Family Of Chromatin Remodelers Research Articles

The chromatin remodelers RSC and ISW1 display functional and chromatin-based promoter antagonism.

ISWI family chromatin remodelers typically organize nucleosome arrays, while SWI/SNF family remodelers (RSC) typically disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for rsc suppressors that yielded mutations in the promoter-focused ISW1a complex or mutations in the 'basic patch' of histone H4 (an epitope that regulates ISWI activity), strongly supporting RSC-ISW1a antagonism in vivo. RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism and reveal different mechanisms at two different promoter architectures.

ALT1, a Snf2 family chromatin remodeling ATPase, negatively regulates alkaline tolerance through enhanced defense against oxidative stress in rice.

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.

Overexpression of SMARCA5 correlates with cell proliferation and migration in breast cancer.

SMARCA5 partners with RSF-1 to compose the RSF complex, which belongs to the ISWI family of chromatin remodelers. Recent studies referred that SMARCA5 was overexpressed in some malignant tumors. However, expression pattern and biological roles of SMARCA5 in breast cancer have not been examined. In the present study, we found that SMARCA5 was overexpressed in breast cancer specimens by immunohistochemistry. Significant association was observed between SMARCA5 overexpression and TNM stage (p = 0.0199), tumor size (p = 0.0066), high proliferation index (p = 0.0366), and poor overall survival (p = 0.0141). SMARCA5 overexpression also correlated with Rsf-1 expression levels (p = 0.0120). Furthermore, colony formation assay and Matrigel invasion assay showed that knockdown of SMARCA5 expression in MDA-MB-231 and MDA-MB-435s cell lines with high endogenous expression decreased cell proliferation and cell invasion. Flow cytometry showed knockdown of SMARCA5-arrested cell cycle. Further analysis of cell cycle and invasion-related molecules showed that SMARCA5 downregulated cyclin A, MMP2 expression and upregulated p21 expression. In conclusion, our study demonstrated that SMARCA5 was overexpressed in human breast cancers and correlated with poor prognosis. SMARCA5 contributes to breast cancer cell proliferation and invasion.

The role of BAF (mSWI/SNF) complexes in mammalian neural development.

The BAF (mammalian SWI/SNF) complexes are a family of multi-subunit ATP-dependent chromatin remodelers that use ATP hydrolysis to alter chromatin structure. Distinct BAF complex compositions are possible through combinatorial assembly of homologous subunit families and can serve non-redundant functions. In mammalian neural development, developmental stage-specific BAF assemblies are found in embryonic stem cells, neural progenitors and postmitotic neurons. In particular, the neural progenitor-specific BAF complexes are essential for controlling the kinetics and mode of neural progenitor cell division, while neuronal BAF function is necessary for the maturation of postmitotic neuronal phenotypes as well as long-term memory formation. The microRNA-mediated mechanism for transitioning from npBAF to nBAF complexes is instructive for the neuronal fate and can even convert fibroblasts into neurons. The high frequency of BAF subunit mutations in neurological disorders underscores the rate-determining role of BAF complexes in neural development, homeostasis, and plasticity.

ACF Takes the Driver’s Seat

ISWI family chromatin remodeling enzymes generate regularly spaced nucleosome arrays. In a recent Nature report, Hwang etal. (2014) describe how ACF gauges the length of linker DNA when deciding to accelerate nucleosome sliding or to put on the brakes.

Architects of the genome: CHD dysfunction in cancer, developmental disorders and neurological syndromes.

Chromatin is vital to normal cells, and its deregulation contributes to a spectrum of human ailments. An emerging concept is that aberrant chromatin regulation culminates in gene expression programs that set the stage for the seemingly diverse pathologies of cancer, developmental disorders and neurological syndromes. However, the mechanisms responsible for such common etiology have been elusive. Recent evidence has implicated lesions affecting chromatin-remodeling proteins in cancer, developmental disorders and neurological syndromes, suggesting a common source for these different pathologies. Here, we focus on the chromodomain helicase DNA binding chromatin-remodeling family and the recent evidence for its deregulation in diverse pathological conditions, providing a new perspective on the underlying mechanisms and their implications for these prevalent human diseases.

INO80 Facilitates Pluripotency Gene Activation in Embryonic Stem Cell Self-Renewal, Reprogramming, and Blastocyst Development

The master transcription factors play integral roles in the pluripotency transcription circuitry of embryonic stem cells (ESCs). How they selectively activate expression of the pluripotency network while simultaneously repressing genes involved in differentiation is not fully understood. Here, we define a requirement for the INO80 complex, a SWI/SNF family chromatin remodeler, in ESC self-renewal, somatic cell reprogramming, and blastocyst development. We show that Ino80, the chromatin remodeling ATPase, co-occupies pluripotency gene promoters with the master transcription factors, and its occupancy is dependent on OCT4 and WDR5. At the pluripotency genes, Ino80 maintains an open chromatin architecture and licenses recruitment of Mediator and RNA polymerase II for gene activation. Our data reveal an essential role for INO80 in the expression of the pluripotency network and illustrate the coordination among chromatin remodeler, transcription factor, and histone-modifying enzyme in the regulation of the pluripotent state.

