Subunit Of Chromatin Remodeling Complex Research Articles

Cancer-associated polybromo-1 bromodomain 4 missense variants variably impact bromodomain ligand binding and cell growth suppression

The Polybromo, BRG1-associated factors (PBAF) chromatin remodeling complex subunit Polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic post-translational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, ∼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches.

A59 EXPLORING THE ROLE OF ARID1A IN COLONIC HOMEOSTASIS AND REGENERATION

Abstract Background Recent advances in cancer genome analysis have revealed frequently mutated epigenetic regulators as a novel feature in cancer development. Within this context, the Arid1a (AT-rich interactive domain-containing protein 1A) gene, a subunit of the BAF chromatin remodeling complex, has emerged as a frequently mutated gene in many cancers. Additionally, repeated colonic injury caused by diseases such as inflammatory bowel disease (IBD), is also a major risk factor leading to colorectal cancer. Therefore, understanding the connection between colonic epigenetic regulation and its role in response to injury is particularly important for understanding the pathobiology of colorectal cancer. Aims Aim 1: Exploring the role of Arid1a during colonic homeostasis. Mouse model with Arid1a conditionally knocked out in the colon will be analysed at different time points to test whether Arid1a is required during colonic homeostasis. Aim 2: Determining the role of Arid1a during colonic injury and regeneration. Mice will be exposed to a known colitis model to induce colonic injury. Arid1a will be deleted after injury to find out its role during regeneration and recovery from the injury. Methods To examine the effect of Arid1a loss on colonic homeostasis, mice between 6-10 weeks old were injected with 2mg/20g tamoxifen for three days to induce Arid1a knockout using the VilCreERT2; Arid1afl/fl mousseline. Mice were then euthanised using CO2 chamber and analysed 10 days, 3 weeks, and 8 weeks post Arid1a deletion. Histological analysis was performed to examine morphological changes in the colon. To examine the effect of Arid1a on injury caused by colitis, mice were treated with dextran sulfate sodium (DSS) to cause colitis-like injury. Then, Arid1a is deleted by injecting 2mg/20g tamoxifen. Changes in histology and specific cell types will be examined both in the short- and long-term. Results To test whether Arid1a loss alone causes morphological changes in the colon, I compared the crypt lengths between VilCreERT2; Arid1afl/fl mutant mice with their littermate controls and saw that there was no difference in both morphology and length of the crypts. My results show that Arid1a loss in the colon after injury resulted in impaired regeneration characterized by the prolonged loss of goblet cells and cryptal structure in the distal colon. In the long term, Arid1a loss before or after DSS treatment both lead to tumor formation, suggesting that Arid1a is required for colonic regeneration and repair while suppressing tumorigenesis. Conclusions Based on my results, I conclude that Arid1a deletion alone does not cause significant morphological changes in the colon, but this loss affects colonic regeneration and recovery compared to colons with Arid1a intact. In the long term, the dysregulated colonic regeneration may lead to tumorigenesis. Funding Agencies University of Toronto Department of Molecular Genetics

Cervical lymphoepithelioma-like carcinoma with deficient mismatch repair and loss of SMARCA4/BRG1: a case report and five related cases

BackgroundWe encountered a cervical lymphoepithelial carcinoma (LEC) possessing a predominantly solid architecture with deficient mismatch repair (dMMR) and loss of expression of the SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complex subunit. This is the first case report of LEC with dMMR and loss of SWI/SNF complex subunit.Case presentationA 34-year-old woman presented at our hospital with menstrual irregularities and abnormal vaginal bleeding. Magnetic resonance imaging revealed an exophytic mass in the posterior uterine cervix. Biopsy specimens confirmed squamous cell carcinoma with a 2018 International Federation of Gynecology and Obstetrics (FIGO) uterine cervical cancer stage of IB2. In a subsequent conization specimen, the tumor appeared exophytic. Microscopically, the tumor cells formed a predominant solid architecture. Abundant lymphocytic infiltration was observed. The pathological diagnosis indicated human papillomavirus (HPV)-associated squamous cell carcinoma with LEC pattern and pT1b2. Immunohistochemically, high programmed death-ligand 1 (PD-L1) expression, dMMR, and loss of the switch/sucrose non-fermentable family-related, matrix-associated, actin-dependent regulator of chromatin subfamily member 4 (SMARCA4)/BRG1, an SWI/SNF complex subunit, were observed. The patient underwent a radical hysterectomy and is alive without disease one year and five months later. Our analysis of five additional LEC cases revealed a consistent association with high-risk HPV and elevated PD-L1 expression. In addition to the present case, another patient exhibited dMMR. The SWI/SNF complex was retained except in the present case. The prognosis was favorable in all cases.ConclusionsThis unique case of LEC with dMMR suggests a distinct clinical entity with potential immunotherapy implications. Analysis of the other five LEC cases revealed that LEC was immune hot, and immune checkpoint inhibitors may be effective. The two dMMR cases showed loss of MLH1 and PMS2 expressions, and prominently high tumor PD-L1 expression. In those cases, dMMR might have contributed to the morphological characteristics of LEC.

