Controlling Chromatin Accessibility Research Articles

Abstract LB008: Integrative dynamic MultiOMICs analysis identifies cell cycle and DNA damage response phenotypes as downstream consequences of using the PRMT5 inhibitor AZD3470

Abstract Background: PRMT5 is an epigenetic enzyme that catalyzes symmetric di-methylation of arginine (SDMA) of multiple substrates that regulate biological processes including RNA splicing and cell cycle. The role of PRMT5 in controlling chromatin accessibility has been investigated in some cancer contexts, but its role in non-small cell lung cancer (NSCLC) global chromatin regulation is not known. AstraZeneca has developed an MTA-cooperative PRMT5 inhibitor (AZD3470) that selectively inhibits PRMT5 in MTAP-null tumors and currently is in Phase I clinical trial (NCT06130553, NCT06130553). Here we applied integrative dynamic MultiOMICs analysis upon AZD3470 treatment in six MTAP-null NSCLC cancer cell lines to inform on PRMT5’s function in maintaining tumor cell growth and survival. Methods: Utilizing an integrative approach, our study explored the effects of AZD3470 on human MTAP-deleted NSCLC cells. MultiOMICs analysis including SDMA post-translational modification (PTM) scans, ATAC-seq, RNA-seq, and mass spectrometry proteomics were employed to delineate the immediate effects and downstream phenotypes of AZD3470 PRMT5 inhibition. Results: AZD3470 induced significant SDMA modification of splicing factors consistent with known roles for PRMT5. PRMT5 inhibition by AZD3470 led to changes in global chromatin accessibility in NSCLC cells. Specific changes in genes involved in the cell cycle pathway were observed including CDK6. Integrative analysis of transcriptomics and proteomics data consistently showed alterations in cell cycle, DNA damage repair and splicing related pathways upon treatment, consistent with the established biological roles of PRMT5. Alternative splicing analysis showed that AZD3470 treatment induced a unique retained intron within the ATM gene and resulted in reduced ATM protein levels. Conclusion: Here we identify AZD3470-induced changes to protein post-translational modification, chromatin accessibility, gene expression and RNA splicing in MTAP-deficient NSCLC cancer cell lines. Different area of MultiOMICs data together pointed to changes in cell cycle and DNA repair pathways. Further, we identified alternative splicing of ATM as a consequence of AZD3470 PRMT5i treatment resulting in significant reduction of ATM protein expression. Keywords: AZD3470, NSCLC, PRMT5 Inhibition, Chromatin Accessibility, Alternative Splicing, DNA Repair, ATM Gene. Citation Format: Ted Hong, Steven Criscione, Andrew Jarnuczak, Jelena Urosevic, Stephanie Ashenden, Ghaith Hamza, Praveen Kumar, Nevena Cvetesic, Lauren Bradshaw, Shaun Moore, Daniel Karl, Tianhui Zhang, Abel Sousa, Jonathan Cairns, Anthony Iannetta, Andrew Zhang, Eric Miele, John Reicha, Chris Chambers, Ramy Elgendy, Julia Lindgren, Daniel Jachimowicz, Maryam Clausen, Graham Belfield, Tony Cheung, Michael Grondine, James T. Lynch, Ho Man Chan, Susan Critchlow, Emma Dean. Integrative dynamic MultiOMICs analysis identifies cell cycle and DNA damage response phenotypes as downstream consequences of using the PRMT5 inhibitor AZD3470 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB008.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Investigation of regenerative nutrology of cells/molecules to the organism: a systematic review

