ATP-dependent Chromatin Remodeling Complexes Research Articles

Chromatin remodeling mediated by ARID1A is indispensable for normal hematopoiesis in mice

Precise regulation of chromatin architecture is vital to physiological processes including hematopoiesis. ARID1A is a core component of the mammalian SWI/SNF complex, which is one of the ATP-dependent chromatin remodeling complexes. To uncover the role of ARID1A in hematopoietic development, we utilized hematopoietic cell-specific deletion of Arid1a in mice. We demonstrate that ARID1A is essential for maintaining the frequency and function of hematopoietic stem cells and its loss impairs the differentiation of both myeloid and lymphoid lineages. ARID1A deficiency led to a global reduction in open chromatin and ensuing transcriptional changes affected key genes involved in hematopoietic development. We also observed that silencing of ARID1A affected ATRA-induced differentiation of NB4 cells, suggesting its role in granulocytic differentiation of human leukemic cells. Overall, our study provides a comprehensive elucidation of the function of ARID1A in hematopoiesis and highlights the central role of ARID1A-containing SWI/SNF complex in maintaining chromatin dynamics in hematopoietic cells.

Epigenetic mechanisms, trauma, and psychopathology: targeting chromatin remodeling complexes.

Environmental pressure affects the genotype throughout different epigenetic processes. There is currently ample evidence on the role of epigenetics in developing various mental disorders. A burden of environmental pressure, such as psychological trauma, and its influence on genotype can lead to a variety of psychopathologies. Thus, this study focuses on the epigenetic activity of the complex protein machinery operating on chromatin - the ATP-dependent chromatin remodeling complexes. Although there are several recent studies on the molecular structure, functions, and taxonomy of ATP-dependent chromatin remodeling complexes, the focus of this paper is to highlight the importance of those 'protein machines' in developing psychiatric disorders. Data were obtained from human preclinical and clinical studies. The results of this review indicate an importance of ATP-dependent chromatin remodeling complexes in the interaction between environmental factors, including traumatic events, and genetic vulnerability to stress. Several studies indicate that ATP-dependent chromatin remodeling complexes play a crucial role in the development and consolidation of memory, in neurodevelopmental processes, and in etiology depressive-like behavior. Thus, the activity of those 'protein machines' emerges as a key factor in the pathophysiology of various psychiatric diseases. It can also be concluded that the limitations of clinical studies may be explained by inappropriate laboratory methods and research paradigms due to the delayed timeframe of biochemical responses to environmental stimuli. Future research in this field may enable a better understanding of the pathophysiology of psychiatric diseases and contribute to the development of novel molecular treatment targets.

MBD3/NuRD loss participates with KDM6A program to promote DOCK5/8 expression and Rac GTPase activation in human acute myeloid leukemia.

Cancer genome sequencing studies have focused on identifying oncogenic mutations. However, mutational profiling alone may not always help dissect underlying epigenetic dependencies in tumorigenesis. Nucleosome remodeling and deacetylase (NuRD) is an ATP-dependent chromatin remodeling complex that regulates transcriptional architecture and is involved in cell fate commitment. We demonstrate that loss of MBD3, an important NuRD scaffold, in human primary acute myeloid leukemia (AML) cells associates with leukemic NuRD. Interestingly, CHD4, an intact ATPase subunit of leukemic NuRD, coimmunoprecipitates and participates with H3K27Me3/2-demethylase KDM6A to induce expression of atypical guanine nucleotide exchange factors, dedicator of cytokinesis (DOCK) 5 and 8 (DOCK5/8), promoting Rac GTPase signaling. Mechanistically, MBD3 deficiency caused loss of histone deacytelase 1 occupancy with a corresponding increase in KDM6A, CBP, and H3K27Ac on DOCK5/8 loci, leading to derepression of gene expression. Importantly, the Cancer Genome Atlas AML cohort reveals that DOCK5/ 8 levels are correlated with MBD3 and KDM6A, and DOCK5/ 8 expression is significantly increased in patients who are MBD3 low and KDM6A high with a poor survival. In addition, pharmacological inhibition of DOCK signaling selectively attenuates AML cell survival. Because MBD3 and KDM6A have been implicated in metastasis, our results may suggest a general phenomenon in tumorigenesis. Collectively, these findings provide evidence for MBD3-deficient NuRD in leukemia pathobiology and inform a novel epistasis between NuRD and KDM6A toward maintenance of oncogenic gene expression in AML.-Biswas, M., Chatterjee, S. S., Boila, L. D., Chakraborty, S., Banerjee, D., Sengupta, A. MBD3/NuRD loss participates with KDM6A program to promote DOCK5/8 expression and Rac GTPase activation in human acute myeloid leukemia.