SWI/SNF factors required for cellular resistance to DNA damage include ARID1A and ARID1B and show interdependent protein stability.

The SWI/SNF chromatin-remodeling family contains various protein complexes, which regulate gene expression during cellular development and influence DNA damage response in an ATP- and complex-dependent manner, of which details remain elusive. Recent human genome sequencing of various cancer cells revealed frequent mutations in SWI/SNF factors, especially ARID1A, a variant subunit in the BRG1-associated factor (BAF) complex of the SWI/SNF family. We combined live-cell analysis and gene-suppression experiments to show that suppression of either ARID1A or its paralog ARID1B led to reduced nonhomologous end joining activity of DNA double-strand breaks (DSB), decreased accumulation of KU70/KU80 proteins at DSB, and sensitivity to ionizing radiation, as well as to cisplatin and UV. Thus, in contrast to transcriptional regulation, both ARID1 proteins are required for cellular resistance to various types of DNA damage, including DSB. The suppression of other SWI/SNF factors, namely SNF5, BAF60a, BAF60c, BAF155, or BAF170, exhibits a similar phenotype. Of these factors, ARID1A, ARID1B, SNF5, and BAF60c are necessary for the immediate recruitment of the ATPase subunit of the SWI/SNF complex to DSB, arguing that both ARID1 proteins facilitate the damage response of the complex. Finally, we found interdependent protein stability among the SWI/SNF factors, suggesting their direct interaction within the complex and the reason why multiple factors are frequently lost in parallel in cancer cells. Taken together, we show that cancer cells lacking in the expression of certain SWI/SNF factors, including ARID1A, are deficient in DNA repair and potentially vulnerable to DNA damage.

Regulating the Chromatin Landscape: Structural and Mechanistic Perspectives

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).

ISWI chromatin remodeling: one primary actor or a coordinated effort?

The ISWI family of ATP-dependent chromatin remodelers regulates transcription of coding and noncoding RNA by mobilizing nucleosomes and controlling the length of linker DNA separating nucleosomes (spacing). Nucleosome movement is tightly coupled to the DNA translocation activity of the helicase domain in the catalytic subunit. There may be other domains besides the helicase domain needed to move DNA in and out of nucleosomes. The C terminus of the ISWI catalytic subunit with the conserved HAND, SANT, and SLIDE domains may be involved in nucleosome spacing. There are several models of how the C terminus may facilitate in ISWI remodeling such as regulating the activity of the helicase domain and causing the helicase domain to translocate more efficiently on DNA or to enhance its selectivity for nucleosomes. Another possibility is that domains like SLIDE promote linker DNA entering into nucleosomes in a coordinated manner with the helicase domain.

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

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. 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. 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. Somatic mutations 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. 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.

Structure and Subunit Topology of the INO80 Chromatin Remodeler and Its Nucleosome Complex

INO80/SWR1 family chromatin remodelers are complexes composed of >15 subunits and molecular masses exceeding 1 MDa. Their important role in transcription and genome maintenance is exchanging the histone variants H2A and H2A.Z. We report the architecture of S. cerevisiae INO80 using an integrative approach of electron microscopy, crosslinking and mass spectrometry. INO80 has an embryo-shaped head-neck-body-foot architecture and shows dynamic open and closed conformations. We can assign an Rvb1/Rvb2 heterododecamer to the head in close contact with the Ino80 Snf2 domain, Ies2, and the Arp5 module at the neck. The high-affinity nucleosome-binding Nhp10 module localizes to the body, whereas the module that contains actin, Arp4, and Arp8 maps to the foot. Structural and biochemical analyses indicate that the nucleosome is bound at the concave surface near the neck, flanked by the Rvb1/2 and Arp8 modules. Our analysis establishes a structural and functional framework for this family of large remodelers.

