Distinct Chromatin Remodeling Complexes Research Articles

The chromatin-remodeling subunit Baf200 promotes homology-directed DNA repair and regulates distinct chromatin-remodeling complexes

The efficiency and type of pathway chosen to repair DNA double-strand breaks (DSBs) are critically influenced by the nucleosome packaging and the chromatin architecture surrounding the DSBs. The Swi/Snf (PBAF and BAF) chromatin-remodeling complexes contribute to DNA damage-induced nucleosome remodeling, but the mechanism by which it contributes to this function is poorly understood. Herein, we report how the Baf200 (Arid2) PBAF-defining subunit regulates DSB repair. We used cytological and biochemical approaches to show that Baf200 plays an important function by facilitating homologous recombination-dependent processes, such as recruitment of Rad51 (a key component of homologous recombination) to DSBs, homology-directed repair, and cell survival after DNA damage. Furthermore, we observed that Baf200 and Rad51 are present in the same complex and that this interaction is mediated by C-terminal sequences in both proteins. It has been recognized previously that the interplay between distinct forms of Swi/Snf has profound functional consequences, but we understand little about the composition of complexes formed by PBAF protein subunits. Our biochemical analyses reveal that Baf200 forms at least two distinct complexes. One is a canonical form of PBAF including the Swi/Snf-associated Brg1 catalytic subunit, and the other contains Baf180 but not Brg1. This distinction of PBAF complexes based on their unique composition provides the foundation for future studies on the specific contributions of the PBAF forms to the regulation of DNA repair.

The SWI/SNF BAF-A complex is essential for neural crest development

Growing evidence indicates that chromatin remodeler mutations underlie the pathogenesis of human neurocristopathies or disorders that affect neural crest cells (NCCs). However, causal relationships among chromatin remodeler subunit mutations and NCC defects remain poorly understood. Here we show that homozygous loss of ARID1A-containing, SWI/SNF chromatin remodeling complexes (BAF-A) in NCCs results in embryonic lethality in mice, with mutant embryos succumbing to heart defects. Strikingly, monoallelic loss of ARID1A in NCCs led to craniofacial defects in adult mice, including shortened snouts and low set ears, and these defects were more pronounced following homozygous loss of ARID1A, with the ventral cranial bones being greatly reduced in size. Early NCC specification and expression of the BRG1 NCC target gene, PLEXINA2, occurred normally in the absence of ARID1A. Nonetheless, mutant embryos displayed incomplete conotruncal septation of the cardiac outflow tract and defects in the posterior pharyngeal arteries, culminating in persistent truncus arteriosus and agenesis of the ductus arteriosus. Consistent with this, migrating cardiac NCCs underwent apoptosis within the circumpharyngeal ridge. Our data support the notion that multiple, distinct chromatin remodeling complexes govern genetically separable events in NCC development and highlight a potential pathogenic role for NCCs in the human BAF complex disorder, Coffin-Siris Syndrome.

Abstract PR02: Transcriptional regulation mediated by biochemically distinct forms of SWI/SNF

Abstract Background: We often consider the SWI/SNF complex as a single entity with a single function in a particular cell type. This model ignores that multiple positions within the SWI/SNF chromatin remodeling complex can be filled by mutually exclusive subunits. Inclusion or exclusion of such subunits gives rise to an array of biochemically distinct chromatin remodeling complexes, some of which have profound functional consequences on gene regulation and differentiation. Here, we examine the functional relationships among three complex-specific ARID (AT-Rich Interacting Domain) subunits of the SWI/SNF complex in a human hepatocellular carcinoma cell line using ChIP-seq, RNA-seq, and public data on transcription factor binding. Results: Using RNAi directed at each of the three subunits, we identify widespread overlap in transcriptional regulation mediated by SWI/SNF. The ARID subunits are commonly bound together at the promoters of active genes, with more variable binding associated with distal regulatory elements. ARID1B and ARID2 cooperate to repress the expression of hundreds of genes. Additionally, we identify many examples of competitive interactions between ARID1A and ARID1B or ARID2, and show that gene expression changes following loss of one ARID are dependent on the function of an alternative ARID. These distinct regulatory modalities are correlated with differential occupancy by transcription factors. Together these data suggest distinct SWI/SNF complexes dictate gene-specific transcription through interactions between different forms of SWI/SNF and specific co-factors. The ARID subunits are among the most frequently mutated chromatin regulators in cancer. Understanding the functional relationship between distinct forms of the complex is a necessary first step for identifying their potentially unique roles in oncogenesis. Citation Format: Jesse R. Raab, Samuel Resnick, Terry Magnuson. Transcriptional regulation mediated by biochemically distinct forms of SWI/SNF. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr PR02.

