticulocytes and erythroleukemia cells. We have now succeeded in demonstrating in soluble extracts and more purified preparations from rabbit skeletal muscle a 12-fold stimulation by ATP of breakdown of endogenous proteins and a 6-fold stimulation of 125I-lysozyme degradation. However, it has still not been possible to demonstrate such large effects of ATP in similar preparations from liver. Nevertheless, after fractionation by DEAE-chromatography and gel filtration, we found that extracts from liver as well as muscle contain both the enzymes which conjugate ubiquitin to 125I-lysozyme and an enzyme which specifically degrades the ubiquitin-protein conjugates. When this proteolytic activity was recombined with the conjugating enzymes, ATP + ubiquitin-dependent degradation of many proteins was observed. This proteinase is unusually large, approx. 1500 kDa, requires ATP hydrolysis for activity and resembles the ubiquitin-protein-conjugate degrading activity isolated from reticulocytes. Thus the ATP + ubiquitin-dependent pathway is likely to be present in all mammalian cells, although certain tissues may contain inhibitory factors. Images Fig. 2. PMID:2820375

The Prefoldin Complex Regulates Chromatin Dynamics during Transcription Elongation

PubMed Central

Millán-Zambrano, Gonzalo; Rodríguez-Gil, Alfonso; Peñate, Xenia; de Miguel-Jiménez, Lola; Morillo-Huesca, Macarena; Krogan, Nevan; Chávez, Sebastián

2013-01-01

Transcriptional elongation requires the concerted action of several factors that allow RNA polymerase II to advance through chromatin in a highly processive manner. In order to identify novel elongation factors, we performed systematic yeast genetic screening based on the GLAM (Gene Length-dependent Accumulation of mRNA) assay, which is used to detect defects in the expression of long transcription units. Apart from well-known transcription elongation factors, we identified mutants in the prefoldin complex subunits, which were among those that caused the most dramatic phenotype. We found that prefoldin, so far involved in the cytoplasmic co-translational assembly of protein complexes, is also present in the nucleus and that a subset of its subunits are recruited to chromatin in a transcription-dependent manner. Prefoldin influences RNA polymerase II the elongation rate in vivo and plays an especially important role in the transcription elongation of long genes and those whose promoter regions contain a canonical TATA box. Finally, we found a specific functional link between prefoldin and histone dynamics after nucleosome remodeling, which is consistent with the extensive network of genetic interactions between this factor and the machinery regulating chromatin function. This study establishes the involvement of prefoldin in transcription elongation, and supports a role for this complex in cotranscriptional histone eviction. PMID:24068951

The prefoldin complex regulates chromatin dynamics during transcription elongation.

PubMed

Millán-Zambrano, Gonzalo; Rodríguez-Gil, Alfonso; Peñate, Xenia; de Miguel-Jiménez, Lola; Morillo-Huesca, Macarena; Krogan, Nevan; Chávez, Sebastián

2013-01-01

Transcriptional elongation requires the concerted action of several factors that allow RNA polymerase II to advance through chromatin in a highly processive manner. In order to identify novel elongation factors, we performed systematic yeast genetic screening based on the GLAM (Gene Length-dependent Accumulation of mRNA) assay, which is used to detect defects in the expression of long transcription units. Apart from well-known transcription elongation factors, we identified mutants in the prefoldin complex subunits, which were among those that caused the most dramatic phenotype. We found that prefoldin, so far involved in the cytoplasmic co-translational assembly of protein complexes, is also present in the nucleus and that a subset of its subunits are recruited to chromatin in a transcription-dependent manner. Prefoldin influences RNA polymerase II the elongation rate in vivo and plays an especially important role in the transcription elongation of long genes and those whose promoter regions contain a canonical TATA box. Finally, we found a specific functional link between prefoldin and histone dynamics after nucleosome remodeling, which is consistent with the extensive network of genetic interactions between this factor and the machinery regulating chromatin function. This study establishes the involvement of prefoldin in transcription elongation, and supports a role for this complex in cotranscriptional histone eviction.

The mammalian INO80 chromatin remodeling complex is required for replication stress recovery

PubMed Central

Vassileva, Ivelina; Yanakieva, Iskra; Peycheva, Michaela; Gospodinov, Anastas; Anachkova, Boyka

2014-01-01

A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart. PMID:25016522

Dual Roles for Ikaros in Regulation of Macrophage Chromatin State and Inflammatory Gene Expression.

PubMed

Oh, Kyu-Seon; Gottschalk, Rachel A; Lounsbury, Nicolas W; Sun, Jing; Dorrington, Michael G; Baek, Songjoon; Sun, Guangping; Wang, Ze; Krauss, Kathleen S; Milner, Joshua D; Dutta, Bhaskar; Hager, Gordon L; Sung, Myong-Hee; Fraser, Iain D C

2018-06-13

Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor families. The transcription factor Ikaros has been shown to play a critical role in lymphoid cell development and differentiation; however, its function in myeloid cells and innate immune responses is less appreciated. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1 -/- macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes, and chromatin immunoprecipitation sequencing analyses support a key role for Ikaros in sustained NF-κB chromatin binding. Decreased Ikaros expression in Ikzf1 +/- mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.

Connecting the dots: chromatin and alternative splicing in EMT

PubMed Central

Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

2015-01-01

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

Connecting the dots: chromatin and alternative splicing in EMT.

PubMed

Warns, Jessica A; Davie, James R; Dhasarathy, Archana

2016-02-01

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

Symmetry broken and rebroken during the ATP hydrolysis cycle of the mitochondrial Hsp90 TRAP1

PubMed Central

Elnatan, Daniel; Betegon, Miguel; Liu, Yanxin; Ramelot, Theresa; Kennedy, Michael A; Agard, David A

2017-01-01

Hsp90 is a homodimeric ATP-dependent molecular chaperone that remodels its substrate ‘client’ proteins, facilitating their folding and activating them for biological function. Despite decades of research, the mechanism connecting ATP hydrolysis and chaperone function remains elusive. Particularly puzzling has been the apparent lack of cooperativity in hydrolysis of the ATP in each protomer. A crystal structure of the mitochondrial Hsp90, TRAP1, revealed that the catalytically active state is closed in a highly strained asymmetric conformation. This asymmetry, unobserved in other Hsp90 homologs, is due to buckling of one of the protomers and is most pronounced at the broadly conserved client-binding region. Here, we show that rather than being cooperative or independent, ATP hydrolysis on the two protomers is sequential and deterministic. Moreover, dimer asymmetry sets up differential hydrolysis rates for each protomer, such that the buckled conformation favors ATP hydrolysis. Remarkably, after the first hydrolysis, the dimer undergoes a flip in the asymmetry while remaining in a closed state for the second hydrolysis. From these results, we propose a model where direct coupling of ATP hydrolysis and conformational flipping rearranges client-binding sites, providing a paradigm of how energy from ATP hydrolysis can be used for client remodeling. DOI: http://dx.doi.org/10.7554/eLife.25235.001 PMID:28742020

Chromatin-remodeling SWI/SNF complex regulates coenzyme Q6 synthesis and a metabolic shift to respiration in yeast.

PubMed

Awad, Agape M; Venkataramanan, Srivats; Nag, Anish; Galivanche, Anoop Raj; Bradley, Michelle C; Neves, Lauren T; Douglass, Stephen; Clarke, Catherine F; Johnson, Tracy L

2017-09-08

Despite its relatively streamlined genome, there are many important examples of regulated RNA splicing in Saccharomyces cerevisiae Here, we report a role for the chromatin remodeler SWI/SNF in respiration, partially via the regulation of splicing. We find that a nutrient-dependent decrease in Snf2 leads to an increase in splicing of the PTC7 transcript. The spliced PTC7 transcript encodes a mitochondrial phosphatase regulator of biosynthesis of coenzyme Q 6 (ubiquinone or CoQ 6 ) and a mitochondrial redox-active lipid essential for electron and proton transport in respiration. Increased splicing of PTC7 increases CoQ 6 levels. The increase in PTC7 splicing occurs at least in part due to down-regulation of ribosomal protein gene expression, leading to the redistribution of spliceosomes from this abundant class of intron-containing RNAs to otherwise poorly spliced transcripts. In contrast, a protein encoded by the nonspliced isoform of PTC7 represses CoQ 6 biosynthesis. Taken together, these findings uncover a link between Snf2 expression and the splicing of PTC7 and establish a previously unknown role for the SWI/SNF complex in the transition of yeast cells from fermentative to respiratory modes of metabolism. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

mTOR signaling regulates myotube hypertrophy by modulating protein synthesis, rDNA transcription, and chromatin remodeling.

PubMed

von Walden, Ferdinand; Liu, Chang; Aurigemma, Nicole; Nader, Gustavo A

2016-10-01

Ribosome production is an early event during skeletal muscle hypertrophy and precedes muscle protein accretion. Signaling via mTOR is crucial for ribosome production and hypertrophy; however, the mechanisms by which it regulates these processes remain to be identified. Herein, we investigated the activation of mTOR signaling in hypertrophying myotubes and determined that mTOR coordinates various aspects of gene expression important for ribosome production. First, inhibition of translation with cycloheximide had a more potent effect on protein synthesis than rapamycin indicating that mTOR function during hypertrophy is not on general, but rather on specific protein synthesis. Second, blocking Pol II transcription had a similar effect as Rapamycin and, unexpectedly, revealed the necessity of Pol II transcription for Pol I transcription, suggesting that mTOR may regulate ribosome production also by controlling Class II genes at the transcriptional level. Third, Pol I activity is essential for rDNA transcription and, surprisingly, for protein synthesis as selective Pol I inhibition blunted rDNA transcription, protein synthesis, and the hypertrophic response of myotubes. Finally, mTOR has nuclear localization in muscle, which is not sensitive to rapamycin. Inhibition of mTOR signaling by rapamycin disrupted mTOR-rDNA promoter interaction and resulted in altered histone marks indicative of repressed transcription and formation of higher-order chromatin structure. Thus mTOR signaling appears to regulate muscle hypertrophy by affecting protein synthesis, Class I and II gene expression, and chromatin remodeling. Copyright © 2016 the American Physiological Society.

SWI/SNF chromatin remodeling complex is critical for the expression of microphthalmia-associated transcription factor in melanoma cells

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

Vachtenheim, Jiri, E-mail: jivach@upn.anet.cz; Ondrusova, Lubica; Borovansky, Jan

2010-02-12

The microphthalmia-associated transcription factor (MITF) is required for melanocyte development, maintenance of the melanocyte-specific transcription, and survival of melanoma cells. MITF positively regulates expression of more than 25 genes in pigment cells. Recently, it has been demonstrated that expression of several MITF downstream targets requires the SWI/SNF chromatin remodeling complex, which contains one of the two catalytic subunits, Brm or Brg1. Here we show that the expression of MITF itself critically requires active SWI/SNF. In several Brm/Brg1-expressing melanoma cell lines, knockdown of Brg1 severely compromised MITF expression with a concomitant dowregulation of MITF targets and decreased cell proliferation. Although Brmmore » was able to substitute for Brg1 in maintaining MITF expression and melanoma cell proliferation, sequential knockdown of both Brm and Brg1 in 501mel cells abolished proliferation. In Brg1-null SK-MEL-5 melanoma cells, depletion of Brm alone was sufficient to abrogate MITF expression and cell proliferation. Chromatin immunoprecipitation confirmed the binding of Brg1 or Brm to the promoter of MITF. Together these results demonstrate the essential role of SWI/SNF for expression of MITF and suggest that SWI/SNF may be a promissing target in melanoma therapy.« less

RSC-dependent constructive and destructive interference between opposing arrays of phased nucleosomes in yeast.

PubMed

Ganguli, Dwaipayan; Chereji, Răzvan V; Iben, James R; Cole, Hope A; Clark, David J

2014-10-01

RSC and SWI/SNF are related ATP-dependent chromatin remodeling machines that move nucleosomes, regulating access to DNA. We addressed their roles in nucleosome phasing relative to transcription start sites in yeast. SWI/SNF has no effect on phasing at the global level. In contrast, RSC depletion results in global nucleosome repositioning: Both upstream and downstream nucleosomal arrays shift toward the nucleosome-depleted region (NDR), with no change in spacing, resulting in a narrower and partly filled NDR. The global picture of RSC-depleted chromatin represents the average of a range of chromatin structures, with most genes showing a shift of the +1 or the -1 nucleosome into the NDR. Using RSC ChIP data reported by others, we show that RSC occupancy is highest on the coding regions of heavily transcribed genes, though not at their NDRs. We propose that RSC has a role in restoring chromatin structure after transcription. Analysis of gene pairs in different orientations demonstrates that phasing patterns reflect competition between phasing signals emanating from neighboring NDRs. These signals may be in phase, resulting in constructive interference and a regular array, or out of phase, resulting in destructive interference and fuzzy positioning. We propose a modified barrier model, in which a stable complex located at the NDR acts as a bidirectional phasing barrier. In RSC-depleted cells, this barrier has a smaller footprint, resulting in narrower NDRs. Thus, RSC plays a critical role in organizing yeast chromatin. Published by Cold Spring Harbor Laboratory Press.

ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair.

PubMed

Tsuda, Masataka; Cho, Kosai; Ooka, Masato; Shimizu, Naoto; Watanabe, Reiko; Yasui, Akira; Nakazawa, Yuka; Ogi, Tomoo; Harada, Hiroshi; Agama, Keli; Nakamura, Jun; Asada, Ryuta; Fujiike, Haruna; Sakuma, Tetsushi; Yamamoto, Takashi; Murai, Junko; Hiraoka, Masahiro; Koike, Kaoru; Pommier, Yves; Takeda, Shunichi; Hirota, Kouji

2017-01-01

ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1's ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1's role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.

The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

PubMed

Ho, James; Sielaff, Hendrik; Nadeem, Aftab; Svanborg, Catharina; Grüber, Gerhard

2015-05-22

HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting

NASA Technical Reports Server (NTRS)

Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)

1993-01-01

Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

Acetyllysine-binding and function of bromodomain-containing proteins in chromatin.

PubMed

Dyson, M H; Rose, S; Mahadevan, L C

2001-08-01

Acetylated histones are generally associated with active chromatin. The bromodomain has recently been identified as a protein module capable of binding to acetylated lysine residues, and hence is able to mediate the recruitment of factors to acetylated chromatin. Functional studies of bromodomain-containing proteins indicate how this domain contributes to the activity of a number of nuclear factors including histone acetyltransferases and chromatin remodelling complexes. Here, we review the characteristics of acetyllysine-binding by bromodomains, discuss associated domains found in these proteins, and address the function of the bromodomain in the context of chromatin. Finally, the modulation of bromodomain binding by neighbouring post-translational modifications within histone tails might provide a mechanism through which combinations of covalent marks could exert control on chromatin function.

DNA Stimulates ATP-Dependent Proteolysis and Protein-Dependent ATPase Activity of Protease La from Escherichia coli

NASA Astrophysics Data System (ADS)

Chung, Chin Ha; Goldberg, Alfred L.

1982-02-01

The product of the lon gene in Escherichia coli is an ATP-dependent protease, protease La, that also binds strongly to DNA. Addition of double-stranded or single-stranded DNA to the protease in the presence of ATP was found to stimulate the hydrolysis of casein or globin 2- to 7-fold, depending on the DNA concentration. Native DNA from several sources (plasmid pBR322, phage T7, or calf thymus) had similar effects, but after denaturation the DNA was 20-100% more effective than the native form. Although poly(rA), globin mRNA, and various tRNAs did not stimulate proteolysis, poly(rC) and poly(rU) were effective. Poly(dT) was stimulatory but (dT)10 was not. In the presence of DNA as in its absence, proteolysis required concomitant ATP hydrolysis, and the addition of DNA also enhanced ATP hydrolysis by protease La 2-fold, but only in the presence of casein. At much higher concentrations, DNA inhibited proteolysis as well as ATP cleavage. Thus, association of this enzyme with DNA may regulate the degradation of cell proteins in vivo.

Chromatin remodeling and stem cell theory of relativity.

PubMed

Cerny, Jan; Quesenberry, Peter J

2004-10-01

The field of stem cell biology is currently being redefined. Stem cell (hematopoietic and non-hematopoietic) differentiation has been considered hierarchical in nature, but recent data suggest that there is no progenitor/stem cell hierarchy, but rather a reversible continuum. The stem cell (hematopoietic and non-hematopoietic) phenotype, the total differentiation capacity (hematopoietic and non-hematopoietic), gene expression as well as other stem cell functional characteristics (homing, receptor and adhesion molecule expression) vary throughout a cell-cycle transit widely. This seems to be dependent on shifting chromatin and gene expression with cell-cycle transit. The published data on DNA methylation, histone acetylation, and also RNAi, the major regulators of gene expression, conjoins very well and provides an explanation for the major issues of stem cell biology. Those features of stem cells mentioned above can be rather difficult to apprehend when a classical hierarchy biology view is applied, but they become clear and easier to understand once they are correlated with the underlining epigenetic changes. We are entering a new era of stem cell biology the era of "chromatinomics." We are one step closer to the practical use of cellular therapy for degenerative diseases.

Soft skills turned into hard facts: nucleosome remodelling at developmental switches.

PubMed

Chioda, M; Becker, P B

2010-07-01

Nucleosome remodelling factors are regulators of DNA accessibility in chromatin and lubricators of all major functions of eukaryotic genomes. Their action is transient and reversible, yet can be decisive for irreversible cell-fate decisions during development. In addition to the well-known local actions of nucleosome remodelling factors during transcription initiation, more global and fundamental roles for remodelling complexes in shaping the epigenome during development are emerging.

Stage-dependent remodeling of the nuclear envelope and lamina during rabbit early embryonic development.

PubMed

Popken, Jens; Schmid, Volker J; Strauss, Axel; Guengoer, Tuna; Wolf, Eckhard; Zakhartchenko, Valeri

2016-04-22

Utilizing 3D structured illumination microscopy, we investigated the quality and quantity of nuclear invaginations and the distribution of nuclear pores during rabbit early embryonic development and identified the exact time point of nucleoporin 153 (NUP153) association with chromatin during mitosis. Contrary to bovine early embryonic nuclei, featuring almost exclusively nuclear invaginations containing a small volume of cytoplasm, nuclei in rabbit early embryonic stages show additionally numerous invaginations containing a large volume of cytoplasm. Small-volume invaginations frequently emanated from large-volume nuclear invaginations but not vice versa, indicating a different underlying mechanism. Large- and small-volume nuclear envelope invaginations required the presence of chromatin, as they were restricted to chromatin-positive areas. The chromatin-free contact areas between nucleolar precursor bodies (NPBs) and large-volume invaginations were free of nuclear pores. Small-volume invaginations were not in contact with NPBs. The number of invaginations and isolated intranuclear vesicles per nucleus peaked at the 4-cell stage. At this stage, the nuclear surface showed highly concentrated clusters of nuclear pores surrounded by areas free of nuclear pores. Isolated intranuclear lamina vesicles were usually NUP153 negative. Cytoplasmic, randomly distributed NUP153-positive clusters were highly abundant at the zygote stage and decreased in number until they were almost absent at the 8-cell stage and later. These large NUP153 clusters may represent a maternally provided NUP153 deposit, but they were not visible as clusters during mitosis. Major genome activation at the 8- to 16-cell stage may mark the switch from a necessity for a deposit to on-demand production. NUP153 association with chromatin is initiated during metaphase before the initiation of the regeneration of the lamina. To our knowledge, the present study demonstrates for the first time major remodeling

From neural development to cognition: unexpected roles for chromatin

PubMed Central

Ronan, Jehnna L.; Wu, Wei

2014-01-01

Recent genome-sequencing studies in human neurodevelopmental and psychiatric disorders have uncovered mutations in many chromatin regulators. These human genetic studies, along with studies in model organisms, are providing insight into chromatin regulatory mechanisms in neural development and how alterations to these mechanisms can cause cognitive deficits, such as intellectual disability. We discuss several implicated chromatin regulators, including BAF (also known as SWI/SNF) and CHD8 chromatin remodellers, HDAC4 and the Polycomb component EZH2. Interestingly, mutations in EZH2 and certain BAF complex components have roles in both neurodevelopmental disorders and cancer, and overlapping point mutations are suggesting functionally important residues and domains. We speculate on the contribution of these similar mutations to disparate disorders. PMID:23568486

The TRPM7 chanzyme is cleaved to release a chromatin modifying kinase

PubMed Central

Krapivinsky, Grigory; Krapivinsky, Luba; Manasian, Yunona; Clapham, David E.

2014-01-01

SUMMARY TRPM7 is a ubiquitous ion channel and kinase, a unique ‘chanzyme’, required for proper early embryonic development. It conducts Zn2+, Mg2+, Ca2+ as well as monovalent cations, and contains a functional serine/threonine kinase at its carboxyl terminus. Here, we show that in normal tissues and cell lines, the kinase is proteolytically cleaved from the channel domain in a cell type-specific manner. These TRPM7 Cleaved Kinase fragments (M7CKs) translocate to the nucleus and bind multiple components of chromatin remodeling complexes, including Polycomb group proteins. In the nucleus, the kinase phosphorylates specific serines/threonines of histones. M7CK-dependent phosphorylation of H3Ser10 at promoters of TRPM7-dependent genes correlates with their activity. We also demonstrate that cytosolic free [Zn2+] is TRPM7-dependent and regulates M7CK binding to transcription factors containing zinc-finger domains. These findings suggest that TRPM7-mediated modulation of intracellular Zn2+ concentration couples ion channel signaling to epigenetic chromatin covalent modifications that affect gene expression patterns. PMID:24855944

Histone arginine methylations: their roles in chromatin dynamics and transcriptional regulation

PubMed Central

LITT, Michael; QIU, Yi; HUANG, Suming

2017-01-01

Synopsis PRMTs (protein arginine N-methyltransferases) specifically modify the arginine residues of key cellular and nuclear proteins as well as histone substrates. Like lysine methylation, transcriptional repression or activation is dependent upon the site and type of arginine methylation on histone tails. Recent discoveries imply that histone arginine methylation is an important modulator of dynamic chromatin regulation and transcriptional controls. However, under the shadow of lysine methylation, the roles of histone arginine methylation have been under-explored. The present review focuses on the roles of histone arginine methylation in the regulation of gene expression, and the interplays between histone arginine methylation, histone acetylation, lysine methylation and chromatin remodelling factors. In addition, we discuss the dynamic regulation of arginine methylation by arginine demethylases, and how dysregulation of PRMTs and their activities are linked to human diseases such as cancer. PMID:19220199

Ectopically tethered CP190 induces large-scale chromatin decondensation

NASA Astrophysics Data System (ADS)

Ahanger, Sajad H.; Günther, Katharina; Weth, Oliver; Bartkuhn, Marek; Bhonde, Ramesh R.; Shouche, Yogesh S.; Renkawitz, Rainer

2014-01-01

Insulator mediated alteration in higher-order chromatin and/or nucleosome organization is an important aspect of epigenetic gene regulation. Recent studies have suggested a key role for CP190 in such processes. In this study, we analysed the effects of ectopically tethered insulator factors on chromatin structure and found that CP190 induces large-scale decondensation when targeted to a condensed lacO array in mammalian and Drosophila cells. In contrast, dCTCF alone, is unable to cause such a decondensation, however, when CP190 is present, dCTCF recruits it to the lacO array and mediates chromatin unfolding. The CP190 induced opening of chromatin may not be correlated with transcriptional activation, as binding of CP190 does not enhance luciferase activity in reporter assays. We propose that CP190 may mediate histone modification and chromatin remodelling activity to induce an open chromatin state by its direct recruitment or targeting by a DNA binding factor such as dCTCF.

Rat Liver Canalicular Membrane Vesicles Contain an ATP-Dependent Bile Acid Transport System

NASA Astrophysics Data System (ADS)

Nishida, Toshirou; Gatmaitan, Zenaida; Che, Mingxin; Arias, Irwin M.

1991-08-01

The secretion of bile by the liver is primarily determined by the ability of the hepatocyte to transport bile acids into the bile canaliculus. A carrier-mediated process for the transport of taurocholate, the major bile acid in humans and rats, was previously demonstrated in canalicular membrane vesicles from rat liver. This process is driven by an outside-positive membrane potential that is, however, insufficient to explain the large bile acid concentration gradient between the hepatocyte and bile. In this study, we describe an ATP-dependent transport system for taurocholate in inside-out canalicular membrane vesicles from rat liver. The transport system is saturable, temperature-dependent, osmotically sensitive, specifically requires ATP, and does not function in sinusoidal membrane vesicles and right side-out canalicular membrane vesicles. Transport was inhibited by other bile acids but not by substrates for the previously demonstrated ATP-dependent canalicular transport systems for organic cations or nonbile acid organic anions. Defects in ATP-dependent canalicular transport of bile acids may contribute to reduced bile secretion (cholestasis) in various developmental, inheritable, and acquired disorders.

The Nucleosome Remodeling and Deacetylase (NuRD) Complex in Development and Disease

PubMed Central

Basta, Jeannine; Rauchman, Michael

2014-01-01

The Nucleosome Remodeling and Deacetylase (NuRD) complex is one of the major chromatin remodeling complexes found in cells. It plays an important role in regulating gene transcription, genome integrity and cell cycle progression. Through its impact on these basic cellular processes, increasing evidence indicates that alterations in the activity of this macromolecular complex can lead to developmental defects, oncogenesis and accelerated ageing. Recent genetic and biochemical studies have elucidated the mechanisms of NuRD action in modifying the chromatin landscape. These advances have the potential to lead to new therapeutic approaches to birth defects and cancer. PMID:24880148

Functional expression of a heterologous nickel-dependent, ATP-independent urease in Saccharomyces cerevisiae.

PubMed

Milne, N; Luttik, M A H; Cueto Rojas, H F; Wahl, A; van Maris, A J A; Pronk, J T; Daran, J M

2015-07-01

In microbial processes for production of proteins, biomass and nitrogen-containing commodity chemicals, ATP requirements for nitrogen assimilation affect product yields on the energy producing substrate. In Saccharomyces cerevisiae, a current host for heterologous protein production and potential platform for production of nitrogen-containing chemicals, uptake and assimilation of ammonium requires 1 ATP per incorporated NH3. Urea assimilation by this yeast is more energy efficient but still requires 0.5 ATP per NH3 produced. To decrease ATP costs for nitrogen assimilation, the S. cerevisiae gene encoding ATP-dependent urease (DUR1,2) was replaced by a Schizosaccharomyces pombe gene encoding ATP-independent urease (ure2), along with its accessory genes ureD, ureF and ureG. Since S. pombe ure2 is a Ni(2+)-dependent enzyme and Saccharomyces cerevisiae does not express native Ni(2+)-dependent enzymes, the S. pombe high-affinity nickel-transporter gene (nic1) was also expressed. Expression of the S. pombe genes into dur1,2Δ S. cerevisiae yielded an in vitro ATP-independent urease activity of 0.44±0.01 µmol min(-1) mg protein(-1) and restored growth on urea as sole nitrogen source. Functional expression of the Nic1 transporter was essential for growth on urea at low Ni(2+) concentrations. The maximum specific growth rates of the engineered strain on urea and ammonium were lower than those of a DUR1,2 reference strain. In glucose-limited chemostat cultures with urea as nitrogen source, the engineered strain exhibited an increased release of ammonia and reduced nitrogen content of the biomass. Our results indicate a new strategy for improving yeast-based production of nitrogen-containing chemicals and demonstrate that Ni(2+)-dependent enzymes can be functionally expressed in S. cerevisiae. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly

PubMed Central

MacAlpine, Heather K.; Lubelsky, Yoav; Hartemink, Alexander J.

2015-01-01

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2–7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we “footprinted” nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2–7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS). PMID:25593310

Genomic and proteomic characterization of ARID1A chromatin remodeller in ampullary tumors

PubMed Central

Nastase, Anca; Teo, Jin Yao; Heng, Hong Lee; Ng, Cedric Chuan Young; Myint, Swe Swe; Rajasegaran, Vikneswari; Loh, Jia Liang; Lee, Ser Yee; Ooi, London Lucien; Chung, Alexander Yaw Fui; Chow, Pierce Kah Hoe; Cheow, Peng Chung; Wan, Wei Keat; Azhar, Rafy; Khoo, Avery; Xiu, Sam Xin; Alkaff, Syed Muhammad Fahmy; Cutcutache, Ioana; Lim, Jing Quan; Ong, Choon Kiat; Herlea, Vlad; Dima, Simona; Duda, Dan G; Teh, Bin Tean; Popescu, Irinel; Lim, Tony Kiat Hon

2017-01-01

AT rich interactive domain 1A (ARID1A) is one of the most commonly mutated genes in a broad variety of tumors. The mechanisms that involve ARID1A in ampullary cancer progression remains elusive. Here, we evaluated the frequency of ARID1A and KRAS mutations in ampullary adenomas and adenocarcinomas and in duodenal adenocarcinomas from two cohorts of patients from Singapore and Romania, correlated with clinical and pathological tumor features, and assessed the functional role of ARID1A. In the ampullary adenocarcinomas, the frequency of KRAS and ARID1A mutations was 34.7% and 8.2% respectively, with a loss or reduction of ARID1A protein in 17.2% of the cases. ARID1A mutational status was significantly correlated with ARID1A protein expression level (P=0.023). There was a significant difference in frequency of ARID1A mutation between Romania and Singapore (2.7% versus 25%, P=0.04), suggestive of different etiologies. One somatic mutation was detected in the ampullary adenoma group. In vitro studies indicated the tumor suppressive role of ARID1A. Our results warrant further investigation of this chromatin remodeller as a potential early biomarker of the disease, as well as identification of therapeutic targets in ARID1A mutated ampullary cancers. PMID:28401006

Genomic and proteomic characterization of ARID1A chromatin remodeller in ampullary tumors.

PubMed

Nastase, Anca; Teo, Jin Yao; Heng, Hong Lee; Ng, Cedric Chuan Young; Myint, Swe Swe; Rajasegaran, Vikneswari; Loh, Jia Liang; Lee, Ser Yee; Ooi, London Lucien; Chung, Alexander Yaw Fui; Chow, Pierce Kah Hoe; Cheow, Peng Chung; Wan, Wei Keat; Azhar, Rafy; Khoo, Avery; Xiu, Sam Xin; Alkaff, Syed Muhammad Fahmy; Cutcutache, Ioana; Lim, Jing Quan; Ong, Choon Kiat; Herlea, Vlad; Dima, Simona; Duda, Dan G; Teh, Bin Tean; Popescu, Irinel; Lim, Tony Kiat Hon

2017-01-01

AT rich interactive domain 1A (ARID1A) is one of the most commonly mutated genes in a broad variety of tumors. The mechanisms that involve ARID1A in ampullary cancer progression remains elusive. Here, we evaluated the frequency of ARID1A and KRAS mutations in ampullary adenomas and adenocarcinomas and in duodenal adenocarcinomas from two cohorts of patients from Singapore and Romania, correlated with clinical and pathological tumor features, and assessed the functional role of ARID1A . In the ampullary adenocarcinomas, the frequency of KRAS and ARID1A mutations was 34.7% and 8.2% respectively, with a loss or reduction of ARID1A protein in 17.2% of the cases. ARID1A mutational status was significantly correlated with ARID1A protein expression level (P=0.023). There was a significant difference in frequency of ARID1A mutation between Romania and Singapore (2.7% versus 25%, P=0.04), suggestive of different etiologies. One somatic mutation was detected in the ampullary adenoma group. In vitro studies indicated the tumor suppressive role of ARID1A . Our results warrant further investigation of this chromatin remodeller as a potential early biomarker of the disease, as well as identification of therapeutic targets in ARID1A mutated ampullary cancers.

Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

PubMed Central

Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

2003-01-01

Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

Synaptic, transcriptional and chromatin genes disrupted in autism.

PubMed

De Rubeis, Silvia; He, Xin; Goldberg, Arthur P; Poultney, Christopher S; Samocha, Kaitlin; Cicek, A Erucment; Kou, Yan; Liu, Li; Fromer, Menachem; Walker, Susan; Singh, Tarinder; Klei, Lambertus; Kosmicki, Jack; Shih-Chen, Fu; Aleksic, Branko; Biscaldi, Monica; Bolton, Patrick F; Brownfeld, Jessica M; Cai, Jinlu; Campbell, Nicholas G; Carracedo, Angel; Chahrour, Maria H; Chiocchetti, Andreas G; Coon, Hilary; Crawford, Emily L; Curran, Sarah R; Dawson, Geraldine; Duketis, Eftichia; Fernandez, Bridget A; Gallagher, Louise; Geller, Evan; Guter, Stephen J; Hill, R Sean; Ionita-Laza, Juliana; Jimenz Gonzalez, Patricia; Kilpinen, Helena; Klauck, Sabine M; Kolevzon, Alexander; Lee, Irene; Lei, Irene; Lei, Jing; Lehtimäki, Terho; Lin, Chiao-Feng; Ma'ayan, Avi; Marshall, Christian R; McInnes, Alison L; Neale, Benjamin; Owen, Michael J; Ozaki, Noriio; Parellada, Mara; Parr, Jeremy R; Purcell, Shaun; Puura, Kaija; Rajagopalan, Deepthi; Rehnström, Karola; Reichenberg, Abraham; Sabo, Aniko; Sachse, Michael; Sanders, Stephan J; Schafer, Chad; Schulte-Rüther, Martin; Skuse, David; Stevens, Christine; Szatmari, Peter; Tammimies, Kristiina; Valladares, Otto; Voran, Annette; Li-San, Wang; Weiss, Lauren A; Willsey, A Jeremy; Yu, Timothy W; Yuen, Ryan K C; Cook, Edwin H; Freitag, Christine M; Gill, Michael; Hultman, Christina M; Lehner, Thomas; Palotie, Aaarno; Schellenberg, Gerard D; Sklar, Pamela; State, Matthew W; Sutcliffe, James S; Walsh, Christiopher A; Scherer, Stephen W; Zwick, Michael E; Barett, Jeffrey C; Cutler, David J; Roeder, Kathryn; Devlin, Bernie; Daly, Mark J; Buxbaum, Joseph D

2014-11-13

The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.

An overproduction of astellolides induced by genetic disruption of chromatin-remodeling factors in Aspergillus oryzae.