Histone deacetylases 1 and 2 maintain S-phase chromatin and DNA replication fork progression

BackgroundHistone deacetylases (HDACs) play a critical role in the maintenance of genome stability. Class I HDACs, histone deacetylase 1 and 2 (Hdac1 and Hdac2) are recruited to the replication fork by virtue of their interactions with the replication machinery. However, functions for Hdac1 and Hdac2 (Hdacs1,2) in DNA replication are not fully understood.ResultsUsing genetic knockdown systems and novel Hdacs1,2-selective inhibitors, we found that loss of Hdacs1,2 leads to a reduction in the replication fork velocity, and an increase in replication stress response culminating in DNA damage. These observed defects are due to a direct role for Hdacs1,2 in DNA replication, as transcription of genes involved in replication was not affected in the absence of Hdacs1,2. We found that loss of Hdacs1,2 functions increases histone acetylation (ac) on chromatin in S-phase cells and affects nascent chromatin structure, as evidenced by the altered sensitivity of newly synthesized DNA to nuclease digestion. Specifically, H4K16ac, a histone modification involved in chromatin decompaction, is increased on nascent chromatin upon abolishing Hdacs1,2 activities. It was previously shown that H4K16ac interferes with the functions of SMARCA5, an ATP-dependent ISWI family chromatin remodeler. We found SMARCA5 also associates with nascent DNA and loss of SMARCA5 decreases replication fork velocity similar to the loss or inhibition of Hdacs1,2.ConclusionsOur studies reveal important roles for Hdacs1,2 in nascent chromatin structure maintenance and regulation of SMARCA5 chromatin-remodeler function, which together are required for proper replication fork progression and genome stability in S-phase.

The ATP-dependent Chromatin Remodeling Enzyme Fun30 Represses Transcription by Sliding Promoter-proximal Nucleosomes

The evolutionarily conserved ATP-dependent chromatin remodeling enzyme Fun30 has recently been shown to play important roles in heterochromatin silencing and DNA repair. However, how Fun30 remodels nucleosomes is not clear. Here we report a nucleosome sliding activity of Fun30 and its role in transcriptional repression. We observed that Fun30 repressed the expression of genes involved in amino acid and carbohydrate metabolism, the stress response, and meiosis. In addition, Fun30 was localized at the 5' and 3' ends of genes and within the open reading frames of its targets. Consistent with its role in gene repression, we observed that Fun30 target genes lacked histone modifications often associated with gene activation and showed an increased level of ubiquitinated histone H2B. Furthermore, a genome-wide nucleosome mapping analysis revealed that the length of the nucleosome-free region at the 5' end of a subset of genes was changed in Fun30-depleted cells. In addition, the positions of the -1, +2, and +3 nucleosomes at the 5' end of target genes were shifted significantly, whereas the position of the +1 nucleosome remained largely unchanged in the fun30Δ mutant. Finally, we demonstrated that affinity-purified, single-component Fun30 exhibited a nucleosome sliding activity in an ATP-dependent manner. These results define a role for Fun30 in the regulation of transcription and indicate that Fun30 remodels chromatin at the 5' end of genes by sliding promoter-proximal nucleosomes.

ATP-Dependent Chromatin Remodeling by Cockayne Syndrome Protein B and NAP1-Like Histone Chaperones Is Required for Efficient Transcription-Coupled DNA Repair

The Cockayne syndrome complementation group B (CSB) protein is essential for transcription-coupled DNA repair, and mutations in CSB are associated with Cockayne syndrome—a devastating disease with complex clinical features, including the appearance of premature aging, sun sensitivity, and numerous neurological and developmental defects. CSB belongs to the SWI2/SNF2 ATP–dependent chromatin remodeler family, but the extent to which CSB remodels chromatin and whether this activity is utilized in DNA repair is unknown. Here, we show that CSB repositions nucleosomes in an ATP–dependent manner in vitro and that this activity is greatly enhanced by the NAP1-like histone chaperones, which we identify as new CSB–binding partners. By mapping functional domains and analyzing CSB derivatives, we demonstrate that chromatin remodeling by the combined activities of CSB and the NAP1-like chaperones is required for efficient transcription-coupled DNA repair. Moreover, we show that chromatin remodeling and repair protein recruitment mediated by CSB are separable activities. The collaboration that we observed between CSB and the NAP1-like histone chaperones adds a new dimension to our understanding of the ways in which ATP–dependent chromatin remodelers and histone chaperones can regulate chromatin structure. Taken together, the results of this study offer new insights into the functions of chromatin remodeling by CSB in transcription-coupled DNA repair as well as the underlying mechanisms of Cockayne syndrome.