Conserved abundance and topological features in chromatin-remodeling protein interaction networks.

The study of conserved protein interaction networks seeks to better understand the evolution and regulation of protein interactions. Here, we present a quantitative proteomic analysis of 18 orthologous baits from three distinct chromatin-remodeling complexes in Saccharomyces cerevisiae and Homo sapiens. We demonstrate that abundance levels of orthologous proteins correlate strongly between the two organisms and both networks have highly similar topologies. We therefore used the protein abundances in one species to cross-predict missing protein abundance levels in the other species. Lastly, we identified a novel conserved low-abundance subnetwork further demonstrating the value of quantitative analysis of networks.

Remodeling and spacing factor 1 (RSF1) deposits centromere proteins at DNA double-strand breaks to promote non-homologous end-joining

The cellular response to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) in native chromatin requires a tight coordination between the activities of DNA repair machineries and factors that modulate chromatin structure. SMARCA5 is an ATPase of the SNF2 family of chromatin remodeling factors that has recently been implicated in the DSB response. It forms distinct chromatin remodeling complexes with several non-canonical subunits, including the remodeling and spacing factor 1 (RSF1) protein. Despite the fact that RSF1 is often overexpressed in tumors and linked to tumorigenesis and genome instability, its role in the DSB response remains largely unclear. Here we show that RSF1 accumulates at DSB sites and protects human cells against IR-induced DSBs by promoting repair of these lesions through homologous recombination (HR) and non-homologous end-joining (NHEJ). Although SMARCA5 regulates the RNF168-dependent ubiquitin response that targets BRCA1 to DSBs, we found RSF1 to be dispensable for this process. Conversely, we found that RSF1 facilitates the assembly of centromere proteins CENP-S and CENP-X at sites of DNA damage, while SMARCA5 was not required for these events. Mechanistically, we uncovered that CENP-S and CENP-X, upon their incorporation by RSF1, promote assembly of the NHEJ factor XRCC4 at damaged chromatin. In contrast, CENP-S and CENP-X were dispensable for HR, suggesting that RSF1 regulates HR independently of these centromere proteins. Our findings reveal distinct functions of RSF1 in the 2 major pathways of DSB repair and explain how RSF1, through the loading of centromere proteins and XRCC4 at DSBs, promotes repair by non-homologous end-joining.

Identification of Novel Domains within the Ioc2 and Ioc3 Subunits of the ISW1 Chromatin Remodeling Complex That Explicitly Distinguishes the Catalytic Activity of ISW1a and ISW1b

In S.cerevisiae, ISW1 forms two distinct chromatin remodeling complexes, ISW1a and ISW1b. ISW1a is composed of Isw1 and Ioc3 while, ISW1b is comprised of Isw1, Ioc2, and Ioc4. Although these complexes share the same ATPase subunit, both exhibit catalytic activity that is distinctive. ISW1a remodels with directionality preferences, spaces nucleosomes, and adapts active/inactive conformational changes dependent on linker DNA length. Meanwhile, ISW1b has substrate preference for short nucleosome arrays, lacks directional remodeling and spacing activity. The differences exhibited in these complexes are attributed to the associated subunits of each complex. Ioc2 has an atypical PHD motif that is often found in chromatin associated proteins and Ioc4 has a PWWP motif, a putative DNA binding domain. Ioc3 has no conserved domains that have been previously characterized, neither does it bear any similarity to proteins which their function has been defined. No work has been done to identify the domain organization or the contribution of these subunits to the activity of the ISW1 complexes. The goals of this project was to characterize the domain architecture of the Ioc2 and Ioc3 subunits of the ISW1 chromatin remodeling complexes, and also identify the key defining features of these two complexes. Using mutational analysis, this work explicitly identifies regions within the subunits that are required for the maintenance of complex integrity, modulation of nucleosome interaction, directional remodeling, and spacing activity. Further characterization of these domains revealed that their deletion results in remodeling and spacing activity reminiscent of its sister complex, suggesting that these domains serve as the distinguishing features that define the ISW1a and ISW1b complexes. This work was supported by Public Health Service grant GM 70864 from the National Institute of Health.