Abstract A048: Novel murine models of ARID1A and PBRM1-deficient cholangiocarcinoma for preclinical discovery and development

Abstract Genes encoding SWI/SNF chromatin remodeling complex subunits are recurrently mutated across human cancers. In cholangiocarcinoma (CCA), AT-rich interaction domain-1A (ARID1A) and polybromo-1 (PBRM1) loss of function mutations occur in approximately 18 and 10% of cases respectively. Yet our mechanistic understanding and ability to therapeutically exploit ARID1A and PBRM1 deficiency are immature. Genetically accurate preclinical models are needed to define the role of ARID1A and PBRM1 and test therapeutic approaches in CCA. Analysis of human CCA tumors showed that loss of function mutations in ARID1A and PBRM1 frequently co-occur with each other and with PI3K/AKT pathway activation. Therefore, we developed novel murine models of cholangiocarcinoma initiated by Arid1a knockout or dual Arid1a/Pbrm1 knockout in the context of AKT activation, employing a surgical biliary transfection method for somatic gene editing with CRISPR/Cas9. Mice with cholangiocyte Arid1a knockout and AKT activation (AAC) or dual Arid1a/Pbrm1 knockout and AKT activation (APAC) formed numerous liver tumors within 8 months. Histologically, tumors were predominantly CCA and mixed HCC/CCA, with a similar spectrum of tumors across AAC and APAC mice. Gene editing with CRISPR/Cas9 was confirmed at the DNA level, and loss of protein expression was confirmed in tumor cells by immunohistochemistry. Similar to human CCA, many tumors were densely infiltrated with cancer-associated fibroblasts and a variable degree of CD45+ immune cells. To generate scalable models for preclinical testing, tumors from AAC and APAC mice were dissociated to create cell lines for in vitro studies and propagated by orthotopic transplantation for in vivo studies. Cell lines and orthotopic tumors were validated and retained the genetic features and histologic characteristics of the parental tumors. Collectively these studies have produced a suite of novel, well-characterized, genetically relevant mouse models of epigenetic dysregulation in CCA due to ARID1A and PBRM1 deficiency, which can be used broadly for discovery research and preclinical testing of novel treatment approaches. Citation Format: Caitlin B. Conboy, Jennifer L. Tomlinson, Ryan D. Watkins, Jayla T. Millender, Danielle M. Carlson, Rory L. Smoot, Keith D. Robertson, Greg J. Gores. Novel murine models of ARID1A and PBRM1-deficient cholangiocarcinoma for preclinical discovery and development [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A048.

PBRM1 mutations might render a subtype of biliary tract cancers sensitive to drugs targeting the DNA damage repair system