PubMed

Shinohara, Yasutomo; Kawatani, Makoto; Futamura, Yushi; Osada, Hiroyuki; Koyama, Yasuji

2016-01-01

The filamentous fungus Aspergillus oryzae is an important industrial mold. Recent genomic analysis indicated that A. oryzae has a large number of biosynthetic genes for secondary metabolites (SMs), but many of the SMs they produce have not been identified. For better understanding of SMs production by A. oryzae, we screened a gene-disruption library of transcription factors including chromatin-remodeling factors and found two gene disruptions that show similarly altered SM production profiles. One is a homolog of Aspergillus nidulans cclA, a component of the histone 3 lysine 4 (H3K4) methyltransferase complex of proteins associated with Set1 complex, and the other, sppA, is an ortholog of Saccharomyces cerevisiae SPP1, another component of a complex of proteins associated with Set1 complex. The cclA and sppA disruptions in A. oryzae are deficient in trimethylation of H3K4. Furthermore, one of the SMs that increased in the cclA disruptant was identified as astellolide F (14-deacetyl astellolide B). These data indicate that both cclA and sppA affect production of SMs including astellolides by affecting the methylation status of H3K4 in A. oryzae.

Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

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

Ostrup, Olga, E-mail: osvarcova@gmail.com; Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo; Norwegian Center for Stem Cell Research, Oslo

Highlights: {yields} Xenopus egg extract remodels nuclei and alter cell growth characteristics. {yields} Ribosomal genes are reprogrammed within 6 h after extract exposure. {yields} rDNA reprogramming involves promoter targeting of SNF2H remodeling complex. {yields} Xenopus egg extract does not initiate stress-related response in somatic cells. {yields} Aza-cytidine elicits a stress-induced response in reprogrammed cells. -- Abstract: Extracts from Xenopus eggs can reprogram gene expression in somatic nuclei, however little is known about the earliest processes associated with the switch in the transcriptional program. We show here that an early reprogramming event is the remodeling of ribosomal chromatin and gene expression.more » This occurs within hours of extract treatment and is distinct from a stress response. Egg extract elicits remodeling of the nuclear envelope, chromatin and nucleolus. Nucleolar remodeling involves a rapid and stable decrease in ribosomal gene transcription, and promoter targeting of the nucleolar remodeling complex component SNF2H without affecting occupancy of the transcription factor UBF and the stress silencers SUV39H1 and SIRT1. During this process, nucleolar localization of UBF and SIRT1 is not altered. On contrary, azacytidine pre-treatment has an adverse effect on rDNA remodeling induced by extract and elicits a stress-type nuclear response. Thus, an early event of Xenopus egg extract-mediated nuclear reprogramming is the remodeling of ribosomal genes involving nucleolar remodeling complex. Condition-specific and rapid silencing of ribosomal genes may serve as a sensitive marker for evaluation of various reprogramming methods.« less

Chromatin- and temperature-dependent modulation of radiation-induced double-strand breaks.

PubMed

Elmroth, K; Nygren, J; Stenerlöw, B; Hultborn, R

2003-10-01

To investigate the influence of chromatin organization and scavenging capacity in relation to irradiation temperature on the induction of double-strand breaks (DSB) in structures derived from human diploid fibroblasts. Agarose plugs with different chromatin structures (intact cells+/-wortmannin, permeabilized cells with condensed chromatin, nucleoids and DNA) were prepared and irradiated with X-rays at 2 or 37 degrees C and lysed using two different lysis protocols (new ice-cold lysis or standard lysis at 37 degrees C). Induction of DSB was determined by constant-field gel electrophoresis. The dose-modifying factor (DMF(temp)) for irradiation at 37 compared with 2 degrees C was 0.92 in intact cells (i.e. more DSB induced at 2 degrees C), but gradually increased to 1.5 in permeabilized cells, 2.2 in nucleoids and 2.6 in naked DNA, suggesting a role of chromatin organization for temperature modulation of DNA damage. In addition, DMF(temp) was influenced by the presence of 0.1 M DMSO or 30 mM glutathione, but not by post-irradiation temperature. The protective effect of low temperature was correlated to the indirect effects of ionizing radiation and was not dependent on post-irradiation temperature. Reasons for a dose modifying factor <1 in intact cells are discussed.

Sucralose, an activator of the glucose-sensing receptor, increases ATP by calcium-dependent and -independent mechanisms.

PubMed

Li, Longfei; Ohtsu, Yoshiaki; Nakagawa, Yuko; Masuda, Katsuyoshi; Kojima, Itaru

2016-08-31

Sucralose is an artificial sweetener and activates the glucose-sensing receptor expressed in pancreatic β-cells. Although sucralose does not enter β-cells nor acts as a substrate for glucokinase, it induces a marked elevation of intracellular ATP ([ATP]c). The present study was conducted to identify the signaling pathway responsible for the elevation of [ATP]c induced by sucralose. Previous studies have shown that sucralose elevates cyclic AMP (cAMP), activates phospholipase C (PLC) and stimulates Ca(2+) entry by a Na(+)-dependent mechanism in MIN6 cells. The addition of forskolin induced a marked elevation of cAMP, whereas it did not affect [ATP]c. Carbachol, an activator of PLC, did not increase [ATP]c. In addition, activation of protein kinase C by dioctanoylglycerol did not affect [ATP]c. In contrast, nifedipine, an inhibitor of the voltage-dependent Ca(2+) channel, significantly reduced [ATP]c response to sucralose. Removal of extracellular Na(+) nearly completely blocked sucralose-induced elevation of [ATP]c. Stimulation of Na(+) entry by adding a Na(+) ionophore monensin elevated [ATP]c. The monensin-induced elevation of [ATP]c was only partially inhibited by nifedipine and loading of BAPTA, both of which completely abolished elevation of [Ca(2+)]c. These results suggest that Na(+) entry is critical for the sucralose-induced elevation of [ATP]c. Both calcium-dependent and -independent mechanisms are involved in the action of sucralose.

Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1

PubMed Central

Butryn, Agata; Schuller, Jan M; Stoehr, Gabriele; Runge-Wollmann, Petra; Förster, Friedrich; Auble, David T; Hopfner, Karl-Peter

2015-01-01

Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex. DOI: http://dx.doi.org/10.7554/eLife.07432.001 PMID:26258880

Epigenetic control of plant immunity.

PubMed

Alvarez, María E; Nota, Florencia; Cambiagno, Damián A

2010-07-01

In eukaryotic genomes, gene expression and DNA recombination are affected by structural chromatin traits. Chromatin structure is shaped by the activity of enzymes that either introduce covalent modifications in DNA and histone proteins or use energy from ATP to disrupt histone-DNA interactions. The genomic 'marks' that are generated by covalent modifications of histones and DNA, or by the deposition of histone variants, are susceptible to being altered in response to stress. Recent evidence has suggested that proteins generating these epigenetic marks play crucial roles in the defence against pathogens. Histone deacetylases are involved in the activation of jasmonic acid- and ethylene-sensitive defence mechanisms. ATP-dependent chromatin remodellers mediate the constitutive repression of the salicylic acid-dependent pathway, whereas histone methylation at the WRKY70 gene promoter affects the activation of this pathway. Interestingly, bacterial-infected tissues show a net reduction in DNA methylation, which may affect the disease resistance genes responsible for the surveillance against pathogens. As some epigenetic marks can be erased or maintained and transmitted to offspring, epigenetic mechanisms may provide plasticity for the dynamic control of emerging pathogens without the generation of genomic lesions.

Shelterin Protects Chromosome Ends by Compacting Telomeric Chromatin

PubMed Central

Bandaria, Jigar N.; Qin, Peiwu; Berk, Veysel; Chu, Steven; Yildiz, Ahmet

2016-01-01

SUMMARY Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, and not by DNA methylation, histone deacetylation or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres become more accessible to telomere-associated proteins and accumulate DDR signals. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery. PMID:26871633

Chromatin regulators as a guide for cancer treatment choice

PubMed Central

Gurard-Levin, Zachary A.; Wilson, Laurence O.W.; Pancaldi, Vera; Postel-Vinay, Sophie; Sousa, Fabricio G.; Reyes, Cecile; Marangoni, Elisabetta; Gentien, David; Valencia, Alfonso; Pommier, Yves; Cottu, Paul; Almouzni, Geneviève

2016-01-01

The limited capacity to predict a patient’s response to distinct chemotherapeutic agents is a major hurdle in cancer management. The efficiency of a large fraction of current cancer therapeutics (radio- and chemotherapies) is influenced by chromatin structure. Reciprocally, alterations in chromatin organization may impact resistance mechanisms. Here, we explore how the mis-expression of chromatin regulators—factors involved in the establishment and maintenance of functional chromatin domains—can inform about the extent of docetaxel response. We exploit gene Affymetrix and NanoString gene expression data for a set of chromatin regulators generated from breast cancer patient-derived xenograft (PDX) models and patient samples treated with docetaxel. Random Forest classification reveals specific panels of chromatin regulators, including key components of the SWI/SNF chromatin remodeler, which readily distinguish docetaxel high-responders and poor-responders. Further exploration of SWI/SNF components in the comprehensive NCI-60 dataset reveals that the expression inversely correlates with docetaxel sensitivity. Finally, we show that loss of the SWI/SNF subunit BRG1 (SMARCA4) in a model cell line leads to enhanced docetaxel sensitivity. Altogether, our findings point towards chromatin regulators as biomarkers for drug response as well as therapeutic targets to sensitize patients towards docetaxel and combat drug resistance. PMID:27196757

HIV-1 Vpr Induces the Degradation of ZIP and sZIP, Adaptors of the NuRD Chromatin Remodeling Complex, by Hijacking DCAF1/VprBP

PubMed Central

Maudet, Claire; Sourisce, Adèle; Dragin, Loïc; Lahouassa, Hichem; Rain, Jean-Christophe; Bouaziz, Serge; Ramirez, Bertha Cécilia; Margottin-Goguet, Florence

2013-01-01

The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered. PMID:24116224

Redox-dependent complex formation by an ATP-dependent activator of the corrinoid/iron-sulfur protein

PubMed Central

Hennig, Sandra E.; Jeoung, Jae-Hun; Goetzl, Sebastian; Dobbek, Holger

2012-01-01

Movement, cell division, protein biosynthesis, electron transfer against an electrochemical gradient, and many more processes depend on energy conversions coupled to the hydrolysis of ATP. The reduction of metal sites with low reduction potentials (E0′ < -500 mV) is possible by connecting an energetical uphill electron transfer with the hydrolysis of ATP. The corrinoid-iron/sulfur protein (CoFeSP) operates within the reductive acetyl-CoA pathway by transferring a methyl group from methyltetrahydrofolate bound to a methyltransferase to the [Ni-Ni-Fe4S4] cluster of acetyl-CoA synthase. Methylation of CoFeSP only occurs in the low-potential Co(I) state, which can be sporadically oxidized to the inactive Co(II) state, making its reductive reactivation necessary. Here we show that an open-reading frame proximal to the structural genes of CoFeSP encodes an ATP-dependent reductive activator of CoFeSP. Our biochemical and structural analysis uncovers a unique type of reductive activator distinct from the electron-transferring ATPases found to reduce the MoFe-nitrogenase and 2-hydroxyacyl-CoA dehydratases. The CoFeSP activator contains an ASKHA domain (acetate and sugar kinases, Hsp70, and actin) harboring the ATP-binding site, which is also present in the activator of 2-hydroxyacyl-CoA dehydratases and a ferredoxin-like [2Fe-2S] cluster domain acting as electron donor. Complex formation between CoFeSP and its activator depends on the oxidation state of CoFeSP, which provides evidence for a unique strategy to achieve unidirectional electron transfer between two redox proteins. PMID:22431597

Novel pedigree analysis implicates DNA repair and chromatin remodeling in multiple myeloma risk

PubMed Central

Curtin, Karen; Rajamanickam, Venkatesh; Jayabalan, David; Atanackovic, Djordje; Rajkumar, S. Vincent; Kumar, Shaji; Slager, Susan; Galia, Perrine; Demangel, Delphine; Salama, Mohamed; Joseph, Vijai; Lipkin, Steven M.; Dumontet, Charles; Vachon, Celine M.

2018-01-01

The high-risk pedigree (HRP) design is an established strategy to discover rare, highly-penetrant, Mendelian-like causal variants. Its success, however, in complex traits has been modest, largely due to challenges of genetic heterogeneity and complex inheritance models. We describe a HRP strategy that addresses intra-familial heterogeneity, and identifies inherited segments important for mapping regulatory risk. We apply this new Shared Genomic Segment (SGS) method in 11 extended, Utah, multiple myeloma (MM) HRPs, and subsequent exome sequencing in SGS regions of interest in 1063 MM / MGUS (monoclonal gammopathy of undetermined significance–a precursor to MM) cases and 964 controls from a jointly-called collaborative resource, including cases from the initial 11 HRPs. One genome-wide significant 1.8 Mb shared segment was found at 6q16. Exome sequencing in this region revealed predicted deleterious variants in USP45 (p.Gln691* and p.Gln621Glu), a gene known to influence DNA repair through endonuclease regulation. Additionally, a 1.2 Mb segment at 1p36.11 is inherited in two Utah HRPs, with coding variants identified in ARID1A (p.Ser90Gly and p.Met890Val), a key gene in the SWI/SNF chromatin remodeling complex. Our results provide compelling statistical and genetic evidence for segregating risk variants for MM. In addition, we demonstrate a novel strategy to use large HRPs for risk-variant discovery more generally in complex traits. PMID:29389935

Novel pedigree analysis implicates DNA repair and chromatin remodeling in multiple myeloma risk.

PubMed

Waller, Rosalie G; Darlington, Todd M; Wei, Xiaomu; Madsen, Michael J; Thomas, Alun; Curtin, Karen; Coon, Hilary; Rajamanickam, Venkatesh; Musinsky, Justin; Jayabalan, David; Atanackovic, Djordje; Rajkumar, S Vincent; Kumar, Shaji; Slager, Susan; Middha, Mridu; Galia, Perrine; Demangel, Delphine; Salama, Mohamed; Joseph, Vijai; McKay, James; Offit, Kenneth; Klein, Robert J; Lipkin, Steven M; Dumontet, Charles; Vachon, Celine M; Camp, Nicola J

2018-02-01

The high-risk pedigree (HRP) design is an established strategy to discover rare, highly-penetrant, Mendelian-like causal variants. Its success, however, in complex traits has been modest, largely due to challenges of genetic heterogeneity and complex inheritance models. We describe a HRP strategy that addresses intra-familial heterogeneity, and identifies inherited segments important for mapping regulatory risk. We apply this new Shared Genomic Segment (SGS) method in 11 extended, Utah, multiple myeloma (MM) HRPs, and subsequent exome sequencing in SGS regions of interest in 1063 MM / MGUS (monoclonal gammopathy of undetermined significance-a precursor to MM) cases and 964 controls from a jointly-called collaborative resource, including cases from the initial 11 HRPs. One genome-wide significant 1.8 Mb shared segment was found at 6q16. Exome sequencing in this region revealed predicted deleterious variants in USP45 (p.Gln691* and p.Gln621Glu), a gene known to influence DNA repair through endonuclease regulation. Additionally, a 1.2 Mb segment at 1p36.11 is inherited in two Utah HRPs, with coding variants identified in ARID1A (p.Ser90Gly and p.Met890Val), a key gene in the SWI/SNF chromatin remodeling complex. Our results provide compelling statistical and genetic evidence for segregating risk variants for MM. In addition, we demonstrate a novel strategy to use large HRPs for risk-variant discovery more generally in complex traits.

A novel germ cell-specific protein, SHIP1, forms a complex with chromatin remodeling activity during spermatogenesis.

PubMed

Choi, Eunyoung; Han, Cecil; Park, Inju; Lee, Boyeon; Jin, Sora; Choi, Heejin; Kim, Do Han; Park, Zee Yong; Eddy, Edward M; Cho, Chunghee

2008-12-12

To determine the mechanisms of spermatogenesis, it is essential to identify and characterize germ cell-specific genes. Here we describe a protein encoded by a novel germ cell-specific gene, Mm.290718/ZFP541, identified from the mouse spermatocyte UniGene library. The protein contains specific motifs and domains potentially involved in DNA binding and chromatin reorganization. An antibody against Mm.290718/ZFP541 revealed the existence of the protein in testicular spermatogenic cells (159 kDa) but not testicular and mature sperm. Immunostaining analysis of cells at various stages of spermatogenesis consistently showed that the protein is present in spermatocytes and round spermatids only. Transfection assays and immunofluorescence studies indicate that the protein is localized specifically in the nucleus. Proteomic analyses performed to explore the functional characteristics of Mm.290718/ZFP541 showed that the protein forms a unique complex. Other major components of the complex included histone deacetylase 1 (HDAC1) and heat-shock protein A2. Disappearance of Mm.290718/ZFP541 was highly correlated with hyperacetylation in spermatids during spermatogenesis, and specific domains of the protein were involved in the regulation of interactions and nuclear localization of HDAC1. Furthermore, we found that premature hyperacetylation, induced by an HDAC inhibitor, is associated with an alteration in the integrity of Mm.290718/ZFP541 in spermatogenic cells. Our results collectively suggest that the Mm.290718/ZFP541 complex is implicated in chromatin remodeling during spermatogenesis, and we provide further information on the previously unknown molecular mechanism. Consequently, we re-designate Mm.290718/ZFP541 as "SHIP1" representing spermatogenic cell HDAC-interacting protein 1.

In silico evidence for sequence-dependent nucleosome sliding

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

Lequieu, Joshua; Schwartz, David C.; de Pablo, Juan J.

Nucleosomes represent the basic building block of chromatin and provide an important mechanism by which cellular processes are controlled. The locations of nucleosomes across the genome are not random but instead depend on both the underlying DNA sequence and the dynamic action of other proteins within the nucleus. These processes are central to cellular function, and the molecular details of the interplay between DNA sequence and nudeosome dynamics remain poorly understood. In this work, we investigate this interplay in detail by relying on a molecular model, which permits development of a comprehensive picture of the underlying free energy surfaces andmore » the corresponding dynamics of nudeosome repositioning. The mechanism of nudeosome repositioning is shown to be strongly linked to DNA sequence and directly related to the binding energy of a given DNA sequence to the histone core. It is also demonstrated that chromatin remodelers can override DNA-sequence preferences by exerting torque, and the histone H4 tail is then identified as a key component by which DNA-sequence, histone modifications, and chromatin remodelers could in fact be coupled.« less

A CAF-1–PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage

PubMed Central

Moggs, Jonathan G.; Grandi, Paola; Quivy, Jean-Pierre; Jónsson, Zophonías O.; Hübscher, Ulrich; Becker, Peter B.; Almouzni, Geneviève

2000-01-01

Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA–CAF-1 interaction in the context of DNA damage processing and checkpoint control. PMID:10648606

Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways

PubMed Central

Okada, Seiko F.; Ribeiro, Carla M. P.; Sesma, Juliana I.; Seminario-Vidal, Lucia; Abdullah, Lubna H.; van Heusden, Catharina; Lazarowski, Eduardo R.

2013-01-01

ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)–associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling–promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca2+ chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca2+-dependent vesicular mechanisms not associated with mucin granule secretion. PMID:23763446

The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif

PubMed Central

Barillà, Daniela; Carmelo, Emma; Hayes, Finbarr

2007-01-01

The ParF protein of plasmid TP228 belongs to the ubiquitous superfamily of ParA ATPases that drive DNA segregation in bacteria. ATP-bound ParF polymerizes into multistranded filaments. The partner protein ParG is dimeric, consisting of C-termini that interweave into a ribbon–helix–helix domain contacting the centromeric DNA and unstructured N-termini. ParG stimulates ATP hydrolysis by ParF ≈30-fold. Here, we establish that the mobile tails of ParG are crucial for this enhancement and that arginine R19 within the tail is absolutely required for activation of ParF nucleotide hydrolysis. R19 is part of an arginine finger-like loop in ParG that is predicted to intercalate into the ParF nucleotide-binding pocket thereby promoting ATP hydrolysis. Significantly, mutations of R19 abrogated DNA segregation in vivo, proving that intracellular stimulation of ATP hydrolysis by ParG is a key regulatory process for partitioning. Furthermore, ParG bundles ParF-ATP filaments as well as promoting nucleotide-independent polymerization. The N-terminal flexible tail is required for both activities, because N-terminal ΔParG polypeptides are defective in both functions. Strikingly, the critical arginine finger-like residue R19 is dispensable for ParG-mediated remodeling of ParF polymers, revealing that the ParG N-terminal tail possesses two separable activities in the interplay with ParF: a catalytic function during ATP hydrolysis and a mechanical role in modulation of polymerization. We speculate that activation of nucleotide hydrolysis via an arginine finger loop may be a conserved, regulatory mechanism of ParA family members and their partner proteins, including ParA-ParB and Soj-Spo0J that mediate DNA segregation and MinD-MinE that determine septum localization. PMID:17261809

The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif.

PubMed

Barillà, Daniela; Carmelo, Emma; Hayes, Finbarr

2007-02-06

The ParF protein of plasmid TP228 belongs to the ubiquitous superfamily of ParA ATPases that drive DNA segregation in bacteria. ATP-bound ParF polymerizes into multistranded filaments. The partner protein ParG is dimeric, consisting of C-termini that interweave into a ribbon-helix-helix domain contacting the centromeric DNA and unstructured N-termini. ParG stimulates ATP hydrolysis by ParF approximately 30-fold. Here, we establish that the mobile tails of ParG are crucial for this enhancement and that arginine R19 within the tail is absolutely required for activation of ParF nucleotide hydrolysis. R19 is part of an arginine finger-like loop in ParG that is predicted to intercalate into the ParF nucleotide-binding pocket thereby promoting ATP hydrolysis. Significantly, mutations of R19 abrogated DNA segregation in vivo, proving that intracellular stimulation of ATP hydrolysis by ParG is a key regulatory process for partitioning. Furthermore, ParG bundles ParF-ATP filaments as well as promoting nucleotide-independent polymerization. The N-terminal flexible tail is required for both activities, because N-terminal DeltaParG polypeptides are defective in both functions. Strikingly, the critical arginine finger-like residue R19 is dispensable for ParG-mediated remodeling of ParF polymers, revealing that the ParG N-terminal tail possesses two separable activities in the interplay with ParF: a catalytic function during ATP hydrolysis and a mechanical role in modulation of polymerization. We speculate that activation of nucleotide hydrolysis via an arginine finger loop may be a conserved, regulatory mechanism of ParA family members and their partner proteins, including ParA-ParB and Soj-Spo0J that mediate DNA segregation and MinD-MinE that determine septum localization.

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages.

PubMed

da Silva-Souza, Hercules Antônio; Lira, Maria Nathalia de; Costa-Junior, Helio Miranda; da Cruz, Cristiane Monteiro; Vasconcellos, Jorge Silvio Silva; Mendes, Anderson Nogueira; Pimenta-Reis, Gabriela; Alvarez, Cora Lilia; Faccioli, Lucia Helena; Serezani, Carlos Henrique; Schachter, Julieta; Persechini, Pedro Muanis

2014-07-01

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca(2+) concentration ([Ca(2+)]i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca(2+)]i. Chelating Ca(2+) ions in the extracellular medium suppressed the intracellular Ca(2+) signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca(2+)- and P2X7-independent transport mechanism in macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.

The nucleosomal acidic patch relieves auto-inhibition by the ISWI remodeler SNF2h

PubMed Central

Gamarra, Nathan; Johnson, Stephanie L; Trnka, Michael J; Burlingame, Alma L

2018-01-01

ISWI family chromatin remodeling motors use sophisticated autoinhibition mechanisms to control nucleosome sliding. Yet how the different autoinhibitory domains are regulated is not well understood. Here we show that an acidic patch formed by histones H2A and H2B of the nucleosome relieves the autoinhibition imposed by the AutoN and the NegC regions of the human ISWI remodeler SNF2h. Further, by single molecule FRET we show that the acidic patch helps control the distance travelled per translocation event. We propose a model in which the acidic patch activates SNF2h by providing a landing pad for the NegC and AutoN auto-inhibitory domains. Interestingly, the INO80 complex is also strongly dependent on the acidic patch for nucleosome sliding, indicating that this substrate feature can regulate remodeling enzymes with substantially different mechanisms. We therefore hypothesize that regulating access to the acidic patch of the nucleosome plays a key role in coordinating the activities of different remodelers in the cell. PMID:29664398

Prefrontal cortex expression of chromatin modifier genes in male WSP and WSR mice changes across ethanol dependence, withdrawal, and abstinence.

PubMed

Hashimoto, Joel G; Gavin, David P; Wiren, Kristine M; Crabbe, John C; Guizzetti, Marina

2017-05-01

Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatin modifications after chronic alcohol exposure, chronic alcohol exposure followed by 8 h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines. We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression. We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitinationhylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter. Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study. Published by Elsevier

Global alterations of the transcriptional landscape during yeast growth and development in the absence of Ume6-dependent chromatin modification.

PubMed

Lardenois, Aurélie; Becker, Emmanuelle; Walther, Thomas; Law, Michael J; Xie, Bingning; Demougin, Philippe; Strich, Randy; Primig, Michael

2015-10-01

Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MAT a/α wild-type and ume6/ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes.

Prefrontal cortex expression of chromatin modifier genes in male WSP and WSR mice changes across ethanol dependence, withdrawal, and abstinence

PubMed Central

Hashimoto, Joel G.; Gavin, David P.; Wiren, Kristine M.; Crabbe, John C.; Guizzetti, Marina

2017-01-01

Alcohol-use disorder (AUD) is a relapsing disorder associated with excessive ethanol consumption. Recent studies support the involvement of epigenetic mechanisms in the development of AUD. Studies carried out so far have focused on a few specific epigenetic modifications. The goal of this project was to investigate gene expression changes of epigenetic regulators that mediate a broad array of chromatin modifications after chronic alcohol exposure, chronic alcohol exposure followed by 8 h withdrawal, and chronic alcohol exposure followed by 21 days of abstinence in Withdrawal-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) selected mouse lines. We found that chronic vapor exposure to highly intoxicating levels of ethanol alters the expression of several chromatin remodeling genes measured by quantitative PCR array analyses. The identified effects were independent of selected lines, which, however, displayed baseline differences in epigenetic gene expression. We reported dysregulation in the expression of genes involved in histone acetylation, deacetylation, lysine and arginine methylation and ubiquitination, and in DNA methylation during chronic ethanol exposure and withdrawal, but not after 21 days of abstinence. Ethanol-induced changes are consistent with decreased histone acetylation and with decreased deposition of the permissive ubiquitination mark H2BK120ub, associated with reduced transcription. On the other hand, ethanol-induced changes in the expression of genes involved in histone lysine methylation are consistent with increased transcription. The net result of these modifications on gene expression is likely to depend on the combination of the specific histone tail modifications present at a given time on a given promoter. Since alcohol does not modulate gene expression unidirectionally, it is not surprising that alcohol does not unidirectionally alter chromatin structure toward a closed or open state, as suggested by the results of this study. PMID

Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodeling

PubMed Central

North, Justin A.; Javaid, Sarah; Ferdinand, Michelle B.; Chatterjee, Nilanjana; Picking, Jonathan W.; Shoffner, Matthew; Nakkula, Robin J.; Bartholomew, Blaine; Ottesen, Jennifer J.; Fishel, Richard; Poirier, Michael G.

2011-01-01

Nucleosomes, the fundamental units of chromatin structure, are regulators and barriers to transcription, replication and repair. Post-translational modifications (PTMs) of the histone proteins within nucleosomes regulate these DNA processes. Histone H3(T118) is a site of phosphorylation [H3(T118ph)] and is implicated in regulation of transcription and DNA repair. We prepared H3(T118ph) by expressed protein ligation and determined its influence on nucleosome dynamics. We find H3(T118ph) reduces DNA–histone binding by 2 kcal/mol, increases nucleosome mobility by 28-fold and increases DNA accessibility near the dyad region by 6-fold. Moreover, H3(T118ph) increases the rate of hMSH2–hMSH6 nucleosome disassembly and enables nucleosome disassembly by the SWI/SNF chromatin remodeler. These studies suggest that H3(T118ph) directly enhances and may reprogram chromatin remodeling reactions. PMID:21576235

Rapid and reversible epigenome editing by endogenous chromatin regulators.

PubMed

Braun, Simon M G; Kirkland, Jacob G; Chory, Emma J; Husmann, Dylan; Calarco, Joseph P; Crabtree, Gerald R

2017-09-15

Understanding the causal link between epigenetic marks and gene regulation remains a central question in chromatin biology. To edit the epigenome we developed the FIRE-Cas9 system for rapid and reversible recruitment of endogenous chromatin regulators to specific genomic loci. We enhanced the dCas9-MS2 anchor for genome targeting with Fkbp/Frb dimerizing fusion proteins to allow chemical-induced proximity of a desired chromatin regulator. We find that mSWI/SNF (BAF) complex recruitment is sufficient to oppose Polycomb within minutes, leading to activation of bivalent gene transcription in mouse embryonic stem cells. Furthermore, Hp1/Suv39h1 heterochromatin complex recruitment to active promoters deposits H3K9me3 domains, resulting in gene silencing that can be reversed upon washout of the chemical dimerizer. This inducible recruitment strategy provides precise kinetic information to model epigenetic memory and plasticity. It is broadly applicable to mechanistic studies of chromatin in mammalian cells and is particularly suited to the analysis of endogenous multi-subunit chromatin regulator complexes.Understanding the link between epigenetic marks and gene regulation requires the development of new tools to directly manipulate chromatin. Here the authors demonstrate a Cas9-based system to recruit chromatin remodelers to loci of interest, allowing rapid, reversible manipulation of epigenetic states.

ALT1, a Snf2 Family Chromatin Remodeling ATPase, Negatively Regulates Alkaline Tolerance through Enhanced Defense against Oxidative Stress in Rice

PubMed Central

Guo, Mingxin; Wang, Ruci; Wang, Juan; Hua, Kai; Wang, Yueming; Liu, Xiaoqiang; Yao, Shanguo

2014-01-01

Alkaline salt stress adversely affects rice growth, productivity and grain quality. However, the mechanism underlying this process remains elusive. We characterized here an alkaline tolerant mutant, alt1 in rice. Map-based cloning revealed that alt1 harbors a mutation in a chromatin remodeling ATPase gene. ALT1-RNAi transgenic plants under different genetic background mimicked the alt1 phenotype, exhibiting tolerance to alkaline stress in a transcript dosage-dependent manner. The predicted ALT1 protein belonged to the Ris1 subgroup of the Snf2 family and was localized in the nucleus, and transcription of ALT1 was transiently suppressed after alkaline treatment. Although the absorption of several metal ions maintained well in the mutant under alkaline stress, expression level of the genes involved in metal ions homeostasis was not altered in the alt1 mutant. Classification of differentially expressed abiotic stress related genes, as revealed by microarray analysis, found that the majority (50/78) were involved in ROS production, ROS scavenging, and DNA repair. This finding was further confirmed by that alt1 exhibited lower levels of H2O2 under alkaline stress and tolerance to methyl viologen treatment. Taken together, these results suggest that ALT1 negatively functions in alkaline tolerance mainly through the defense against oxidative damage, and provide a potential two-step strategy for improving the tolerance of rice plants to alkaline stress. PMID:25473841

H4 replication-dependent diacetylation and Hat1 promote S-phase chromatin assembly in vivo

PubMed Central

Ejlassi-Lassallette, Aïda; Mocquard, Eloïse; Arnaud, Marie-Claire; Thiriet, Christophe

2011-01-01

While specific posttranslational modification patterns within the H3 and H4 tail domains are associated with the S-phase, their actual functions in replication-dependent chromatin assembly have not yet been defined. Here we used incorporation of trace amounts of recombinant proteins into naturally synchronous macroplasmodia of Physarum polycephalum to examine the function of H3 and H4 tail domains in replication-coupled chromatin assembly. We found that the H3/H4 complex lacking the H4 tail domain was not efficiently recovered in nuclei, whereas depletion of the H3 tail domain did not impede nuclear import but chromatin assembly failed. Furthermore, our results revealed that the proper pattern of acetylation on the H4 tail domain is required for nuclear import and chromatin assembly. This is most likely due to binding of Hat1, as coimmunoprecipitation experiments showed Hat1 associated with predeposition histones in the cytoplasm and with replicating chromatin. These results suggest that the type B histone acetyltransferase assists in shuttling the H3/H4 complex from cytoplasm to the replication forks. PMID:21118997

Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

PubMed

Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

2017-01-05

The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Targeting the chromatin remodeling enzyme BRG1 increases the efficacy of chemotherapy drugs in breast cancer cells

PubMed Central

Wu, Qiong; Sharma, Soni; Cui, Hang; LeBlanc, Scott E.; Zhang, Hong; Muthuswami, Rohini; Nickerson, Jeffrey A.; Imbalzano, Anthony N.

2016-01-01

Brahma related gene product 1 (BRG1) is an ATPase that drives the catalytic activity of a subset of the mammalian SWI/SNF chromatin remodeling enzymes. BRG1 is overexpressed in most human breast cancer tumors without evidence of mutation and is required for breast cancer cell proliferation. We demonstrate that knockdown of BRG1 sensitized triple negative breast cancer cells to chemotherapeutic drugs used to treat breast cancer. An inhibitor of the BRG1 bromodomain had no effect on breast cancer cell viability, but an inhibitory molecule that targets the BRG1 ATPase activity recapitulated the increased drug efficacy observed in the presence of BRG1 knockdown. We further demonstrate that inhibition of BRG1 ATPase activity blocks the induction of ABC transporter genes by these chemotherapeutic drugs and that BRG1 binds to ABC transporter gene promoters. This inhibition increased intracellular concentrations of the drugs, providing a likely mechanism for the increased chemosensitivity. Since ABC transporters and their induction by chemotherapy drugs are a major cause of chemoresistance and treatment failure, these results support the idea that targeting the enzymatic activity of BRG1 would be an effective adjuvant therapy for breast cancer. PMID:27029062

Atomic force microscopy of chromatin arrays reveal non-monotonic salt dependence of array compaction in solution

PubMed Central

Krzemien, Katarzyna M.; Beckers, Maximilian; Quack, Salina; Michaelis, Jens

2017-01-01

Compaction of DNA in chromatin is a hallmark of the eukaryotic cell and unravelling its structure is required for an understanding of DNA involving processes. Despite strong experimental efforts, many questions concerning the DNA packing are open. In particular, it is heavily debated whether an ordered structure referred to as the “30 nm fibre” exist in vivo. Scanning probe microscopy has become a cutting edge technology for the high-resolution imaging of DNA- protein complexes. Here, we perform high-resolution atomic force microscopy of non-cross-linked chromatin arrays in liquid, under different salt conditions. A statistical analysis of the data reveals that array compaction is salt dependent in a non-monotonic fashion. A simple physical model can qualitatively explain the observed findings due to the opposing effects of salt dependent stiffening of DNA, nucleosome stability and histone-histone interactions. While for different salt concentrations different compaction states are observed, our data do not provide support for the existence of regular chromatin fibres. Our studies add new insights into chromatin structure, and with that contribute to a further understanding of the DNA condensation. PMID:28296908

Functional interdependence at the chromatin level between the MKK6/p38 and IGF1/Pi3K/AKT pathways during muscle differentiation

PubMed Central

Carlo, Serra; Daniela, Palacios; Chiara, Mozzetta; Sonia, Forcales; Ianessa, Morantte; Meri, Ripani; Jones David, R.; Keyong, Du; Jhala Ulupi, S.; Cristiano, Simone; Lorenzo, Puri Pier

2009-01-01

During muscle regeneration, the mechanism integrating environmental cues at the chromatin of muscle progenitors is unknown. We show that inflammation-activated MKK6-p38 and IGF1-induced Pi3K/AKT pathways converge on the chromatin of muscle genes to target distinct components of the muscle transcriptosome. p38 α/β kinases recruit the SWI/SNF chromatin-remodeling complex; AKT 1 and 2 promote the association of MyoD with p300 and PCAF acetyltransferases, via direct phosphorylation of p300. Pharmacological or genetic interference with either pathway led to partial assembly of discrete chromatin-bound complexes, which reflected two reversible and distinct cellular phenotypes. Remarkably, Pi3K/AKT blockade was permissive for chromatin recruitment of MEF2-SWI/SNF complex, whose remodeling activity was compromised in the absence of MyoD and acetyltransferases. The functional interdependence between p38 and IGF1/Pi3K/AKT pathways was further established by the evidence that blockade of AKT chromatin targets was sufficient to prevent the activation of the myogenic program triggered by deliberate activation of p38 signaling PMID:17964260

Chromatin remodelling and DNA repair genes are frequently mutated in endometrioid endometrial carcinoma.