Phosphorylation of Williams Syndrome Transcription Factor by MAPK Induces a Switching between Two Distinct Chromatin Remodeling Complexes

Changes in the environment of a cell precipitate extracellular signals and sequential cascades of protein modification and elicit nuclear transcriptional responses. However, the functional links between intracellular signaling-dependent gene regulation and epigenetic regulation by chromatin-modifying proteins within the nucleus are largely unknown. Here, we describe novel epigenetic regulation by MAPK cascades that modulate formation of an ATP-dependent chromatin remodeling complex, WINAC (WSTF Including Nucleosome Assembly Complex), an SWI/SNF-type complex containing Williams syndrome transcription factor (WSTF). WSTF, a specific component of two chromatin remodeling complexes (SWI/SNF-type WINAC and ISWI-type WICH), was phosphorylated by the stimulation of MAPK cascades in vitro and in vivo. Ser-158 residue in the WAC (WSTF/Acf1/cbpq46) domain, located close to the N terminus of WSTF, was identified as a major phosphorylation target. Using biochemical analysis of a WSTF mutant (WSTF-S158A) stably expressing cell line, the phosphorylation of this residue (Ser-158) was found to be essential for maintaining the association between WSTF and core BAF complex components, thereby maintaining the ATPase activity of WINAC. WINAC-dependent transcriptional regulation of vitamin D receptor was consequently impaired by this WSTF mutation, but the recovery from DNA damage mediated by WICH was not impaired. Our results suggest that WSTF serves as a nuclear sensor of the extracellular signals to fine-tune the chromatin remodeling activity of WINAC. WINAC mediates a previously unknown MAPK-dependent step in epigenetic regulation, and this MAPK-dependent switching mechanism between the two functionally distinct WSTF-containing complexes might underlie the diverse functions of WSTF in various nuclear events.

Identification and characterization of the IRD motif, a novel domain identified in the Ioc3 subunit of the ISW1a chromatin remodeling complex

ISW1 forms two distinct chromatin remodeling complexes in S. cerevisiae, ISW1a and ISW1b. ISW1a is composed of Isw1 and Ioc3 while, ISW1b is comprised of Isw1, Ioc2, and Ioc4. Isw1, functions as the catalytic subunit of the complex. Similar to drosophila ISWI, Isw1 contains HAND, SANT, and SLIDE domains. Ioc2 has an atypical PHD motif that is often found in chromatin associated proteins and Ioc4 has a PWWP motif, a putative DNA binding domain, which is often found in enzymes that function in transcription regulation. Ioc3 has no conserved domains that have been previously characterized in S. cerevisiae; neither does it bear any similarity to proteins which their function has been defined.The aim of this work was to determine the domain architecture of Ioc3 of the ISW1a chromatin remodeling complex. This work explicitly identifies the regions of Ioc3 responsible for maintenance of complex integrity, modulation of directionality during remodeling, and nucleosome spacing. More significantly, the IRD domain (ISW1a‐Regulator of Directionality domain), a novel domain within Ioc3 that has not been previously characterized in S. cerevisiae was identified. The IRD domain was found to be required for the proper regulation of ISW1a remodeling vitro and is a distinguishing feature between the ISW1a and ISW1b complexes.This work was supported by Public Health Service grant GM 70864 from the National Institute of Health.

Essential Role for the SANT Domain in the Functioning of Multiple Chromatin Remodeling Enzymes

The SANT domain is a novel motif found in a number of eukaryotic transcriptional regulatory proteins that was identified based on its homology to the DNA binding domain of c-myb. Here we show that the SANT domain is essential for the in vivo functions of yeast Swi3p, Ada2p, and Rsc8p, subunits of three distinct chromatin remodeling complexes. We also find that the Ada2p SANT domain is essential for histone acetyltransferase activity of native, Gcn5p-containing HAT complexes. Furthermore, kinetic analyses indicate that an intact SANT domain is required for an Ada2p-dependent enhancement of histone tail binding and enzymatic catalysis by Gcn5p. Our results are consistent with a general role for SANT domains in functional interactions with histone N-terminal tails.

The Isw2 Chromatin Remodeling Complex Represses Early Meiotic Genes upon Recruitment by Ume6p

The ISWI class of chromatin remodeling factors exhibits potent chromatin remodeling activities in vitro. However, the in vivo functions of this class of factors are unknown at a molecular level. We have found that S. cerevisiae Isw2 complex represses transcription of early meiotic genes during mitotic growth in a parallel pathway to Rpd3-Sin3 histone deacetylase complex. This repressor function of Isw2 complex is largely dependent upon Ume6p, which recruits the complex to target genes. Nuclease digestion analyses revealed that Isw2 complex establishes nuclease-inaccessible chromatin structure near the Ume6p binding site in vivo. Based on these findings, we propose a model for the mechanism of transcriptional repression by two distinct chromatin remodeling complexes.