SMARCB1/INI1 Is Diagnostically Useful in Distinguishing α-Fetoprotein-producing Gastric Carcinoma from Hepatocellular Carcinoma.

Switch/sucrose non-fermentable (SWI/SNF)-related, matrix-associated, actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1), also named integrase interactor 1, is one of the core subunit proteins in the SWI/SNF ATP-dependent chromatin remodeling complex encoded at chromosomal position 22q11.2. Complete loss of SMARCB1 expression has been reported in various malignant tumors. Immunohistochemistry has demonstrated that SMARCB1 mutation/inactivation correlates well with loss of nuclear SMARCB1 expression. This study investigated SMARCB1 expression in hepatocellular carcinomas (HCCs) and α-fetoprotein (AFP)-producing gastric carcinomas by immunohistochemistry. For comparison, SMARCB1 immunostaining in intrahepatic cholangiocarcinoma (ICC) was also performed. Thirty classical HCCs, 30 ICCs and 10 AFP-producing gastric carcinomas were analyzed. Only one case of HCC had focal labeling of SMARCB1 in the nuclei. Twelve cases of ICC were immunopositive for SMARCB1, with either focal or diffuse reactivity. All AFP-producing gastric carcinomas were immunopositive for SMARCB1 in the nuclei, and the reactivity was consistently diffuse. SMARCB1 is a potential marker for distinguishing metastatic AFP-producing gastric carcinoma from HCC.

Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes

Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.

From the Editor’s Desk…: October 2018

From the Editor’s Desk…: October 2018

Modulating the Expression Strength of the Baculovirus/Insect Cell Expression System: A Toolbox Applied to the Human Tumor Suppressor SMARCB1/SNF5.

The human tumor suppressor SMARCB1/INI1/SNF5/BAF47 (SNF5) is a core subunit of the multi-subunit ATP-dependent chromatin remodeling complex SWI/SNF, also known as Brahma/Brahma-related gene 1 (BRM/BRG1)-associated factor (BAF). Experimental studies of SWI/SNF are currently considerably limited by the low cellular abundance of this complex; thus, recombinant protein production represents a key to obtain the SWI/SNF proteins for molecular and structural studies. While the expression of mammalian proteins in bacteria is often difficult, the baculovirus/insect cell expression system can overcome limitations of prokaryotic expression systems and facilitate the co-expression of multiple proteins. Here, we demonstrate that human full-length SNF5 tagged with a C-terminal 3 × FLAG can be expressed and purified from insect cell extracts in monomeric and dimeric forms. To this end, we constructed a set of donor and acceptor vectors for the expression of individualproteins and protein complexes in the baculovirus/insect cell expression system under the control of a polyhedrin (polh), p10, or a minimal Drosophila melanogaster Hsp70 promoter. We show that the SNF5 expression level could be modulated by the selection of the promoter used to control expression. The vector set also comprises vectors that encode a 3 × FLAG tag, Twin-Strep tag, or CBP-3 × FLAG-TEV-ProteinA triple tag to facilitate affinity selection and detection. By gel filtration and split-ubiquitin assays, we show that human full-length SNF5 has the ability to self-interact. Overall, the toolbox developed herein offers the possibility to flexibly select the promoter strength as well as the affinity tag and is suggested to advance the recombinant expression of chromatin remodeling factors and other challenging proteins.