PubMed

García-Sanz, Pablo; Triviño, Juan Carlos; Mota, Alba; Pérez López, María; Colás, Eva; Rojo-Sebastián, Alejandro; García, Ángel; Gatius, Sonia; Ruiz, María; Prat, Jaime; López-López, Rafael; Abal, Miguel; Gil-Moreno, Antonio; Reventós, Jaume; Matias-Guiu, Xavier; Moreno-Bueno, Gema

2017-04-01

In developed countries, endometrial carcinoma is the most common cancer that affects the female genital tract. Endometrial carcinoma is divided into two main histological types, type I or endometrioid and type II or non-endometrioid, each of which have characteristic, although not exclusive, molecular alterations and mutational profiles. Nevertheless, information about the implication and relevance of some of these genes in this disease is lacking. We sought here to identify new recurrently mutated genes in endometrioid cancers that play a role in tumourigenesis and that influence the clinical outcome. We focused on low-grade, non-ultramutated tumours as these tumours have a worse prognosis than the ultramutated POLE-positive endometrioid endometrial carcinomas (EECs). We performed exome-sequencing of 11 EECs with matched normal tissue and subsequently validated 15 candidate genes in 76 samples. For the first time, we show that mutations in chromatin remodelling-related genes (KMT2D, KMT2C, SETD1B and BCOR) and in DNA-repair-related genes (BRCA1, BRCA2, RAD50 and CHD4) are frequent in this subtype of endometrial cancer. The alterations to these genes occurred with frequencies ranging from 35.5% for KMT2D to 10.5% for BRCA1 and BCOR, with some showing a tendency toward co-occurrence (RAD50-KMT2D and RAD50-SETD1B). All these genes harboured specific mutational hotspots. In addition, the mutational status of KMT2C, KMT2D and SETD1B helps to predict the degree of myometrial invasion, a critical prognostic feature. These results highlight the possible implication of these genes in this disease, creating opportunities for new therapeutic approaches. © 2016 UICC.

SIRT6 stabilizes DNA-dependent Protein Kinase at chromatin for DNA double-strand break repair

PubMed Central

McCord, Ronald A.; Michishita, Eriko; Hong, Tao; Berber, Elisabeth; Boxer, Lisa D.; Kusumoto, Rika; Guan, Shenheng; Shi, Xiaobing; Gozani, Or; Burlingame, Alma L.; Bohr, Vilhelm A.; Chua, Katrin F.

2009-01-01

The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor with chromatin impacts on the efficiency of repair, and establish a link between chromatin regulation, DNA repair, and a mammalian Sir2 factor. PMID:20157594

Regulation of ATP production: dependence on calcium concentration and respiratory state.

PubMed

Fink, Brian D; Bai, Fan; Yu, Liping; Sivitz, William I

2017-08-01

Nanomolar free calcium enhances oxidative phosphorylation. However, the effects over a broad concentration range, at different respiratory states, or on specific energy substrates are less clear. We examined the action of varying [Ca 2+ ] over respiratory states ranging 4 to 3 on skeletal muscle mitochondrial respiration, potential, ATP production, and H 2 O 2 production using ADP recycling to clamp external [ADP]. Calcium at 450 nM enhanced respiration in mitochondria energized by the complex I substrates, glutamate/malate (but not succinate), at [ADP] of 4-256 µM, but more substantially at intermediate respiratory states and not at all at state 4. Using varied [Ca 2+ ], we found that the stimulatory effects on respiration and ATP production were most prominent at nanomolar concentrations, but inhibitory at 10 µM or higher. ATP production decreased more than respiration at 10 µM calcium. However, potential continued to increase up to 10 µM; suggesting a calcium-induced inability to utilize potential for phosphorylation independent of opening of the mitochondrial permeability transition pore (MTP). This effect of 10 µM calcium was confirmed by direct determination of ATP production over a range of potential created by differing substrate concentrations. Consistent with past reports, nanomolar [Ca 2+ ] had a stimulatory effect on utilization of potential for phosphorylation. Increasing [Ca 2+ ] was positively and continuously associated with H 2 O 2 production. In summary, the stimulatory effect of calcium on mitochondrial function is substrate dependent and most prominent over intermediate respiratory states. Calcium stimulates or inhibits utilization of potential for phosphorylation dependent on concentration with inhibition at higher concentration independent of MTP opening.

The LSH/HELLS homolog Irc5 contributes to cohesin association with chromatin in yeast

PubMed Central

Bakowski, Tomasz; Maciaszczyk-Dziubinska, Ewa; Wysocki, Robert

2017-01-01

Abstract Accurate chromosome segregation is essential for every living cell as unequal distribution of chromosomes during cell division may result in genome instability that manifests in carcinogenesis and developmental disorders. Irc5 from Saccharomyces cerevisiae is a member of the conserved Snf2 family of ATP-dependent DNA translocases and its function is poorly understood. Here, we identify Irc5 as a novel interactor of the cohesin complex. Irc5 associates with Scc1 cohesin subunit and contributes to cohesin binding to chromatin. Disruption of IRC5 decreases cohesin levels at centromeres and chromosome arms, causing premature sister chromatid separation. Moreover, reduced cohesin occupancy at the rDNA region in cells lacking IRC5 leads to the loss of rDNA repeats. We also show that the translocase activity of Irc5 is required for its function in cohesion pathway. Finally, we demonstrate that in the absence of Irc5 both the level of chromatin-bound Scc2, a member of cohesin loading complex, and physical interaction between Scc1 and Scc2 are reduced. Our results suggest that Irc5 is an auxiliary factor that is involved in cohesin association with chromatin. PMID:28383696

Epigenetic regulation of open chromatin in pluripotent stem cells

PubMed Central

Kobayashi, Hiroshi; Kikyo, Nobuaki

2014-01-01

The recent progress in pluripotent stem cell research has opened new avenues of disease modeling, drug screening, and transplantation of patient-specific tissues that had been unimaginable until a decade ago. The central mechanism underlying pluripotency is epigenetic gene regulation; the majority of cell signaling pathways, both extracellular and cytoplasmic, eventually alter the epigenetic status of their target genes during the process of activating or suppressing the genes to acquire or maintain pluripotency. It has long been thought that the chromatin of pluripotent stem cells is globally open to enable the timely activation of essentially all genes in the genome during differentiation into multiple lineages. The current article reviews descriptive observations and the epigenetic machinery relevant to what is supposed to be globally open chromatin in pluripotent stem cells. This includes microscopic appearance, permissive gene transcription, chromatin remodeling complexes, histone modifications, DNA methylation, noncoding RNAs, dynamic movement of chromatin proteins, nucleosome accessibility and positioning, and long-range chromosomal interactions. Detailed analyses of each element, however, have revealed that the globally open chromatin hypothesis is not necessarily supported by some of the critical experimental evidence, such as genome-wide nucleosome accessibility and nucleosome positioning. Further understanding of the epigenetic gene regulation is expected to determine the true nature of the so-called globally open chromatin in pluripotent stem. PMID:24695097

Global alterations of the transcriptional landscape during yeast growth and development in the absence of Ume6-dependent chromatin modification

PubMed Central

Lardenois, Aurélie; Becker, Emmanuelle; Walther, Thomas; Law, Michael J.; Xie, Bingning; Demougin, Philippe; Strich, Randy

2017-01-01

Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MATa/α wild-type and ume6/ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes. PMID:25957495

Genome-wide chromatin state transitions associated with developmental and environmental cues.

PubMed

Zhu, Jiang; Adli, Mazhar; Zou, James Y; Verstappen, Griet; Coyne, Michael; Zhang, Xiaolan; Durham, Timothy; Miri, Mohammad; Deshpande, Vikram; De Jager, Philip L; Bennett, David A; Houmard, Joseph A; Muoio, Deborah M; Onder, Tamer T; Camahort, Ray; Cowan, Chad A; Meissner, Alexander; Epstein, Charles B; Shoresh, Noam; Bernstein, Bradley E

2013-01-31

Differences in chromatin organization are key to the multiplicity of cell states that arise from a single genetic background, yet the landscapes of in vivo tissues remain largely uncharted. Here, we mapped chromatin genome-wide in a large and diverse collection of human tissues and stem cells. The maps yield unprecedented annotations of functional genomic elements and their regulation across developmental stages, lineages, and cellular environments. They also reveal global features of the epigenome, related to nuclear architecture, that also vary across cellular phenotypes. Specifically, developmental specification is accompanied by progressive chromatin restriction as the default state transitions from dynamic remodeling to generalized compaction. Exposure to serum in vitro triggers a distinct transition that involves de novo establishment of domains with features of constitutive heterochromatin. We describe how these global chromatin state transitions relate to chromosome and nuclear architecture, and discuss their implications for lineage fidelity, cellular senescence, and reprogramming. Copyright © 2013 Elsevier Inc. All rights reserved.

Targeting of the Fun30 nucleosome remodeller by the Dpb11 scaffold facilitates cell cycle-regulated DNA end resection

PubMed Central

Bantele, Susanne CS; Ferreira, Pedro; Gritenaite, Dalia; Boos, Dominik; Pfander, Boris

2017-01-01

DNA double strand breaks (DSBs) can be repaired by either recombination-based or direct ligation-based mechanisms. Pathway choice is made at the level of DNA end resection, a nucleolytic processing step, which primes DSBs for repair by recombination. Resection is thus under cell cycle control, but additionally regulated by chromatin and nucleosome remodellers. Here, we show that both layers of control converge in the regulation of resection by the evolutionarily conserved Fun30/SMARCAD1 remodeller. Budding yeast Fun30 and human SMARCAD1 are cell cycle-regulated by interaction with the DSB-localized scaffold protein Dpb11/TOPBP1, respectively. In yeast, this protein assembly additionally comprises the 9-1-1 damage sensor, is involved in localizing Fun30 to damaged chromatin, and thus is required for efficient long-range resection of DSBs. Notably, artificial targeting of Fun30 to DSBs is sufficient to bypass the cell cycle regulation of long-range resection, indicating that chromatin remodelling during resection is underlying DSB repair pathway choice. DOI: http://dx.doi.org/10.7554/eLife.21687.001 PMID:28063255

The physical size of transcription factors is key to transcriptional regulation in chromatin domains

NASA Astrophysics Data System (ADS)

Maeshima, Kazuhiro; Kaizu, Kazunari; Tamura, Sachiko; Nozaki, Tadasu; Kokubo, Tetsuro; Takahashi, Koichi

2015-02-01

Genetic information, which is stored in the long strand of genomic DNA as chromatin, must be scanned and read out by various transcription factors. First, gene-specific transcription factors, which are relatively small (˜50 kDa), scan the genome and bind regulatory elements. Such factors then recruit general transcription factors, Mediators, RNA polymerases, nucleosome remodellers, and histone modifiers, most of which are large protein complexes of 1-3 MDa in size. Here, we propose a new model for the functional significance of the size of transcription factors (or complexes) for gene regulation of chromatin domains. Recent findings suggest that chromatin consists of irregularly folded nucleosome fibres (10 nm fibres) and forms numerous condensed domains (e.g., topologically associating domains). Although the flexibility and dynamics of chromatin allow repositioning of genes within the condensed domains, the size exclusion effect of the domain may limit accessibility of DNA sequences by transcription factors. We used Monte Carlo computer simulations to determine the physical size limit of transcription factors that can enter condensed chromatin domains. Small gene-specific transcription factors can penetrate into the chromatin domains and search their target sequences, whereas large transcription complexes cannot enter the domain. Due to this property, once a large complex binds its target site via gene-specific factors it can act as a ‘buoy’ to keep the target region on the surface of the condensed domain and maintain transcriptional competency. This size-dependent specialization of target-scanning and surface-tethering functions could provide novel insight into the mechanisms of various DNA transactions, such as DNA replication and repair/recombination.

Activity-dependent ATP-waves in the mouse neocortex are independent from astrocytic calcium waves.

PubMed

Haas, Brigitte; Schipke, Carola G; Peters, Oliver; Söhl, Goran; Willecke, Klaus; Kettenmann, Helmut

2006-02-01

In the corpus callosum, astrocytic calcium waves propagate via a mechanism involving ATP-release but not gap junctional coupling. In the present study, we report for the neocortex that calcium wave propagation depends on functional astrocytic gap junctions but is still accompanied by ATP-release. In acute slices obtained from the neocortex of mice deficient for astrocytic expression of connexin43, the calcium wave did not propagate. In contrast, in the corpus callosum and hippocampus of these mice, the wave propagated as in control animals. In addition to calcium wave propagation in astrocytes, ATP-release was recorded as a calcium signal from 'sniffer cells', a cell line expressing high-affinity purinergic receptors placed on the surface of the slice. The astrocyte calcium wave in the neocortex was accompanied by calcium signals in the 'sniffer cell' population. In the connexin43-deficient mice we recorded calcium signals from sniffer cells also in the absence of an astrocytic calcium wave. Our findings indicate that astrocytes propagate calcium signals by two separate mechanisms depending on the brain region and that ATP release can propagate within the neocortex independent from calcium waves.

Locus-specific epigenetic remodeling controls addiction- and depression-related behaviors.

PubMed

Heller, Elizabeth A; Cates, Hannah M; Peña, Catherine J; Sun, Haosheng; Shao, Ningyi; Feng, Jian; Golden, Sam A; Herman, James P; Walsh, Jessica J; Mazei-Robison, Michelle; Ferguson, Deveroux; Knight, Scott; Gerber, Mark A; Nievera, Christian; Han, Ming-Hu; Russo, Scott J; Tamminga, Carol S; Neve, Rachael L; Shen, Li; Zhang, H Steve; Zhang, Feng; Nestler, Eric J

2014-12-01

Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin-modifying enzymes and histone post-translational modifications in discrete brain regions. Given the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. We investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo. We found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allowed us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior.

Functional interdependence at the chromatin level between the MKK6/p38 and IGF1/PI3K/AKT pathways during muscle differentiation.

PubMed

Serra, Carlo; Palacios, Daniela; Mozzetta, Chiara; Forcales, Sonia V; Morantte, Ianessa; Ripani, Meri; Jones, David R; Du, Keyong; Jhala, Ulupi S; Simone, Cristiano; Puri, Pier Lorenzo

2007-10-26

During muscle regeneration, the mechanism integrating environmental cues at the chromatin of muscle progenitors is unknown. We show that inflammation-activated MKK6-p38 and insulin growth factor 1 (IGF1)-induced PI3K/AKT pathways converge on the chromatin of muscle genes to target distinct components of the muscle transcriptosome. p38 alpha/beta kinases recruit the SWI/SNF chromatin-remodeling complex; AKT1 and 2 promote the association of MyoD with p300 and PCAF acetyltransferases, via direct phosphorylation of p300. Pharmacological or genetic interference with either pathway led to partial assembly of discrete chromatin-bound complexes, which reflected two reversible and distinct cellular phenotypes. Remarkably, PI3K/AKT blockade was permissive for chromatin recruitment of MEF2-SWI/SNF complex, whose remodeling activity was compromised in the absence of MyoD and acetyltransferases. The functional interdependence between p38 and IGF1/PI3K/AKT pathways was further established by the evidence that blockade of AKT chromatin targets was sufficient to prevent the activation of the myogenic program triggered by deliberate activation of p38 signaling.

Chromatin remodeling modulates radiosensitivity of the daughter cells derived from cell population exposed to low- and high-LET irradiation

PubMed Central

Chen, Xiaoyan; Zhu, Lin; Zhang, Hang; Wang, Chen; Shao, Chunlin

2017-01-01

Radiation effects are dependent of linear energy transfer (LET), but it is still obscure whether the daughter cells (DCs) derived from irradiated population are radioresistance and much less the underlying mechanism. With the measurements of survival, proliferation and γH2AX foci, this study shows that the DCs from γ-ray irradiated cells (DCs-γ) became more radioresistant than its parent control without irradiation, but the radiosensitivity of DCs from α-particle irradiated cells (DCs-α) was not altered. After irradiation with equivalent doses of γ-rays and α-particles, the foci number of histone H3 lysine 9 dimethylation (H3K9me3) and the activity of histone deacetylase (HDAC) in DCs-γ was extensively higher than these in DCs-α and its parent control, indicating that a higher level of heterochromatin was formed in DCs-γ but not in DCs-α. Treatment of cells with SAHA (an inhibitor of HDAC) decreased the level of heterochromatin domains by inhibiting the expressions of H3K9m3 and HP-1a proteins and triggering the expression of acetylated core histone H3 (Ac-H3). When cells were treated with SAHA, the radioresistance phenotype of DCs-γ was eliminated so that the radiosensitivities of DCs-γ, DCs-α and their parent cells approached to same levels. Our current results reveal that γ-rays but not α-particles could induce chromatin remodeling and heterochromatinization which results in the occurrence of radioresistance of DCs, indicating that the combination treatment of irradiation and HDAC inhibitor could serve as a potential cancer therapy strategy, especially for the fraction radiotherapy of low-LET irradiation. PMID:28881774

Chromatin Structure and Replication Origins: Determinants Of Chromosome Replication And Nuclear Organization

PubMed Central

Smith, Owen K.; Aladjem, Mirit I.

2014-01-01

The DNA replication program is, in part, determined by the epigenetic landscape that governs local chromosome architecture and directs chromosome duplication. Replication must coordinate with other biochemical processes occurring concomitantly on chromatin, such as transcription and remodeling, to insure accurate duplication of both genetic and epigenetic features and to preserve genomic stability. The importance of genome architecture and chromatin looping in coordinating cellular processes on chromatin is illustrated by two recent sets of discoveries. First, chromatin-associated proteins that are not part of the core replication machinery were shown to affect the timing of DNA replication. These chromatin-associated proteins could be working in concert, or perhaps in competition, with the transcriptional machinery and with chromatin modifiers to determine the spatial and temporal organization of replication initiation events. Second, epigenetic interactions are mediated by DNA sequences that determine chromosomal replication. In this review we summarize recent findings and current models linking spatial and temporal regulation of the replication program with epigenetic signaling. We discuss these issues in the context of the genome’s three-dimensional structure with an emphasis on events occurring during the initiation of DNA replication. PMID:24905010

Sanguinarine interacts with chromatin, modulates epigenetic modifications, and transcription in the context of chromatin.

PubMed

Selvi B, Ruthrotha; Pradhan, Suman Kalyan; Shandilya, Jayasha; Das, Chandrima; Sailaja, Badi Sri; Shankar G, Naga; Gadad, Shrikanth S; Reddy, Ashok; Dasgupta, Dipak; Kundu, Tapas K

2009-02-27

DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. We report the dynamic interaction of the DNA intercalator and potential anticancer plant alkaloid, sanguinarine (SGR), with chromatin. Association of SGR with different levels of chromatin structure was enthalpy driven with micromolar dissociation constant. Apart from DNA, it binds with comparable affinity with core histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR-mediated repression of epigenetic marks and the alteration of chromatin geography (nucleography) also result in the modulation of global gene expression. These data, conclusively, show an anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context.

Large-Scale ATP-Independent Nucleosome Unfolding by a Histone Chaperone

PubMed Central

Valieva, Maria E.; Armeev, Grigoriy A.; Kudryashova, Kseniya S.; Gerasimova, Nadezhda S.; Shaytan, Alexey K.; Kulaeva, Olga I.; McCullough, Laura L.; Formosa, Tim; Georgiev, Pavel G.; Kirpichnikov, Mikhail P.; Studitsky, Vasily M.; Feofanov, Alexey V.

2017-01-01

DNA accessibility to regulatory proteins is significantly affected by nucleosome structure and dynamics. FACT (facilitates chromatin transcription) increases the accessibility of nucleosomal DNA but the mechanism and extent of this nucleosome reorganization are unknown. We report here the effects of FACT on single nucleosomes revealed with spFRET microscopy. FACT binding results in a dramatic, ATP-independent, and reversible uncoiling of DNA that affects at least 70% of the DNA in a nucleosome. A mutated version of FACT is defective in this uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this is an important function of FACT in vivo. PMID:27820806

Phosphatidylserine translocation to the mitochondrion is an ATP-dependent process in permeabilized animal cells

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

Voelker, D.R.

1989-12-01

Chinese hamster ovary (CHO-K1) cells were pulse labeled with ({sup 3}H)serine, and the synthesis of phosphatidyl({sup 3}H)ethanolamine from phosphatidyl({sup 3}H)serine during the subsequent chase was used as a measure of lipid translocation to the mitochondria. When the CHO-K1 cells were pulse labeled and subsequently permeabilized with 50 {mu}g of saponin per ml, there was no significant turnover of nascent phosphatidyl({sup 3}H)serine to form phosphatidyl({sup 3}H)ethanolamine during an ensuring chase. Supplementation of the permeabilized cells with 2 mM ATP resulted in significant phosphatidyl({sup 3}H)ethanolamine synthesis (83% of that found in intact cells) from phosphatidyl({sup 3}H)serine during a subsequent 2-hr chase. Phosphatidyl({supmore » 3}H)ethanolamine synthesis essentially ceased after 2 hr in the permeabilized cells. The translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine was a saturable process with respect to ATP concentration in permeabilized cells. The conversion of phosphatidyl({sup 3}H)serine to phosphatidyl({sup 3}H)ethanolamine did not occur in saponin-treated cultures supplemented with 2 mM AMP, 2 mM 5{prime}-adenylyl imidodiphosphate, or apyrase plus 2 mM ATP. ATP was the most effective nucleotide, but the addition of GTP, CTP, UTP, and ADP also supported the translocation-dependent synthesis of phosphatidyl({sup 3}H)ethanolamine albeit to a lesser extent. These data provide evidence that the interorganelle translocation of phosphatidylserine requires ATP and is largely independent of soluble cytosolic proteins.« less

The LSH/HELLS homolog Irc5 contributes to cohesin association with chromatin in yeast.

PubMed

Litwin, Ireneusz; Bakowski, Tomasz; Maciaszczyk-Dziubinska, Ewa; Wysocki, Robert

2017-06-20

Accurate chromosome segregation is essential for every living cell as unequal distribution of chromosomes during cell division may result in genome instability that manifests in carcinogenesis and developmental disorders. Irc5 from Saccharomyces cerevisiae is a member of the conserved Snf2 family of ATP-dependent DNA translocases and its function is poorly understood. Here, we identify Irc5 as a novel interactor of the cohesin complex. Irc5 associates with Scc1 cohesin subunit and contributes to cohesin binding to chromatin. Disruption of IRC5 decreases cohesin levels at centromeres and chromosome arms, causing premature sister chromatid separation. Moreover, reduced cohesin occupancy at the rDNA region in cells lacking IRC5 leads to the loss of rDNA repeats. We also show that the translocase activity of Irc5 is required for its function in cohesion pathway. Finally, we demonstrate that in the absence of Irc5 both the level of chromatin-bound Scc2, a member of cohesin loading complex, and physical interaction between Scc1 and Scc2 are reduced. Our results suggest that Irc5 is an auxiliary factor that is involved in cohesin association with chromatin. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Maintenance of Xist Imprinting Depends on Chromatin Condensation State and Rnf12 Dosage in Mice.

PubMed

Fukuda, Atsushi; Mitani, Atsushi; Miyashita, Toshiyuki; Sado, Takashi; Umezawa, Akihiro; Akutsu, Hidenori

2016-10-01

In female mammals, activation of Xist (X-inactive specific transcript) is essential for establishment of X chromosome inactivation. During early embryonic development in mice, paternal Xist is preferentially expressed whereas maternal Xist (Xm-Xist) is silenced. Unlike autosomal imprinted genes, Xist imprinting for Xm-Xist silencing was erased in cloned or parthenogenetic but not fertilized embryos. However, the molecular mechanism underlying the variable nature of Xm-Xist imprinting is poorly understood. Here, we revealed that Xm-Xist silencing depends on chromatin condensation states at the Xist/Tsix genomic region and on Rnf12 expression levels. In early preimplantation, chromatin decondensation via H3K9me3 loss and histone acetylation gain caused Xm-Xist derepression irrespective of embryo type. Although the presence of the paternal genome during pronuclear formation impeded Xm-Xist derepression, Xm-Xist was robustly derepressed when the maternal genome was decondensed before fertilization. Once Xm-Xist was derepressed by chromatin alterations, the derepression was stably maintained and rescued XmXpΔ lethality, indicating that loss of Xm-Xist imprinting was irreversible. In late preimplantation, Oct4 served as a chromatin opener to create transcriptional permissive states at Xm-Xist/Tsix genomic loci. In parthenogenetic embryos, Rnf12 overdose caused Xm-Xist derepression via Xm-Tsix repression; physiological Rnf12 levels were essential for Xm-Xist silencing maintenance in fertilized embryos. Thus, chromatin condensation and fine-tuning of Rnf12 dosage were crucial for Xist imprint maintenance by silencing Xm-Xist.

Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

PubMed Central

De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

2014-01-01

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

Protein and Genetic Composition of Four Chromatin Types in Drosophila melanogaster Cell Lines.

PubMed

Boldyreva, Lidiya V; Goncharov, Fyodor P; Demakova, Olga V; Zykova, Tatyana Yu; Levitsky, Victor G; Kolesnikov, Nikolay N; Pindyurin, Alexey V; Semeshin, Valeriy F; Zhimulev, Igor F

2017-04-01

Recently, we analyzed genome-wide protein binding data for the Drosophila cell lines S2, Kc, BG3 and Cl.8 (modENCODE Consortium) and identified a set of 12 proteins enriched in the regions corresponding to interbands of salivary gland polytene chromosomes. Using these data, we developed a bioinformatic pipeline that partitioned the Drosophila genome into four chromatin types that we hereby refer to as aquamarine, lazurite, malachite and ruby. Here, we describe the properties of these chromatin types across different cell lines. We show that aquamarine chromatin tends to harbor transcription start sites (TSSs) and 5' untranslated regions (5'UTRs) of the genes, is enriched in diverse "open" chromatin proteins, histone modifications, nucleosome remodeling complexes and transcription factors. It encompasses most of the tRNA genes and shows enrichment for non-coding RNAs and miRNA genes. Lazurite chromatin typically encompasses gene bodies. It is rich in proteins involved in transcription elongation. Frequency of both point mutations and natural deletion breakpoints is elevated within lazurite chromatin. Malachite chromatin shows higher frequency of insertions of natural transposons. Finally, ruby chromatin is enriched for proteins and histone modifications typical for the "closed" chromatin. Ruby chromatin has a relatively low frequency of point mutations and is essentially devoid of miRNA and tRNA genes. Aquamarine and ruby chromatin types are highly stable across cell lines and have contrasting properties. Lazurite and malachite chromatin types also display characteristic protein composition, as well as enrichment for specific genomic features. We found that two types of chromatin, aquamarine and ruby, retain their complementary protein patterns in four Drosophila cell lines.

Flightless I (Drosophila) homolog facilitates chromatin accessibility of the estrogen receptor α target genes in MCF-7 breast cancer cells

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

Jeong, Kwang Won, E-mail: kwjeong@gachon.ac.kr

2014-04-04

Highlights: • H3K4me3 and Pol II binding at TFF1 promoter were reduced in FLII-depleted MCF-7 cells. • FLII is required for chromatin accessibility of the enhancer of ERalpha target genes. • Depletion of FLII causes inhibition of proliferation of MCF-7 cells. - Abstract: The coordinated activities of multiple protein complexes are essential to the remodeling of chromatin structure and for the recruitment of RNA polymerase II (Pol II) to the promoter in order to facilitate the initiation of transcription in nuclear receptor-mediated gene expression. Flightless I (Drosophila) homolog (FLII), a nuclear receptor coactivator, is associated with the SWI/SNF-chromatin remodeling complexmore » during estrogen receptor (ER)α-mediated transcription. However, the function of FLII in estrogen-induced chromatin opening has not been fully explored. Here, we show that FLII plays a critical role in establishing active histone modification marks and generating the open chromatin structure of ERα target genes. We observed that the enhancer regions of ERα target genes are heavily occupied by FLII, and histone H3K4me3 and Pol II binding induced by estrogen are decreased in FLII-depleted MCF-7 cells. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments showed that depletion of FLII resulted in reduced chromatin accessibility of multiple ERα target genes. These data suggest FLII as a key regulator of ERα-mediated transcription through its role in regulating chromatin accessibility for the binding of RNA Polymerase II and possibly other transcriptional coactivators.« less

Mechanisms of charge transfer in human copper ATPases ATP7A and ATP7B.

PubMed

Tadini-Buoninsegni, Francesco; Smeazzetto, Serena

2017-04-01

ATP7A and ATP7B are Cu + -transporting ATPases of subclass IB and play a fundamental role in intracellular copper homeostasis. ATP7A/B transfer Cu + ions across the membrane from delivery to acceptor proteins without establishing a free Cu + gradient. Transfer of copper across the membrane is coupled to ATP hydrolysis. Current measurements on solid supported membranes (SSM) were performed to investigate the mechanism of copper-related charge transfer across ATP7A and ATP7B. SSM measurements demonstrated that electrogenic copper displacement occurs within ATP7A/B following addition of ATP and formation of the phosphorylated intermediate. Comparison of the time constants for cation displacement in ATP7A/B and sarcoplasmic reticulum Ca 2+ -ATPase is consistent with the slower phosphoenzyme formation in copper ATPases. Moreover, ATP-dependent copper transfer in ATP7A/B is not affected by varying the pH, suggesting that net proton counter-transport may not occur in copper ATPases. Platinum anticancer drugs activate ATP7A/B and are subjected to ATP-dependent vectorial displacement with a mechanism analogous to that of copper. © 2016 IUBMB Life, 69(4):218-225, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq).

PubMed

Soyer, Jessica L; Möller, Mareike; Schotanus, Klaas; Connolly, Lanelle R; Galazka, Jonathan M; Freitag, Michael; Stukenbrock, Eva H

2015-06-01

The presence or absence of specific transcription factors, chromatin remodeling machineries, chromatin modification enzymes, post-translational histone modifications and histone variants all play crucial roles in the regulation of pathogenicity genes. Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) provides an important tool to study genome-wide protein-DNA interactions to help understand gene regulation in the context of native chromatin. ChIP-seq is a convenient in vivo technique to identify, map and characterize occupancy of specific DNA fragments with proteins against which specific antibodies exist or which can be epitope-tagged in vivo. We optimized existing ChIP protocols for use in the wheat pathogen Zymoseptoria tritici and closely related sister species. Here, we provide a detailed method, underscoring which aspects of the technique are organism-specific. Library preparation for Illumina sequencing is described, as this is currently the most widely used ChIP-seq method. One approach for the analysis and visualization of representative sequence is described; improved tools for these analyses are constantly being developed. Using ChIP-seq with antibodies against H3K4me2, which is considered a mark for euchromatin or H3K9me3 and H3K27me3, which are considered marks for heterochromatin, the overall distribution of euchromatin and heterochromatin in the genome of Z. tritici can be determined. Our ChIP-seq protocol was also successfully applied to Z. tritici strains with high levels of melanization or aberrant colony morphology, and to different species of the genus (Z. ardabiliae and Z. pseudotritici), suggesting that our technique is robust. The methods described here provide a powerful framework to study new aspects of chromatin biology and gene regulation in this prominent wheat pathogen. Copyright © 2015 Elsevier Inc. All rights reserved.

The chromatin-binding protein Smyd1 restricts adult mammalian heart growth

PubMed Central

Kimball, Todd; Rasmussen, Tara L.; Rosa-Garrido, Manuel; Chen, Haodong; Tran, Tam; Miller, Mickey R.; Gray, Ricardo; Jiang, Shanxi; Ren, Shuxun; Wang, Yibin; Tucker, Haley O.; Vondriska, Thomas M.

2016-01-01

All terminally differentiated organs face two challenges, maintaining their cellular identity and restricting organ size. The molecular mechanisms responsible for these decisions are of critical importance to organismal development, and perturbations in their normal balance can lead to disease. A hallmark of heart failure, a condition affecting millions of people worldwide, is hypertrophic growth of cardiomyocytes. The various forms of heart failure in human and animal models share conserved transcriptome remodeling events that lead to expression of genes normally silenced in the healthy adult heart. However, the chromatin remodeling events that maintain cell and organ size are incompletely understood; insights into these mechanisms could provide new targets for heart failure therapy. Using a quantitative proteomics approach to identify muscle-specific chromatin regulators in a mouse model of hypertrophy and heart failure, we identified upregulation of the histone methyltransferase Smyd1 during disease. Inducible loss-of-function studies in vivo demonstrate that Smyd1 is responsible for restricting growth in the adult heart, with its absence leading to cellular hypertrophy, organ remodeling, and fulminate heart failure. Molecular studies reveal Smyd1 to be a muscle-specific regulator of gene expression and indicate that Smyd1 modulates expression of gene isoforms whose expression is associated with cardiac pathology. Importantly, activation of Smyd1 can prevent pathological cell growth. These findings have basic implications for our understanding of cardiac pathologies and open new avenues to the treatment of cardiac hypertrophy and failure by modulating Smyd1. PMID:27663768

Contraction of small mesenteric arteries induced by micromolar concentrations of ATP released from caged ATP.