Polybromo-1 (PBRM1) loss of function mutations are present in a fraction of biliary tract cancers (BTCs). PBRM1, a subunit of the PBAF chromatin-remodeling complex, is involved in DNA damage repair. Herein, we aimed to decipher the molecular landscape of PBRM1 mutated (mut) BTCs and to define potential translational aspects. Totally, 1848 BTC samples were analyzed using next-generation DNA-sequencing and immunohistochemistry (Caris Life Sciences, Phoenix, AZ). siRNA-mediated knockdown of PBRM1 was performed in the BTC cell line EGI1 to assess the therapeutic vulnerabilities of ATR and PARP inhibitors in vitro. PBRM1 mutations were identified in 8.1% (n = 150) of BTCs and were more prevalent in intrahepatic BTCs (9.9%) compared to gallbladder cancers (6.0%) or extrahepatic BTCs (4.5%). Higher rates of co-mutations in chromatin-remodeling genes (e.g., ARID1A 31% vs. 16%) and DNA damage repair genes (e.g., ATRX 4.4% vs. 0.3%) were detected in PBRM1-mutated (mut) vs. PBRM1-wildtype (wt) BTCs. No difference in real-world overall survival was observed between PBRM1-mut and PBRM1-wt patients (HR 1.043, 95% CI 0.821–1.325, p = 0.731). In vitro, experiments suggested that PARP ± ATR inhibitors induce synthetic lethality in the PBRM1 knockdown BTC model. Our findings served as the scientific rationale for PARP inhibition in a heavily pretreated PBRM1-mut BTC patient, which induced disease control. This study represents the largest and most extensive molecular profiling study of PBRM1-mut BTCs, which in vitro sensitizes to DNA damage repair inhibiting compounds. Our findings might serve as a rationale for future testing of PARP/ATR inhibitors in PBRM1-mut BTCs.

Knockdown of DOM/Tip60 Complex Subunits Impairs Male Meiosis of Drosophila melanogaster.

ATP-dependent chromatin remodeling complexes are involved in nucleosome sliding and eviction and/or the incorporation of histone variants into chromatin to facilitate several cellular and biological processes, including DNA transcription, replication and repair. The DOM/TIP60 chromatin remodeling complex of Drosophila melanogaster contains 18 subunits, including the DOMINO (DOM), an ATPase that catalyzes the exchange of the canonical H2A with its variant (H2A.V), and TIP60, a lysine-acetyltransferase that acetylates H4, H2A and H2A.V histones. In recent decades, experimental evidence has shown that ATP-dependent chromatin remodeling factors, in addition to their role in chromatin organization, have a functional relevance in cell division. In particular, emerging studies suggested the direct roles of ATP-dependent chromatin remodeling complex subunits in controlling mitosis and cytokinesis in both humans and D. melanogaster. However, little is known about their possible involvement during meiosis. The results of this work show that the knockdown of 12 of DOM/TIP60 complex subunits generates cell division defects that, in turn, cause total/partial sterility in Drosophila males, providing new insights into the functions of chromatin remodelers in cell division control during gametogenesis.

Zebrafish smarcc1a mutants reveal requirements for BAF chromatin remodeling complexes in distinguishing the atrioventricular canal from the cardiac chambers.

Essential patterning processes transform the heart tube into a compartmentalized organ with distinct chambers separated by an atrioventricular canal (AVC). This transition involves the refinement of expression of genes that are first found broadly throughout the heart tube and then become restricted to the AVC. Despite the importance of cardiac patterning, we do not fully understand the mechanisms that limit gene expression to the AVC. We show that the zebrafish gene smarcc1a, encoding a BAF chromatin remodeling complex subunit homologous to mammalian BAF155, is critical for cardiac patterning. In smarcc1a mutants, myocardial differentiation and heart tube assembly appear to proceed normally. Subsequently, the smarcc1a mutant heart fails to exhibit refinement of gene expression patterns to the AVC, and the persistence of broad gene expression is accompanied by failure of chamber expansion. In addition to their cardiac defects, smarcc1a mutants lack pectoral fins, indicating similarity to tbx5a mutants. However, comparison of smarcc1a and tbx5a mutants suggests that perturbation of tbx5a function is not sufficient to cause the smarcc1a mutant phenotype. Our data indicate an important role for Smarcc1a-containing chromatin remodeling complexes in regulating the changes in gene expression and morphology that distinguish the AVC from the cardiac chambers.

Histone chaperones AtChz1A and AtChz1B are required for H2A.Z deposition and interact with the SWR1 chromatin-remodeling complex in Arabidopsis thaliana.