Binding of TMPRSS2-ERG to BAF Chromatin Remodeling Complexes Mediates Prostate Oncogenesis

Chromosomal rearrangements resulting in the fusion of TMPRSS2, an androgen-regulated gene, and the ETS family transcription factor ERG occur in over half of prostate cancers. However, the mechanism by which ERG promotes oncogenic gene expression and proliferation remains incompletely understood. Here, we identify a binding interaction between ERG and the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complex, which is conserved among other oncogenic ETS factors, including ETV1, ETV4, and ETV5. We find that ERG drives genome-wide retargeting of BAF complexes in a manner dependent on binding of ERG to the ETS DNA motif. Moreover, ERG requires intact BAF complexes for chromatin occupancy and BAF complex ATPase activity for target gene regulation. In a prostate organoid model, BAF complexes are required for ERG-mediated basal-to-luminal transition, a hallmark of ERG activity in prostate cancer. These observations suggest a fundamental interdependence between ETS transcription factors and BAF chromatin remodeling complexes in cancer.

ATP-Dependent Chromatin Remodeling During Cortical Neurogenesis.

The generation of individual neurons (neurogenesis) during cortical development occurs in discrete steps that are subtly regulated and orchestrated to ensure normal histogenesis and function of the cortex. Notably, various gene expression programs are known to critically drive many facets of neurogenesis with a high level of specificity during brain development. Typically, precise regulation of gene expression patterns ensures that key events like proliferation and differentiation of neural progenitors, specification of neuronal subtypes, as well as migration and maturation of neurons in the developing cortex occur properly. ATP-dependent chromatin remodeling complexes regulate gene expression through utilization of energy from ATP hydrolysis to reorganize chromatin structure. These chromatin remodeling complexes are characteristically multimeric, with some capable of adopting functionally distinct conformations via subunit reconstitution to perform specific roles in major aspects of cortical neurogenesis. In this review, we highlight the functions of such chromatin remodelers during cortical development. We also bring together various proposed mechanisms by which ATP-dependent chromatin remodelers function individually or in concert, to specifically modulate vital steps in cortical neurogenesis.

SWI/SNF subunit expression heterogeneity in human aplastic anemia stem/progenitors

Acquired aplastic anemia (AA) is a bone marrow (BM) failure associated with autoimmune destruction of hematopoietic stem cells (HSCs). Although somatic mutations have been identified in AA patients, mutations alone do not explain AA pathophysiology. SWI/SNF is an evolutionarily conserved, multi-subunit, ATP-dependent chromatin-remodeling protein complex that plays an important role in mammalian hematopoiesis. Herein, gene expression analysis identified a significant loss of the SWI/SNF core component SMARCC1, along with ARID1B, ACTL6A, and SMARCD1, in human AA BM CD34+ HSCs and hematopoietic stem and progenitor cells (HSPCs) compared with normal HSPCs. However, expression of SMARCA4, SMARCB1, SMARCD3, and DPF2 remained intact in our AA cohort. PBRM1, BRD7, and SMARCA2 expression were significantly upregulated in both untreated and follow-up AA patients. Clonal hematopoiesis in AA is associated with evolution to late clonal disorders, including myelodysplastic syndromes (MDS). Apart from SMARCD1 loss, we did not observe significant alteration of SWI/SNF expression in MDS HSPCs, indicating SWI/SNF differential expression in AA and MDS. In addition, except for ACTL6A, SWI/SNF expression was unaltered in aged HSPCs. Importantly, our results provide evidence for loss of SWI/SNF in AA, and may implicate AA HSPC-autonomous defective SWI/SNF regulation as an integral component of BM failure, in addition to autoimmune destruction of AA HSCs. These findings illustrate for the first time SWI/SNF subunit expression heterogeneity in human AA HSPCs and require prognostic validation in a larger cohort.