PubMed

Sjöblom-Widfeldt, N; Arner, A; Nilsson, H

1993-01-01

The concentration dependence of ATP-induced contractions in isolated resistance arteries was estimated using photolysis of caged ATP. Rat mesenteric vessels were isolated and mounted for force registration in a small chamber allowing illumination from a xenon-flash lamp. Photolysis of 100 microM caged ATP, which released about 20 microM ATP within a few milliseconds in the vessel, induced a transient contraction with an amplitude approximately 40-50% of the response induced by 10 microM noradrenaline. The responses could neither be induced by the light flash as such nor by caged ATP alone nor by photolysis of caged phosphate. The amplitude of the contractions was dependent on the concentration of caged ATP, and the effective concentration for ATP was estimated to be in the range of 1-10 microM. In contrast, when ATP was introduced by diffusion, about a 100-fold higher concentration was required. Thus photolytic release of ATP minimizes metabolism before its action on receptors and reveals action of ATP in a concentration range consistent with a role of ATP as a transmitter in nervous regulation of the tone of resistance vessels.

Chromatin and RNAi factors protect the C. elegans germline against repetitive sequences

PubMed Central

Robert, Valérie J.P.; Sijen, Titia; van Wolfswinkel, Josien; Plasterk, Ronald H.A.

2005-01-01

Protection of genomes against invasion by repetitive sequences, such as transposons, viruses, and repetitive transgenes, involves strong and selective silencing of these sequences. During silencing of repetitive transgenes, a trans effect (“cosuppression”) occurs that results in silencing of cognate endogenous genes. Here we report RNA interference (RNAi) screens performed to catalog genes required for cosuppression in the Caenorhabditis elegans germline. We find factors with a putative role in chromatin remodeling and factors involved in RNAi. Together with molecular data also presented in this study, these results suggest that in C. elegans repetitive sequences trigger transcriptional gene silencing using RNAi and chromatin factors. PMID:15774721

The difference in LET and ion species dependence for induction of initially measured and non-rejoined chromatin breaks in normal human fibroblasts.

PubMed

Tsuruoka, Chizuru; Suzuki, Masao; Hande, M Prakash; Furusawa, Yoshiya; Anzai, Kazunori; Okayasu, Ryuichi

2008-08-01

We studied the LET and ion species dependence of the induction of chromatin breaks measured immediately after irradiation as initially measured breaks and after 24 h postirradiation incubation (37 degrees C) as non-rejoined breaks in normal human fibroblasts with different heavy ions, such as carbon, neon, silicon and iron, generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS). Chromatin breaks were measured as an excess number of fragments of prematurely condensed chromosomes using premature chromosome condensation (PCC). The results showed that the number of excess fragments per cell per Gy for initially measured chromatin breaks was dependent on LET in the range from 13.3 to 113.1 keV/mum but was not dependent on ion species. On the other hand, the number of non-rejoined chromatin breaks detected after 24 h postirradiation incubation was clearly dependent on both LET and ion species. No significant difference was observed in the cross section for initially measured breaks, but a statistically significant difference was observed in the cross section for non-rejoined breaks among carbon, neon, silicon and iron ions. This suggests that the LET-dependent structure in the biological effects is reflected in biological consequences of repair processes.

The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex.

PubMed

Bele, Tanja; Fabbretti, Elsa

2016-08-01

P2X3 receptors, gated by extracellular ATP, are expressed by sensory neurons and are involved in peripheral nociception and pain sensitization. The ability of P2X3 receptors to transduce extracellular stimuli into neuronal signals critically depends on the dynamic molecular partnership with the calcium/calmodulin-dependent serine protein kinase (CASK). The present work used trigeminal sensory neurons to study the impact that activation of P2X3 receptors (evoked by the agonist α,β-meATP) has on the release of endogenous ATP and how CASK modulates this phenomenon. P2X3 receptor function was followed by ATP efflux via Pannexin1 (Panx1) hemichannels, a mechanism that was blocked by the P2X3 receptor antagonist A-317491, and by P2X3 silencing. ATP efflux was enhanced by nerve growth factor, a treatment known to potentiate P2X3 receptor function. Basal ATP efflux was not controlled by CASK, and carbenoxolone or Pannexin silencing reduced ATP release upon P2X3 receptor function. CASK-controlled ATP efflux followed P2X3 receptor activity, but not depolarization-evoked ATP release. Molecular biology experiments showed that CASK was essential for the transactivation of Panx1 upon P2X3 receptor activation. These data suggest that P2X3 receptor function controls a new type of feed-forward purinergic signaling on surrounding cells, with consequences at peripheral and spinal cord level. Thus, P2X3 receptor-mediated ATP efflux may be considered for the future development of pharmacological strategies aimed at containing neuronal sensitization. P2X3 receptors are involved in sensory transduction and associate to CASK. We have studied in primary sensory neurons the molecular mechanisms downstream P2X3 receptor activation, namely ATP release and partnership with CASK or Panx1. Our data suggest that CASK and P2X3 receptors are part of an ATP keeper complex, with important feed-forward consequences at peripheral and central level. © 2016 International Society for Neurochemistry.

Recruitment by the Repressor Freud-1 of Histone Deacetylase-Brg1 Chromatin Remodeling Complexes to Strengthen HTR1A Gene Repression.

PubMed

Souslova, Tatiana; Mirédin, Kim; Millar, Anne M; Albert, Paul R

2017-12-01

Five-prime repressor element under dual repression binding protein-1 (Freud-1)/CC2D1A is genetically linked to intellectual disability and implicated in neuronal development. Freud-1 represses the serotonin-1A (5-HT1A) receptor gene HTR1A by histone deacetylase (HDAC)-dependent or HDAC-independent mechanisms in 5-HT1A-negative (e.g., HEK-293) or 5-HT1A-expressing cells (SK-N-SH), respectively. To identify the underlying mechanisms, Freud-1-associated proteins were affinity-purified from HEK-293 nuclear extracts and members of the Brg1/SMARCCA chromatin remodeling and Sin3A-HDAC corepressor complexes were identified. Pull-down assays using recombinant proteins showed that Freud-1 interacts directly with the Brg1 carboxyl-terminal domain; interaction with Brg1 required the carboxyl-terminal of Freud-1. Freud-1 complexes in HEK-293 and SK-N-SH cells differed, with low levels of BAF170/SMARCC2 and BAF57/SMARCE1 in HEK-293 cells and low-undetectable BAF155/SMARCC1, Sin3A, and HDAC1/2 in SK-N-SH cells. Similarly, by quantitative chromatin immunoprecipitation, Brg1-BAF170/57 and Sin3A-HDAC complexes were observed at the HTR1A promoter in HEK-293 cells, whereas in SK-N-SH cells, Sin3A-HDAC proteins were not detected. Quantifying 5-HT1A receptor mRNA levels in cells treated with siRNA to Freud-1, Brg1, or both RNAs addressed the functional role of the Freud-1-Brg1 complex. In HEK-293 cells, 5-HT1A receptor mRNA levels were increased only when both Freud-1 and Brg1 were depleted, but in SK-N-SH cells, depletion of either protein upregulated 5-HT1A receptor RNA. Thus, recruitment by Freud-1 of Brg1, BAF155, and Sin3A-HDAC complexes appears to strengthen repression of the HTR1A gene to prevent its expression inappropriate cell types, while recruitment of the Brg1-BAF170/57 complex is permissive to 5-HT1A receptor expression. Alterations in Freud-1-Brg1 interactions in mutants associated with intellectual disability could impair gene repression leading to altered neuronal

Recruitment by the Repressor Freud-1 of Histone Deacetylase-Brg1 Chromatin Remodeling Complexes to Strengthen HTR1A Gene Repression

PubMed Central

Souslova, Tatiana; Mirédin, Kim; Millar, Anne M.

2017-01-01

Five-prime repressor element under dual repression binding protein-1 (Freud-1)/CC2D1A is genetically linked to intellectual disability and implicated in neuronal development. Freud-1 represses the serotonin-1A (5-HT1A) receptor gene HTR1A by histone deacetylase (HDAC)-dependent or HDAC-independent mechanisms in 5-HT1A-negative (e.g., HEK-293) or 5-HT1A-expressing cells (SK-N-SH), respectively. To identify the underlying mechanisms, Freud-1-associated proteins were affinity-purified from HEK-293 nuclear extracts and members of the Brg1/SMARCCA chromatin remodeling and Sin3A-HDAC corepressor complexes were identified. Pull-down assays using recombinant proteins showed that Freud-1 interacts directly with the Brg1 carboxyl-terminal domain; interaction with Brg1 required the carboxyl-terminal of Freud-1. Freud-1 complexes in HEK-293 and SK-N-SH cells differed, with low levels of BAF170/SMARCC2 and BAF57/SMARCE1 in HEK-293 cells and low-undetectable BAF155/SMARCC1, Sin3A, and HDAC1/2 in SK-N-SH cells. Similarly, by quantitative chromatin immuno-precipitation, Brg1-BAF170/57 and Sin3A-HDAC complexes were observed at the HTR1A promoter in HEK-293 cells, whereas in SK-N-SH cells, Sin3A-HDAC proteins were not detected. Quantifying 5-HT1A receptor mRNA levels in cells treated with siRNA to Freud-1, Brg1, or both RNAs addressed the functional role of the Freud-1-Brg1 complex. In HEK-293 cells, 5-HT1A receptor mRNA levels were increased only when both Freud-1 and Brg1 were depleted, but in SK-N-SH cells, depletion of either protein upregulated 5-HT1A receptor RNA. Thus, recruitment by Freud-1 of Brg1, BAF155, and Sin3A-HDAC complexes appears to strengthen repression of the HTR1A gene to prevent its expression inappropriate cell types, while recruitment of the Brg1-BAF170/57 complex is permissive to 5-HT1A receptor expression. Alterations in Freud-1-Brg1 interactions in mutants associated with intellectual disability could impair gene repression leading to altered neuronal

Role of ND10 nuclear bodies in the chromatin repression of HSV-1.

PubMed

Gu, Haidong; Zheng, Yi

2016-04-05

Herpes simplex virus (HSV) is a neurotropic virus that establishes lifelong latent infection in human ganglion sensory neurons. This unique life cycle necessitates an intimate relation between the host defenses and virus counteractions over the long course of infection. Two important aspects of host anti-viral defense, nuclear substructure restriction and epigenetic chromatin regulation, have been intensively studied in the recent years. Upon viral DNA entering the nucleus, components of discrete nuclear bodies termed nuclear domain 10 (ND10), converge at viral DNA and place restrictions on viral gene expression. Meanwhile the infected cell mobilizes its histones and histone-associated repressors to force the viral DNA into nucleosome-like structures and also represses viral transcription. Both anti-viral strategies are negated by various HSV countermeasures. One HSV gene transactivator, infected cell protein 0 (ICP0), is a key player in antagonizing both the ND10 restriction and chromatin repression. On one hand, ICP0 uses its E3 ubiquitin ligase activity to target major ND10 components for proteasome-dependent degradation and thereafter disrupts the ND10 nuclear bodies. On the other hand, ICP0 participates in de-repressing the HSV chromatin by changing histone composition or modification and therefore activates viral transcription. Involvement of a single viral protein in two seemingly different pathways suggests that there is coordination in host anti-viral defense mechanisms and also cooperation in viral counteraction strategies. In this review, we summarize recent advances in understanding the role of chromatin regulation and ND10 dynamics in both lytic and latent HSV infection. We focus on the new observations showing that ND10 nuclear bodies play a critical role in cellular chromatin regulation. We intend to find the connections between the two major anti-viral defense pathways, chromatin remodeling and ND10 structure, in order to achieve a better

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

PubMed

Kudlow, J E; Leung, Y

1984-06-15

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP

Global Quantitative Modeling of Chromatin Factor Interactions

PubMed Central

Zhou, Jian; Troyanskaya, Olga G.

2014-01-01

Chromatin is the driver of gene regulation, yet understanding the molecular interactions underlying chromatin factor combinatorial patterns (or the “chromatin codes”) remains a fundamental challenge in chromatin biology. Here we developed a global modeling framework that leverages chromatin profiling data to produce a systems-level view of the macromolecular complex of chromatin. Our model ultilizes maximum entropy modeling with regularization-based structure learning to statistically dissect dependencies between chromatin factors and produce an accurate probability distribution of chromatin code. Our unsupervised quantitative model, trained on genome-wide chromatin profiles of 73 histone marks and chromatin proteins from modENCODE, enabled making various data-driven inferences about chromatin profiles and interactions. We provided a highly accurate predictor of chromatin factor pairwise interactions validated by known experimental evidence, and for the first time enabled higher-order interaction prediction. Our predictions can thus help guide future experimental studies. The model can also serve as an inference engine for predicting unknown chromatin profiles — we demonstrated that with this approach we can leverage data from well-characterized cell types to help understand less-studied cell type or conditions. PMID:24675896

A futile cycle, formed between two ATP-dependant gamma-glutamyl cycle enzymes, gamma-glutamyl cysteine synthetase and 5-oxoprolinase: the cause of cellular ATP depletion in nephrotic cystinosis?

PubMed

Kumar, Akhilesh; Bachhawat, Anand Kumar

2010-03-01

Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model that explains how a futile cycle that is generated between two ATP-utilizing enzymes of the gamma-glutamyl cycle leads to ATP depletion. The enzyme gamma-glutamyl cysteine synthetase (gamma-GCS), in the absence of cysteine, forms 5-oxoproline (instead of the normal substrate, gamma-glutamyl cysteine) and the 5-oxoproline is converted into glutamate by the ATP-dependant enzyme, 5-oxoprolinase. Thus, in cysteine-limiting conditions, glutamate is cycled back into glutamate via 5-oxoproline at the cost of two ATP molecules without production of glutathione and is the cause of the decreased levels of glutathione synthesis, as well as the ATP depletion observed in these cells. The model is also compatible with the differences seen in the human patients and the mouse model of cystinosis, where renal failure is not observed.

Nitric oxide deficiency determines global chromatin changes in Duchenne muscular dystrophy.

PubMed

Colussi, Claudia; Gurtner, Aymone; Rosati, Jessica; Illi, Barbara; Ragone, Gianluca; Piaggio, Giulia; Moggio, Maurizio; Lamperti, Costanza; D'Angelo, Grazia; Clementi, Emilio; Minetti, Giulia; Mozzetta, Chiara; Antonini, Annalisa; Capogrossi, Maurizio C; Puri, Pier Lorenzo; Gaetano, Carlo

2009-07-01

The present study provides evidence that abnormal patterns of global histone modification are present in the skeletal muscle nuclei of mdx mice and Duchenne muscular dystrophy (DMD) patients. A combination of specific histone H3 modifications, including Ser-10 phosphorylation, acetylation of Lys 9 and 14, and Lys 79 methylation, were found enriched in muscle biopsies from human patients affected by DMD and in late-term fetuses, early postnatal pups, or adult mdx mice. In this context, chromatin immunoprecipitation experiments showed an enrichment of these modifications at the loci of genes involved in proliferation or inflammation, suggesting a regulatory effect on gene expression. Remarkably, the reexpression of dystrophin induced by gentamicin treatment or the administration of nitric oxide (NO) donors reversed the abnormal pattern of H3 histone modifications. These findings suggest an unanticipated link between the dystrophin-activated NO signaling and the remodeling of chromatin. In this context, the regulation of class IIa histone deacetylases (HDACs) 4 and 5 was found altered as a consequence of the reduced NO-dependent protein phosphatase 2A activity, indicating that both NO and class IIa HDACs are important for satellite cell differentiation and gene expression in mdx mice. In conclusion, this work provides the first evidence of a role for NO as an epigenetic regulator in DMD.

Chromatin remodeling gene EZH2 involved in the genetic etiology of autism in Chinese Han population.

PubMed

Li, Jun; You, Yang; Yue, Weihua; Yu, Hao; Lu, Tianlan; Wu, Zhiliu; Jia, Meixiang; Ruan, Yanyan; Liu, Jing; Zhang, Dai; Wang, Lifang

2016-01-01

Autism spectrum disorder (ASD) is a group of severe neurodevelopmental disorders. Epigenetic factors play a critical role in the etiology of ASD. Enhancer of zest homolog 2 (EZH2), which encodes a histone methyltransferase, plays an important role in the process of chromatin remodeling during neurodevelopment. Further, EZH2 is located in chromosome 7q35-36, which is one of the linkage regions for autism. However, the genetic relationship between autism and EZH2 remains unclear. To investigate the association between EZH2 and autism in Chinese Han population, we performed a family-based association study between autism and three tagged single nucleotide polymorphisms (SNPs) that covered 95.4% of the whole region of EZH2. In the discovery cohort of 239 trios, two SNPs (rs740949 and rs6464926) showed a significant association with autism. To decrease false positive results, we expanded the sample size to 427 trios. A SNP (rs6464926) was significantly associated with autism even after Bonferroni correction (p=0.008). Haplotype G-T (rs740949 and rs6464926) was a risk factor for autism (Z=2.655, p=0.008, Global p=0.024). In silico function prediction for SNPs indicated that these two SNPs might be regulatory SNPs. Expression pattern of EZH2 showed that it is highly expressed in human embryonic brains. In conclusion, our findings demonstrate that EZH2 might contribute to the genetic etiology of autism in Chinese Han population. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Remodeling by fibroblasts alters the rate-dependent mechanical properties of collagen.

PubMed

Babaei, Behzad; Davarian, Ali; Lee, Sheng-Lin; Pryse, Kenneth M; McConnaughey, William B; Elson, Elliot L; Genin, Guy M

2016-06-01

The ways that fibroblasts remodel their environment are central to wound healing, development of musculoskeletal tissues, and progression of pathologies such as fibrosis. However, the changes that fibroblasts make to the material around them and the mechanical consequences of these changes have proven difficult to quantify, especially in realistic, viscoelastic three-dimensional culture environments, leaving a critical need for quantitative data. Here, we observed the mechanisms and quantified the mechanical effects of fibroblast remodeling in engineered tissue constructs (ETCs) comprised of reconstituted rat tail (type I) collagen and human fibroblast cells. To study the effects of remodeling on tissue mechanics, stress-relaxation tests were performed on ETCs cultured for 24, 48, and 72h. ETCs were treated with deoxycholate and tested again to assess the ECM response. Viscoelastic relaxation spectra were obtained using the generalized Maxwell model. Cells exhibited viscoelastic damping at two finite time constants over which the ECM showed little damping, approximately 0.2s and 10-30s. Different finite time constants in the range of 1-7000s were attributed to ECM relaxation. Cells remodeled the ECM to produce a relaxation time constant on the order of 7000s, and to merge relaxation finite time constants in the 0.5-2s range into a single time content in the 1s range. Results shed light on hierarchical deformation mechanisms in tissues, and on pathologies related to collagen relaxation such as diastolic dysfunction. As fibroblasts proliferate within and remodel a tissue, they change the tissue mechanically. Quantifying these changes is critical for understanding wound healing and the development of pathologies such as cardiac fibrosis. Here, we characterize for the first time the spectrum of viscoelastic (rate-dependent) changes arising from the remodeling of reconstituted collagen by fibroblasts. The method also provides estimates of the viscoelastic spectra of

miR-151-5p, targeting chromatin remodeler SMARCA5, as a marker for the BRCAness phenotype

PubMed Central

Tommasi, Stefania; Pinto, Rosamaria; Danza, Katia; Pilato, Brunella; Palumbo, Orazio; Micale, Lucia; Summa, Simona De

2016-01-01

In recent years, the assessment of biomarkers useful for “precision medicine” has been a hot topic in research. The involvement of microRNAs in the pathogenesis of breast cancer has been highly investigated with the aim of being able to molecularly stratify this highly heterogeneous disease. Our aim was to identify microRNAs targeting DNA repair machinery, through Affymetrix GeneChip miRNA Arrays, in a cohort of BRCA-related and sporadic breast cancers. Moreover, we analyzed microRNA expression taking into account our previous results on the expression of PARP1, because of its importance in targeted therapy. miR-361-5p and miR-151-5p were found to be overexpressed in PARP1-upregulating BRCA-germline mutated and sporadic breast tumors. Pathway enrichment analysis was performed to identify potential target genes to be analyzed in the validation step in an independent cohort. Our results confirmed the overexpression of miR-151-5p and, interestingly, its role in the targeting of SMARCA5, a chromatin remodeler. This result was also confirmed in vitro, both through luciferase assay and by analyzing endogenous levels of SMARCA5 in MCF-7 cell lines using miR-151-5p mimic and inhibitor. In conclusion, our data showed the possibility of considering the overexpression of PARP1 and miR-151-5p as biomarkers useful to correctly treat sporadic breast cancers, which eventually could be considered as BRCAness tumors, with PARP-inhibitors. PMID:27385001

BHD-associated kidney cancer exhibits unique molecular characteristics and a wide variety of variants in chromatin remodeling genes.

PubMed

Hasumi, Hisashi; Furuya, Mitsuko; Tatsuno, Kenji; Yamamoto, Shogo; Baba, Masaya; Hasumi, Yukiko; Isono, Yasuhiro; Suzuki, Kae; Jikuya, Ryosuke; Otake, Shinji; Muraoka, Kentaro; Osaka, Kimito; Hayashi, Narihiko; Makiyama, Kazuhide; Miyoshi, Yasuhide; Kondo, Keiichi; Nakaigawa, Noboru; Kawahara, Takashi; Izumi, Koji; Teranishi, Junichi; Yumura, Yasushi; Uemura, Hiroji; Nagashima, Yoji; Metwalli, Adam R; Schmidt, Laura S; Aburatani, Hiroyuki; Linehan, W Marston; Yao, Masahiro

2018-05-14

Birt-Hogg-Dubé (BHD) syndrome is a hereditary kidney cancer syndrome, which predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas and pulmonary cysts. The responsible gene FLCN is a tumor suppressor for kidney cancer which plays an important role in energy homeostasis through the regulation of mitochondrial oxidative metabolism. However, the process by which FLCN-deficiency leads to renal tumorigenesis is unclear. In order to clarify molecular pathogenesis of BHD-associated kidney cancer, we conducted whole-exome sequencing analysis using next-generation sequencing technology as well as metabolite analysis using LC/MS and GC/MS. Whole-exome sequencing analysis of BHD-associated kidney cancer revealed that copy number variations (CNV) of BHD-associated kidney cancer are considerably different from those already reported in sporadic cases. In somatic variant analysis, very few variants were commonly observed in BHD-associated kidney cancer; however, variants in chromatin remodeling genes were frequently observed in BHD-associated kidney cancer (17/29 tumors, 59%). Metabolite analysis of BHD-associated kidney cancer revealed metabolic reprogramming towards upregulated redox regulation which may neutralize reactive oxygen species potentially produced from mitochondria with increased respiratory capacity under FLCN-deficiency. BHD-associated kidney cancer displays unique molecular characteristics which are completely different from sporadic kidney cancer, providing mechanistic insight into tumorigenesis under FLCN-deficiency as well as a foundation for development of novel therapeutics for kidney cancer.

The chromatin-binding protein Smyd1 restricts adult mammalian heart growth.

PubMed

Franklin, Sarah; Kimball, Todd; Rasmussen, Tara L; Rosa-Garrido, Manuel; Chen, Haodong; Tran, Tam; Miller, Mickey R; Gray, Ricardo; Jiang, Shanxi; Ren, Shuxun; Wang, Yibin; Tucker, Haley O; Vondriska, Thomas M

2016-11-01

All terminally differentiated organs face two challenges, maintaining their cellular identity and restricting organ size. The molecular mechanisms responsible for these decisions are of critical importance to organismal development, and perturbations in their normal balance can lead to disease. A hallmark of heart failure, a condition affecting millions of people worldwide, is hypertrophic growth of cardiomyocytes. The various forms of heart failure in human and animal models share conserved transcriptome remodeling events that lead to expression of genes normally silenced in the healthy adult heart. However, the chromatin remodeling events that maintain cell and organ size are incompletely understood; insights into these mechanisms could provide new targets for heart failure therapy. Using a quantitative proteomics approach to identify muscle-specific chromatin regulators in a mouse model of hypertrophy and heart failure, we identified upregulation of the histone methyltransferase Smyd1 during disease. Inducible loss-of-function studies in vivo demonstrate that Smyd1 is responsible for restricting growth in the adult heart, with its absence leading to cellular hypertrophy, organ remodeling, and fulminate heart failure. Molecular studies reveal Smyd1 to be a muscle-specific regulator of gene expression and indicate that Smyd1 modulates expression of gene isoforms whose expression is associated with cardiac pathology. Importantly, activation of Smyd1 can prevent pathological cell growth. These findings have basic implications for our understanding of cardiac pathologies and open new avenues to the treatment of cardiac hypertrophy and failure by modulating Smyd1. Copyright © 2016 the American Physiological Society.

Telomerase Reverse Transcriptase Deficiency Prevents Neointima Formation Through Chromatin Silencing of E2F1 Target Genes.

PubMed

Endorf, Elizabeth B; Qing, Hua; Aono, Jun; Terami, Naoto; Doyon, Geneviève; Hyzny, Eric; Jones, Karrie L; Findeisen, Hannes M; Bruemmer, Dennis

2017-02-01

Aberrant proliferation of smooth muscle cells (SMC) in response to injury induces pathological vascular remodeling during atherosclerosis and neointima formation. Telomerase is rate limiting for tissue renewal and cell replication; however, the physiological role of telomerase in vascular diseases remains to be determined. The goal of the present study was to determine whether telomerase reverse transcriptase (TERT) affects proliferative vascular remodeling and to define the molecular mechanism by which TERT supports SMC proliferation. We first demonstrate high levels of TERT expression in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program and not reversed by ectopic overexpression of E2F1. Finally, chromatin immunoprecipitation and accessibility assays revealed that TERT is recruited to E2F1 target sites and promotes chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. These data indicate a previously unrecognized role for TERT in neointima formation through epigenetic regulation of proliferative gene expression in SMC. © 2016 American Heart Association, Inc.

Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells.

PubMed

Shiraishi, Takumi; Verdone, James E; Huang, Jessie; Kahlert, Ulf D; Hernandez, James R; Torga, Gonzalo; Zarif, Jelani C; Epstein, Tamir; Gatenby, Robert; McCartney, Annemarie; Elisseeff, Jennifer H; Mooney, Steven M; An, Steven S; Pienta, Kenneth J

2015-01-01

The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease.

Glycolysis is the primary bioenergetic pathway for cell motility and cytoskeletal remodeling in human prostate and breast cancer cells

PubMed Central

Shiraishi, Takumi; Verdone, James E.; Huang, Jessie; Kahlert, Ulf D.; Hernandez, James R.; Torga, Gonzalo; Zarif, Jelani C.; Epstein, Tamir; Gatenby, Robert; McCartney, Annemarie; Elisseeff, Jennifer H.; Mooney, Steven M.; An, Steven S.; Pienta, Kenneth J.

2015-01-01

The ability of a cancer cell to detach from the primary tumor and move to distant sites is fundamental to a lethal cancer phenotype. Metabolic transformations are associated with highly motile aggressive cellular phenotypes in tumor progression. Here, we report that cancer cell motility requires increased utilization of the glycolytic pathway. Mesenchymal cancer cells exhibited higher aerobic glycolysis compared to epithelial cancer cells while no significant change was observed in mitochondrial ATP production rate. Higher glycolysis was associated with increased rates of cytoskeletal remodeling, greater cell traction forces and faster cell migration, all of which were blocked by inhibition of glycolysis, but not by inhibition of mitochondrial ATP synthesis. Thus, our results demonstrate that cancer cell motility and cytoskeleton rearrangement is energetically dependent on aerobic glycolysis and not oxidative phosphorylation. Mitochondrial derived ATP is insufficient to compensate for inhibition of the glycolytic pathway with regard to cellular motility and CSK rearrangement, implying that localization of ATP derived from glycolytic enzymes near sites of active CSK rearrangement is more important for cell motility than total cellular ATP production rate. These results extend our understanding of cancer cell metabolism, potentially providing a target metabolic pathway associated with aggressive disease. PMID:25426557

ATP depletion during mitotic arrest induces mitotic slippage and APC/CCdh1-dependent cyclin B1 degradation.

PubMed

Park, Yun Yeon; Ahn, Ju-Hyun; Cho, Min-Guk; Lee, Jae-Ho

2018-04-27

ATP depletion inhibits cell cycle progression, especially during the G1 phase and the G2 to M transition. However, the effect of ATP depletion on mitotic progression remains unclear. We observed that the reduction of ATP after prometaphase by simultaneous treatment with 2-deoxyglucose and NaN 3 did not arrest mitotic progression. Interestingly, ATP depletion during nocodazole-induced prometaphase arrest resulted in mitotic slippage, as indicated by a reduction in mitotic cells, APC/C-dependent degradation of cyclin B1, increased cell attachment, and increased nuclear membrane reassembly. Additionally, cells successfully progressed through the cell cycle after mitotic slippage, as indicated by EdU incorporation and time-lapse imaging. Although degradation of cyclin B during normal mitotic progression is primarily regulated by APC/C Cdc20 , we observed an unexpected decrease in Cdc20 prior to degradation of cyclin B during mitotic slippage. This decrease in Cdc20 was followed by a change in the binding partner preference of APC/C from Cdc20 to Cdh1; consequently, APC/C Cdh1 , but not APC/C Cdc20 , facilitated cyclin B degradation following ATP depletion. Pulse-chase analysis revealed that ATP depletion significantly abrogated global translation, including the translation of Cdc20 and Cdh1. Additionally, the half-life of Cdh1 was much longer than that of Cdc20. These data suggest that ATP depletion during mitotic arrest induces mitotic slippage facilitated by APC/C Cdh1 -dependent cyclin B degradation, which follows a decrease in Cdc20 resulting from reduced global translation and the differences in the half-lives of the Cdc20 and Cdh1 proteins.

Biophysical Regulation of Chromatin Architecture Instills a Mechanical Memory in Mesenchymal Stem Cells

PubMed Central

Heo, Su-Jin; Thorpe, Stephen D.; Driscoll, Tristan P.; Duncan, Randall L.; Lee, David A.; Mauck, Robert L.

2015-01-01

Mechanical cues direct the lineage commitment of mesenchymal stem cells (MSCs). In this study, we identified the operative molecular mechanisms through which dynamic tensile loading (DL) regulates changes in chromatin organization and nuclear mechanics in MSCs. Our data show that, in the absence of exogenous differentiation factors, short term DL elicits a rapid increase in chromatin condensation, mediated by acto-myosin based cellular contractility and the activity of the histone-lysine N-methyltransferase EZH2. The resulting change in chromatin condensation stiffened the MSC nucleus, making it less deformable when stretch was applied to the cell. We also identified stretch induced ATP release and purinergic calcium signaling as a central mediator of this chromatin condensation process. Further, we showed that DL, through differential stabilization of the condensed chromatin state, established a ‘mechanical memory’ in these cells. That is, increasing strain levels and number of loading events led to a greater degree of chromatin condensation that persisted for longer periods of time after the cessation of loading. These data indicate that, with mechanical perturbation, MSCs develop a mechanical memory encoded in structural changes in the nucleus which may sensitize them to future mechanical loading events and define the trajectory and persistence of their lineage specification. PMID:26592929

An ATP-dependent ligase with substrate flexibility involved in assembly of the peptidyl nucleoside antibiotic polyoxin.

PubMed

Gong, Rong; Qi, Jianzhao; Wu, Pan; Cai, You-Sheng; Ma, Hongmin; Liu, Yang; Duan, He; Wang, Meng; Deng, Zixin; Price, Neil P J; Chen, Wenqing

2018-04-27

Polyoxin (POL) is an unusual peptidyl nucleoside antibiotic, in which peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A polG mutant is capable of accumulating multiple intermediates, including the peptidyl moiety (carbamoylpolyoxamic acid, CPOAA) and the nucleosides skeletons (POL-C and the previously overlooked thymine POL-C). We further demonstrated that PolG employs an ATP-dependent mechanism for amide bond formation, and that the generation of the hybrid nucleoside antibiotic, POL-N, is also governed by PolG. Finally, we determined that the deduced ATP-binding sites are functionally essential for PolG, and that they are highly conserved in a number of related ATP-dependent ligases. These insights have allowed us proposed a catalytic mechanism for the assembly of peptidyl nucleoside antibiotic via an acyl-phosphate intermediate, and have opened the way for the combinatorial biosynthesis/pathway engineering of this group of nucleoside antibiotics. Importance POL is well known for its remarkable antifungal bioactivities and unusual structural features. Actually, elucidation of the POL assembly logic not only provides the enzymatic basis for further biosynthetic understanding of related peptidyl nucleoside antibiotics, but also contributes to the rational generation of more hybrid nucleoside antibiotics via synthetic biology strategy. Copyright © 2018 American Society for Microbiology.

PRMT4 Is a Novel Coactivator of c-Myb-Dependent Transcription in Haematopoietic Cell Lines

PubMed Central

Berberich, Hannah; Zeller, Marc S.; Teichmann, Sophia; Adamkiewicz, Jürgen; Müller, Rolf; Klempnauer, Karl-Heinz; Bauer, Uta-Maria

2013-01-01

Protein arginine methyltransferase 4 (PRMT4)–dependent methylation of arginine residues in histones and other chromatin-associated proteins plays an important role in the regulation of gene expression. However, the exact mechanism of how PRMT4 activates transcription remains elusive. Here, we identify the chromatin remodeller Mi2α as a novel interaction partner of PRMT4. PRMT4 binds Mi2α and its close relative Mi2β, but not the other components of the repressive Mi2-containing NuRD complex. In the search for the biological role of this interaction, we find that PRMT4 and Mi2α/β interact with the transcription factor c-Myb and cooperatively coactivate c-Myb target gene expression in haematopoietic cell lines. This coactivation requires the methyltransferase and ATPase activity of PRMT4 and Mi2, respectively. Chromatin immunoprecipitation analysis shows that c-Myb target genes are direct transcriptional targets of PRMT4 and Mi2. Knockdown of PRMT4 or Mi2α/β in haematopoietic cells of the erythroid lineage results in diminished transcriptional induction of c-Myb target genes, attenuated cell growth and survival, and deregulated differentiation resembling the effects caused by c-Myb depletion. These findings reveal an important and so far unknown connection between PRMT4 and the chromatin remodeller Mi2 in c-Myb signalling. PMID:23505388

PRMT4 is a novel coactivator of c-Myb-dependent transcription in haematopoietic cell lines.