Introduction: In the context of tissue regeneration and nutrology, stem cells from adult tissue mediate homeostasis and regeneration of tissues and organs. These decisions are directly integrated with the body's energy balance and nutritional status. Endogenous metabolites and dietary nutrients can directly influence epigenetic enzymes. Objective: It was to carry out a systematic review of the regenerative nutrology of cells and molecules in the body. Methods: The systematic review rules of the PRISMA Platform were followed. The research was carried out from April to June 2023 in Scopus, PubMed, Science Direct, Scielo, and Google Scholar databases. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed according to the Cochrane instrument. Results and Conclusion: A total of 112 articles were found. A total of 45 articles were evaluated in full and 25 were included and developed in this systematic review study. Considering the Cochrane tool for risk of bias, the overall assessment resulted in 18 studies with a high risk of bias and 18 studies that did not meet GRADE. It was evidenced that the metabolism of stem cells was centered on the central metabolism of carbon and the balance between glycolysis versus oxidative phosphorylation in the regulation of cell destiny. Epigenetic modifications to DNA and histones, proteins that alter cellular fate, control chromatin accessibility and downstream gene expression. In addition to the connection between metabolism and epigenetic pathways, nutrients can impact cellular status by modulating signaling pathway activity. A clear example is through the mechanistic targeting of the rapamycin (mTOR) signaling pathway, and in particular the mTOR 1 complex. Nutritional supplementation accelerated the healing of skin ulcers and reduced the intensity of wound care in non-malnourished patients.

MED16 Negatively Regulates Erythropoiesis and Myelopoiesis through Modulation of Chromatin Accessibility

Tissue-specific transcription factors work in conjunction with numerous coregulators to modulate transcription networks during hematopoiesis; disruptions in this process may lead to hematopoietic disorders, including myelodysplastic syndromes (MDS). While the Mediator complex is known to regulate various aspects of hematopoiesis, the mechanisms underlying subunit-specific functions have not been fully understood. Here, we uncovered that MED16, a core component of Mediator tail module, serves as a crucial negative regulator of erythropoiesis and myelopoiesis by controlling chromatin accessibility at specific target genes. To investigate the function of MED16 in vivo, we transplanted control (shSCR) or MED16-deficient (shMED16) human cord blood CD34+ hematopoietic stem and progenitor cells (HSPCs) into immunodeficient mice. Mice receiving shMED16 HSPCs showed a higher proportion of human erythroid and myeloid cells in the bone marrow (BM), while B cell and T cell reconstitution remained unaffected. MED16 deficiency led to an increased number of myeloid colonies and a more pronounced increase in erythroid colonies in the colony-forming assay. During erythroid differentiation, MED16 deficiency resulted in an elevated ratio of CD235a+ cells and increased enucleation. Furthermore, hematopoietic-specific Med16 knockout ( Mx1-Cre × Med16 -/- Med16 KO)mice showed normal BM and spleen cellularity, with an increased frequency of common myeloid progenitors (CMPs). Med16 KO mice exhibited a higher frequency of CD11b+ myeloid cells and Ter119+ erythroid cells in the BM and spleen. A higher percentage of enucleated erythrocytes were observed in the BM of Med16 KO mice. Additionally, in the model of phenylhydrazine-induced hemolytic anemia, Med16 KO mice showed accelerated red blood cell recovery and pronounced splenomegaly, highlighting the crucial role of Med16 in stress erythropoiesis. Our findings demonstrate that MED16 plays a repressive role in hematopoietic development. To elucidate how MED16 regulates gene expression, we conducted single-cell RNA-seq and ATAC-seq of cultured CD34+ cells after MED16 knockdown in primary human CD34+ HSPCs. MED16 deficiency resulted in upregulation of erythroid and innate immunity genes in erythroid and myeloid cells, respectively. Interestingly, MED16 deficiency led to significantly increased chromatin accessibility around the transcription start sites of upregulated genes. Considering the Mediator complex often interacts with transcription factors to convey information, we conducted MED16 chromatin IP sequencing (ChIP-seq). The majority of MED16 occupancy peaks were located at promoters (59.68%). Colocalization analysis of MED16 with chromatin occupancy of hematopoietic-related transcription factors revealed significant overlap with RUNX1. To unveil the mechanism of MED16-mediated transcriptional repression, we conducted immunoprecipitation coupled with mass spectrometry on cultured CD34+ cells to identify MED16-interacting nuclear proteins. We identified the FAcilitates Chromatin Transcription (FACT) complex as a potential candidate. ChIP-seq of the FACT complex subunit SPT16 showed weakened chromatin occupancy signals upon MED16 deficiency. Similar results were observed in mouse BM Ter119+ erythroblasts. These findings indicate that MED16 plays a crucial role in modulating chromatin accessibility and transcriptional repression, potentially through cooperation with RUNX1 and the FACT complex. Furthermore, we found that MED16 overexpression impeded erythroid cell enucleation, similar to MDS erythroid dysplasia. We observed higher MED16 expression in BM-derived CD34+ cells from 183 MDS patients across different subtypes compared to healthy individuals. Interestingly, knocking down MED16 in bone marrow mononuclear cells from MDS patients restored normal erythropoiesis. Transcriptome analysis revealed that MED16 deficiency upregulated innate immunity and erythroid genes. Together, these findings shed light on the previously unknown role of MED16 in hematopoiesis and its association with MDS. Our study provides mechanistic insights into the lineage commitment and developmental progression of hematopoiesis governed by Mediator and may have implications for the treatment of hematological disorders.