The histone variant H2A.Z plays key functions in transcription and genome stability in all eukaryotes ranging from yeast to human, but the molecular mechanisms by which H2A.Z is incorporated into chromatin remain largely obscure. Here, we characterized the two homologs of yeast Chaperone for H2A.Z-H2B (Chz1) in Arabidopsis thaliana, AtChz1A and AtChz1B. AtChz1A/AtChz1B were verified to bind to H2A.Z-H2B and facilitate nucleosome assembly invitro. Simultaneous knockdown of AtChz1A and AtChz1B, which exhibit redundant functions, led to a genome-wide reduction in H2A.Z and phenotypes similar to those of the H2A.Z-deficient mutant hta9-1hta11-2, including early flowering and abnormal flower morphologies. Interestingly, AtChz1A was found to physically interact with ACTIN-RELATED PROTEIN 6 (ARP6), an evolutionarily conserved subunit of the SWR1 chromatin-remodeling complex. Genetic interaction analyses showed that atchz1a-1atchz1b-1 was hypostatic to arp6-1. Consistently, genome-wide profiling analyses revealed partially overlapping genes and fewer misregulated genes and H2A.Z-reduced chromatin regions in atchz1a-1atchz1b-1 compared with arp6-1. Together, our results demonstrate that AtChz1A and AtChz1B act as histone chaperones to assist the deposition of H2A.Z into chromatin via interacting with SWR1, thereby playing critical roles in the transcription of genes involved in flowering and many other processes.

Mutual Regulation of ncRNAs and Chromatin Remodeling Complexes in Normal and Pathological Conditions.

Chromatin remodeling is the one of the main epigenetic mechanisms of gene expression regulation both in normal cells and in pathological conditions. In recent years, a growing number of investigations have confirmed that epigenetic regulators are tightly connected and form a comprehensive network of regulatory pathways and feedback loops. Genes encoding protein subunits of chromatin remodeling complexes are often mutated and change their expression in diseases, as well as non-coding RNAs (ncRNAs). Moreover, different mechanisms of their mutual regulation have already been described. Further understanding of these processes may help apply their clinical potential for establishment of the diagnosis, prognosis, and treatment of the diseases. The therapeutic targeting of the chromatin structure has many limitations because of the complexity of its regulation, with the involvement of a large number of genes, proteins, non-coding transcripts, and other intermediary molecules. However, several successful strategies have been proposed to target subunits of chromatin remodeling complexes and genes encoding them, as well as the ncRNAs that regulate the operation of these complexes and direct them to the target gene regions. In our review, we focus on chromatin remodeling complexes and ncRNAs, their mutual regulation, role in cellular processes and potential clinical application.

Abstract 3884: Targeting the ARID1A mutations overcomes primary resistance to ALK inhibitors in EML4-ALK positive NSCLC

Abstract Introduction: Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitors (TKIs) have improved an initial clinical response of non-small-cell lung cancer (NSCLC) patients with ALK-rearrangements. However, a subset of patients shows a poor response to ALK-TKIs. Here, we aimed to identify novel mechanism of primary resistance in preclinical model from a patient who presented an impressive resistance to sequential treatment with ALK TKIs. Experimental design: The PDC YU-1076 cells were established from pleural effusion of the patient at the time he was receiving chemotherapy and radiotherapy after failure of ALK-TKI treatment. Sanger sequencing confirmed ML4-ALK variant 1 detected at the initial diagnosis. No resistance mutations in the ALK kinase domain were detected. To investigate co-occurring genetic alterations, we performed whole exome sequencing (WES) on YU-1076 cells and the matched blood sample. We further investigated the molecular profile of YU-1076 cells using RNA-sequencing analysis. Results: YU-1076 cells exhibited cross-resistance to clinically available ALK-TKIs including crizotinib, ceritinib, alectinib, and lorlatinib. However, immunoblot analysis of YU-1076 cells treated with incremental doses of TKIs unexpectedly revealed an effective reduction of ALK activity and downstream signals. We noticed that YU-1076 cells gradually changed from a small, round shape to a fibroblast-like shape following the treatment of ALK-TKIs. Transcriptome analysis confirmed enrichment for gene signatures related epithelial-to-mesenchymal transition (EMT) in ALK TKI-treated YU-1076 cells, suggesting that ALK-TKIs treatment promotes YU-1076 cells toward a more mesenchymal phenotype. WES analysis identified a major chromatin remodeling complex subunit, AT-rich interacting domain 1A (ARID1A), as well as MYC amplification, CDKN2A loss, and TP53 mutations. We focused on a synthetic lethal strategies using the SFK inhibitor dasatinib and the EZH2 inhibitor GSK126 in ARID1A mutant cancers. The combination of dasatinib with ALK-TKIs restored the sensitivity to the ALK TKIs in YU-1076 cells, accompanied by suppression of EMT marker genes VIM and CDH2 and increase of apoptotic markers BIM, PARP, and CAS3. Congruently, dasatinib significantly impaired tumor growth in YU-1076-xenografts. GSK126 also induced synergistic inhibition of cell growth with upregulation of apoptosis marker genes in YU-1076 cells, but did not affect the expression of EMT marker genes. Conclusion: Our data indicate that ARID1A could be potentially used as a predictive biomarker for unfavorable ALK-TKI response. In this context, a combination strategy of ALK TKI with dasatinib may be effective in overcoming primary resistance. Citation Format: Seung Yeon Oh, You Won Lee, Eun Ji Lee, Ju Young Kim, Sewon Park, Ju Yeon Park, Su-Jin Choi, Mi Ra Yu, Sowon Aum, Jiyun Lee, Chang Gon Kim, Jii Bum Lee, Sun Min Lim, Min Hee Hong, Mi Ran Yun, Byoung Chul Cho. Targeting the ARID1A mutations overcomes primary resistance to ALK inhibitors in EML4-ALK positive NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3884.