PubMed

Streubel, Gundula; Bouchard, Caroline; Berberich, Hannah; Zeller, Marc S; Teichmann, Sophia; Adamkiewicz, Jürgen; Müller, Rolf; Klempnauer, Karl-Heinz; Bauer, Uta-Maria

2013-01-01

Protein arginine methyltransferase 4 (PRMT4)-dependent methylation of arginine residues in histones and other chromatin-associated proteins plays an important role in the regulation of gene expression. However, the exact mechanism of how PRMT4 activates transcription remains elusive. Here, we identify the chromatin remodeller Mi2α as a novel interaction partner of PRMT4. PRMT4 binds Mi2α and its close relative Mi2β, but not the other components of the repressive Mi2-containing NuRD complex. In the search for the biological role of this interaction, we find that PRMT4 and Mi2α/β interact with the transcription factor c-Myb and cooperatively coactivate c-Myb target gene expression in haematopoietic cell lines. This coactivation requires the methyltransferase and ATPase activity of PRMT4 and Mi2, respectively. Chromatin immunoprecipitation analysis shows that c-Myb target genes are direct transcriptional targets of PRMT4 and Mi2. Knockdown of PRMT4 or Mi2α/β in haematopoietic cells of the erythroid lineage results in diminished transcriptional induction of c-Myb target genes, attenuated cell growth and survival, and deregulated differentiation resembling the effects caused by c-Myb depletion. These findings reveal an important and so far unknown connection between PRMT4 and the chromatin remodeller Mi2 in c-Myb signalling.

ATP-dependent human RISC assembly pathways.

PubMed

Yoda, Mayuko; Kawamata, Tomoko; Paroo, Zain; Ye, Xuecheng; Iwasaki, Shintaro; Liu, Qinghua; Tomari, Yukihide

2010-01-01

The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1-4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3'-mid (guide position 12-15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.

The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

PubMed Central

Fenton, Aron W.; Hutchinson, Myra

2009-01-01

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37°C. As a function of increasing pH, hL-PYK's affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and Ala slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to Ala is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for Ala inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response. PMID:19467627

Colorectal Carcinomas With CpG Island Methylator Phenotype 1 Frequently Contain Mutations in Chromatin Regulators

PubMed Central

Tahara, Tomomitsu; Yamamoto, Eiichiro; Madireddi, Priyanka; Suzuki, Hiromu; Maruyama, Reo; Chung, Woonbok; Garriga, Judith; Jelinek, Jaroslav; Yamano, Hiro-o; Sugai, Tamotsu; Kondo, Yutaka; Toyota, Minoru; Issa, Jean-Pierre J.; Estécio, Marcos R. H.

2014-01-01

BACKGROUND & AIMS Subgroups of colorectal carcinomas (CRCs) characterized by DNA methylation anomalies are termed CpG island methylator phenotype (CIMP)1, CIMP2, or CIMP-negative. The pathogenesis of CIMP1 colorectal carcinomas, and their effects on patients’ prognoses and responses to treatment, differ from those of other CRCs. We sought to identify genetic somatic alterations associated with CIMP1 CRCs. METHODS We examined genomic DNA samples from 100 primary CRCs, 10 adenomas, and adjacent normal-appearing mucosae from patients undergoing surgery or colonoscopy at 3 tertiary medical centers. We performed exome sequencing of 16 colorectal tumors and their adjacent normal tissues. Extensive comparison with known somatic alterations in CRCs allowed segregation of CIMP1-exclusive alterations. The prevalence of mutations in selected genes was determined from an independent cohort. RESULTS We found that genes that regulate chromatin were mutated in CIMP1 CRCs; the highest rates of mutation were observed in CHD7 and CHD8, which encode members of the chromodomain helicase/adenosine triphosphate—dependent chromatin remodeling family. Somaticmutations in these 2 genes were detected in 5 of 9 CIMP1 CRCs. A prevalence screen showed that nonsilencing mutations in CHD7 and CHD8 occurred significantly more frequently in CIMP1 tumors (18 of 42 [43%]) than in CIMP2 (3 of 34 [9%]; P < .01) or CIMP-negative tumors (2 of 34 [6%]; P < .001). CIMP1 markers had increased binding by CHD7, compared with all genes. Genes altered in patients with CHARGE syndrome (congenital malformations involving the central nervous system, eye, ear, nose, and mediastinal organs) who had CHD7 mutations were also altered in CRCs with mutations in CHD7. CONCLUSIONS Aberrations in chromatin remodeling could contribute to the development of CIMP1 CRCs. A better understanding of the biological determinants of CRCs can be achieved when these tumors are categorized according to their epigenetic status. PMID

A Ca2+-dependent remodelled actin network directs vesicle trafficking to build wall ingrowth papillae in transfer cells.

PubMed

Zhang, Hui-Ming; Colyvas, Kim; Patrick, John W; Offler, Christina E

2017-10-13

The transport function of transfer cells is conferred by an enlarged plasma membrane area, enriched in nutrient transporters, that is supported on a scaffold of wall ingrowth (WI) papillae. Polarized plumes of elevated cytosolic Ca2+ define loci at which WI papillae form in developing adaxial epidermal transfer cells of Vicia faba cotyledons that are induced to trans-differentiate when the cotyledons are placed on culture medium. We evaluated the hypothesis that vesicle trafficking along a Ca2+-regulated remodelled actin network is the mechanism that underpins this outcome. Polarized to the outer periclinal cytoplasm, a Ca2+-dependent remodelling of long actin bundles into short, thin bundles was found to be essential for assembling WI papillae but not the underlying uniform wall layer. The remodelled actin network directed polarized vesicle trafficking to sites of WI papillae construction, and a pharmacological study indicated that both exo- and endocytosis contributed to assembly of the papillae. Potential candidates responsible for the Ca2+-dependent actin remodelling, along with those underpinning polarized exo- and endocyotosis, were identified in a transcriptome RNAseq database generated from the trans-differentiating epidermal cells. Of most significance, endocytosis was controlled by up-regulated expression of a dynamin-like isoform. How a cycle of localized exo- and endocytosis, regulated by Ca2+-dependent actin remodelling, assembles WI papillae is discussed. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX

PubMed Central

Clynes, David; Jelinska, Clare; Xella, Barbara; Ayyub, Helena; Scott, Caroline; Mitson, Matthew; Taylor, Stephen; Higgs, Douglas R.; Gibbons, Richard J.

2015-01-01

Fifteen per cent of cancers maintain telomere length independently of telomerase by the homologous recombination (HR)-associated alternative lengthening of telomeres (ALT) pathway. A unifying feature of these tumours are mutations in ATRX. Here we show that expression of ectopic ATRX triggers a suppression of the pathway and telomere shortening. Importantly ATRX-mediated ALT suppression is dependent on the histone chaperone DAXX. Re-expression of ATRX is associated with a reduction in replication fork stalling, a known trigger for HR and loss of MRN from telomeres. A G-quadruplex stabilizer partially reverses the effect of ATRX, inferring ATRX may normally facilitate replication through these sequences that, if they persist, promote ALT. We propose that defective telomere chromatinization through loss of ATRX promotes the persistence of aberrant DNA secondary structures, which in turn present a barrier to DNA replication, leading to replication fork stalling, collapse, HR and subsequent recombination-mediated telomere synthesis in ALT cancers. PMID:26143912

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

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

Sreenath, Kadreppa; Pavithra, Lakshminarasimhan; Singh, Sandeep

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 RNAmore » 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.« less

Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state.

PubMed

Moreira, Otacilio C; Rios, Priscila F; Barrabin, Hector

2005-07-15

The bidentate complex of ATP with Cr(3+), CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca(2+)-ATPase and the Na(+),K(+)-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca(2+) and Na(+), respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca(2+)-ATPase. The complex inhibited with similar efficiency the Ca(2+)-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T(1/2)=30 min at 37 degrees C) with a K(i)=28+/-9 microM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg(2+) but unaltered when Ca(2+) was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca(2+) occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La(3+) with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca(2+) at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca(2+) promoted by the plasma membrane Ca(2+)-ATPase goes through an enzymatic phospho-intermediate that maintains Ca(2+) ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.

Knockdown of the Chromatin Remodeling Gene Brahma by RNA Interference Reduces Reproductive Fitness and Lifespan in Common Bed Bug (Hemiptera: Cimicidae).

PubMed

Basnet, Sanjay; Kamble, Shripat T

2018-05-04

The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) is a nuisance household pest causing significant medical and economic impacts. RNA interference (RNAi) of genes that are involved in vital physiological processes can serve as potential RNAi targets for insect control. Brahma is an ATPase subunit of a chromatin-remodeling complex involved in transcription of several genes for cellular processes, most importantly the homeotic genes. In this study, we used a microinjection technique to deliver double stranded RNA into female bed bugs. Delivery of 0.05 and 0.5 µg/insect of brahma dsRNA directly into hemocele resulted substantial reduction in oviposition. Eggs laid by bed bugs receiving both doses of brahma dsRNA exhibited significantly lower hatching percentage as compared to controls. In addition, brahma RNAi in female bed bugs caused significant mortality. Our results disclosed the potential of brahma RNAi to suppress bed bug population through injection of specific dsRNA, suggesting a critical function of this gene in bed bugs' reproduction and survival. Based on our data, brahma can be a promising RNAi target for suppression of bed bug population.

The DEG/ENaC cation channel protein UNC-8 drives activity-dependent synapse removal in remodeling GABAergic neurons

PubMed Central

Miller-Fleming, Tyne W; Petersen, Sarah C; Manning, Laura; Matthewman, Cristina; Gornet, Megan; Beers, Allison; Hori, Sayaka; Mitani, Shohei; Bianchi, Laura; Richmond, Janet; Miller, David M

2016-01-01

Genetic programming and neural activity drive synaptic remodeling in developing neural circuits, but the molecular components that link these pathways are poorly understood. Here we show that the C. elegans Degenerin/Epithelial Sodium Channel (DEG/ENaC) protein, UNC-8, is transcriptionally controlled to function as a trigger in an activity-dependent mechanism that removes synapses in remodeling GABAergic neurons. UNC-8 cation channel activity promotes disassembly of presynaptic domains in DD type GABA neurons, but not in VD class GABA neurons where unc-8 expression is blocked by the COUP/TF transcription factor, UNC-55. We propose that the depolarizing effect of UNC-8-dependent sodium import elevates intracellular calcium in a positive feedback loop involving the voltage-gated calcium channel UNC-2 and the calcium-activated phosphatase TAX-6/calcineurin to initiate a caspase-dependent mechanism that disassembles the presynaptic apparatus. Thus, UNC-8 serves as a link between genetic and activity-dependent pathways that function together to promote the elimination of GABA synapses in remodeling neurons. DOI: http://dx.doi.org/10.7554/eLife.14599.001 PMID:27403890

Regulation of Egr1 Target Genes by the Nurd Chromatin Remodeling Complex

DTIC Science & Technology

2008-06-01

Wade for providing a Mi2 antibody , and Megan Santarius for excellent technical assistance. REFERENCES 1. Russo, M. W., Sevetson, B. R., and Milbrandt...achieve crosslinking. Chromatin was then sonicated and immunoprecipitated with antibodies directed against Egr1, MTA2 or IgG control. After...promoter of IGF2, although the effects are more subtle. Control ChIP assays employing an EGR1 antibody show correlated increased binding of EGR1

Comparative Genomics Reveals Chd1 as a Determinant of Nucleosome Spacing in Vivo.

PubMed

Hughes, Amanda L; Rando, Oliver J

2015-07-14

Packaging of genomic DNA into nucleosomes is nearly universally conserved in eukaryotes, and many features of the nucleosome landscape are quite conserved. Nonetheless, quantitative aspects of nucleosome packaging differ between species because, for example, the average length of linker DNA between nucleosomes can differ significantly even between closely related species. We recently showed that the difference in nucleosome spacing between two Hemiascomycete species-Saccharomyces cerevisiae and Kluyveromyces lactis-is established by trans-acting factors rather than being encoded in cis in the DNA sequence. Here, we generated several S. cerevisiae strains in which endogenous copies of candidate nucleosome spacing factors are deleted and replaced with the orthologous factors from K. lactis. We find no change in nucleosome spacing in such strains in which H1 or Isw1 complexes are swapped. In contrast, the K. lactis gene encoding the ATP-dependent remodeler Chd1 was found to direct longer internucleosomal spacing in S. cerevisiae, establishing that this remodeler is partially responsible for the relatively long internucleosomal spacing observed in K. lactis. By analyzing several chimeric proteins, we find that sequence differences that contribute to the spacing activity of this remodeler are dispersed throughout the coding sequence, but that the strongest spacing effect is linked to the understudied N-terminal end of Chd1. Taken together, our data find a role for sequence evolution of a chromatin remodeler in establishing quantitative aspects of the chromatin landscape in a species-specific manner. Copyright © 2015 Hughes and Rando.

Epigenetic Heterogeneity of B-Cell Lymphoma: DNA Methylation, Gene Expression and Chromatin States

PubMed Central

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

2015-01-01

Mature B-cell lymphoma is a clinically and biologically highly diverse disease. Its diagnosis and prognosis is a challenge due to its molecular heterogeneity and diverse regimes of biological dysfunctions, which are partly driven by epigenetic mechanisms. We here present an integrative analysis of DNA methylation and gene expression data of several lymphoma subtypes. Our study confirms previous results about the role of stemness genes during development and maturation of B-cells and their dysfunction in lymphoma locking in more proliferative or immune-reactive states referring to B-cell functionalities in the dark and light zone of the germinal center and also in plasma cells. These dysfunctions are governed by widespread epigenetic effects altering the promoter methylation of the involved genes, their activity status as moderated by histone modifications and also by chromatin remodeling. We identified four groups of genes showing characteristic expression and methylation signatures among Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma and multiple myeloma. These signatures are associated with epigenetic effects such as remodeling from transcriptionally inactive into active chromatin states, differential promoter methylation and the enrichment of targets of transcription factors such as EZH2 and SUZ12. PMID:26371046

Isolation of active regulatory elements from eukaryotic chromatin using FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements)

PubMed Central

Giresi, Paul G.; Lieb, Jason D.

2009-01-01

The binding of sequence-specific regulatory factors and the recruitment of chromatin remodeling activities cause nucleosomes to be evicted from chromatin in eukaryotic cells. Traditionally, these active sites have been identified experimentally through their sensitivity to nucleases. Here we describe the details of a simple procedure for the genome-wide isolation of nucleosome-depleted DNA from human chromatin, termed FAIRE (Formaldehyde Assisted Isolation of Regulatory Elements). We also provide protocols for different methods of detecting FAIRE-enriched DNA, including use of PCR, DNA microarrays, and next-generation sequencing. FAIRE works on all eukaryotic chromatin tested to date. To perform FAIRE, chromatin is crosslinked with formaldehyde, sheared by sonication, and phenol-chloroform extracted. Most genomic DNA is crosslinked to nucleosomes and is sequestered to the interphase, whereas DNA recovered in the aqueous phase corresponds to nucleosome-depleted regions of the genome. The isolated regions are largely coincident with the location of DNaseI hypersensitive sites, transcriptional start sites, enhancers, insulators, and active promoters. Given its speed and simplicity, FAIRE has utility in establishing chromatin profiles of diverse cell types in health and disease, isolating DNA regulatory elements en masse for further characterization, and as a screening assay for the effects of small molecules on chromatin organization. PMID:19303047

Dietary protein deficiency reduces lysosomal and nonlysosomal ATP-dependent proteolysis in muscle

NASA Technical Reports Server (NTRS)

Tawa, N. E. Jr; Kettelhut, I. C.; Goldberg, A. L.

1992-01-01

When rats are fed a protein deficient (PD) diet for 7 days, rates of proteolysis in skeletal muscle decrease by 40-50% (N. E. Tawa, Jr., and A. L. Goldberg. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E317-325, 1992). To identify the underlying biochemical adaptations, we measured different proteolytic processes in incubated muscles. The capacity for intralysosomal proteolysis, as shown by sensitivity to methylamine or lysosomal protease inhibitors, fell 55-75% in muscles from PD rats. Furthermore, extracts of muscles of PD rats showed 30-70% lower activity of many lysosomal proteases, including cathepsins B, H, and C, and carboxypeptidases A and C, as well as other lysosomal hydrolases. The fall in cathepsin B and proteolysis was evident by 3 days on the PD diet, and both returned to control levels 3 days after refeeding of the normal diet. In muscles maintained under optimal conditions, 80-90% of protein breakdown occurs by nonlysosomal pathways. In muscles of PD rats, this ATP-dependent process was also 40-60% slower. Even though overall proteolysis decreased in muscles of PD rats, their capacity for Ca(2+)-dependent proteolysis increased (by 66%), as did the activity of the calpains (+150-250%). Thus the lysosomal and the ATP-dependent processes decrease coordinately and contribute to the fall in muscle proteolysis in PD animals.

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus , a New Twist on ATP Formation

DOE PAGES

James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.; ...

2016-08-16

Syntrophus aciditrophicusis a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation byS. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome ofS. aciditrophicusleaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show thatS. aciditrophicususes AMP-forming, acetyl-CoA synthetase (Acs1)more » for ATP synthesis from acetyl-CoA.acs1mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, ofS. aciditrophicusgrown in pure culture and coculture. Cell extracts ofS. aciditrophicushad low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified fromS. aciditrophicusand recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) inS. aciditrophicuscells support the operation of Acs1 in the acetate-forming direction. Thus,S. aciditrophicushas a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. We find bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA.Syntrophus aciditrophicusapparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as

Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus , a New Twist on ATP Formation

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

James, Kimberly L.; Ríos-Hernández, Luis A.; Wofford, Neil Q.

Syntrophus aciditrophicusis a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation byS. aciditrophicus. However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome ofS. aciditrophicusleaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show thatS. aciditrophicususes AMP-forming, acetyl-CoA synthetase (Acs1)more » for ATP synthesis from acetyl-CoA.acs1mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, ofS. aciditrophicusgrown in pure culture and coculture. Cell extracts ofS. aciditrophicushad low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified fromS. aciditrophicusand recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) inS. aciditrophicuscells support the operation of Acs1 in the acetate-forming direction. Thus,S. aciditrophicushas a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. We find bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA.Syntrophus aciditrophicusapparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as

Modeling epigenome folding: formation and dynamics of topologically associated chromatin domains

PubMed Central

Jost, Daniel; Carrivain, Pascal; Cavalli, Giacomo; Vaillant, Cédric

2014-01-01

Genomes of eukaryotes are partitioned into domains of functionally distinct chromatin states. These domains are stably inherited across many cell generations and can be remodeled in response to developmental and external cues, hence contributing to the robustness and plasticity of expression patterns and cell phenotypes. Remarkably, recent studies indicate that these 1D epigenomic domains tend to fold into 3D topologically associated domains forming specialized nuclear chromatin compartments. However, the general mechanisms behind such compartmentalization including the contribution of epigenetic regulation remain unclear. Here, we address the question of the coupling between chromatin folding and epigenome. Using polymer physics, we analyze the properties of a block copolymer model that accounts for local epigenomic information. Considering copolymers build from the epigenomic landscape of Drosophila, we observe a very good agreement with the folding patterns observed in chromosome conformation capture experiments. Moreover, this model provides a physical basis for the existence of multistability in epigenome folding at sub-chromosomal scale. We show how experiments are fully consistent with multistable conformations where topologically associated domains of the same epigenomic state interact dynamically with each other. Our approach provides a general framework to improve our understanding of chromatin folding during cell cycle and differentiation and its relation to epigenetics. PMID:25092923

Dynamics of the metal binding domains and regulation of the human copper transporters ATP7B and ATP7A.

PubMed

Yu, Corey H; Dolgova, Natalia V; Dmitriev, Oleg Y

2017-04-01

Copper transporters ATP7A and ATP7B regulate copper levels in the human cells and deliver copper to the biosynthetic pathways. ATP7A and ATP7B belong to the P-type ATPases and share much of the domain architecture and the mechanism of ATP hydrolysis with the other, well-studied, enzymes of this type. A unique structural feature of the copper ATPases is the chain of six cytosolic metal-binding domains (MBDs), which are believed to be involved in copper-dependent regulation of the activity and intracellular localization of these enzymes. Although the structures of all the MBDs have been solved, the mechanism of copper-dependent regulation of ATP7B and ATP7A, the roles of individual MBDs, and the relationship between the regulatory and catalytic copper binding are still unknown. We describe the structure and dynamics of the MBDs, review the current knowledge about their functional roles and propose a mechanism of regulation of ATP7B by copper-dependent changes in the dynamics and conformation of the MBD chain. Transient interactions between the MBDs, rather than transitions between distinct static conformations are likely to form the structural basis of regulation of the ATP-dependent copper transporters in human cells. © 2016 IUBMB Life, 69(4):226-235, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

Phosphorylation of the chromatin remodeling factor DPF3a induces cardiac hypertrophy through releasing HEY repressors from DNA.

PubMed

Cui, Huanhuan; Schlesinger, Jenny; Schoenhals, Sophia; Tönjes, Martje; Dunkel, Ilona; Meierhofer, David; Cano, Elena; Schulz, Kerstin; Berger, Michael F; Haack, Timm; Abdelilah-Seyfried, Salim; Bulyk, Martha L; Sauer, Sascha; Sperling, Silke R

2016-04-07

DPF3 (BAF45c) is a member of the BAF chromatin remodeling complex. Two isoforms have been described, namely DPF3a and DPF3b. The latter binds to acetylated and methylated lysine residues of histones. Here, we elaborate on the role of DPF3a and describe a novel pathway of cardiac gene transcription leading to pathological cardiac hypertrophy. Upon hypertrophic stimuli, casein kinase 2 phosphorylates DPF3a at serine 348. This initiates the interaction of DPF3a with the transcriptional repressors HEY, followed by the release of HEY from the DNA. Moreover, BRG1 is bound by DPF3a, and is thus recruited to HEY genomic targets upon interaction of the two components. Consequently, the transcription of downstream targets such as NPPA and GATA4 is initiated and pathological cardiac hypertrophy is established. In human, DPF3a is significantly up-regulated in hypertrophic hearts of patients with hypertrophic cardiomyopathy or aortic stenosis. Taken together, we show that activation of DPF3a upon hypertrophic stimuli switches cardiac fetal gene expression from being silenced by HEY to being activated by BRG1. Thus, we present a novel pathway for pathological cardiac hypertrophy, whose inhibition is a long-term therapeutic goal for the treatment of the course of heart failure. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

ATPase activity and light scattering of acto-heavy meromyosin: dependence on ATP concentration and on ionic strength.

PubMed

Dancker, P

1975-01-01

1. The dependence on ATP concentration of ATPase activity and light scattering decrease of acto-HMM could be described at very low ionic strength by one hyperbolic adsorption isotherm with a dissociation constant of 3 X 10(-6)M. Hence the increase of ATP ase activity was paralleled by a decrease in light scattering. At higher values of ionic strength ATPase activity stopped rising before HMM was completely saturated with ATP. Higher ionic strength prevented ATPase activity from further increasing when the rigor links (links between actin and nucleotide-free myosin), which have formerly protected the ATPase against the suppressing action of higher ionic strength have fallen below a certain amount. This protecting influence of rigor links did not require tropomyosin-troponin. 2. For complete activation of ATPase activity by actin less actin was needed when HMM was incompletely saturated with ATP than when it was completely saturated with ATP. 3. The apparent affinity of ATP to regulated acto-HMM (which contained tropomyosin-troponin) was lower than to unregulated acto-HMM (which was devoid of tropomyosin-troponin). In the presence of rigor complexes (indicated by an incomplete decrease of light scattering) the ATPase activity of regulated acto-HMM was higher than that of unregulated acto-HMM. At increasing ATP concentrations the ATPase activity of regulated acto-HMM stopped rising at a similar degree of saturation with ATP as the ATPase activity of unregulated acto-HMM at the same ionic strength.

The Involvement of Hydrogen-producing and ATP-dependent NADPH-consuming Pathways in Setting the Redox Poise in the Chloroplast of Chlamydomonas reinhardtii in Anoxia

PubMed Central

Clowez, Sophie; Godaux, Damien; Cardol, Pierre; Wollman, Francis-André; Rappaport, Fabrice

2015-01-01

Photosynthetic microalgae are exposed to changing environmental conditions. In particular, microbes found in ponds or soils often face hypoxia or even anoxia, and this severely impacts their physiology. Chlamydomonas reinhardtii is one among such photosynthetic microorganisms recognized for its unusual wealth of fermentative pathways and the extensive remodeling of its metabolism upon the switch to anaerobic conditions. As regards the photosynthetic electron transfer, this remodeling encompasses a strong limitation of the electron flow downstream of photosystem I. Here, we further characterize the origin of this limitation. We show that it stems from the strong reducing pressure that builds up upon the onset of anoxia, and this pressure can be relieved either by the light-induced synthesis of ATP, which promotes the consumption of reducing equivalents, or by the progressive activation of the hydrogenase pathway, which provides an electron transfer pathway alternative to the CO2 fixation cycle. PMID:25691575

Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase

PubMed Central

Johnson, Larry; Atanasova, Kalina R.; Bui, Phuong Q.; Lee, Jungnam; Hung, Shu-Chen; Yilmaz, Özlem; Ojcius, David M.

2015-01-01

Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the proinflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs. PMID:25828169

The effects of gamma radiation, UV and visible light on ATP levels in yeast cells depend on cellular melanization.

PubMed

Bryan, Ruth; Jiang, Zewei; Friedman, Matthew; Dadachova, Ekaterina

2011-10-01

Previously we have shown that growth of melanized fungi is stimulated by low levels of gamma radiation. The goal of this study was to examine the effects of visible light, UV light, and gamma radiation on the energy level (ATP concentration) in melanized Cryptococcus neoformans cells. Melanized C. neoformans cells as well as non-melanized controls were subjected to visible, UV or gamma radiation, and ATP was quantified by measuring the amount of light emitted by the ATP-dependent reaction of luciferase with luciferin. We found that all three forms of radiation led to a reduction in the ATP levels in melanized C. neoformans cells. This points to a universal melanin-related mechanism underlying observation of ATP decrease in irradiated melanized cells. In contrast, in non-melanized cells visible light led to increase in ATP levels; gamma radiation did not cause any changes while UV exposure resulted in some ATP decrease, however, much less pronounced than in melanized cells. Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Ca2+ and MgATP2- dependence of shortening in skinned single smooth muscle cells.

PubMed

Warshaw, D M; McBride, W J; Hubbard, M S

1987-04-01

Most studies of skinned smooth muscle have been performed in whole tissue preparations. In this study, we report the development of a chemically skinned single smooth muscle cell preparation from the toad, Bufo marinus, stomach. Isolated smooth muscle cells were skinned using saponin. The effect of various ionic environments (i.e., changing free Ca2+ and MgATP2-) on skinned cell contractile response was assessed by measuring cell lengths from populations of cells using a computer-assisted length-measuring system. Comparison of cell length histograms were used to determine the extent of cell shortening in response to a given ionic perturbation. Once skinned, the single cells shortened with a sensitivity to free calcium (ED50 = 1.5 microM Ca2+) that was three orders of magnitude lower than potassium depolarized cells (ED50 = 1.5 mM Ca2+). In addition to the calcium sensitivity, the effect of free MgATP2- on the extent of cell shortening was investigated. The extent of cell shortening was dependent on free MgATP2- with the maximum shortening response occurring at MgATP2- greater than 1 mM.

Sirtuins: molecular traffic lights in the crossroad of oxidative stress, chromatin remodeling, and transcription.

PubMed

Rajendran, Ramkumar; Garva, Richa; Krstic-Demonacos, Marija; Demonacos, Constantinos

2011-01-01

Transcription is regulated by acetylation/deacetylation reactions of histone and nonhistone proteins mediated by enzymes called KATs and HDACs, respectively. As a major mechanism of transcriptional regulation, protein acetylation is a key controller of physiological processes such as cell cycle, DNA damage response, metabolism, apoptosis, and autophagy. The deacetylase activity of class III histone deacetylases or sirtuins depends on the presence of NAD(+) (nicotinamide adenine dinucleotide), and therefore, their function is closely linked to cellular energy consumption. This activity of sirtuins connects the modulation of chromatin dynamics and transcriptional regulation under oxidative stress to cellular lifespan, glucose homeostasis, inflammation, and multiple aging-related diseases including cancer. Here we provide an overview of the recent developments in relation to the diverse biological activities associated with sirtuin enzymes and stress responsive transcription factors, DNA damage, and oxidative stress and relate the involvement of sirtuins in the regulation of these processes to oncogenesis. Since the majority of the molecular mechanisms implicated in these pathways have been described for Sirt1, this sirtuin family member is more extensively presented in this paper.

Transcription and chromatin determinants of de novo DNA methylation timing in oocytes.

PubMed

Gahurova, Lenka; Tomizawa, Shin-Ichi; Smallwood, Sébastien A; Stewart-Morgan, Kathleen R; Saadeh, Heba; Kim, Jeesun; Andrews, Simon R; Chen, Taiping; Kelsey, Gavin

2017-01-01

Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally

Pontin functions as an essential coactivator for Oct4-dependent lincRNA expression in mouse embryonic stem cells.

PubMed

Boo, Kyungjin; Bhin, Jinhyuk; Jeon, Yoon; Kim, Joomyung; Shin, Hi-Jai R; Park, Jong-Eun; Kim, Kyeongkyu; Kim, Chang Rok; Jang, Hyonchol; Kim, In-Hoo; Kim, V Narry; Hwang, Daehee; Lee, Ho; Baek, Sung Hee

2015-04-10

The actions of transcription factors, chromatin modifiers and noncoding RNAs are crucial for the programming of cell states. Although the importance of various epigenetic machineries for controlling pluripotency of embryonic stem (ES) cells has been previously studied, how chromatin modifiers cooperate with specific transcription factors still remains largely elusive. Here, we find that Pontin chromatin remodelling factor plays an essential role as a coactivator for Oct4 for maintenance of pluripotency in mouse ES cells. Genome-wide analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in ES cell maintenance. Intriguingly, we find that the Oct4-dependent coactivator function of Pontin extends to the transcription of large intergenic noncoding RNAs (lincRNAs) and in particular linc1253, a lineage programme repressing lincRNA, is a Pontin-dependent Oct4 target lincRNA. Together, our findings demonstrate that the Oct4-Pontin module plays critical roles in the regulation of genes involved in ES cell fate determination.

Epigenetic remodelling and dysregulation of DLGAP4 is linked with early-onset cerebellar ataxia

PubMed Central

Minocherhomji, Sheroy; Hansen, Claus; Kim, Hyung-Goo; Mang, Yuan; Bak, Mads; Guldberg, Per; Papadopoulos, Nickolas; Eiberg, Hans; Doh, Gerald Dayebga; Møllgård, Kjeld; Hertz, Jens Michael; Nielsen, Jørgen E.; Ropers, Hans-Hilger; Tümer, Zeynep; Tommerup, Niels; Kalscheuer, Vera M.; Silahtaroglu, Asli

2014-01-01

Genome instability, epigenetic remodelling and structural chromosomal rearrangements are hallmarks of cancer. However, the coordinated epigenetic effects of constitutional chromosomal rearrangements that disrupt genes associated with congenital neurodevelopmental diseases are poorly understood. To understand the genetic–epigenetic interplay at breakpoints of chromosomal translocations disrupting CG-rich loci, we quantified epigenetic modifications at DLGAP4 (SAPAP4), a key post-synaptic density 95 (PSD95) associated gene, truncated by the chromosome translocation t(8;20)(p12;q11.23), co-segregating with cerebellar ataxia in a five-generation family. We report significant epigenetic remodelling of the DLGAP4 locus triggered by the t(8;20)(p12;q11.23) translocation and leading to dysregulation of DLGAP4 expression in affected carriers. Disruption of DLGAP4 results in monoallelic hypermethylation of the truncated DLGAP4 promoter CpG island. This induced hypermethylation is maintained in somatic cells of carriers across several generations in a t(8;20) dependent-manner however, is erased in the germ cells of the translocation carriers. Subsequently, chromatin remodelling of the locus-perturbed monoallelic expression of DLGAP4 mRNAs and non-coding RNAs in haploid cells having the translocation. Our results provide new mechanistic insight into the way a balanced chromosomal rearrangement associated with a neurodevelopmental disorder perturbs allele-specific epigenetic mechanisms at breakpoints leading to the deregulation of the truncated locus. PMID:24986922

Molecular architecture of the ATP-dependent CodWX protease having an N-terminal serine active site

PubMed Central

Kang, Min Suk; Kim, Soon Rae; Kwack, Pyeongsu; Lim, Byung Kook; Ahn, Sung Won; Rho, Young Min; Seong, Ihn Sik; Park, Seong-Chul; Eom, Soo Hyun; Cheong, Gang-Won; Chung, Chin Ha

2003-01-01

CodWX in Bacillus subtilis is an ATP-dependent, N-terminal serine protease, consisting of CodW peptidase and CodX ATPase. Here we show that CodWX is an alkaline protease and has a distinct molecular architecture. ATP hydrolysis is required for the formation of the CodWX complex and thus for its proteolytic function. Remarkably, CodX has a ‘spool-like’ structure that is formed by interaction of the intermediate domains of two hexameric or heptameric rings. In the CodWX complex, CodW consisting of two stacked hexameric rings (WW) binds to either or both ends of a CodX double ring (XX), forming asymmetric (WWXX) or symmetric cylindrical particles (WWXXWW). CodWX can also form an elongated particle, in which an additional CodX double ring is bound to the symmetric particle (WWXXWWXX). In addition, CodWX is capable of degrading EzrA, an inhibitor of FtsZ ring formation, implicating it in the regulation of cell division. Thus, CodWX appears to constitute a new type of protease that is distinct from other ATP-dependent proteases in its structure and proteolytic mechanism. PMID:12805205

Chromatin Structure and the Cell Cycle

PubMed Central

Pederson, Thoru

1972-01-01

Pancreatic DNase I is used to probe the structure of chromatin isolated from synchronized HeLa cells. The degree to which DNA in chromatin is protected from DNase attack varies during the G1, S, and G2 phases of the cell cycle. In addition, the DNase sensitivity of chromatin from contact-inhibited African green monkey kidney cells differs from that of actively dividing, subconfluent cultures. These cell cycle-dependent chromatin changes were observed consistently at all enzyme concentrations (5000-fold range) and incubation times (15 min-2 hr) tested. The results indicate that the degree of complexing between DNA and chromosomal proteins changes during interphase, and they suggest that the chromosome coiling cycle of visible mitosis may extend in more subtle form over the entire cell cycle. PMID:4626402

Stretch-induced Ca2+ independent ATP release in hippocampal astrocytes.