Disruption of mammalian SWI/SNF and polycomb complexes in human sarcomas: mechanisms and therapeutic opportunities.

Soft-tissue sarcomas are increasingly characterized and subclassified by genetic abnormalities that represent underlying drivers of their pathology. Hallmark tumor suppressor gene mutations and pathognomonic gene fusions collectively account for approximately one-third of all sarcomas. These genetic abnormalities most often result in global transcriptional misregulation via disruption of protein regulatory complexes which govern chromatin architecture. Specifically, alterations to mammalian SWI/SNF (mSWI/SNF or BAF) ATP-dependent chromatin remodeling complexes and polycomb repressive complexes cause disease-specific changes in chromatin architecture and gene expression across a number of sarcoma subtypes. Understanding the functions of chromatin regulatory complexes and the mechanisms underpinning their roles in oncogenesis will be required for the design and development of new therapeutic strategies in sarcomas. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The CUE1 domain of the SNF2-like chromatin remodeler SMARCAD1 mediates its association with KRAB-associated protein 1 (KAP1) and KAP1 target genes

Chromatin in embryonic stem cells (ESCs) differs markedly from that in somatic cells, with ESCs exhibiting a more open chromatin configuration. Accordingly, ATP-dependent chromatin remodeling complexes are important regulators of ESC homeostasis. Depletion of the remodeler SMARCAD1, an ATPase of the SNF2 family, has been shown to affect stem cell state, but the mechanistic explanation for this effect is unknown. Here, we set out to gain further insights into the function of SMARCAD1 in mouse ESCs. We identified KRAB-associated protein 1 (KAP1) as the stoichiometric binding partner of SMARCAD1 in ESCs. We found that this interaction occurs on chromatin and that SMARCAD1 binds to different classes of KAP1 target genes, including zinc finger protein (ZFP) and imprinted genes. We also found that the RING B-box coiled-coil (RBCC) domain in KAP1 and the proximal coupling of ubiquitin conjugation to ER degradation (CUE) domain in SMARCAD1 mediate their direct interaction. Of note, retention of SMARCAD1 in the nucleus depended on KAP1 in both mouse ESCs and human somatic cells. Mutations in the CUE1 domain of SMARCAD1 perturbed the binding to KAP1 in vitro and in vivo Accordingly, an intact CUE1 domain was required for tethering this remodeler to the nucleus. Moreover, mutation of the CUE1 domain compromised SMARCAD1 binding to KAP1 target genes. Taken together, our results reveal a mechanism that localizes SMARCAD1 to genomic sites through the interaction of SMARCAD1's CUE1 motif with KAP1.

Hepatic SMARCA4 predicts HCC recurrence and promotes tumour cell proliferation by regulating SMAD6 expression

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is typically diagnosed at advanced stages. Identification and characterisation of genes within amplified and deleted chromosomal loci can provide new insights into the pathogenesis of cancer and lead to new approaches for diagnosis and therapy. In our previous study, we found a recurrent region of copy number amplification at 19p13.2 in hepatocellular carcinoma (HCC). In the present study, we performed integrated copy number analysis and expression profiling at this locus and a putative cancer gene, SMARCA4/BRG1, was uncovered in this region. BRG1 is a part of the large ATP-dependent chromatin remodelling complex SWI/SNF. The function of BRG1 in various cancers is unclear, including its role in HCC tumorigenesis. Here, we found that BRG1 is upregulated in HCC and that its level significantly correlates with cancer progression in HCC patients. Importantly, we also found that nuclear expression of BRG1 predicts early recurrence for HCC patients. Furthermore, we demonstrated that BRG1 promotes HCC cell proliferation in vitro and in vivo. BRG1 was observed not only to facilitate S-phase entry but also to attenuate cell apoptosis. Finally, we discovered that one of the mechanisms by which BRG1 promotes cell proliferation is the upregulation of SMAD6. These findings highlight the important role of BRG1 in the regulation of HCC proliferation and provide valuable information for cancer prognosis and treatment.