PBRM1 bromodomains associate with RNA to facilitate chromatin association.

PBRM1 is a subunit of the PBAF chromatin remodeling complex, which is mutated in 40-50% of clear cell renal cell carcinoma patients. It is thought to largely function as a chromatin binding subunit of the PBAF complex, but the molecular mechanism underlying this activity is not fully known. PBRM1 contains six tandem bromodomains which are known to cooperate in binding of nucleosomes acetylated at histone H3 lysine 14 (H3K14ac). Here, we demonstrate that the second and fourth bromodomains from PBRM1 also bind nucleic acids, selectively associating with double stranded RNA elements. Disruption of the RNA binding pocket is found to compromise PBRM1 chromatin binding and inhibit PBRM1-mediated cellular growth effects.

PlantCHRs: A comprehensive database of plant chromatin remodeling factors

The Snf2 protein family is a group of ATP-dependent chromatin remodeling factors (CHRs) that play an essential role in gene expression regulation. In plants, Snf2 is involved in growth, development, as well as stress resistance. However, only a very limited number of experimentally validated Snf2 have been identified and reported, while the majority remaining undiscovered in most species . In this study, we predicted 3135 Snf2 proteins and 8398 chromatin remodeling complex (CRC) subunits in diverse plant species, and constructed the Plant Chromatin Remodeling Factors Database (PlantCHRs, http://www.functionalgenomics.cn/PlantCHRs/), which provide a comprehensive resource for researchers to access information about plant CHRs. We also developed an online tool capable of predicting CHRs and CRC subunits. Moreover, we investigated the distribution of Snf2 proteins in different species and observed a significant increase in the number of Snf2 proteins and the diversity of the Snf2 subfamily during the evolution, highlighting their evolutionary importance. By analyzing the expression patterns of the Snf2 genes in different tissues of maize and Arabidopsis, we found that the Snf2 proteins may show some conservation across different species in regulating plant growth and development. Over the all, we established a comprehensive database for plant CHRs, which will facilitate the researches on plant chromatin remodeling.

Selective and Cell-Active PBRM1 Bromodomain Inhibitors Discovered through NMR Fragment Screening.

PBRM1 is a subunit of the PBAF chromatin remodeling complex that uniquely contains six bromodomains. PBRM1 can operate as a tumor suppressor or tumor promoter. PBRM1 is a tumor promoter in prostate cancer, contributing to migratory and immunosuppressive phenotypes. Selective chemical probes targeting PBRM1 bromodomains are desired to elucidate the association between aberrant PBRM1 chromatin binding and cancer pathogenesis and the contributions of PBRM1 to immunotherapy. Previous PBRM1 inhibitors unselectively bind SMARCA2 and SMARCA4 bromodomains with nanomolar potency. We used our protein-detected NMR screening pipeline to screen 1968 fragments against the second PBRM1 bromodomain, identifying 17 hits with Kd values from 45 μM to >2 mM. Structure-activity relationship studies on the tightest-binding hit resulted in nanomolar inhibitors with selectivity for PBRM1 over SMARCA2 and SMARCA4. These chemical probes inhibit the association of full-length PBRM1 to acetylated histone peptides and selectively inhibit growth of a PBRM1-dependent prostate cancer cell line.