Genetic syndromes caused by mutations in epigenetic genes

The orchestrated organization of epigenetic factors that control chromatin dynamism, including DNA methylation, histone marks, non-coding RNAs (ncRNAs) and chromatin-remodeling proteins, is essential for the proper function of tissue homeostasis, cell identity and development. Indeed, deregulation of epigenetic profiles has been described in several human pathologies, including complex diseases (such as cancer, cardiovascular and neurological diseases), metabolic pathologies (type 2 diabetes and obesity) and imprinting disorders. Over the last decade it has become increasingly clear that mutations of genes involved in epigenetic mechanism, such as DNA methyltransferases, methyl-binding domain proteins, histone deacetylases, histone methylases and members of the SWI/SNF family of chromatin remodelers are linked to human disorders, including Immunodeficiency Centromeric instability Facial syndrome 1, Rett syndrome, Rubinstein-Taybi syndrome, Sotos syndrome or alpha-thalassemia/mental retardation X-linked syndrome, among others. As new members of the epigenetic machinery are described, the number of human syndromes associated with epigenetic alterations increases. As recent examples, mutations of histone demethylases and members of the non-coding RNA machinery have recently been associated with Kabuki syndrome, Claes-Jensen X-linked mental retardation syndrome and Goiter syndrome. In this review, we describe the variety of germline mutations of epigenetic modifiers that are known to be associated with human disorders, and discuss the therapeutic potential of epigenetic drugs as palliative care strategies in the treatment of such disorders.

Nucleosome mobilization by ISW2 requires the concerted action of the ATPase and SLIDE domains

The ISWI family of ATP-dependent chromatin remodelers represses transcription by changing nucleosome positioning. The interactions with extranucleosomal DNA and the requirement of a minimal length of extranucleosomal DNA by ISWI mediate the spacing of nucleosomes. ISW2 from Saccharomyces cerevisiae, a member of the ISWI family, has a conserved domain called SLIDE (SANT-like ISWI domain), whose binding to extranucleosomal DNA ~19 bp from the edge of nucleosomes is required for efficiently pushing DNA into nucleosomes and maintaining the unidirectional movement of nucleosomes, as reported here. Loss of SLIDE binding does not perturb ATPase domain binding to the SHL2 site of nucleosomes or its initial movement of DNA inside of nucleosomes. ISW2 has therefore two distinct roles in mobilizing nucleosomes, with the ATPase domain translocating and moving DNA inside nucleosomes, and the SLIDE domain facilitating the entry of linker DNA into nucleosomes.

Overcoming the chromatin barrier to end resection

Repair of double-strand breaks by homologous recombination requires Repair of double-strand breaks by homologous recombination requires 5'-3' resection of the DNA ends to create 3' single-stranded DNA tails. While much progress has been made in identifying the proteins that directly participate in end resection, how this process occurs in the context of chromatin is not well understood. Two papers in Nature report that Fun30, a poorly characterized member of the Swi2/Snf2 family of chromatin remodelers, plays a role in end processing by facilitating the Exo1 and Sgs1-Dna2 resection pathways.

Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer

Gastric cancer is a heterogeneous disease with multiple environmental etiologies and alternative pathways of carcinogenesis. Beyond mutations in TP53, alterations in other genes or pathways account for only small subsets of the disease. We performed exome sequencing of 22 gastric cancer samples and identified previously unreported mutated genes and pathway alterations; in particular, we found genes involved in chromatin modification to be commonly mutated. A downstream validation study confirmed frequent inactivating mutations or protein deficiency of ARID1A, which encodes a member of the SWI-SNF chromatin remodeling family, in 83% of gastric cancers with microsatellite instability (MSI), 73% of those with Epstein-Barr virus (EBV) infection and 11% of those that were not infected with EBV and microsatellite stable (MSS). The mutation spectrum for ARID1A differs between molecular subtypes of gastric cancer, and mutation prevalence is negatively associated with mutations in TP53. Clinically, ARID1A alterations were associated with better prognosis in a stage-independent manner. These results reveal the genomic landscape, and highlight the importance of chromatin remodeling, in the molecular taxonomy of gastric cancer.

CHD6 chromatin remodeler is a negative modulator of influenza virus replication that relocates to inactive chromatin upon infection

The influenza virus establishes close functional and structural connections with the nucleus of the infected cell. Thus, viral ribonucleoproteins (RNPs) are closely bound to chromatin components and the main constituent of viral RNPs, the nucleoprotein (NP) protein, interacts with histone tails. Using a yeast two-hybrid screening, we previously found that the PA influenza virus polymerase subunit interacts with the CHD6 protein, a member of the CHD family of chromatin remodelers. Here we show that CHD6 also interacts with the viral polymerase complex and colocalizes with viral RNPs in the infected cells. To study the relationships between RNPs, chromatin and CHD6, we have analysed whether NP and CHD6 binds to peptides representing trimethylated lysines of histone 3 tails that mark transcriptionally active or inactive chromatin. Upon infection, NP binds to marks of repressed chromatin and, interestingly an important recruitment of CHD6 to these heterochromatin marks occurs in this situation. Silencing experiments indicate that CHD6 acts as a negative modulator of influenza virus replication. Hence, the CHD6 association with inactive chromatin could be part of a process where the influenza virus triggers modifications of chromatin-associated proteins that could contribute to the pathogenic events used by the virus to induce host cell shut-off.