Molecular Mechanisms of Lupus Susceptibility Allele PBX1D.

Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.

Molecular mechanisms of PBX1 regulation of CD4 +T cell inflammatory phenotypes

Abstract The pre-B cell leukemia homeobox1 (Pbx1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, Pbx1-d that lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4 +T cells from lupus-prone mice and SLE patients than healthy controls. We have shown that overexpression of Pbx1-d in CD4 +T cells impaired Treg cell homeostasis and expanded inflammatory CD4 +T cells. Here, we investigated the molecular mechanisms by which PBX1 regulates T cells. We showed that PBX1 is preferentially expressed in Treg cells over other CD4+ T cell subsets, and that it is negatively regulated by TCR activation. Furthermore, PBX1-D protein was less stable than the normal isoform, PBX1-B, in a pulse chase experiment. PBX1-B but not PBX1-D has two predictive ubiquitin binding sites, K292 and K293, located in the DNA binding domain. Mutation of these two sites reduced PBX1-B stability to a level similar to PBX1-D, suggesting that a loss of function in the lupus associated allele through the ubiquitin-proteasome dependent pathway. In addition, contrary to PBX1-B, PBX1-D is retained in the cytoplasm without the help of the MEIS or PREP1 co-factors, indicating different requirements for nuclear translocation. Overall, these findings suggest that multiple transcriptional and post-transcriptional mechanisms are responsible for PBX1-D loss of function and induction of CD4 +T cell inflammatory phenotypes in SLE. This work was supported by National Institutes of Health Grants AI045050

Abstract 2122: The dual BRM/BRG1 (SMARCA2/4) inhibitor FHD-286 induces differentiation in preclinical models of AML