PubMed

Xiong, Yingfei; Teng, Sasa; Zheng, Lianghong; Sun, Suhua; Li, Jie; Guo, Ning; Li, Mingli; Wang, Li; Zhu, Feipeng; Wang, Changhe; Rao, Zhiren; Zhou, Zhuan

2018-02-28

Similar to neurons, astrocytes actively participate in synaptic transmission via releasing gliotransmitters. The Ca 2+ -dependent release of gliotransmitters includes glutamate and ATP. Following an 'on-cell-like' mechanical stimulus to a single astrocyte, Ca 2+ independent single, large, non-quantal, ATP release occurs. Astrocytic ATP release is inhibited by either selective antagonist treatment or genetic knockdown of P2X7 receptor channels. Our work suggests that ATP can be released from astrocytes via two independent pathways in hippocampal astrocytes; in addition to the known Ca 2+ -dependent vesicular release, larger non-quantal ATP release depends on P2X7 channels following mechanical stretch. Astrocytic ATP release is essential for brain functions such as synaptic long-term potentiation for learning and memory. However, whether and how ATP is released via exocytosis remains hotly debated. All previous studies of non-vesicular ATP release have used indirect assays. By contrast, two recent studies report vesicular ATP release using more direct assays. In the present study, using patch clamped 'ATP-sniffer cells', we re-investigated astrocytic ATP release at single-vesicle resolution in hippocampal astrocytes. Following an 'on-cell-like' mechanical stimulus of a single astrocyte, a Ca 2+ independent single large non-quantal ATP release occurred, in contrast to the Ca 2+ -dependent multiple small quantal ATP release in a chromaffin cell. The mechanical stimulation-induced ATP release from an astrocyte was inhibited by either exposure to a selective antagonist or genetic knockdown of P2X7 receptor channels. Functional P2X7 channels were expressed in astrocytes in hippocampal brain slices. Thus, in addition to small quantal ATP release, larger non-quantal ATP release depends on P2X7 channels in astrocytes. © 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.

A role for the nucleoporin Nup170p in chromatin structure and gene silencing

PubMed Central

Van de Vosse, David W.; Wan, Yakun; Lapetina, Diego L.; Chen, Wei-Ming; Chiang, Jung-Hsien; Aitchison, John D.; Wozniak, Richard W.

2013-01-01

Embedded in the nuclear envelope, nuclear pore complexes (NPCs) not only regulate nuclear transport, but also interface with transcriptionally active euchromatin, largely silenced heterochromatin, as well as the boundaries between these regions. It is unclear what functional role NPCs play in establishing or maintaining these distinct chromatin domains. We report that the yeast NPC protein Nup170p interacts with regions of the genome containing ribosomal protein and subtelomeric genes. Here, it functions in nucleosome positioning and as a repressor of transcription. We show that the role of Nup170p in subtelomeric gene silencing is linked to its association with the RSC chromatin-remodeling complex and the silencing factor Sir4p, and that the binding of Nup170p and Sir4p to subtelomeric chromatin is cooperative and necessary for the association of telomeres with the nuclear envelope. Our results establish the NPC as an active participant in silencing and the formation of peripheral heterochromatin. PMID:23452847

Epigenetic remodeling and modification to preserve skeletogenesis in vivo.

PubMed

Godfrey, Tanner C; Wildman, Benjamin J; Javed, Amjad; Lengner, Christopher J; Hassan, Mohammad Quamarul

2018-12-01

Current studies offer little insight on how epigenetic remodeling of bone-specific chromatin maintains bone mass in vivo. Understanding this gap and precise mechanism is pivotal for future therapeutic innovation to prevent bone loss. Recently, we found that low bone mass is associated with decreased H3K27 acetylation (activating histone modification) of bone specific gene promoters. Here, we aim to elucidate the epigenetic mechanisms by which a miRNA cluster controls bone synthesis and homeostasis by regulating chromatin accessibility and H3K27 acetylation. In order to decipher the epigenetic axis that regulates osteogenesis, we studied a drug inducible anti-miR-23a cluster (miR-23a Cl ZIP ) knockdown mouse model. MiR-23a cluster knockdown (heterozygous) mice developed high bone mass. These mice displayed increased expression of Runx2 and Baf45a, essential factors for skeletogenesis; and decreased expression of Ezh2, a chromatin repressor indispensable for skeletogenesis. ChIP assays using miR-23a Cl knockdown calvarial cells revealed a BAF45A-EZH2 epigenetic antagonistic mechanism that maintains bone formation. Together, our findings support that the miR-23a Cl connection with tissue-specific RUNX2-BAF45A-EZH2 function is a novel molecular epigenetic axis through which a miRNA cluster orchestrates chromatin modification to elicit major effects on osteogenesis in vivo.

Condensins exert force on chromatin-nuclear envelope tethers to mediate nucleoplasmic reticulum formation in Drosophila melanogaster.

PubMed

Bozler, Julianna; Nguyen, Huy Q; Rogers, Gregory C; Bosco, Giovanni

2014-12-30

Although the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology. Copyright © 2015 Bozler et al.

Condensins Exert Force on Chromatin-Nuclear Envelope Tethers to Mediate Nucleoplasmic Reticulum Formation in Drosophila melanogaster

PubMed Central

Bozler, Julianna; Nguyen, Huy Q.; Rogers, Gregory C.; Bosco, Giovanni

2014-01-01

Although the nuclear envelope is known primarily for its role as a boundary between the nucleus and cytoplasm in eukaryotes, it plays a vital and dynamic role in many cellular processes. Studies of nuclear structure have revealed tissue-specific changes in nuclear envelope architecture, suggesting that its three-dimensional structure contributes to its functionality. Despite the importance of the nuclear envelope, the factors that regulate and maintain nuclear envelope shape remain largely unexplored. The nuclear envelope makes extensive and dynamic interactions with the underlying chromatin. Given this inexorable link between chromatin and the nuclear envelope, it is possible that local and global chromatin organization reciprocally impact nuclear envelope form and function. In this study, we use Drosophila salivary glands to show that the three-dimensional structure of the nuclear envelope can be altered with condensin II-mediated chromatin condensation. Both naturally occurring and engineered chromatin-envelope interactions are sufficient to allow chromatin compaction forces to drive distortions of the nuclear envelope. Weakening of the nuclear lamina further enhanced envelope remodeling, suggesting that envelope structure is capable of counterbalancing chromatin compaction forces. Our experiments reveal that the nucleoplasmic reticulum is born of the nuclear envelope and remains dynamic in that they can be reabsorbed into the nuclear envelope. We propose a model where inner nuclear envelope-chromatin tethers allow interphase chromosome movements to change nuclear envelope morphology. Therefore, interphase chromatin compaction may be a normal mechanism that reorganizes nuclear architecture, while under pathological conditions, such as laminopathies, compaction forces may contribute to defects in nuclear morphology. PMID:25552604

Deficiency of ATP6V1H Causes Bone Loss by Inhibiting Bone Resorption and Bone Formation through the TGF-β1 Pathway

PubMed Central

Duan, Xiaohong; Liu, Jin; Zheng, Xueni; Wang, Zhe; Zhang, Yanli; Hao, Ying; Yang, Tielin; Deng, Hongwen

2016-01-01

Vacuolar-type H +-ATPase (V-ATPase) is a highly conserved, ancient enzyme that couples the energy of ATP hydrolysis to proton transport across vesicular and plasma membranes of eukaryotic cells. Previously reported mutations of various V-ATPase subunits are associated with increased bone density. We now show that haploinsufficiency for the H subunit of the V1 domain (ATP6V1H) is associated with osteoporosis in humans and mice. A genome-wide SNP array analysis of 1625 Han Chinese found that 4 of 15 tag SNPs (26.7%) within ATP6V1H were significantly associated with low spine bone mineral density. Atp6v1h+/- knockout mice generated by the CRISPR/Cas9 technique had decreased bone remodeling and a net bone matrix loss. Atp6v1h+/- osteoclasts showed impaired bone formation and increased bone resorption. The increased intracellular pH of Atp6v1h+/- osteoclasts downregulated TGF-β1 activation, thereby reducing induction of osteoblast formation but the bone mineralization was not altered. However, bone formation was reduced more than bone resorption. Our data provide evidence that partial loss of ATP6V1H function results in osteoporosis/osteopenia. We propose that defective osteoclast formation triggers impaired bone formation by altering bone remodeling. In the future, ATP6V1H might, therefore, serve as a target for the therapy of osteoporosis. PMID:27924156

Time-dependent LXR/RXR pathway modulation characterizes capillary remodeling in inflammatory corneal neovascularization.

PubMed

Mukwaya, Anthony; Lennikov, Anton; Xeroudaki, Maria; Mirabelli, Pierfrancesco; Lachota, Mieszko; Jensen, Lasse; Peebo, Beatrice; Lagali, Neil

2018-05-01

Inflammation in the normally immune-privileged cornea can initiate a pathologic angiogenic response causing vision-threatening corneal neovascularization. Inflammatory pathways, however, are numerous, complex and are activated in a time-dependent manner. Effective resolution of inflammation and associated angiogenesis in the cornea requires knowledge of these pathways and their time dependence, which has, to date, remained largely unexplored. Here, using a model of endogenous resolution of inflammation-induced corneal angiogenesis, we investigate the time dependence of inflammatory genes in effecting capillary regression and the return of corneal transparency. Endogenous capillary regression was characterized by a progressive thinning and remodeling of angiogenic capillaries and inflammatory cell retreat in vivo in the rat cornea. By whole-genome longitudinal microarray analysis, early suppression of VEGF ligand-receptor signaling and inflammatory pathways preceded an unexpected later-phase preferential activation of LXR/RXR, PPARα/RXRα and STAT3 canonical pathways, with a concurrent attenuation of LPS/IL-1 inhibition of RXR function and Wnt/β-catenin signaling pathways. Potent downstream inflammatory cytokines such as Cxcl5, IL-1β, IL-6 and Ccl2 were concomitantly downregulated during the remodeling phase. Upstream regulators of the inflammatory pathways included Socs3, Sparc and ApoE. A complex and coordinated time-dependent interplay between pro- and anti-inflammatory signaling pathways highlights a potential anti-inflammatory role of LXR/RXR, PPARα/RXRα and STAT3 signaling pathways in resolving inflammatory corneal angiogenesis.

ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1

PubMed Central

Renes, Johan; de Vries, Elisabeth G E; Nienhuis, Edith F; Jansen, Peter L M; Müller, Michael

1999-01-01

The present study was performed to investigate the ability of the multidrug resistance protein (MRP1) to transport different cationic substrates in comparison with MDR1-P-glycoprotein (MDR1). Transport studies were performed with isolated membrane vesicles from in vitro selected multidrug resistant cell lines overexpressing MDR1 (A2780AD) or MRP1 (GLC4/Adr) and a MRP1-transfected cell line (S1(MRP)). As substrates we used 3H-labelled derivatives of the hydrophilic monoquaternary cation N-(4′,4′-azo-n-pentyl)-21-deoxy-ajmalinium (APDA), the basic drug vincristine and the more hydrophobic basic drug daunorubicin. All three are known MDR1-substrates. MRP1 did not mediate transport of these substrates per se. In the presence of reduced glutathione (GSH), there was an ATP-dependent uptake of vincristine and daunorubicin, but not of APDA, into GLC4/Adr and S1(MRP) membrane vesicles which could be inhibited by the MRP1-inhibitor MK571. ATP- and GSH-dependent transport of daunorubicin and vincristine into GLC4/Adr membrane vesicles was inhibited by the MRP1-specific monoclonal antibody QCRL-3. MRP1-mediated daunorubicin transport rates were dependent on the concentration of GSH and were maximal at concentrations ⩾10 mM. The apparent KM value for GSH was 2.7 mM. Transport of daunorubicin in the presence of 10 mM GSH was inhibited by MK571 with an IC50 of 0.4 μM. In conclusion, these results demonstrate that MRP1 transports vincristine and daunorubicin in an ATP- and GSH-dependent manner. APDA is not a substrate for MRP1. PMID:10188979

Mediator-dependent Nuclear Receptor Functions

PubMed Central

Chen, Wei; Roeder, Robert

2011-01-01

As gene-specific transcription factors, nuclear hormone receptors are broadly involved in many important biological processes. Their function on target genes requires the stepwise assembly of different coactivator complexes that facilitate chromatin remodeling and subsequent preinitiation complex (PIC) formation and function. Mediator has proved to be a crucial, and general, nuclear receptor-interacting coactivator, with demonstrated functions in transcription steps ranging from chromatin remodeling to subsequent PIC formation and function. Here we discuss (i) our current understanding of pathways that nuclear receptors and other interacting cofactors employ to recruit Mediator to target gene enhancers and promoters, including conditional requirements for the strong NR-Mediator interactions mediated by the NR AF2 domain and the MED1 LXXLLL motifs and (ii) mechanisms by which Mediator acts to transmit signals from enhancer-bound nuclear receptors to the general transcription machinery at core promoters to effect PIC formation and function. PMID:21854863

The major architects of chromatin: architectural proteins in bacteria, archaea and eukaryotes.

PubMed

Luijsterburg, Martijn S; White, Malcolm F; van Driel, Roel; Dame, Remus Th

2008-01-01

The genomic DNA of all organisms across the three kingdoms of life needs to be compacted and functionally organized. Key players in these processes are DNA supercoiling, macromolecular crowding and architectural proteins that shape DNA by binding to it. The architectural proteins in bacteria, archaea and eukaryotes generally do not exhibit sequence or structural conservation especially across kingdoms. Instead, we propose that they are functionally conserved. Most of these proteins can be classified according to their architectural mode of action: bending, wrapping or bridging DNA. In order for DNA transactions to occur within a compact chromatin context, genome organization cannot be static. Indeed chromosomes are subject to a whole range of remodeling mechanisms. In this review, we discuss the role of (i) DNA supercoiling, (ii) macromolecular crowding and (iii) architectural proteins in genome organization, as well as (iv) mechanisms used to remodel chromosome structure and to modulate genomic activity. We conclude that the underlying mechanisms that shape and remodel genomes are remarkably similar among bacteria, archaea and eukaryotes.

[Mg2+, ATP-dependent plasma membrane calcium pump of smooth muscle cells. I. Structural organization and properties].

PubMed

Veklich, T O; Mazur, Iu Iu; Kosterin, S O

2015-01-01

Tight control of cytoplasm Ca2+ concentration is essential in cell functioning. Changing of Ca2+ concentration is thorough in smooth muscle cells, because it determines relaxation/constraint process. One of key proteins which control Ca2+ concentration in cytoplasm is Mg2+, ATP-dependent plasma membrane calcium pump. Thus, it is important to find compoumds which allowed one to change Mg2+, ATP-dependent plasma membrane calcium pump activity, as long as this topic is of current interest in biochemical research which regards energy and pharmacomechanical coupling mechanism of muscle excitation and contraction. In this article we generalized literatute and own data about properties of smooth muscle cell plasma membrane Ca(2+)-pump. Stuctural oganization, kinetical properties and molecular biology are considered.

Long range epigenetic silencing is a trans-species mechanism that results in cancer specific deregulation by overriding the chromatin domains of normal cells.

PubMed

Forn, Marta; Muñoz, Mar; Tauriello, Daniele V F; Merlos-Suárez, Anna; Rodilla, Verónica; Bigas, Anna; Batlle, Eduard; Jordà, Mireia; Peinado, Miguel A

2013-12-01

DNA methylation and chromatin remodeling are frequently implicated in the silencing of genes involved in carcinogenesis. Long Range Epigenetic Silencing (LRES) is a mechanism of gene inactivation that affects multiple contiguous CpG islands and has been described in different human cancer types. However, it is unknown whether there is a coordinated regulation of the genes embedded in these regions in normal cells and in early stages of tumor progression. To better characterize the molecular events associated with the regulation and remodeling of these regions we analyzed two regions undergoing LRES in human colon cancer in the mouse model. We demonstrate that LRES also occurs in murine cancer in vivo and mimics the molecular features of the human phenomenon, namely, downregulation of gene expression, acquisition of inactive histone marks, and DNA hypermethylation of specific CpG islands. The genes embedded in these regions showed a dynamic and autonomous regulation during mouse intestinal cell differentiation, indicating that, in the framework considered here, the coordinated regulation in LRES is restricted to cancer. Unexpectedly, benign adenomas in Apc(Min/+) mice showed overexpression of most of the genes affected by LRES in cancer, which suggests that the repressive remodeling of the region is a late event. Chromatin immunoprecipitation analysis of the transcriptional insulator CTCF in mouse colon cancer cells revealed disrupted chromatin domain boundaries as compared with normal cells. Malignant regression of cancer cells by in vitro differentiation resulted in partial reversion of LRES and gain of CTCF binding. We conclude that genes in LRES regions are plastically regulated in cell differentiation and hyperproliferation, but are constrained to a coordinated repression by abolishing boundaries and the autonomous regulation of chromatin domains in cancer cells. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All

PHF6 Degrees of Separation: The Multifaceted Roles of a Chromatin Adaptor Protein.

PubMed

Todd, Matthew A M; Ivanochko, Danton; Picketts, David J

2015-06-19

The importance of chromatin regulation to human disease is highlighted by the growing number of mutations identified in genes encoding chromatin remodeling proteins. While such mutations were first identified in severe developmental disorders, or in specific cancers, several genes have been implicated in both, including the plant homeodomain finger protein 6 (PHF6) gene. Indeed, germline mutations in PHF6 are the cause of the Börjeson-Forssman-Lehmann X-linked intellectual disability syndrome (BFLS), while somatic PHF6 mutations have been identified in T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Studies from different groups over the last few years have made a significant impact towards a functional understanding of PHF6 protein function. In this review, we summarize the current knowledge of PHF6 with particular emphasis on how it interfaces with a distinct set of interacting partners and its functional roles in the nucleoplasm and nucleolus. Overall, PHF6 is emerging as a key chromatin adaptor protein critical to the regulation of neurogenesis and hematopoiesis.

Restraint of angiogenesis by zinc finger transcription factor CTCF-dependent chromatin insulation

PubMed Central

Tang, Ming; Chen, Bo; Pardo, Carolina; Pampo, Christine; Chen, Jing; Lien, Ching-Ling; Wu, Lizi; Wang, Heiman; Yao, Kai; Oh, S. Paul; Seto, Edward; Smith, Lois E. H.; Siemann, Dietmar W.; Kladde, Michael P.; Cepko, Constance L.; Lu, Jianrong

2011-01-01

Angiogenesis is meticulously controlled by a fine balance between positive and negative regulatory activities. Vascular endothelial growth factor (VEGF) is a predominant angiogenic factor and its dosage is precisely regulated during normal vascular formation. In cancer, VEGF is commonly overproduced, resulting in abnormal neovascularization. VEGF is induced in response to various stimuli including hypoxia; however, very little is known about the mechanisms that confine its induction to ensure proper angiogenesis. Chromatin insulation is a key transcription mechanism that prevents promiscuous gene activation by interfering with the action of enhancers. Here we show that the chromatin insulator-binding factor CTCF binds to the proximal promoter of VEGF. Consistent with the enhancer-blocking mode of chromatin insulators, CTCF has little effect on basal expression of VEGF but specifically affects its activation by enhancers. CTCF knockdown cells are sensitized for induction of VEGF and exhibit elevated proangiogenic potential. Cancer-derived CTCF missense mutants are mostly defective in blocking enhancers at the VEGF locus. Moreover, during mouse retinal development, depletion of CTCF causes excess angiogenesis. Therefore, CTCF-mediated chromatin insulation acts as a crucial safeguard against hyperactivation of angiogenesis. PMID:21896759

Chromatin Computation

PubMed Central

Bryant, Barbara

2012-01-01

In living cells, DNA is packaged along with protein and RNA into chromatin. Chemical modifications to nucleotides and histone proteins are added, removed and recognized by multi-functional molecular complexes. Here I define a new computational model, in which chromatin modifications are information units that can be written onto a one-dimensional string of nucleosomes, analogous to the symbols written onto cells of a Turing machine tape, and chromatin-modifying complexes are modeled as read-write rules that operate on a finite set of adjacent nucleosomes. I illustrate the use of this “chromatin computer” to solve an instance of the Hamiltonian path problem. I prove that chromatin computers are computationally universal – and therefore more powerful than the logic circuits often used to model transcription factor control of gene expression. Features of biological chromatin provide a rich instruction set for efficient computation of nontrivial algorithms in biological time scales. Modeling chromatin as a computer shifts how we think about chromatin function, suggests new approaches to medical intervention, and lays the groundwork for the engineering of a new class of biological computing machines. PMID:22567109

Small angle x-ray scattering of chromatin. Radius and mass per unit length depend on linker length.

PubMed Central

Williams, S P; Langmore, J P

1991-01-01

Analyses of low angle x-ray scattering from chromatin, isolated by identical procedures but from different species, indicate that fiber diameter and number of nucleosomes per unit length increase with the amount of nucleosome linker DNA. Experiments were conducted at physiological ionic strength to obtain parameters reflecting the structure most likely present in living cells. Guinier analyses were performed on scattering from solutions of soluble chromatin from Necturus maculosus erythrocytes (linker length 48 bp), chicken erythrocytes (linker length 64 bp), and Thyone briareus sperm (linker length 87 bp). The results were extrapolated to infinite dilution to eliminate interparticle contributions to the scattering. Cross-sectional radii of gyration were found to be 10.9 +/- 0.5, 12.1 +/- 0.4, and 15.9 +/- 0.5 nm for Necturus, chicken, and Thyone chromatin, respectively, which are consistent with fiber diameters of 30.8, 34.2, and 45.0 nm. Mass per unit lengths were found to be 6.9 +/- 0.5, 8.3 +/- 0.6, and 11.8 +/- 1.4 nucleosomes per 10 nm for Necturus, chicken, and Thyone chromatin, respectively. The geometrical consequences of the experimental mass per unit lengths and radii of gyration are consistent with a conserved interaction among nucleosomes. Cross-linking agents were found to have little effect on fiber external geometry, but significant effect on internal structure. The absolute values of fiber diameter and mass per unit length, and their dependencies upon linker length agree with the predictions of the double-helical crossed-linker model. A compilation of all published x-ray scattering data from the last decade indicates that the relationship between chromatin structure and linker length is consistent with data obtained by other investigators. Images FIGURE 1 PMID:2049522

Butyl benzyl phthalate suppresses the ATP-induced cell proliferation in human osteosarcoma HOS cells

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

Liu, P.-S., E-mail: pslediting@mail.scu.edu.t; Chen, C.-Y.

2010-05-01

Butyl benzyl phthalate (BBP), an endocrine disruptor present in the environment, exerts its genomic effects via intracellular steroid receptors and elicits non-genomic effects by interfering with membrane ion-channel receptors. We previously found that BBP blocks the calcium signaling coupled with P2X receptors in PC12 cells (Liu and Chen, 2006). Osteoblast P2X receptors were recently reported to play a role in cell proliferation and bone remodeling. In this present study, the effects of BBP on ATP-induced responses were investigated in human osteosarcoma HOS cells. These receptors mRNA had been detected, named P2X4, P2X7, P2Y2, P2Y4, P2Y5, P2Y9, and P2Y11, in humanmore » osteosarcoma HOS cells by RT-PCR. The enhancement of cell proliferation and the decrease of cytoviability had both been shown to be coupled to stimulation via different concentrations of ATP. BBP suppressed the ATP-induced calcium influx (mainly coupled with P2X) and cell proliferation but not the ATP-induced intracellular calcium release (mainly coupled with P2Y) and cytotoxicity in human osteosarcoma HOS cells. Suramin, a common P2 receptor's antagonist, blocked the ATP-induced calcium signaling, cell proliferation, and cytotoxicity. We suggest that P2X is mainly responsible for cell proliferation, and P2Y might be partially responsible for the observed cytotoxicity. BBP suppressed the calcium signaling coupled with P2X, suppressing cell proliferation. Since the importance of P2X receptors during bone metastasis has recently become apparent, the possible toxic risk of environmental BBP during bone remodeling is a public problem of concern.« less

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

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

Deutscher, J.; Pevec, B.; Beyreuther, K.

1986-10-21

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolyptic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system,more » HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction. The site of ATP-dependent phosphorylation in HPr of S faecalis has now been determined. (/sup 32/P)P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, they obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, they isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-. Thus, the site of ATP-dependent phosphorylation was determined to be Ser-46 within the primary structure of HPr.« less

The BAF (BRG1/BRM-Associated Factor) chromatin-remodeling complex exhibits ethanol sensitivity in fetal neural progenitor cells and regulates transcription at the miR-9-2 encoding gene locus.

PubMed

Burrowes, Sasha G; Salem, Nihal A; Tseng, Alexander M; Balaraman, Sridevi; Pinson, Marisa R; Garcia, Cadianna; Miranda, Rajesh C

2017-05-01

Fetal alcohol spectrum disorders are a leading cause of intellectual disability worldwide. Previous studies have shown that developmental ethanol exposure results in loss of microRNAs (miRNAs), including miR-9, and loss of these miRNAs, in turn, mediates some of ethanol's teratogenic effects in the developing brain. We previously found that ethanol increased methylation at the miR-9-2 encoding gene locus in mouse fetal neural stem cells (NSC), advancing a mechanism for epigenetic silencing of this locus and consequently, miR-9 loss in NSCs. Therefore, we assessed the role of the BAF (BRG1/BRM-Associated Factor) complex, which disassembles nucleosomes to facilitate access to chromatin, as an epigenetic mediator of ethanol's effects on miR-9. Chromatin immunoprecipitation and DNAse I-hypersensitivity analyses showed that the BAF complex was associated with both transcriptionally accessible and heterochromatic regions of the miR-9-2 locus, and that disintegration of the BAF complex by combined knockdown of BAF170 and BAF155 resulted in a significant decrease in miR-9. We hypothesized that ethanol exposure would result in loss of BAF-complex function at the miR-9-2 locus. However, ethanol exposure significantly increased mRNA transcripts for maturation-associated BAF-complex members BAF170, SS18, ARID2, BAF60a, BRM/BAF190b, and BAF53b. Ethanol also significantly increased BAF-complex binding within an intron containing a CpG island and in the terminal exon encoding precursor (pre)-miR-9-2. These data suggest that the BAF complex may adaptively respond to ethanol exposure to protect against a complete loss of miR-9-2 in fetal NSCs. Chromatin remodeling factors may adapt to the presence of a teratogen, to maintain transcription of critical miRNA regulatory pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

Ethanol Dose- and Time-dependently Increases α and β Subunits of Mitochondrial ATP Synthase of Cultured Neonatal Rat Cardiomyocytes.

PubMed

Mashimo, Keiko; Arthur, Peter G; Ohno, Youkichi

2015-01-01

Mitochondria are target subcellular organelles of ethanol. In this study, the effects of ethanol on protein composition was examined with 2-dimensional electrophoresis of protein extracts from cultured neonatal rat cardiomyocytes exposed to 100 mM ethanol for 24 hours. A putative β subunit of mitochondrial ATP synthase was increased, which was confirmed by Western blot. The cellular protein abundances in the α and β subunits of ATP synthase increased in dose (0, 10, 50, and 100 mM) - and time (0.5 hour and 24 hours) -dependent manners. The DNA microarray analysis of total RNA extract demonstrated that gene expression of the corresponding messenger RNAs of these subunit proteins did not significantly alter due to 24-hour ethanol exposure. Therefore, protein expression of these nuclear-encoded mitochondrial proteins may be regulated at the translational, rather than the transcriptional, level. Alternatively, degradation of these subunit proteins might be decreased. Additionally, cellular ATP content of cardiomyocytes scarcely decreased following 24-hour exposure to any examined concentrations of ethanol. Previous studies, together with this study, have demonstrated that protein abundance of the α subunit or β subunit or both subunits of ATP synthase after ethanol exposure or dysfunctional conditions might differ according to tissue: significant increases in heart but decreases in liver and brain. Thus, it is suggested that the abundance of subunit proteins of mitochondrial ATP synthase in the ethanol-exposed heart, being different from that in the liver and brain, should increase dose-dependently through either translational upregulation or decreased degradation or both to maintain ATP production, as the heart requires much more energy than other tissues for continuing sustained contractions.

Knockdown Brm and Baf170, components of chromatin remodeling complex, facilitates reprogramming of somatic cells

USDA-ARS?s Scientific Manuscript database

The SWI/SNF (SWItch/Sucrose NonFermentable or BAF, Brg/Brahma-associated factors) complexes are epigenetic modifiers of chromatin structure and undergo progressive changes in subunit composition during cellular differentiation. For example, in embryonic stem cells (ESCs) esBAF contains Brg1 and Baf...

Crystal Structure of the Chromodomain Helicase DNA-binding Protein 1 (Chd1) DNA-binding Domain in Complex with DNA*

PubMed Central

Sharma, Amit; Jenkins, Katherine R.; Héroux, Annie; Bowman, Gregory D.

2011-01-01

Chromatin remodelers are ATP-dependent machines that dynamically alter the chromatin packaging of eukaryotic genomes by assembling, sliding, and displacing nucleosomes. The Chd1 chromatin remodeler possesses a C-terminal DNA-binding domain that is required for efficient nucleosome sliding and believed to be essential for sensing the length of DNA flanking the nucleosome core. The structure of the Chd1 DNA-binding domain was recently shown to consist of a SANT and SLIDE domain, analogous to the DNA-binding domain of the ISWI family, yet the details of how Chd1 recognized DNA were not known. Here we present the crystal structure of the Saccharomyces cerevisiae Chd1 DNA-binding domain in complex with a DNA duplex. The bound DNA duplex is straight, consistent with the preference exhibited by the Chd1 DNA-binding domain for extranucleosomal DNA. Comparison of this structure with the recently solved ISW1a DNA-binding domain bound to DNA reveals that DNA lays across each protein at a distinct angle, yet contacts similar surfaces on the SANT and SLIDE domains. In contrast to the minor groove binding seen for Isw1 and predicted for Chd1, the SLIDE domain of the Chd1 DNA-binding domain contacts the DNA major groove. The majority of direct contacts with the phosphate backbone occur only on one DNA strand, suggesting that Chd1 may not strongly discriminate between major and minor grooves. PMID:22033927

An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon iosAsp formation but is restored by Protein Isoaspartyl Methltransferase

USDA-ARS?s Scientific Manuscript database

Arabidopsis thaliana PLANT RNA HELICASE75 (AtPRH75) demonstrated an ATP-dependent, RNA duplex unwinding capacity and an ATP-independent, RNA duplex reforming ability. It is known to accumulate isoAsp, but the consequences of isoAsp formation in AtPRH75 are unknown. Duplex unwinding was abolished by ...

N terminus of Swr1 binds to histone H2AZ and provides a platform for subunit assembly in the chromatin remodeling complex.

PubMed

Wu, Wei-Hua; Wu, Chwen-Huey; Ladurner, Andreas; Mizuguchi, Gaku; Wei, Debbie; Xiao, Hua; Luk, Ed; Ranjan, Anand; Wu, Carl

2009-03-06

Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.

β-Nicotinamide Adenine Dinucleotide (β-NAD) Inhibits ATP-Dependent IL-1β Release from Human Monocytic Cells.

PubMed

Hiller, Sebastian Daniel; Heldmann, Sarah; Richter, Katrin; Jurastow, Innokentij; Küllmar, Mira; Hecker, Andreas; Wilker, Sigrid; Fuchs-Moll, Gabriele; Manzini, Ivan; Schmalzing, Günther; Kummer, Wolfgang; Padberg, Winfried; McIntosh, J Michael; Damm, Jelena; Zakrzewicz, Anna; Grau, Veronika

2018-04-10

While interleukin-1β (IL-1β) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1β maturation, is released from damaged cells along with β-nicotinamide adenine dinucleotide (β-NAD). Here, we tested the hypothesis that β-NAD controls ATP-signaling and, hence, IL-1β release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')- O -(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of β-NAD. IL-1β was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca 2+ -independent phospholipase A2 (iPLA2β, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous β-NAD signaled via P2Y receptors and dose-dependently (IC 50 = 15 µM) suppressed the BzATP-induced IL-1β release. Signaling involved iPLA2β, release of a soluble mediator, and nAChR subunit α9. Patch-clamp experiments revealed that β-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular β-NAD that suppresses ATP-induced release of IL-1β by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.

Regulation of Egr1 Target Genes by the NuRD Chromatin Remodeling Complex

DTIC Science & Technology

2006-12-01

the M12 metastatic subline of P69SV40T prostate epithelial cells (Bae et al. 1998), Western analysis using an antibody directed against CHD4 revealed...Chromatin was then sonicated and immunoprecipitated with antibodies directed against EGR1, MTA2 or IgG control. The specificity of the assay is tested...subtle. Control ChIP assays employing an EGR1 antibody show correlated increased binding of EGR1 upon EGR1 overexpression, but this level of EGR1

Dynamics of passive and active particles in the cell nucleus.

PubMed

Hameed, Feroz M; Rao, Madan; Shivashankar, G V

2012-01-01

Inspite of being embedded in a dense meshwork of nuclear chromatin, gene loci and large nuclear components are highly dynamic at 37°C. To understand this apparent unfettered movement in an overdense environment, we study the dynamics of a passive micron size bead in live cell nuclei at two different temperatures (25 and 37°C) with and without external force. In the absence of a force, the beads are caged over large time scales. On application of a threshold uniaxial force (about 10(2) pN), the passive beads appear to hop between cages; this large scale movement is absent upon ATP-depletion, inhibition of chromatin remodeling enzymes and RNAi of lamin B1 proteins. Our results suggest that the nucleus behaves like an active solid with a finite yield stress when probed at a micron scale. Spatial analysis of histone fluorescence anisotropy (a measure of local chromatin compaction, defined as the volume fraction of tightly bound chromatin) shows that the bead movement correlates with regions of low chromatin compaction. This suggests that the physical mechanism of the observed yielding is the active opening of free-volume in the nuclear solid via chromatin remodeling. Enriched transcription sites at 25°C also show caging in the absence of the applied force and directed movement beyond a yield stress, in striking contrast with the large scale movement of transcription loci at 37°C in the absence of a force. This suggests that at physiological temperatures, the loci behave as active particles which remodel the nuclear mesh and reduce the local yield stress.

An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking

PubMed Central

Hoffman, Elizabeth A.; Zaidi, Hussain; Shetty, Savera J.; Bekiranov, Stefan; Auble, David T.