Human EWS-FLI protein recapitulates in Drosophila the neomorphic functions that induce Ewing sarcoma tumorigenesis.

Ewing sarcoma (EwS) is a human malignant tumor typically driven by the Ewing sarcoma-Friend leukemia integration (EWS-FLI) fusion protein. A paucity of genetically modified animal models, partially owed to the high toxicity of EWS-FLI, hinders research on EwS. Here, we report a spontaneous mutant variant, EWS-FLI1FS, that circumvents the toxicity issue in Drosophila. Through proteomic and genomic analyses, we show that human EWS-FLI1FS interacts with the Drosophila homologues of EWS-FLI human protein partners, including core subunits of chromatin remodeling complexes, the transcription machinery, and the spliceosome; brings about a massive dysregulation of transcription that affects a significant fraction of known targets of EWS-FLI in human cells; and modulates splicing. We also show that EWS-FLI1FS performs in Drosophila the two major neomorphic activities that it is known to have in human cells: activation of transcription from GGAA microsatellites and out competition of ETS transcription factors. We conclude that EWS-FLI1FS reproduces in Drosophila the known oncogenic activities of EWS-FLI that drive EwS tumorigenesis in humans. These results open up an unprecedented opportunity to investigate EWS-FLI's oncogenic pathways in vivo in a genetically tractable organism.

The Drosophila BEAF insulator protein interacts with the polybromo subunit of the PBAP chromatin remodeling complex.

The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. Here, we show that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing and micrococcal nuclease sequencing after RNAi-mediated knockdown in cultured S2 cells. Our results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were downregulated, accompanied by fill-in of the promoter nucleosome-depleted region and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused downregulation of several hundred genes and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. Micrococcal nuclease sequencing supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in nucleosome-depleted region nucleosome occupancy after knockdown of either protein. We discuss the possibility of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. We identify Facilitates Chromatin Transcription (FACT) and Nucleosome Remodeling Factor (NURF) as candidate redundant factors.

The SWC4 subunit of the SWR1 chromatin remodeling complex is involved in varying virulence of Metarhizium brunneum isolates offering role of epigenetic regulation of pathogenicity

ABSTRACT The host – pathogen interaction is a multifactorial process subject to a co-evolutionary arms race consisting of rapid changes in both host and pathogen, controlled at the genetic and epigenetic levels. Previously, we showed intra-species variation in disease progression and pathogenicity in aphids for Metarhizium brunneum isolates MbK and Mb7. Herein, we compared genomic, epigenetic, and metabolomic variations between these isolates and their effects on pathogenicity. Genomic variation could not completely explain the observed differences between the isolates. However, differential N6-adenine methylation (6 mA) and its correlation to reduced expression of the essential SWC4 subunit of SWR1 chromatin-remodelling complex (SWR1-C) led us to hypothesize a role for swc4 in the varying pathogenicity. Mutagenesis of the essential swc4 gene in MbKisolate resulted in reduction of secondary-metabolite (SM) secretion and impaired virulence in Galleria mellonella. Our results suggest the role of SWC4 in the regulation of SMs and the role of both SWC4 and SWR1-C in virulence of M. brunneum isolates. A better understanding of epigenetic regulation of SM production and secretion in entomopathogenic fungi may enable theirmanipulation for better biocontrol performance, and expand possibilities for environmentally friendly pest control.

Endometrial Epithelial ARID1A Is Required for Uterine Immune Homeostasis during Early Pregnancy.

A growing body of work suggests epigenetic dysregulation contributes to endometriosis pathophysiology and female infertility. The chromatin remodeling complex subunit AT-rich interaction domain 1A (ARID1A) must be properly expressed to maintain normal uterine function. Endometrial epithelial ARID1A is indispensable for pregnancy establishment in mice through regulation of endometrial gland function; however, ARID1A expression is decreased in infertile women with endometriosis. We hypothesized that ARID1A performs critical operations in the endometrial epithelium necessary for fertility besides maintaining gland function. To identify alterations in uterine gene expression resulting from loss of epithelial ARID1A, we performed RNA-sequencing analysis on pre-implantation uteri from LtfiCre/+Arid1af/f and control mice. Differential expression analysis identified 4181 differentially expressed genes enriched for immune-related ingenuity canonical pathways including agranulocyte adhesion and diapedesis and natural killer cell signaling. RT-qPCR confirmed an increase in pro-inflammatory cytokine and macrophage-related gene expression but a decrease in natural killer cell signaling. Immunostaining confirmed a uterus-specific increase in macrophage infiltration. Flow cytometry delineated an increase in inflammatory macrophages and a decrease in uterine dendritic cells in LtfiCre/+Arid1af/f uteri. These findings demonstrate a role for endometrial epithelial ARID1A in suppressing inflammation and maintaining uterine immune homeostasis, which are required for successful pregnancy and gynecological health.