Abstract The BRG1/BRM-associated factors (BAF) complex (also known as mSWI/SNF) is a critical regulator of the chromatin landscape of the genome. By controlling chromatin accessibility, BAF regulates lineage-specific transcriptional programs, including those important for AML blast cell growth and survival. FHD-286 is a highly potent inhibitor of the BAF catalytic subunits BRM and BRG1 (SMARCA2/4), which demonstrates strong tumor growth inhibition in several AML CDX and PDX models. FHD-286 is being developed for the treatment of relapsed/refractory AML and MDS (see Foghorn website for current status).Investigation of FHD-286 in a broad panel of AML cell lines showed that BAF inhibition affects hematopoietic transcriptional programs important for blast cell self-renewal and identity, agnostic of mutational background. Similarly, FHD-286 demonstrated broad efficacy in ex vivo treatment of AML patient-derived samples from diverse genetic backgrounds, including those with difficult to treat mutational profiles, including mtNPM1, FLT3 ITD, and Inv(3) with EVI1 overexpression. Interestingly, while higher concentrations (≥90 nM) of FHD-286 predominantly induced cytoreduction, lower concentrations (≤30 nM) predominantly led to differentiation-like responses. To investigate this differentiation effect, we performed immunophenotyping of cell lines and primary AML samples following prolonged treatment with FHD-286. Extended treatment (7+ days) with relevant concentrations (5-20 nM) of FHD-286 led to time- and dose-dependent upregulation of the myeloid differentiation marker CD11b, and acquisition of monocyte/metamyelocyte morphology. CD11b+ cells expressed lower levels of the hematopoietic transcription factor PU.1, as well as proliferation and survival proteins Ki67, Myc and BCL2. Additionally, primary patient samples showed a loss of CD34 positivity in blasts and GMP-like cells after 7 days of ex vivo treatment, indicating a decrease in stemness in these populations. Finally, upregulation of CD11b coincided with decreased BRG1 protein, suggesting that immature blasts are characterized by high levels of BRG1. These results suggest that BAF functions to drive transcriptional programs required to maintain AML cells in an undifferentiated state, and that FHD-286 may inhibit AML cell growth by overcoming this differentiation block. Expanding on these findings, we have also demonstrated synergistic activity with multiple combination partners, including cytarabine and decitabine, in vitro. Elaboration of this in both CDX and PDX in vivo models also shows significant survival benefit in difficult to treat mutational backgrounds. Taken together, these findings suggest that FHD-286 is able to target blast progenitor populations that are heavily BRM/BRG1-dependent, and that combination with standard of care agents can achieve profound, mutationally agnostic antitumor activity in AML. Citation Format: Mike Collins, Astrid Thomsen, Ashley Gartin, Gabriel J. Sandoval, Ammar Adam, Sarah Reilly, Laure Delestre, Virginie Penard-Lacronique, Warren Fiskus, Kapil Bhalla, Stephane de Botton, Sam Agresta, Jessica Piel, Murphy Hentemann. The dual BRM/BRG1 (SMARCA2/4) inhibitor FHD-286 induces differentiation in preclinical models of AML [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 2122.

DNA dioxygenases Tet2/3 regulate gene promoter accessibility and chromatin topology in lineage-specific loci to control epithelial differentiation.

Execution of lineage-specific differentiation programs requires tight coordination between many regulators including Ten-eleven translocation (TET) family enzymes, catalyzing 5-methylcytosine oxidation in DNA. Here, by using Keratin 14-Cre-driven ablation of Tet genes in skin epithelial cells, we demonstrate that ablation of Tet2/Tet3 results in marked alterations of hair shape and length followed by hair loss. We show that, through DNA demethylation, Tet2/Tet3 control chromatin accessibility and Dlx3 binding and promoter activity of the Krt25 and Krt28 genes regulating hair shape, as well as regulate interactions between the Krt28 gene promoter and distal enhancer. Moreover, Tet2/Tet3 also control three-dimensional chromatin topology in Keratin type I/II gene loci via DNA methylation-independent mechanisms. These data demonstrate the essential roles for Tet2/3 in establishment of lineage-specific gene expression program and control of Dlx3/Krt25/Krt28 axis in hair follicle epithelial cells and implicate modulation of DNA methylation as a novel approach for hair growth control.