Mitochondrially produced ATP affects stem cell pluripotency via Actl6a-mediated histone acetylation.

ATP is mainly generated by glycolysis in pluripotent stem cells (PSCs) and is consumed to maintain cell viability. Differences in mitochondrial activity among induced (i)PSCs with different degrees of pluripotency are poorly understood. In this study, by comparing gene expression and mitochondrial activity among iPSCs with different degrees of pluripotency, we found that mitochondrial complex I gene expression, complex I activity, and cellular ATP levels were much higher in fully pluripotent stem cell lines than in partially pluripotent stem cell lines. Actin-like protein 6a (Actl6a), a component of ATP-dependent chromatin remodeling and histone acetylation complexes, was more highly expressed in fully pluripotent stem cell lines. ATP promoted Actl6a expression and histone acetylation. Actl6a knockdown reduced the pluripotency of embryonic stem cells (ESCs), and this reduction could not be rescued by the addition of ATP. Furthermore, inhibiting ATP formation by treatment with rotenone reduced the pluripotency of ESCs. These data suggest that the abundance of mitochondrially produced ATP affects stem cell pluripotency via Actl6a-mediated histone acetylation.-Zhang, Y., Cui, P., Li, Y., Feng, G., Tong, M., Guo, L., Li, T., Liu, L., Li, W., Zhou, Q. Mitochondrially produced ATP affects stem cell pluripotency via Actl6a-mediated histone acetylation.

Abstract PR15: BRD9 defines a novel mammalian SWI/SNF (BAF) complex configuration which supports proliferation in AML

Abstract Genes encoding subunits of the mammalian SWI/SNF (BAF) ATP-dependent chromatin remodeling complexes are mutated in over 20% of human cancer. Specific subunits are mutated in specific malignancies, highlighting their tissue-specific protective roles; moreover, synthetic lethal screens have uncovered genetic- and lineage-based features that confer dependence on specific mSWI/SNF subunits. As combinatorial complexity represents a major challenge, identification of specialized mSWI/SNF configurations, subunit-specific functions, binding restrictions, and exclusivity relationships is critical for understanding oncogenic mechanisms and for the selection of appropriate therapeutic agents targeting mSWI/SNF complex subunits. Here, we discover that BRD9, a recently identified mSWI/SNF subunit, defines a novel complex configuration distinct from BAF and PBAF, which we term noncanonical BAF, or ncBAF. We used biochemical methods to isolate BRD9-containing complexes and find that BRD9 selectively marks a sub-stoichiometric group of mSWI/SNF complexes of smaller molecular weight that lack several members of canonical BAF complexes such as BAF47 and ARID1A. Moreover, chemoproteomics using a BRD9 inhibitor (I-BRD9, GSK) isolates only ncBAF and does not resolve BAF-specific or PBAF-specific components, including the highly related bromodomain-containing subunit BRD7. We further identified regions of BRD9 and BRD7 that confer specificity of these subunits to ncBAF and PBAF complexes, respectively, resolving their mSWI/SNF binding domains. Using genome-wide ChIP-seq and RNA-seq experiments, we determined that ncBAF complexes target a subset of all mSWI/SNF complex target genes, and that associated GO terms for these genes are involved in epigenetic regulation, hemopoiesis, and differentiation. Finally, we applied a newly generated approach to deriving functional relationships within and between protein complex families from shRNA and CRISPR-based genetic screening datasets across hundreds of cancer cell lines to explore ncBAF-specific subunits. We find that ncBAF-specific complex subunits form a distinct functional module, supporting biochemical studies and pointing to the specific and divergent functions of the ncBAF configuration. Cancers of hematologic origin collectively exhibit the most significant responses to perturbation of ncBAF subunits, substantiating previous small-molecule screening efforts using BRD9 bromodomain inhibitors. These data demonstrate that ncBAF complexes represent a novel mSWI/SNF complex composition with distinct function in cancer. This abstract is also being presented as Poster 14. Citation Format: Brittany C. Michel, Andrew R. D'Avino, Josh Pan, Robin M. Meyers, Aviad Tsherniak, Paola Grandi, Phillip Humphreys, Neil Garton, Rab K. Prinja, Cigall Kadoch. BRD9 defines a novel mammalian SWI/SNF (BAF) complex configuration which supports proliferation in AML [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR15.