2018-01-01

Formaldehyde crosslinking is widely used in combination with chromatin immunoprecipitation (ChIP) to measure the locations along DNA and relative levels of transcription factor (TF)-DNA interactions in vivo. However, the measurements that are typically made do not provide unambiguous information about the dynamic properties of these interactions. We have developed a method to estimate binding kinetic parameters from time-dependent formaldehyde crosslinking data, called crosslinking kinetics (CLK) analysis. Cultures of yeast cells are crosslinked with formaldehyde for various periods of time, yielding the relative ChIP signal at particular loci. We fit the data using the mass-action CLK model to extract kinetic parameters of the TF-chromatin interaction, including the on- and off-rates and crosslinking rate. From the on- and off-rate we obtain the occupancy and residence time. The following protocol is the second iteration of this method, CLKv2, updated with improved crosslinking and quenching conditions, more information about crosslinking rates, and systematic procedures for modeling the observed kinetic regimes. CLKv2 analysis has been applied to investigate the binding behavior of the TATA-binding protein (TBP), and a selected subset of other TFs. The protocol was developed using yeast cells, but may be applicable to cells from other organisms as well. PMID:29682595

Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium.

PubMed

Platt, James L; Kent, Nicholas A; Kimmel, Alan R; Harwood, Adrian J

2017-04-01

Nucleosome placement and repositioning can direct transcription of individual genes; however, the precise interactions of these events are complex and largely unresolved at the whole-genome level. The Chromodomain-Helicase-DNA binding (CHD) Type III proteins are a subfamily of SWI2/SNF2 proteins that control nucleosome positioning and are associated with several complex human disorders, including CHARGE syndrome and autism. Type III CHDs are required for multicellular development of animals and Dictyostelium but are absent in plants and yeast. These CHDs can mediate nucleosome translocation in vitro, but their in vivo mechanism is unknown. Here, we use genome-wide analysis of nucleosome positioning and transcription profiling to investigate the in vivo relationship between nucleosome positioning and gene expression during development of wild-type (WT) Dictyostelium and mutant cells lacking ChdC, a Type III CHD protein ortholog. We demonstrate major nucleosome positional changes associated with developmental gene regulation in WT. Loss of chdC caused an increase of intragenic nucleosome spacing and misregulation of gene expression, affecting ∼50% of the genes that are repositioned during WT development. These analyses demonstrate active nucleosome repositioning during Dictyostelium multicellular development, establish an in vivo function of CHD Type III chromatin remodeling proteins in this process, and reveal the detailed relationship between nucleosome positioning and gene regulation, as cells transition between developmental states. © 2017 Platt et al.; Published by Cold Spring Harbor Laboratory Press.

Simulated remodeling of loaded collagen networks via strain-dependent enzymatic degradation and constant-rate fiber growth

PubMed Central

Hadi, M.F.; Sander, E.A.; Ruberti, J.W.; Barocas, V. H.

2011-01-01

Recent work has demonstrated that enzymatic degradation of collagen fibers exhibits strain-dependent kinetics. Conceptualizing how the strain dependence affects remodeling of collagenous tissues is vital to our understanding of collagen management in native and bioengineered tissues. As a first step towards this goal, the current study puts forward a multiscale model for enzymatic degradation and remodeling of collagen networks for two sample geometries we routinely use in experiments as model tissues. The multiscale model, driven by microstructural data from an enzymatic decay experiment, includes an exponential strain-dependent kinetic relation for degradation and constant growth. For a dogbone sample under uniaxial load, the model predicted that the distribution of fiber diameters would spread over the course of degradation because of variation in individual fiber load. In a cross-shaped sample, the central region, which experiences smaller, more isotropic loads, showed more decay and less spread in fiber diameter compared to the arms. There was also a slight shift in average orientation in different regions of the cruciform. PMID:22180691

Mutations in chromatin regulators functionally link Cornelia de Lange syndrome and clinically overlapping phenotypes.

PubMed

Parenti, Ilaria; Teresa-Rodrigo, María E; Pozojevic, Jelena; Ruiz Gil, Sara; Bader, Ingrid; Braunholz, Diana; Bramswig, Nuria C; Gervasini, Cristina; Larizza, Lidia; Pfeiffer, Lutz; Ozkinay, Ferda; Ramos, Feliciano; Reiz, Benedikt; Rittinger, Olaf; Strom, Tim M; Watrin, Erwan; Wendt, Kerstin; Wieczorek, Dagmar; Wollnik, Bernd; Baquero-Montoya, Carolina; Pié, Juan; Deardorff, Matthew A; Gillessen-Kaesbach, Gabriele; Kaiser, Frank J

2017-03-01

The coordinated tissue-specific regulation of gene expression is essential for the proper development of all organisms. Mutations in multiple transcriptional regulators cause a group of neurodevelopmental disorders termed "transcriptomopathies" that share core phenotypical features including growth retardation, developmental delay, intellectual disability and facial dysmorphism. Cornelia de Lange syndrome (CdLS) belongs to this class of disorders and is caused by mutations in different subunits or regulators of the cohesin complex. Herein, we report on the clinical and molecular characterization of seven patients with features overlapping with CdLS who were found to carry mutations in chromatin regulators previously associated to other neurodevelopmental disorders that are frequently considered in the differential diagnosis of CdLS. The identified mutations affect the methyltransferase-encoding genes KMT2A and SETD5 and different subunits of the SWI/SNF chromatin-remodeling complex. Complementary to this, a patient with Coffin-Siris syndrome was found to carry a missense substitution in NIPBL. Our findings indicate that mutations in a variety of chromatin-associated factors result in overlapping clinical phenotypes, underscoring the genetic heterogeneity that should be considered when assessing the clinical and molecular diagnosis of neurodevelopmental syndromes. It is clear that emerging molecular mechanisms of chromatin dysregulation are central to understanding the pathogenesis of these clinically overlapping genetic disorders.

Voltage- and ATP-dependent structural rearrangements of the P2X2 receptor associated with the gating of the pore

PubMed Central

Keceli, Batu; Kubo, Yoshihiro

2014-01-01

P2X2 is an extracellular ATP-gated cation channel which has a voltage-dependent gating property even though it lacks a canonical voltage sensor. It is a trimer in which each subunit has two transmembrane helices and a large extracellular domain. The three inter-subunit ATP binding sites are linked to the pore forming transmembrane (TM) domains by β-strands. We analysed structural rearrangements of the linker strands between the ATP binding site and TM domains upon ligand binding and voltage change, electrophysiologically in Xenopus oocytes, using mutants carrying engineered thiol-modifiable cysteine residues. (1) We demonstrated that the double mutant D315C&I67C (at β-14 and β-1, respectively) shows a 2- to 4-fold increase in current amplitude after treatment with a reducing reagent, dithiothreitol (DTT). Application of the thiol-reactive metal Cd2+ induced current decline due to bond formation between D315C and I67C. This effect was not observed in wild type (WT) or in single point mutants. (2) Cd2+-induced current decline was analysed in hyperpolarized and depolarized conditions with different pulse protocols, and also in the presence and absence of ATP. (3) Current decline induced by Cd2+ could be clearly observed in the presence of ATP, but was not clear in the absence of ATP, showing a state-dependent modification. (4) In the presence of ATP, Cd2+ modification was significantly faster in hyperpolarized than in depolarized conditions, showing voltage-dependent structural rearrangements of the linker strands. (5) Experiments using tandem trimeric constructs (TTCs) with controlled number and position of mutations in the trimer showed that the bridging by Cd2+ between 315 and 67 was not intra- but inter-subunit. (6) Finally, we performed similar analyses of a pore mutant T339S, which makes the channel activation voltage insensitive. Cd2+ modification rates of T339S were similar in hyperpolarized and depolarized conditions. Taking these results together, we

ATP-dependent export of neutral amino acids by vacuolar membrane vesicles of Saccharomyces cerevisiae.

PubMed

Ishimoto, Masaya; Sugimoto, Naoko; Sekito, Takayuki; Kawano-Kawada, Miyuki; Kakinuma, Yoshimi

2012-01-01

Amino acid analysis of Saccharomyces cerevisiae cells indicated that neutral amino acids such as glycine and alanine were probably excluded from the vacuoles, and that vacuolar H(+)-ATPase (V-ATPase) was involved in the vacuolar compartmentalization of these amino acids. We found that vacuolar membrane vesicles export neutral amino acids in an ATP-dependent manner. This is important in identifying vacuolar transporters for neutral amino acids.

Variable effects of the mitoK(ATP) channel modulators diazoxide and 5-HD in ATP-depleted renal epithelial cells.

PubMed

Nilakantan, Vani; Liang, Huanling; Mortensen, Jordan; Taylor, Erin; Johnson, Christopher P

2010-02-01

The role of mitochondrial K(ATP) (mitoK(ATP)) channels in renal ischemia-reperfusion injury is controversial with studies showing both protective and deleterious effects. In this study, we compared the effects of the putative mitoK(ATP) opener, diazoxide, and the mitoK(ATP) blocker, 5-hydroxydecanoate (5-HD) on cytotoxicity and apoptosis in tubular epithelial cells derived from rat (NRK-52E) and pig (LLC-PK1) following in vitro ischemic injury. Following ATP depletion-recovery, there was a significant increase in cytotoxicity in both NRK cells and LLC-PK1 cells although NRK cells were more sensitive to the injury. Diazoxide treatment attenuated cytotoxicity in both cell types and 5-HD treatment-increased cytotoxicity in the sensitive NRK cells in a superoxide-dependant manner. The protective effect of diazoxide was also reversed in the presence of 5-HD in ATP-depleted NRK cells. The ATP depletion-mediated increase in superoxide was enhanced by both diazoxide and 5-HD with the effect being more pronounced in the cells undergoing 5-HD treatment. Further, ATP depletion-induced activation of caspase-3 was decreased by diazoxide in NRK cells. In order to determine the signaling pathways involved in apoptosis, we examined the activation of Erk and JNK in ATP-depleted NRK cells. Diazoxide-activated Erk in ATP-depleted cells, but did not have any effect on JNK activation. In contrast, 5-HD did not impact Erk levels but increased JNK activation even under controlled conditions. Further, the use of a JNK inhibitor with 5-HD reversed the deleterious effects of 5-HD. This study demonstrates that in cells that are sensitive to ATP depletion-recovery, mitoK(ATP) channels protect against ATP depletion-mediated cytotoxicity and apoptosis through Erk- and JNK-dependant mechanisms.

The effects of lubrol WX on brain membrane Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ uptake activity following acute and chronic ethanol.

PubMed

Ross, D H; Garrett, K M; Cardenas, H L

1985-02-01

Acute administration of ethanol (2.5 gm/kg, i.p.) to rats inhibits the cytosolic buffering of Ca2+ in nerve terminals. Ca2+ ATPase and ATP-dependent Ca2+ uptake are both inhibited 30 min after a single dose of ethanol. Chronic ethanol administration (6%, 14 days) did not inhibit Ca2+ ATPase but significantly stimulated ATP-dependent Ca2+ uptake. Lubrol WX treatment of acute ethanolic membranes reverses the inhibition of Ca2+ ATPase seen following ethanol. Lubrol WX treatment of chronic ethanolic membranes prevents the increase in ATP-dependent Ca2+ uptake seen in ethanolic membranes. Both acute and chronic ethanol-induced changes in Ca2+ transport within nerve terminals may involve lipid-dependent parameters of the membrane which may underlie neuronal adaptation.

25-Hydroxycholesterol promotes fibroblast-mediated tissue remodeling through NF-κB dependent pathway

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

Ichikawa, Tomohiro; Sugiura, Hisatoshi, E-mail: sugiura@rm.med.tohoku.ac.jp; Koarai, Akira

2013-05-01

Abnormal structural alterations termed remodeling, including fibrosis and alveolar wall destruction, are important features of the pathophysiology of chronic airway diseases such as chronic obstructive pulmonary disease (COPD) and asthma. 25-hydroxycholesterol (25-HC) is enzymatically produced by cholesterol 25-hydorxylase (CH25H) in macrophages and is reported to be involved in the formation of arteriosclerosis. We previously demonstrated that the expression of CH25H and production of 25HC were increased in the lungs of COPD. However, the role of 25-HC in lung tissue remodeling is unknown. In this study, we investigated the effect of 25-HC on fibroblast-mediated tissue remodeling using human fetal lung fibroblastsmore » (HFL-1) in vitro. 25-HC significantly augmented α-smooth muscle actin (SMA) (P<0.001) and collagen I (P<0.001) expression in HFL-1. 25-HC also significantly enhanced the release and activation of matrix metallaoproteinase (MMP)-2 (P<0.001) and MMP-9 (P<0.001) without any significant effect on the production of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. 25-HC stimulated transforming growth factor (TGF)-β{sub 1} production (P<0.01) and a neutralizing anti-TGF-β antibody restored these 25-HC-augmented pro-fibrotic responses. 25-HC significantly promoted the translocation of nuclear factor (NF)-κB p65 into the nuclei (P<0.01), but not phospholylated-c-jun, a complex of activator protein-1. Pharmacological inhibition of NF-κB restored the 25-HC-augmented pro-fibrotic responses and TGF-β{sub 1} release. These results suggest that 25-HC could contribute to fibroblast-mediated lung tissue remodeling by promoting myofibroblast differentiation and the excessive release of extracellular matrix protein and MMPs via an NF-κB-TGF-β dependent pathway.« less

Acetone and Butanone Metabolism of the Denitrifying Bacterium “Aromatoleum aromaticum” Demonstrates Novel Biochemical Properties of an ATP-Dependent Aliphatic Ketone Carboxylase

PubMed Central

Schühle, Karola

2012-01-01

The anaerobic and aerobic metabolism of acetone and butanone in the betaproteobacterium “Aromatoleum aromaticum” is initiated by their ATP-dependent carboxylation to acetoacetate and 3-oxopentanoic acid, respectively. Both reactions are catalyzed by the same enzyme, acetone carboxylase, which was purified and characterized. Acetone carboxylase is highly induced under growth on acetone or butanone and accounts for at least 5.5% of total cell protein. The enzyme consists of three subunits of 85, 75, and 20 kDa, respectively, in a (αβγ)2 composition and contains 1 Zn and 2 Fe per heterohexamer but no organic cofactors. Chromatographic analysis of the ATP hydrolysis products indicated that ATP was exclusively cleaved to AMP and 2 Pi. The stoichiometry was determined to be 2 ATP consumed per acetone carboxylated. Purified acetone carboxylase from A. aromaticum catalyzes the carboxylation of acetone and butanone as the only substrates. However, the enzyme shows induced (uncoupled) ATPase activity with many other substrates that were not carboxylated. Acetone carboxylase is a member of a protein family that also contains acetone carboxylases of various other organisms, acetophenone carboxylase of A. aromaticum, and ATP-dependent hydantoinases/oxoprolinases. While the members of this family share several characteristic features, they differ with respect to the products of ATP hydrolysis, subunit composition, and metal content. PMID:22020645

Structured illumination to spatially map chromatin motions.

PubMed

Bonin, Keith; Smelser, Amanda; Moreno, Naike Salvador; Holzwarth, George; Wang, Kevin; Levy, Preston; Vidi, Pierre-Alexandre

2018-05-01

We describe a simple optical method that creates structured illumination of a photoactivatable probe and apply this method to characterize chromatin motions in nuclei of live cells. A laser beam coupled to a diffractive optical element at the back focal plane of an excitation objective generates an array of near diffraction-limited beamlets with FWHM of 340  ±  30  nm, which simultaneously photoactivate a 7  ×  7 matrix pattern of GFP-labeled histones, with spots 1.70  μm apart. From the movements of the photoactivated spots, we map chromatin diffusion coefficients at multiple microdomains of the cell nucleus. The results show correlated motions of nearest chromatin microdomain neighbors, whereas chromatin movements are uncorrelated at the global scale of the nucleus. The method also reveals a DNA damage-dependent decrease in chromatin diffusion. The diffractive optical element instrumentation can be easily and cheaply implemented on commercial inverted fluorescence microscopes to analyze adherent cell culture models. A protocol to measure chromatin motions in nonadherent human hematopoietic stem and progenitor cells is also described. We anticipate that the method will contribute to the identification of the mechanisms regulating chromatin mobility, which influences most genomic processes and may underlie the biogenesis of genomic translocations associated with hematologic malignancies. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Effects of chromatin decondensation on alternative NHEJ.

PubMed

Moscariello, Mario; Iliakis, George

2013-11-01

In cells of higher eukaryotes, repair of DNA double strand breaks (DSBs) utilizes different forms of potentially error-prone non-homologous end joining (NHEJ): canonical DNA-PK-dependent (C-NHEJ) and alternative backup pathways (A-NHEJ). In contrast to C-NHEJ, A-NHEJ shows pronounced efficiency fluctuations throughout the cell cycle and is severely compromised as cells cease proliferating and enter the plateau phase (Windhofer et al., 2007 [23]). The molecular mechanisms underpinning this response remain unknown but changes in chromatin structure are prime candidate-A-NHEJ-modulators. Since parameters beyond chromatin acetylation appear to determine A-NHEJ efficiency (Manova et al., 2012 [42,76]), we study here the role of chromatin decondensation mediated either by treatment with 5'-aza-2'-deoxycytidine (AzadC) or growth in hypotonic conditions, on A-NHEJ. We report that both treatments have no detectable effect on C-NHEJ but provoke, specifically for A-NHEJ, cell-growth-dependent effects. These results uncover for the first time a link between A-NHEJ and chromatin organization and provide means for understanding the regulatory mechanisms underpinning the growth-state dependency of A-NHEJ. A-NHEJ is implicated in the formation of chromosomal translocations and in chromosome fusions that underlie genomic instability and carcinogenesis. The observations reported here may therefore contribute to the development of drug-based A-NHEJ suppression-strategies aiming at optimizing cancer treatment outcomes and possibly also at suppressing carcinogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

3D FISH to analyse gene domain-specific chromatin re-modeling in human cancer cell lines.

PubMed

Kocanova, Silvia; Goiffon, Isabelle; Bystricky, Kerstin

2018-06-01

Fluorescence in situ hybridization (FISH) is a common technique used to label DNA and/or RNA for detection of a genomic region of interest. However, the technique can be challenging, in particular when applied to single genes in human cancer cells. Here, we provide a step-by-step protocol for analysis of short (35 kb-300 kb) genomic regions in three dimensions (3D). We discuss the experimental design and provide practical considerations for 3D imaging and data analysis to determine chromatin folding. We demonstrate that 3D FISH using BACs (Bacterial Artificial Chromosomes) or fosmids can provide detailed information of the architecture of gene domains. More specifically, we show that mapping of specific chromatin landscapes informs on changes associated with estrogen stimulated gene activity in human breast cancer cell lines. Copyright © 2018 Elsevier Inc. All rights reserved.

The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mt-Hsp70-Tim44 interaction driving mitochondrial protein import.

PubMed Central

Schneider, H C; Westermann, B; Neupert, W; Brunner, M

1996-01-01

Import of preproteins into the mitochondrial matrix is driven by the ATP-dependent interaction of mt-Hsp70 with the peripheral inner membrane import protein Tim44 and the preprotein in transit. We show that Mge1p, a co-chaperone of mt-Hsp70, plays a key role in the ATP-dependent import reaction cycle in yeast. Our data suggest a cycle in which the mt-Hsp70-Tim44 complex forms with ATP: Mge1p promotes assembly of the complex in the presence of ATP. Hydrolysis of ATP by mt-Hsp70 occurs in complex with Tim44. Mge1p is then required for the dissociation of the ADP form of mt-Hsp70 from Tim44 after release of inorganic phosphate but before release of ADP. ATP hydrolysis and complex dissociation are accompanied by tight binding of mt-Hsp70 to the preprotein in transit. Subsequently, the release of mt-Hsp70 from the polypeptide chain is triggered by Mge1p which promotes release of ADP from mt-Hsp70. Rebinding of ATP to mt-Hsp70 completes the reaction cycle. Images PMID:8918457

An ATP-dependent ligase with substrate flexibility involved in assembly of the peptidyl nucleoside antibiotic polyoxin

USDA-ARS?s Scientific Manuscript database

Polyoxin (POL) is an unusual nucleoside antibiotic, in which peptidyl moiety and nucleoside skeleton are linked by an amide bond. However, their biosynthesis remains poorly understood. Here, we report the deciphering of PolG as an ATP-dependent ligase responsible for the assembly of POL. A polG muta...

Large-scale chromatin remodeling at the immunoglobulin heavy chain locus: a paradigm for multigene regulation.

PubMed

Bolland, Daniel J; Wood, Andrew L; Corcoran, Anne E

2009-01-01

complementary processes involved in this large-scale locus organisation. We will examine the structure of the Igh locus and the large-scale and higher-order chromatin remodelling processes associated with V(D)J recombination, at the level of the locus itself, its conformational changes and its dynamic localisation within the nucleus.

Role of Flightless-I (Drosophila) homolog in the transcription activation of type I collagen gene mediated by transforming growth factor beta

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

Lim, Mi-Sun; Jeong, Kwang Won, E-mail: kwjeong@gachon.ac.kr

Highlights: • FLII activates TGFβ-mediated expression of COL1A2 gene. • TGFβ induces the association of FLII with SMAD3 and BRG1 in A549 cells. • FLII is required for the recruitment of SWI/SNF complex and chromatin accessibility to COL1A2 promoter. - Abstract: Flightless-I (Drosophila) homolog (FLII) is a nuclear receptor coactivator that is known to interact with other transcriptional regulators such as the SWI/SNF complex, an ATP-dependent chromatin-remodeling complex, at the promoter or enhancer region of estrogen receptor (ER)-α target genes. However, little is known about the role of FLII during transcription initiation in the transforming growth factor beta (TGFβ)/SMAD-dependent signalingmore » pathway. Here, we demonstrate that FLII functions as a coactivator in the expression of type I collagen gene induced by TGFβ in A549 cells. FLII activates the reporter gene driven by COL1A2 promoter in a dose-dependent manner. Co-expression of GRIP1, CARM1, or p300 did not show any synergistic activation of transcription. Furthermore, the level of COL1A2 expression correlated with the endogenous level of FLII mRNA level. Depletion of FLII resulted in a reduction of TGFβ-induced expression of COL1A2 gene. In contrast, over-expression of FLII caused an increase in the endogenous expression of COL1A2. We also showed that FLII is associated with Brahma-related gene 1 (BRG1) as well as SMAD in A549 cells. Notably, the recruitment of BRG1 to the COL1A2 promoter region was decreased in FLII-depleted A549 cells, suggesting that FLII is required for TGFβ-induced chromatin remodeling, which is carried out by the SWI/SNF complex. Furthermore, formaldehyde-assisted isolation of regulatory elements (FAIRE)-quantitative polymerase chain reaction (qPCR) experiments revealed that depletion of FLII caused a reduction in chromatin accessibility at the COL1A2 promoter. These results suggest that FLII plays a critical role in TGFβ/SMAD-mediated transcription of the COL1A2

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

PubMed

Chan, Tung O; Zhang, Jin; Rodeck, Ulrich; Pascal, John M; Armen, Roger S; Spring, Maureen; Dumitru, Calin D; Myers, Valerie; Li, Xue; Cheung, Joseph Y; Feldman, Arthur M

2011-11-15

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

Hormone-dependent control of developmental timing through regulation of chromatin accessibility

PubMed Central

Uyehara, Christopher M.; Nystrom, Spencer L.; Niederhuber, Matthew J.; Leatham-Jensen, Mary; Ma, Yiqin; Buttitta, Laura A.

2017-01-01

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. Here, we show that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, we demonstrate that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. We also uncover a temporal cascade of transcription factors following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, we show that the ecdysone-induced transcription factor E93 controls temporal identity by directly regulating chromatin accessibility across the genome. Notably, we found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility. PMID:28536147

Genome-wide identification of physically clustered genes suggests chromatin-level co-regulation in male reproductive development in Arabidopsis thaliana

PubMed Central

Reimegård, Johan; Kundu, Snehangshu; Pendle, Ali; Irish, Vivian F.; Shaw, Peter

2017-01-01

Abstract Co-expression of physically linked genes occurs surprisingly frequently in eukaryotes. Such chromosomal clustering may confer a selective advantage as it enables coordinated gene regulation at the chromatin level. We studied the chromosomal organization of genes involved in male reproductive development in Arabidopsis thaliana. We developed an in-silico tool to identify physical clusters of co-regulated genes from gene expression data. We identified 17 clusters (96 genes) involved in stamen development and acting downstream of the transcriptional activator MS1 (MALE STERILITY 1), which contains a PHD domain associated with chromatin re-organization. The clusters exhibited little gene homology or promoter element similarity, and largely overlapped with reported repressive histone marks. Experiments on a subset of the clusters suggested a link between expression activation and chromatin conformation: qRT-PCR and mRNA in situ hybridization showed that the clustered genes were up-regulated within 48 h after MS1 induction; out of 14 chromatin-remodeling mutants studied, expression of clustered genes was consistently down-regulated only in hta9/hta11, previously associated with metabolic cluster activation; DNA fluorescence in situ hybridization confirmed that transcriptional activation of the clustered genes was correlated with open chromatin conformation. Stamen development thus appears to involve transcriptional activation of physically clustered genes through chromatin de-condensation. PMID:28175342

ATP can be dispensable for prespliceosome formation in yeast

PubMed Central

Perriman, Rhonda; Ares, Manuel

2000-01-01

The first ATP-dependent step in pre-mRNA splicing involves the stable binding of U2 snRNP to form the prespliceosome. We show that a prespliceosome-like complex forms in the absence of ATP in yeast extracts lacking the U2 suppressor protein CUS2. These complexes display the same pre-mRNA and U snRNA requirements as authentic prespliceosomes and can be chased through the splicing pathway, indicating that they are a functional intermediate in the spliceosome assembly pathway. ATP-independent prespliceosome-like complexes are also observed in extracts containing a mutant U2 snRNA. Loss of CUS2 does not bypass the role of PRP5, an RNA helicase family member required for ATP-dependent prespliceosome formation. Genetic interactions between CUS2 and a heat-sensitive prp5 allele parallel those observed between CUS2 and U2, and suggest that CUS2 mediates functional interactions between U2 RNA and PRP5. We propose that CUS2 enforces ATP dependence during formation of the prespliceosome by brokering an interaction between PRP5 and the U2 snRNP that depends on correct U2 RNA structure. PMID:10640279

Modeling steady state SO2-dependent changes in capillary ATP concentration using novel O2 micro-delivery methods

PubMed Central

Ghonaim, Nour W.; Fraser, Graham M.; Ellis, Christopher G.; Yang, Jun; Goldman, Daniel

2013-01-01

Adenosine triphosphate (ATP) is known to be released from the erythrocyte in an oxygen (O2) dependent manner. Since ATP is a potent vasodilator, it is proposed to be a key regulator in the pathway that mediates micro-vascular response to varying tissue O2 demand. We propose that ATP signaling mainly originates in the capillaries due to the relatively long erythrocyte transit times in the capillary and the short ATP diffusion distance to the electrically coupled endothelium. We have developed a computational model to investigate the effect of delivering or removing O2 to limited areas at the surface of a tissue with an idealized parallel capillary array on total ATP concentration. Simulations were conducted when exposing full surface to perturbations in tissue O2 tension (PO2) or locally using a circular micro-outlet (~100 μm in diameter), a square micro-slit (200 × 200 μm), or a rectangular micro-slit (1000 μm wide × 200 μm long). Results indicated the rectangular micro-slit has the optimal dimensions for altering hemoglobin saturations (SO2) in sufficient number capillaries to generate effective changes in total [ATP]. This suggests a threshold for the minimum number of capillaries that need to be stimulated in vivo by imposed tissue hypoxia to induce a conducted micro-vascular response. SO2 and corresponding [ATP] changes were also modeled in a terminal arteriole (9 μm in diameter) that replaces 4 surface capillaries in the idealized network geometry. Based on the results, the contribution of terminal arterioles to the net change in [ATP] in the micro-vascular network is minimal although they would participate as O2 sources thus influencing the O2 distribution. The modeling data presented here provide important insights into designing a novel micro-delivery device for studying micro-vascular O2 regulation in the capillaries in vivo. PMID:24069001

Age-Dependent Neurochemical Remodeling of Hypothalamic Astrocytes.

PubMed

Santos, Camila Leite; Roppa, Paola Haack Amaral; Truccolo, Pedro; Fontella, Fernanda Urruth; Souza, Diogo Onofre; Bobermin, Larissa Daniele; Quincozes-Santos, André

2017-10-04

The hypothalamus is a crucial integrative center in the central nervous system, responsible for the regulation of homeostatic activities, including systemic energy balance. Increasing evidence has highlighted a critical role of astrocytes in orchestrating hypothalamic functions; they participate in the modulation of synaptic transmission, metabolic and trophic support to neurons, immune defense, and nutrient sensing. In this context, disturbance of systemic energy homeostasis, which is a common feature of obesity and the aging process, involves inflammatory responses. This may be related to dysfunction of hypothalamic astrocytes. In this regard, the aim of this study was to evaluate the neurochemical properties of hypothalamic astrocyte cultures from newborn, adult, and aged Wistar rats. Age-dependent changes in the regulation of glutamatergic homeostasis, glutathione biosynthesis, amino acid profile, glucose metabolism, trophic support, and inflammatory response were observed. Additionally, signaling pathways including nuclear factor erythroid-derived 2-like 2/heme oxygenase-1 p38 mitogen-activated protein kinase, nuclear factor kappa B, phosphatidylinositide 3-kinase/Akt, and leptin receptor expression may represent putative mechanisms associated with the cellular alterations. In summary, our findings indicate that as age increases, hypothalamic astrocytes remodel and exhibit changes in their neurochemical properties. This process may play a role in the onset and/or progression of metabolic disorders.

Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone

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

Guo, Feng; Stanevich, Vitali; Wlodarchak, Nathan

Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATPγS reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A activemore » site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.« less

RNAi pathways contribute to developmental history-dependent phenotypic plasticity in C. elegans

PubMed Central

Hall, Sarah E.; Chirn, Gung-Wei; Lau, Nelson C.; Sengupta, Piali

2013-01-01

Early environmental experiences profoundly influence adult phenotypes through complex mechanisms that are poorly understood. We previously showed that adult Caenorhabditis elegans that transiently passed through the stress-induced dauer larval stage (post-dauer adults) exhibit significant changes in gene expression profiles, chromatin states, and life history traits when compared with adults that bypassed the dauer stage (control adults). These wild-type, isogenic animals of equivalent developmental stages exhibit different signatures of molecular marks that reflect their distinct developmental trajectories. To gain insight into the mechanisms that contribute to these developmental history-dependent phenotypes, we profiled small RNAs from post-dauer and control adults by deep sequencing. RNA interference (RNAi) pathways are known to regulate genome-wide gene expression both at the chromatin and post-transcriptional level. By quantifying changes in endogenous small interfering RNA (endo-siRNA) levels in post-dauer as compared with control animals, our analyses identified a subset of genes that are likely targets of developmental history-dependent reprogramming through a complex RNAi-mediated mechanism. Mutations in specific endo-siRNA pathways affect expected gene expression and chromatin state changes for a subset of genes in post-dauer animals, as well as disrupt their increased brood size phenotype. We also find that both chromatin state and endo-siRNA distribution in dauers are unique, and suggest that remodeling in dauers provides a template for the subsequent establishment of adult post-dauer profiles. Our results indicate a role for endo-siRNA pathways as a contributing mechanism to early experience-dependent phenotypic plasticity in adults, and describe how developmental history can program adult physiology and behavior via epigenetic mechanisms. PMID:23329696

Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ–mediated host defenses

PubMed Central

Brenier-Pinchart, Marie-Pierre; Bertini, Rose-Laurence; Varesano, Aurélie; De Bock, Pieter-Jan

2016-01-01

An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii–targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ–stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1+ inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ–mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism. PMID:27503074

FasL-triggered death of Jurkat cells requires caspase 8-induced, ATP-dependent cross-talk between Fas and the purinergic receptor P2X(7).

PubMed

Aguirre, Adam; Shoji, Kenji F; Sáez, Juan C; Henríquez, Mauricio; Quest, Andrew F G

2013-02-01

Fas ligation via the ligand FasL activates the caspase-8/caspase-3-dependent extrinsic death pathway. In so-called type II cells, an additional mechanism involving tBid-mediated caspase-9 activation is required to efficiently trigger cell death. Other pathways linking FasL-Fas interaction to activation of the intrinsic cell death pathway remain unknown. However, ATP release and subsequent activation of purinergic P2X(7) receptors (P2X(7)Rs) favors cell death in some cells. Here, we evaluated the possibility that ATP release downstream of caspase-8 via pannexin1 hemichannels (Panx1 HCs) and subsequent activation of P2X(7)Rs participate in FasL-stimulated cell death. Indeed, upon FasL stimulation, ATP was released from Jurkat cells in a time- and caspase-8-dependent manner. Fas and Panx1 HCs colocalized and inhibition of the latter, but not connexin hemichannels, reduced FasL-induced ATP release. Extracellular apyrase, which hydrolyzes ATP, reduced FasL-induced death. Also, oxidized-ATP or Brilliant Blue G, two P2X(7)R blockers, reduced FasL-induced caspase-9 activation and cell death. These results represent the first evidence indicating that the two death receptors, Fas and P2X(7)R connect functionally via caspase-8 and Panx1 HC-mediated ATP release to promote caspase-9/caspase-3-dependent cell death in lymphoid cells. Thus, a hitherto unsuspected route was uncovered connecting the extrinsic to the intrinsic pathway to amplify death signals emanating from the Fas receptor in type II cells. Copyright © 2012 Wiley Periodicals, Inc.

ICI 182,780 induces P-cadherin overexpression in breast cancer cells through chromatin remodelling at the promoter level: a role for C/EBPbeta in CDH3 gene activation.

PubMed

Albergaria, André; Ribeiro, Ana Sofia; Pinho, Sandra; Milanezi, Fernanda; Carneiro, Vítor; Sousa, Bárbara; Sousa, Sónia; Oliveira, Carla; Machado, José Carlos; Seruca, Raquel; Paredes, Joana; Schmitt, Fernando

2010-07-01

CDH3/P-cadherin is a classical cadherin. Overexpression of which has been associated with proliferative lesions of high histological grade, decreased cell polarity and poor survival of patients with breast cancer. In vitro studies showed that it can be up-regulated by ICI 182,780, suggesting that the lack of ERalpha signalling is responsible for the aberrant P-cadherin overexpression and for its role in inducing breast cancer cell invasion and migration. However, the mechanism by which ER-signalling inhibition leads to P-cadherin expression is still unknown. The aim of this study was to explore the molecular mechanism linking the ERalpha-signalling and P-cadherin-regulated expression in breast cancer cell lines. This study showed that ICI 182,780 is able to increase CDH3 promoter activity, inducing high levels of the active chromatin mark H3 lysine 4 dimethylation. We also observed, for the first time, that the transcription factor C/EBPbeta is able to up-regulate CDH3 promoter activity in breast cancer cells. Moreover, we showed that the expression of P-cadherin and C/EBPbeta are highly associated in human breast carcinomas and linked with a worse prognosis of breast cancer patients. This study demonstrates the existence of an epigenetic regulation by which ICI 182,780 up-regulates P-cadherin expression in MCF-7/AZ breast cancer cells through chromatin remodelling at CDH3 promoter, bringing forward the growing evidence that ERalpha signalling-abrogation by anti-oestrogens is able to induce the expression of ERalpha-repressed genes which, in the appropriate cell biology context, may contribute to a breast cancer cell invasion phenotype.CDH3 GenBank accession no. NT_010498.

Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts

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

Watanabe, Tomonobu M.; World Premier Initiative, iFREC, Osaka University, Osaka 565-0871; Higuchi, Sayaka

Highlights: Black-Right-Pointing-Pointer Change in the epigenetic landscape during myogenesis was optically investigated. Black-Right-Pointing-Pointer Mobility of nuclear proteins was used to state the epigenetic status of the cell. Black-Right-Pointing-Pointer Mobility of nuclear proteins decreased as myogenesis progressed in C2C12. Black-Right-Pointing-Pointer Differentiation state diagram was developed using parameters obtained. -- Abstract: Skeletal muscle undergoes complicated differentiation steps that include cell-cycle arrest, cell fusion, and maturation, which are controlled through sequential expression of transcription factors. During muscle differentiation, remodeling of the epigenetic landscape is also known to take place on a large scale, determining cell fate. In an attempt to determine the extentmore » of epigenetic remodeling during muscle differentiation, we characterized the plasticity of the chromatin structure using C2C12 myoblasts. Differentiation of C2C12 cells was induced by lowering the serum concentration after they had reached full confluence, resulting in the formation of multi-nucleated myotubes. Upon induction of differentiation, the nucleus size decreased whereas the aspect ratio increased, indicating the presence of force on the nucleus during differentiation. Movement of the nucleus was also suppressed when differentiation was induced, indicating that the plasticity of chromatin changed upon differentiation. To evaluate the histone dynamics during differentiation, FRAP experiment was performed, which showed an increase in the immobile fraction of histone proteins when differentiation was induced. To further evaluate the change in the histone dynamics during differentiation, FCS was performed, which showed a decrease in histone mobility on differentiation. We here show that the plasticity of chromatin decreases upon differentiation, which takes place in a stepwise manner, and that it can be used as an index for the differentiation

Fragile X Mental Retardation Protein Regulates Activity-Dependent Membrane Trafficking and Trans-Synaptic Signaling Mediating Synaptic Remodeling

PubMed Central

Sears, James C.; Broadie, Kendal

2018-01-01

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. The disease arises through loss of fragile X mental retardation protein (FMRP), which normally exhibits peak expression levels in early-use critical periods, and is required for activity-dependent synaptic remodeling during this transient developmental window. FMRP canonically binds mRNA to repress protein translation, with targets that regulate cytoskeleton dynamics, membrane trafficking, and trans-synaptic signaling. We focus here on recent advances emerging in these three areas from the Drosophila disease model. In the well-characterized central brain mushroom body (MB) olfactory learning/memory circuit, FMRP is required for activity-dependent synaptic remodeling of projection neurons innervating the MB calyx, with function tightly restricted to an early-use critical period. FMRP loss is phenocopied by conditional removal of FMRP only during this critical period, and rescued by FMRP conditional expression only during this critical period. Consistent with FXS hyperexcitation, FMRP loss defects are phenocopied by heightened sensory experience and targeted optogenetic hyperexcitation during this critical period. FMRP binds mRNA encoding Drosophila ESCRTIII core component Shrub (human CHMP4 homolog) to restrict Shrub translation in an activity-dependent mechanism only during this same critical period. Shrub mediates endosomal membrane trafficking, and perturbing Shrub expression is known to interfere with neuronal process pruning. Consistently, FMRP loss and Shrub overexpression targeted to projection neurons similarly causes endosomal membrane trafficking defects within synaptic boutons, and genetic reduction of Shrub strikingly rescues Drosophila FXS model defects. In parallel work on the well-characterized giant fiber (GF) circuit, FMRP limits iontophoretic dye loading into central interneurons, demonstrating an FMRP role controlling core neuronal properties through the

Vimentin filament precursors exchange subunits in an ATP-dependent manner

PubMed Central

Robert, Amélie; Rossow, Molly J.; Hookway, Caroline; Adam, Stephen A.; Gelfand, Vladimir I.

2015-01-01

Intermediate filaments (IFs) are a component of the cytoskeleton capable of profound reorganization in response to specific physiological situations, such as differentiation, cell division, and motility. Various mechanisms were proposed to be responsible for this plasticity depending on the type of IF polymer and the biological context. For example, recent studies suggest that mature vimentin IFs (VIFs) undergo rearrangement by severing and reannealing, but direct subunit exchange within the filament plays little role in filament dynamics at steady state. Here, we studied the dynamics of subunit exchange in VIF precursors, called unit-length filaments (ULFs), formed by the lateral association of eight vimentin tetramers. To block vimentin assembly at the ULF stage, we used the Y117L vimentin mutant (vimentinY117L). By tagging vimentinY117L with a photoconvertible protein mEos3.2 and photoconverting ULFs in a limited area of the cytoplasm, we found that ULFs, unlike mature filaments, were highly dynamic. Subunit exchange among ULFs occurred within seconds and was limited by the diffusion of soluble subunits in the cytoplasm rather than by the association and dissociation of subunits from ULFs. Our data demonstrate that cells expressing vimentinY117L contained a large pool of soluble vimentin tetramers that was in rapid equilibrium with ULFs. Furthermore, vimentin exchange in ULFs required ATP, and ATP depletion caused a dramatic reduction of the soluble tetramer pool. We believe that the dynamic exchange of subunits plays a role in the regulation of ULF assembly and the maintenance of a soluble vimentin pool during the reorganization of filament networks. PMID:26109569

Transcription upregulation via force-induced direct stretching of chromatin

NASA Astrophysics Data System (ADS)

Tajik, Arash; Zhang, Yuejin; Wei, Fuxiang; Sun, Jian; Jia, Qiong; Zhou, Wenwen; Singh, Rishi; Khanna, Nimish; Belmont, Andrew S.; Wang, Ning

2016-12-01

Mechanical forces play critical roles in the function of living cells. However, the underlying mechanisms of how forces influence nuclear events remain elusive. Here, we show that chromatin deformation as well as force-induced transcription of a green fluorescent protein (GFP)-tagged bacterial-chromosome dihydrofolate reductase (DHFR) transgene can be visualized in a living cell by using three-dimensional magnetic twisting cytometry to apply local stresses on the cell surface via an Arg-Gly-Asp-coated magnetic bead. Chromatin stretching depended on loading direction. DHFR transcription upregulation was sensitive to load direction and proportional to the magnitude of chromatin stretching. Disrupting filamentous actin or inhibiting actomyosin contraction abrogated or attenuated force-induced DHFR transcription, whereas activating endogenous contraction upregulated force-induced DHFR transcription. Our findings suggest that local stresses applied to integrins propagate from the tensed actin cytoskeleton to the LINC complex and then through lamina-chromatin interactions to directly stretch chromatin and upregulate transcription.

Simultaneous estimation of intracellular free Mg2+ and pH by use of a new pH-dependent dissociation constant of MgATP.

PubMed

Nakayama, Shinsuke; Nomura, Hideki; Smith, Lorraine M; Clark, Joseph F

2002-06-01

In the present technical note for 31P-NMR, we used a new pH-dependent dissociation constant of MgATP, and re-estimated changes in the intracellular free Mg2+ concentration and pH from the chemical shifts of beta- and gamma-ATP during Na+-removal in smooth muscle. We confirmed the role of Na+ - Mg2+ exchange.

FIND: difFerential chromatin INteractions Detection using a spatial Poisson process

PubMed Central

Chen, Yang; Zhang, Michael Q.

2018-01-01

Polymer-based simulations and experimental studies indicate the existence of a spatial dependency between the adjacent DNA fibers involved in the formation of chromatin loops. However, the existing strategies for detecting differential chromatin interactions assume that the interacting segments are spatially independent from the other segments nearby. To resolve this issue, we developed a new computational method, FIND, which considers the local spatial dependency between interacting loci. FIND uses a spatial Poisson process to detect differential chromatin interactions that show a significant difference in their interaction frequency and the interaction frequency of their neighbors. Simulation and biological data analysis show that FIND outperforms the widely used count-based methods and has a better signal-to-noise ratio. PMID:29440282

CRTC1 Nuclear Translocation Following Learning Modulates Memory Strength via Exchange of Chromatin Remodeling Complexes on the Fgf1 Gene.

PubMed

Uchida, Shusaku; Teubner, Brett J W; Hevi, Charles; Hara, Kumiko; Kobayashi, Ayumi; Dave, Rutu M; Shintaku, Tatsushi; Jaikhan, Pattaporn; Yamagata, Hirotaka; Suzuki, Takayoshi; Watanabe, Yoshifumi; Zakharenko, Stanislav S; Shumyatsky, Gleb P

2017-01-10

Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. Here we find that the transcription cofactor CRTC1 is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB, and histone acetyltransferase CBP induces transient transcription. Strong training later substitutes KAT5 for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins.

PubMed

del Valle, Luis J

2005-06-01

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg-jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø(o) (sperm-specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca(2+), pH, and this activity could be a serine-proteinase. Thermal denaturalization of the oocyte extracts (80 degrees C, 10-15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø(o)) will be a first condition in the process of sperm chromatin remodeling.

TRPC5-eNOS Axis Negatively Regulates ATP-Induced Cardiomyocyte Hypertrophy.

PubMed

Sunggip, Caroline; Shimoda, Kakeru; Oda, Sayaka; Tanaka, Tomohiro; Nishiyama, Kazuhiro; Mangmool, Supachoke; Nishimura, Akiyuki; Numaga-Tomita, Takuro; Nishida, Motohiro

2018-01-01

Cardiac hypertrophy, induced by neurohumoral factors, including angiotensin II and endothelin-1, is a major predisposing factor for heart failure. These ligands can induce hypertrophic growth of neonatal rat cardiomyocytes (NRCMs) mainly through Ca 2+ -dependent calcineurin/nuclear factor of activated T cell (NFAT) signaling pathways activated by diacylglycerol-activated transient receptor potential canonical 3 and 6 (TRPC3/6) heteromultimer channels. Although extracellular nucleotide, adenosine 5'-triphosphate (ATP), is also known as most potent Ca 2+ -mobilizing ligand that acts on purinergic receptors, ATP never induces cardiomyocyte hypertrophy. Here we show that ATP-induced production of nitric oxide (NO) negatively regulates hypertrophic signaling mediated by TRPC3/6 channels in NRCMs. Pharmacological inhibition of NO synthase (NOS) potentiated ATP-induced increases in NFAT activity, protein synthesis, and transcriptional activity of brain natriuretic peptide. ATP significantly increased NO production and protein kinase G (PKG) activity compared to angiotensin II and endothelin-1. We found that ATP-induced Ca 2+ signaling requires inositol 1,4,5-trisphosphate (IP 3 ) receptor activation. Interestingly, inhibition of TRPC5, but not TRPC6 attenuated ATP-induced activation of Ca 2+ /NFAT-dependent signaling. As inhibition of TRPC5 attenuates ATP-stimulated NOS activation, these results suggest that NO-cGMP-PKG axis activated by IP 3 -mediated TRPC5 channels underlies negative regulation of TRPC3/6-dependent hypertrophic signaling induced by ATP stimulation.

Magnetic field affects enzymatic ATP synthesis.

PubMed

Buchachenko, Anatoly L; Kuznetsov, Dmitry A

2008-10-01

The rate of ATP synthesis by creatine kinase extracted from V. xanthia venom was shown to depend on the magnetic field. The yield of ATP produced by enzymes with 24Mg2+ and 26Mg2+ ions in catalytic sites increases by 7-8% at 55 mT and then decreases at 80 mT. For enzyme with 25Mg2+ ion in a catalytic site, the ATP yield increases by 50% and 70% in the fields 55 and 80 mT, respectively. In the Earth field the rate of ATP synthesis by enzyme, in which Mg2+ ion has magnetic nucleus 25Mg, is 2.5 times higher than that by enzymes, in which Mg2+ ion has nonmagnetic, spinless nuclei 24Mg or 26Mg. Both magnetic field effect and magnetic isotope effect demonstrate that the ATP synthesis is an ion-radical process, affected by Zeeman interaction and hyperfine coupling in the intermediate ion-radical pair.

Cell fate determination by ubiquitin-dependent regulation of translation

PubMed Central

Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T.; Rape, Michael

2015-01-01

Metazoan development depends on accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates 1. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell fate determination is less well understood. Here, we have identified the vertebrate-specific ubiquitin ligase CUL3KBTBD8 as an essential regulator of neural crest specification. CUL3KBTBD8 monoubiquitylates NOLC1 and its paralog TCOF1, whose mutation underlies the neurocristopathy Treacher Collins Syndrome 2,3. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favor of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell fate determination. PMID:26399832

RNA polymerase III transcription - regulated by chromatin structure and regulator of nuclear chromatin organization.

PubMed

Pascali, Chiara; Teichmann, Martin

2013-01-01

RNA polymerase III (Pol III) transcription is regulated by modifications of the chromatin. DNA methylation and post-translational modifications of histones, such as acetylation, phosphorylation and methylation have been linked to Pol III transcriptional activity. In addition to being regulated by modifications of DNA and histones, Pol III genes and its transcription factors have been implicated in the organization of nuclear chromatin in several organisms. In yeast, the ability of the Pol III transcription system to contribute to nuclear organization seems to be dependent on direct interactions of Pol III genes and/or its transcription factors TFIIIC and TFIIIB with the structural maintenance of chromatin (SMC) protein-containing complexes cohesin and condensin. In human cells, Pol III genes and transcription factors have also been shown to colocalize with cohesin and the transcription regulator and genome organizer CCCTC-binding factor (CTCF). Furthermore, chromosomal sites have been identified in yeast and humans that are bound by partial Pol III machineries (extra TFIIIC sites - ETC; chromosome organizing clamps - COC). These ETCs/COC as well as Pol III genes possess the ability to act as boundary elements that restrict spreading of heterochromatin.

Distinct modes of DNA accessibility in plant chromatin.

PubMed

Shu, Huan; Wildhaber, Thomas; Siretskiy, Alexey; Gruissem, Wilhelm; Hennig, Lars

2012-01-01

The accessibility of DNA to regulatory proteins is a major property of the chromatin environment that favours or hinders transcription. Recent studies in flies reported that H3K9me2-marked heterochromatin is accessible while H3K27me3-marked chromatin forms extensive domains of low accessibility. Here we show that plants regulate DNA accessibility differently. H3K9me2-marked heterochromatin is the least accessible in the Arabidopsis thaliana genome, and H3K27me3-marked chromatin also has low accessibility. We see that very long genes without H3K9me2 or H3K27me3 are often inaccessible and generated significantly lower amounts of antisense transcripts than other genes, suggesting that reduced accessibility is associated with reduced recognition of alternative promoters. Low accessibility of H3K9me2-marked heterochromatin and long genes depend on cytosine methylation, explaining why chromatin accessibility differs between plants and flies. Together, we conclude that restriction of DNA accessibility is a local property of chromatin and not necessarily a consequence of microscopically visible compaction.

A Bayesian mixture model for chromatin interaction data.

PubMed

Niu, Liang; Lin, Shili

2015-02-01

Chromatin interactions mediated by a particular protein are of interest for studying gene regulation, especially the regulation of genes that are associated with, or known to be causative of, a disease. A recent molecular technique, Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), that uses chromatin immunoprecipitation (ChIP) and high throughput paired-end sequencing, is able to detect such chromatin interactions genomewide. However, ChIA-PET may generate noise (i.e., pairings of DNA fragments by random chance) in addition to true signal (i.e., pairings of DNA fragments by interactions). In this paper, we propose MC_DIST based on a mixture modeling framework to identify true chromatin interactions from ChIA-PET count data (counts of DNA fragment pairs). The model is cast into a Bayesian framework to take into account the dependency among the data and the available information on protein binding sites and gene promoters to reduce false positives. A simulation study showed that MC_DIST outperforms the previously proposed hypergeometric model in terms of both power and type I error rate. A real data study showed that MC_DIST may identify potential chromatin interactions between protein binding sites and gene promoters that may be missed by the hypergeometric model. An R package implementing the MC_DIST model is available at http://www.stat.osu.edu/~statgen/SOFTWARE/MDM.

CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases

PubMed Central

Toliusis, Paulius; Silanskas, Arunas; Szczelkun, Mark D.

2017-01-01

Abstract The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5′-GCCGC-3′ site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes. PMID:28854738

Transcriptional reprogramming of gene expression in bovine somatic cell chromatin transfer embryos

PubMed Central

Rodriguez-Osorio, Nelida; Wang, Zhongde; Kasinathan, Poothappillai; Page, Grier P; Robl, James M; Memili, Erdogan

2009-01-01

Background Successful reprogramming of a somatic genome to produce a healthy clone by somatic cells nuclear transfer (SCNT) is a rare event and the mechanisms involved in this process are poorly defined. When serial or successive rounds of cloning are performed, blastocyst and full term development rates decline even further with the increasing rounds of cloning. Identifying the "cumulative errors" could reveal the epigenetic reprogramming blocks in animal cloning. Results Bovine clones from up to four generations of successive cloning were produced by chromatin transfer (CT). Using Affymetrix bovine microarrays we determined that the transcriptomes of blastocysts derived from the first and the fourth rounds of cloning (CT1 and CT4 respectively) have undergone an extensive reprogramming and were more similar to blastocysts derived from in vitro fertilization (IVF) than to the donor cells used for the first and the fourth rounds of chromatin transfer (DC1 and DC4 respectively). However a set of transcripts in the cloned embryos showed a misregulated pattern when compared to IVF embryos. Among the genes consistently upregulated in both CT groups compared to the IVF embryos were genes involved in regulation of cytoskeleton and cell shape. Among the genes consistently upregulated in IVF embryos compared to both CT groups were genes involved in chromatin remodelling and stress coping. Conclusion The present study provides a data set that could contribute in our understanding of epigenetic errors in somatic cell chromatin transfer. Identifying "cumulative errors" after serial cloning could reveal some of the epigenetic reprogramming blocks shedding light on the reprogramming process, important for both basic and applied research. PMID:19393066

Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

PubMed Central

Śledź, Paweł; Unverdorben, Pia; Beck, Florian; Pfeifer, Günter; Schweitzer, Andreas; Förster, Friedrich; Baumeister, Wolfgang

2013-01-01

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6. PMID:23589842

Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

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

Xu, Ren; Nelson, Celeste M.; Muschler, John L.

2009-06-03

Epithelial cells, once dissociated and placed in two-dimensional (2D) cultures, rapidly lose tissue-specific functions. We showed previously that in addition to prolactin, signaling by laminin-111 was necessary to restore functional differentiation of mammary epithelia. Here, we elucidate two additional aspects of laminin-111 action. We show that in 2D cultures, the prolactin receptor is basolaterally localized and physically segregated from its apically placed ligand. Detachment of the cells exposes the receptor to ligation by prolactin leading to signal transducers and activators of transcription protein 5 (STAT5) activation, but only transiently and not sufficiently for induction of milk protein expression. We showmore » that laminin-111 reorganizes mammary cells into polarized acini, allowing both the exposure of the prolactin receptor and sustained activation of STAT5. The use of constitutively active STAT5 constructs showed that the latter is necessary and sufficient for chromatin reorganization and {beta}-casein transcription. These results underscore the crucial role of continuous laminin signaling and polarized tissue architecture in maintenance of transcription factor activation, chromatin organization, and tissue-specific gene expression.« less

Roles of chromatin insulator proteins in higher-order chromatin organization and transcription regulation

PubMed Central

Vogelmann, Jutta; Valeri, Alessandro; Guillou, Emmanuelle; Cuvier, Olivier; Nollmann, Marcelo

2013-01-01

Eukaryotic chromosomes are condensed into several hierarchical levels of complexity: DNA is wrapped around core histones to form nucleosomes, nucleosomes form a higher-order structure called chromatin, and chromatin is subsequently compartmentalized in part by the combination of multiple specific or unspecific long-range contacts. The conformation of chromatin at these three levels greatly influences DNA metabolism and transcription. One class of chromatin regulatory proteins called insulator factors may organize chromatin both locally, by setting up barriers between heterochromatin and euchromatin, and globally by establishing platforms for long-range interactions. Here, we review recent data revealing a global role of insulator proteins in the regulation of transcription through the formation of clusters of long-range interactions that impact different levels of chromatin organization. PMID:21983085

Critical Involvement of Extracellular ATP Acting on P2RX7 Purinergic Receptors in Photoreceptor Cell Death

PubMed Central

Notomi, Shoji; Hisatomi, Toshio; Kanemaru, Takaaki; Takeda, Atsunobu; Ikeda, Yasuhiro; Enaida, Hiroshi; Kroemer, Guido; Ishibashi, Tatsuro

2011-01-01

Stressed cells release ATP, which participates in neurodegenerative processes through the specific ligation of P2RX7 purinergic receptors. Here, we demonstrate that extracellular ATP and the more specific P2RX7 agonist, 2′- and 3′-O-(4-benzoylbenzoyl)-ATP, both induce photoreceptor cell death when added to primary retinal cell cultures or when injected into the eyes from wild-type mice, but not into the eyes from P2RX7−/− mice. Photoreceptor cell death was accompanied by the activation of caspase-8 and -9, translocation of apoptosis-inducing factor from mitochondria to nuclei, and TUNEL-detectable chromatin fragmentation. All hallmarks of photoreceptor apoptosis were prevented by premedication or co-application of Brilliant Blue G, a selective P2RX7 antagonist that is already approved for the staining of internal limiting membranes during ocular surgery. ATP release is up-regulated by nutrient starvation in primary retinal cell cultures and seems to be an initializing event that triggers primary and/or secondary cell death via the positive feedback loop on P2RX7. Our results encourage the potential application of Brilliant Blue G as a novel neuroprotective agent in retinal diseases or similar neurodegenerative pathologies linked to excessive extracellular ATP. PMID:21983632

A histone H3K36 chromatin switch coordinates DNA double-strand break repair pathway choice.

PubMed

Pai, Chen-Chun; Deegan, Rachel S; Subramanian, Lakxmi; Gal, Csenge; Sarkar, Sovan; Blaikley, Elizabeth J; Walker, Carol; Hulme, Lydia; Bernhard, Eric; Codlin, Sandra; Bähler, Jürg; Allshire, Robin; Whitehall, Simon; Humphrey, Timothy C

2014-06-09

DNA double-strand break (DSB) repair is a highly regulated process performed predominantly by non-homologous end joining (NHEJ) or homologous recombination (HR) pathways. How these pathways are coordinated in the context of chromatin is unclear. Here we uncover a role for histone H3K36 modification in regulating DSB repair pathway choice in fission yeast. We find Set2-dependent H3K36 methylation reduces chromatin accessibility, reduces resection and promotes NHEJ, while antagonistic Gcn5-dependent H3K36 acetylation increases chromatin accessibility, increases resection and promotes HR. Accordingly, loss of Set2 increases H3K36Ac, chromatin accessibility and resection, while Gcn5 loss results in the opposite phenotypes following DSB induction. Further, H3K36 modification is cell cycle regulated with Set2-dependent H3K36 methylation peaking in G1 when NHEJ occurs, while Gcn5-dependent H3K36 acetylation peaks in S/G2 when HR prevails. These findings support an H3K36 chromatin switch in regulating DSB repair pathway choice.

Toxic effects of lead and nickel nitrate on rat liver chromatin components.

PubMed

Rabbani-Chadegani Iii, Azra; Fani, Nesa; Abdossamadi, Sayeh; Shahmir, Nosrat

2011-01-01

The biological activity of heavy metals is related to their physicochemical interaction with biological receptors. In the present study, the effect of low concentrations of nickel nitrate and lead nitrate (<0.3 mM) on rat liver soluble chromatin and histone proteins was examined. The results showed that addition of various concentrations of metals to chromatin solution preceded the chromatin into aggregation and precipitation in a dose-dependant manner; however, the extent of absorbance changes at 260 and 400 nm was different between two metals. Gel electrophoresis of histone proteins and DNA of the supernatants obtained from the metal-treated chromatin and the controls revealed higher affinity of lead nitrate to chromatin compared to nickel nitrate. Also, the binding affinity of lead nitrate to histone proteins free in solution was higher than nickel. On the basis of the results, it is concluded that lead reacts with chromatin components even at very low concentrations and induce chromatin aggregation through histone-DNA cross-links. Whereas, nickel nitrate is less effective on chromatin at low concentrations, suggesting higher toxicity of lead nitrate on chromatin compared to nickel. Copyright © 2010 Wiley Periodicals, Inc.

ATP forms a stable complex with the essential histidine kinase WalK (YycG) domain

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

Celikel, Reha; Veldore, Vidya Harini; Mathews, Irimpan

The histidine WalK (YycG) plays a crucial role in coordinating murein synthesis with cell division and the crystal structure of its ATP binding domain has been determined. Interestingly the bound ATP was not hydrolyzed during crystallization and remains intact in the crystal lattice. In Bacillus subtilis, the WalRK (YycFG) two-component system coordinates murein synthesis with cell division. It regulates the expression of autolysins that function in cell-wall remodeling and of proteins that modulate autolysin activity. The transcription factor WalR is activated upon phosphorylation by the histidine kinase WalK, a multi-domain homodimer. It autophosphorylates one of its histidine residues by transferringmore » the γ-phosphate from ATP bound to its ATP-binding domain. Here, the high-resolution crystal structure of the ATP-binding domain of WalK in complex with ATP is presented at 1.61 Å resolution. The bound ATP remains intact in the crystal lattice. It appears that the strong binding interactions and the nature of the binding pocket contribute to its stability. The triphosphate moiety of ATP wraps around an Mg{sup 2+} ion, providing three O atoms for coordination in a near-ideal octahedral geometry. The ATP molecule also makes strong interactions with the protein. In addition, there is a short contact between the exocyclic O3′ of the sugar ring and O2B of the β-phosphate, implying an internal hydrogen bond. The stability of the WalK–ATP complex in the crystal lattice suggests that such a complex may exist in vivo poised for initiation of signal transmission. This feature may therefore be part of the sensing mechanism by which the WalRK two-component system is so rapidly activated when cells encounter conditions conducive for growth.« less

Deleting HDAC3 Rescues Long-Term Memory Impairments Induced by Disruption of the Neuron-Specific Chromatin Remodeling Subunit BAF53b

ERIC Educational Resources Information Center

Shu, Guanhua; Kramár, Enikö A.; López, Alberto J.; Huynh, Grace; Wood, Marcelo A.; Kwapis, Janine L.

2018-01-01

Multiple epigenetic mechanisms, including histone acetylation and nucleosome remodeling, are known to be involved in long-term memory formation. Enhancing histone acetylation by deleting histone deacetylases, like HDAC3, typically enhances long-term memory formation. In contrast, disrupting nucleosome remodeling by blocking the neuron-specific…

The Role of Bacterial Enhancer Binding Proteins as Specialized Activators of σ54-Dependent Transcription

PubMed Central

2012-01-01

Summary: Bacterial enhancer binding proteins (bEBPs) are transcriptional activators that assemble as hexameric rings in their active forms and utilize ATP hydrolysis to remodel the conformation of RNA polymerase containing the alternative sigma factor σ54. We present a comprehensive and detailed summary of recent advances in our understanding of how these specialized molecular machines function. The review is structured by introducing each of the three domains in turn: the central catalytic domain, the N-terminal regulatory domain, and the C-terminal DNA binding domain. The role of the central catalytic domain is presented with particular reference to (i) oligomerization, (ii) ATP hydrolysis, and (iii) the key GAFTGA motif that contacts σ54 for remodeling. Each of these functions forms a potential target of the signal-sensing N-terminal regulatory domain, which can act either positively or negatively to control the activation of σ54-dependent transcription. Finally, we focus on the DNA binding function of the C-terminal domain and the enhancer sites to which it binds. Particular attention is paid to the importance of σ54 to the bacterial cell and its unique role in regulating transcription. PMID:22933558

BAF200 is required for heart morphogenesis and coronary artery development.

PubMed

He, Lingjuan; Tian, Xueying; Zhang, Hui; Hu, Tianyuan; Huang, Xiuzhen; Zhang, Libo; Wang, Zhong; Zhou, Bin

2014-01-01

ATP-dependent SWI/SNF chromatin remodeling complexes utilize ATP hydrolysis to non-covalently change nucleosome-DNA interactions and are essential in stem cell development, organogenesis, and tumorigenesis. Biochemical studies show that SWI/SNF in mammalian cells can be divided into two subcomplexes BAF and PBAF based on the subunit composition. ARID2 or BAF200 has been defined as an intrinsic subunit of PBAF complex. However, the function of BAF200 in vivo is not clear. To dissect the possible role of BAF200 in regulating embryogenesis and organ development, we generated BAF200 mutant mice and found they were embryonic lethal. BAF200 mutant embryos exhibited multiple cardiac defects including thin myocardium, ventricular septum defect, common atrioventricular valve, and double outlet right ventricle around E14.5. Moreover, we also detected reduced intramyocardial coronary arteries in BAF200 mutants, suggesting that BAF200 is required for proper migration and differentiation of subepicardial venous cells into arterial endothelial cells. Our work revealed that PBAF complex plays a critical role in heart morphogenesis and coronary artery angiogenesis.

Stromal remodeling by the BET bromodomain inhibitor JQ1 suppresses the progression of human pancreatic cancer.

PubMed

Yamamoto, Keisuke; Tateishi, Keisuke; Kudo, Yotaro; Hoshikawa, Mayumi; Tanaka, Mariko; Nakatsuka, Takuma; Fujiwara, Hiroaki; Miyabayashi, Koji; Takahashi, Ryota; Tanaka, Yasuo; Ijichi, Hideaki; Nakai, Yousuke; Isayama, Hiroyuki; Morishita, Yasuyuki; Aoki, Taku; Sakamoto, Yoshihiro; Hasegawa, Kiyoshi; Kokudo, Norihiro; Fukayama, Masashi; Koike, Kazuhiko

2016-09-20

Inhibitors of bromodomain and extraterminal domain (BET) proteins, a family of chromatin reader proteins, have therapeutic efficacy against various malignancies. However, the detailed mechanisms underlying the anti-tumor effects in distinct tumor types remain elusive. Here, we show a novel antitumor mechanism of BET inhibition in pancreatic ductal adenocarcinoma (PDAC). We found that JQ1, a BET inhibitor, decreased desmoplastic stroma, a hallmark of PDAC, and suppressed the growth of patient-derived tumor xenografts (PDX) of PDACs. In vivo antitumor effects of JQ1 were not always associated with the JQ1 sensitivity of respective PDAC cells, and were rather dependent on the suppression of tumor-promoting activity in cancer-associated fibroblasts (CAFs). JQ1 inhibited Hedgehog and TGF-β pathways as potent regulators of CAF activation and suppressed the expression of α-SMA, extracellular matrix, cytokines, and growth factors in human primary CAFs. Consistently, conditioned media (CM) from CAFs promoted the proliferation of PDAC cells along with the activation of ERK, AKT, and STAT3 pathways, though these effects were suppressed when CM from JQ1-treated CAFs was used. Mechanistically, chromatin immunoprecipitation experiments revealed that JQ1 reduced TGF-β-dependent gene expression by disrupting the recruitment of the transcriptional machinery containing BET proteins. Finally, combination therapy with gemcitabine plus JQ1 showed greater efficacy than gemcitabine monotherapy against PDAC in vivo. Thus, our results reveal BET proteins as the critical regulators of CAF-activation and also provide evidence that stromal remodeling by epigenetic modulators can be a novel therapeutic option for PDAC.

Polyphosphate-dependent synthesis of ATP and ADP by the family-2 polyphosphate kinases in bacteria.

PubMed

Nocek, Boguslaw; Kochinyan, Samvel; Proudfoot, Michael; Brown, Greg; Evdokimova, Elena; Osipiuk, Jerzy; Edwards, Aled M; Savchenko, Alexei; Joachimiak, Andrzej; Yakunin, Alexander F

2008-11-18

Inorganic polyphosphate (polyP) is a linear polymer of tens or hundreds of phosphate residues linked by high-energy bonds. It is found in all organisms and has been proposed to serve as an energy source in a pre-ATP world. This ubiquitous and abundant biopolymer plays numerous and vital roles in metabolism and regulation in prokaryotes and eukaryotes, but the underlying molecular mechanisms for most activities of polyP remain unknown. In prokaryotes, the synthesis and utilization of polyP are catalyzed by 2 families of polyP kinases, PPK1 and PPK2, and polyphosphatases. Here, we present structural and functional characterization of the PPK2 family. Proteins with a single PPK2 domain catalyze polyP-dependent phosphorylation of ADP to ATP, whereas proteins containing 2 fused PPK2 domains phosphorylate AMP to ADP. Crystal structures of 2 representative proteins, SMc02148 from Sinorhizobium meliloti and PA3455 from Pseudomonas aeruginosa, revealed a 3-layer alpha/beta/alpha sandwich fold with an alpha-helical lid similar to the structures of microbial thymidylate kinases, suggesting that these proteins share a common evolutionary origin and catalytic mechanism. Alanine replacement mutagenesis identified 9 conserved residues, which are required for activity and include the residues from both Walker A and B motifs and the lid. Thus, the PPK2s represent a molecular mechanism, which potentially allow bacteria to use polyP as an intracellular energy reserve for the generation of ATP and survival.

Oxidative stress signaling to chromatin in health and disease

PubMed Central

Kreuz, Sarah; Fischle, Wolfgang

2016-01-01

Oxidative stress has a significant impact on the development and progression of common human pathologies, including cancer, diabetes, hypertension and neurodegenerative diseases. Increasing evidence suggests that oxidative stress globally influences chromatin structure, DNA methylation, enzymatic and non-enzymatic post-translational modifications of histones and DNA-binding proteins. The effects of oxidative stress on these chromatin alterations mediate a number of cellular changes, including modulation of gene expression, cell death, cell survival and mutagenesis, which are disease-driving mechanisms in human pathologies. Targeting oxidative stress-dependent pathways is thus a promising strategy for the prevention and treatment of these diseases. We summarize recent research developments connecting oxidative stress and chromatin regulation. PMID:27319358