Atherosclerosis licenses for an exceeding immune response in COVID-19 disease

Abstract Background COVID-19 is characterized by emergency hematopoiesis with a dysregulated myeloid compartment, comprising proinflammatory and immunosuppressive immune cells. Preexisting cardiovascular disease (CVD) is a major risk factor for severe and fatal COVID-19 outcomes. Individuals with atherosclerosis are known to have a proinflammatory immune cell phenotype. However, the mechanisms of how CVD causes worse outcomes during SARS-Cov2 infection remain unknown. Purpose To investigate the mechanisms of how immune cells link atherosclerosis to worse COVID-19 outcomes Methods Single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) derived from hospitalized SARS-Cov2 infected patients in an uncomplicated phase of the disease not requiring intensive-care treatment with (n=5) and without (n=6) preexisting atherosclerosis was performed. Results Baseline characteristics between the two groups were similar (atherosclerosis vs. no atherosclerosis: mean age 75 vs. 70 years, oxygen requirement 2.2 vs. 3.2 l/min, CRP 10.7 vs. 6.6 mg/dl, IL-6 61.6 vs. 60.6 pg/ml, all p>0.05). In accordance with previous COVID-19 scRNA-seq studies, we found low-density neutrophils, immature neutrophils, neutrophil like plasmablasts and mostly classical monocytes in the myeloid compartment. Low-density neutrophils from patients with atherosclerosis demonstrated an increased expression of proinflammatory (IL18R1 fold change (fc) = 3.3, IL18RAP fc=1.9, HMGB2 fc=1.8, S100A12 fc=1.7, TLR2 fc=1.5, S100A9 fc=1.4 C3AR1 fc=1.8, TLR4 fc= 1.4, all adjusted p-values <1.3x10–98) and immunosuppressive genes (IL1R2 fc=2.6, ARG1 fc=1.7, ANXA1 fc= 1.6, all adjusted p-values <4.1x10–67). Interestingly, we found an enrichment of proinflammatory COVID-19 specific neutrophil like plasmablasts in patients with atherosclerosis (p=0.049) with an increased expression of inflammatory genes (S100A12 fc=2.5, S100A9 fc=2.5, S100A8 fc=1.8, HMGB2 fc=2.8, IL18R1 fc=3.9 S100A10 fc=2, all adjusted p-values <1.1x10–54). In accordance, monocytes from patients with atherosclerosis showed an enrichment of inflammatory (S100A9 fc=1.6, NEAT1 fc=1.8, C3AR1 fc= 1.5, TLR2 fc= 1.5, IL13RA1 q=1.3, CCR2 fc=1.2, all p-values <1.3x10–60) and immunomodulatory genes (IL1R2 fc=3.5, CD163 fc=2.2, all adjusted p-values <2.7x10–87). Conclusions Our data show for the first time that patients with atherosclerosis have a dysregulated myeloid immune response already in the uncomplicated phase of SARS-CoV-2 infection. Upregulated genes and cell populations found in this study have previously been associated with severe COVID-19. Therefore, the enhanced inflammatory response may contribute to the worse outcome of patients with CVD and might be addressed by antiinflammatory drugs. Further efforts are needed to understand how atherosclerosis may control chromatin accessibility to predispose for an enhanced inflammatory response. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): German Heart Foundation

Opportunistic binding of EcR to open chromatin drives tissue-specific developmental responses

Steroid hormones perform diverse biological functions in developing and adult animals. However, the mechanistic basis for their tissue specificity remains unclear. In Drosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its nuclear receptor, a heterodimer of the EcR and ultraspiracle proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. Here, we investigate the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates morphogenesis. We find that EcR functions bimodally, with both gene repressive and activating functions, even at the same developmental stage. EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Finally, despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, we find that EcR does not control chromatin accessibility at genomic targets. We conclude that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors.