Deletion of SMARCA4 impairs alveolar epithelial type II cells proliferation and aggravates pulmonary fibrosis in mice

Alveolar epithelial cells (AECs) injury and failed reconstitution of the AECs barrier are both integral to alveolar flooding and subsequent pulmonary fibrosis (PF). Nevertheless, the exact mechanisms regulating the regeneration of AECs post-injury still remain unclear. SMARCA4 is a part of the large ATP-dependent chromatin remodelling complex SWI/SNF, which is essential for kidney and heart fibrosis. We investigates SMARCA4 function in lung fibrosis by establishing PF mice model with bleomycin firstly and found that the expression of SMARCA4 was mainly enhanced in alveolar type II (ATII) cells. Moreover, we established an alveolar epithelium-specific SMARCA4-deleted SP-C-rtTA/(tetO)7-Cre/SMARCA4f/f mice (SOSM4Δ/Δ) model, as well as a new SMARCA4-deleted alveolar type II (ATII)-like mle-12 cell line. We found that the bleomycin-induced PF was more aggressive in SOSM4Δ/Δ mice. Also, the proliferation of ATII cells was decreased with the loss of SMARCA4 in vivo and in vitro. In addition, we observed increased proliferation of ATII cells accompanied by abnormally high expression of SMARCA4 in human PF lung sections. These data uncovered the indispensable role of SMARCA4 in the proliferation of ATII cells, which might affect the progression of PF.

CHD8short, a naturally-occurring truncated form of a chromatin remodeler lacking the helicase domain, is a potent transcriptional coregulator

Chromodomain-Helicase-DNA binding protein 8 (CHD8) is a member of a large family of eukaryotic ATP-dependent chromatin remodeling complexes. Loss of function alleles of human chd8 are correlated with autism spectrum disorder. The CHD subfamily members contain a tandem pair of chromodomains that are adjacent to a centrally located Snf2-like helicase domain. An alternatively spliced variant mRNA of CHD8 was identified years ago in mammals that encode a truncated form of the protein, called Duplin, that lacks the helicase domain and everything else in the carboxyl direction. We are using zebrafish to explore the functions of CHD8, especially the truncated form that we refer to as CHD8short (CHD8S). The mRNA for CHD8S is expressed differentially during embryonic development. Using a PCR assay we detected expression of putative zebrafish chd8s mRNA that is barely detectable during early embryogenesis (shield stage at 6h), but increases markedly soon thereafter at 80–90% epiboly (9h) and bud stages (10h), with a return to low levels in 16-somite (17h) and 24hpf embryos. Except for high expression during the shield stage, steady-state levels of chd8l (long) mRNA are relatively constant during the same period of development. We subcloned both chd8l and chd8s cDNAs into expression vector plasmids for use in transient transfection experiments in zebrafish ZF4 cells. In some experiments the luciferase reporter gene was driven by a synthetic promoter that is responsive to activation by ZNF143 activator protein, a known interacting protein with CHD8 in mammalian cells. Whereas CHD8L was a modest coactivator, CHD8S was a potent coactivator, a surprising result since CHD8S is lacking a critical domain to function as a chromatin remodeler enzyme. CHD8S coactivator function is dependent on a region of the protein within the first 50 amino-terminal amino acids. In transient transfection experiments using a Lef1/β-catenin reporter gene, CHD8S was a modest repressor, but deletion of 50 or more amino-terminal amino acids converted it to a coactivator. When synthetic chd8s mRNA was injected into zebrafish embryos in order to overexpress CHD8S, we observed significant brain disruption phenotypes.

SWI/SNF Infobase-An exclusive information portal for SWI/SNF remodeling complex subunits.