OsDDM1b Controls Grain Size by Influencing Cell Cycling and Regulating Homeostasis and Signaling of Brassinosteroid in Rice.

Snf2 family proteins are the crucial subunits of chromatin-remodeling complexes (CRCs), which contributes to the biological processes of transcription, replication, and DNA repair using ATP as energy. Some CRC subunits have been confirmed to be the critical regulators in various aspects of plant growth and development and in epigenetic mechanisms such as histone modification, DNA methylation, and histone variants. However, the functions of Snf2 family genes in rice were poorly investigated. In this study, the relative expression profile of 40 members of Snf2 family in rice was studied at certain developmental stages of seed. Our results revealed that OsCHR741/OsDDM1b (Decrease in DNA methylation 1) was accumulated highly in the early developmental stage of seeds. We further analyzed the OsDDM1b T-DNA insertion loss-of-function of mutant, which exhibited dwarfism, smaller organ size, and shorter and wider grain size than the wild type (Hwayoung, HY), yet no difference in 1,000-grain weight. Consistent with the grain size, the outer parenchyma cell layers of lemma in osddm1b developed more cells with decreased size. OsDDM1b encoded a nucleus, membrane-localized protein and was distributed predominately in young spikelets and seeds, asserting its role in grain size. Meanwhile, the osddm1b was less sensitive to brassinosteroids (BRs) while the endogenous BR levels increased. We detected changes in the expression levels of the BR signaling pathway and feedback-inhibited genes with and without exogenous BR application, and the alterations of expression were also observed in grain size-related genes in the osddm1b. Altogether, our results suggest that OsDDM1b plays a crucial role in grain size via influencing cell proliferation and regulating BR signaling and homeostasis.

Znhit1 controls meiotic initiation in male germ cells by coordinating with Stra8 to activate meiotic gene expression.

The switch from mitosis to meiosis ensures the successive formation of gametes. However, it remains unclear how meiotic initiation occurs within the context of chromatin. Recent studies have shown that zinc finger HIT-type containing 1 (Znhit1), a subunit of the SRCAP chromatin remodeling complex, plays essential roles in modulating the chromatin structure. Herein, we report that the germline-conditional deletion of Znhit1 in male mice specifically blocks meiotic initiation. We show that Znhit1 is required for meiotic prophase events, including synapsis, DNA double-strand break formation, and meiotic DNA replication. Mechanistically, Znhit1 controls the histone variant H2A.Z deposition, which facilitates the expression of meiotic genes, such as Meiosin, but not the expression of Stra8. Interestingly, Znhit1 deficiency disrupts the transcription bubbles of meiotic genes. Thus, our findings identify the essential role of Znhit1-dependent H2A.Z deposition in allowing activation of meiotic gene expression, thereby controlling the initiation of meiosis.

Chromatin-remodeling complexes: Conserved and plant-specific subunits in Arabidopsis.

Adenosine triphosphate-dependent chromatin remodeling complexes are important for the regulation of transcription, DNA replication, and genome stability in eukaryotes. Although genetic studies have illustrated various biological functions of core and accessory subunits of chromatin-remodeling complexes in plants, the identification and characterization of chromatin-remodeling complexes in plants is lagging behind that in yeast and animals. Recent studies determined whether and how the Arabidopsis SWI/SNF, ISWI, INO80, SWR1, and CHD chromatin remodelers function in multi-subunit complexes in Arabidopsis. Both conserved and plant-specific subunits of chromatin-remodeling complexes have been identified and characterized. These findings provide a basis for further studies of the molecular mechanisms by which the chromatin-remodeling complexes function in plants.