Chromatin remodeling complexes facilitate the access of condensed genomic DNA during transcription, replication, and repair, by altering the histone-DNA contacts in the nucleosome structures. SWI/SNF (SWItch/Sucrose Non-Fermentable) family of ATP dependent chromatin remodeling complexes have been documented for their tumour suppressor function. Recent studies have reported the high frequency of cancer causing mutations in this protein family. There exist multiple subunits for this complex and can form context-dependent sub-complexes. The cataloguing of individual subunits of this complex is essential for understanding their specific functions and their mechanism of action during chromatin remodeling. This would also facilitate further studies to characterize cancer causing mutations in SWI/SNF subunits. In the current study, a database containing information on the subunits of SWI/SNF-α (BRG1/BRM-Associated Factors (BAF)) and SWI/SNF-β (Polybromo-Associated BAF (PBAF)) sub classes of SWI/SNF family has been curated and catalogued. The database hosts information on 27 distinct SWI/SNF subunits from 20 organisms spanning a wide evolutionary range of eukaryotes. A non-redundant set of 522 genes coding for SWI/SNF subunits have been documented in the database. A detailed annotation on each subunit, including basic protein/gene information, protein sequence, functional domains, homologs and missense mutations of human proteins have been provided with a user-friendly graphical interface. The SWI/SNF Infobase presented here, would be a first of its kind exclusive information portal on SWI/SNF complex subunits and would be a valuable resource for the research community working on chromatin remodeling. The database is available at http://scbt.sastra.edu/swisnfdb/index.php.

SKIP controls flowering time via the alternative splicing of SEF pre-mRNA in Arabidopsis

BackgroundSimilar to other eukaryotes, splicing is emerging as an important process affecting development and stress tolerance in plants. Ski-interacting protein (SKIP), a splicing factor, is essential for circadian clock function and abiotic stress tolerance; however, the mechanisms whereby it regulates flowering time are unknown.ResultsIn this study, we found that SKIP is required for the splicing of serratedleaves and early flowering (SEF) pre-messenger RNA (mRNA), which encodes a component of the ATP-dependent SWR1 chromatin remodeling complex (SWR1-C). Defects in the splicing of SEF pre-mRNA reduced H2A.Z enrichment at FLC, MAF4, and MAF5, suppressed the expression of these genes, and produced an early flowering phenotype in skip-1 plants.ConclusionsOur findings indicate that SKIP regulates SWR1-C function via alternative splicing to control the floral transition in Arabidopsis thaliana.

The nuclear mitotic apparatus protein NuMA controls rDNA transcription and mediates the nucleolar stress response in a p53-independent manner.

The nuclear mitotic apparatus protein, NuMA, is involved in major cellular events such as DNA damage response, apoptosis and p53-mediated growth-arrest, all of which are under the control of the nucleolus upon stress. Proteomic investigation has identified NuMA among hundreds of nucleolar proteins. Yet, the precise link between NuMA and nucleolar function remains undetermined. We confirm that NuMA is present in the nucleolus and reveal redistribution of NuMA upon actinomycin D or doxorubicin-induced nucleolar stress. NuMA coimmunoprecipitates with RNA polymerase I, with ribosomal proteins RPL26 and RPL24, and with components of B-WICH, an ATP-dependent chromatin remodeling complex associated with rDNA transcription. NuMA also binds to 18S and 28S rRNAs and localizes to rDNA promoter regions. Downregulation of NuMA expression triggers nucleolar stress, as shown by decreased nascent pre-rRNA synthesis, fibrillarin perinucleolar cap formation and upregulation of p27kip1, but not p53. Physiologically relevant nucleolar stress induction with reactive oxygen species reaffirms a p53-independent p27kip1 response pathway and leads to nascent pre-rRNA reduction. It also promotes the decrease in the amount of NuMA. This previously uncharacterized function of NuMA in rDNA transcription and p53-independent nucleolar stress response supports a central role for this nuclear structural protein in cellular homeostasis.