723 DNA dioxygenases Tet2/3 regulate gene promoter accessibility and three-dimensional chromatin topology in lineage-specific loci to control hair growth

Lineage-specific and functionally-related genes frequently form conserved clusters or loci in the mammalian genomes. Execution of lineage-specific differentiation programs requires tight coordination between many regulators including Ten-eleven translocation (TET) family enzymes catalyzing DNA oxidation. However, the role of Tet genes in the control of cell differentiation at the levels of lineage-specific gene loci is unknown. Epithelial keratin genes are organized in the genome into two distinct (Keratin type I and II) loci on mouse chromosomes 11 and 15. Here, by using Keratin 14-driven ablation of Tet genes in skin epithelial cells, we demonstrate that Tet enzymes play essential roles in the control of gene expression in Keratin type I/II gene loci and execution of hair-specific differentiation program. Mice with Keratin 14-driven ablation of Tet2/Tet3 or all three Tet genes exhibit marked alterations of hair shape and length followed by hair loss. We show that through DNA demethylation, Tet2 and Tet3 control chromatin accessibility, Dlx3 binding and promoter activity of the Krt25 and Krt28 genes regulating hair shape, as well as regulate interactions between the Krt28 gene promoter and distal enhancer. However, Tet2/3 also control three-dimensional chromatin topology in Keratin type I/II gene loci via catalytic activity-independent mechanisms. These data demonstrate for the first time the essential roles for Tet2/3 in establishment of lineage-specific gene expression program and control of Dlx3/Krt25/Krt28 axis in hair follicle epithelial cells and implicate modulation of DNA methylation as a novel approach for hair growth control.

RPA1 controls chromatin architecture and maintains lipid metabolic homeostasis.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease, with a prevalence of 25% worldwide. However, the underlying molecular mechanism involved in the development and progression of the NAFLD spectrum remains unclear. Single-stranded DNA-binding protein replication protein A1 (RPA1) participates in DNA replication, recombination, and damage repair. Here, we show that Rpa1+/- mice develop fatty liver disease during aging and in response to a high-fat diet. Liver-specific deletion of Rpa1 results in downregulation of genes related to fatty acid oxidation and impaired fatty acid oxidation, which leads to hepatic steatosis and hepatocellular carcinoma. Mechanistically, RPA1 binds gene regulatory regions, chromatin-remodeling factors, and HNF4A and remodels chromatin architecture, through which RPA1 promotes HNF4A transcriptional activity and fatty acid β oxidation. Collectively, our data demonstrate that RPA1 is an important regulator of NAFLD through controlling chromatin accessibility.

SUMOylation of the chromodomain factor MRG-1 in C. elegans affects chromatin-regulatory dynamics.

Epigenetic mechanisms control chromatin accessibility and gene expression to ensure proper cell fate specification. Histone proteins are integral chromatin components, and their modification promotes gene expression regulation. Specific proteins recognize modified histones such as the chromodomain protein MRG-1. MRG-1 is the Caenorhabditis elegans ortholog of mammalian MRG15, which is involved in DNA repair. MRG-1 binds methylated histone H3 and is important for germline maturation and safeguarding. To elucidate interacting proteins that modulate MRG-1 activity, we performed in-depth protein-protein interaction analysis using immunoprecipitations coupled with massspectrometry. We detected strong association with the Small ubiquitin-like modifier SUMO, and found that MRG-1 is post-translationally modified by SUMO. SUMOylation affects chromatin-binding dynamics of MRG-1, suggesting an epigenetic regulation pathway, which may be conserved.

Regulation of chromatin accessibility by the histone chaperone CAF-1 sustains lineage fidelity

Cell fate commitment is driven by dynamic changes in chromatin architecture and activity of lineage-specific transcription factors (TFs). The chromatin assembly factor-1 (CAF-1) is a histone chaperone that regulates chromatin architecture by facilitating nucleosome assembly during DNA replication. Accumulating evidence supports a substantial role of CAF-1 in cell fate maintenance, but the mechanisms by which CAF-1 restricts lineage choice remain poorly understood. Here, we investigate how CAF-1 influences chromatin dynamics and TF activity during lineage differentiation. We show that CAF-1 suppression triggers rapid differentiation of myeloid stem and progenitor cells into a mixed lineage state. We find that CAF-1 sustains lineage fidelity by controlling chromatin accessibility at specific loci, and limiting the binding of ELF1 TF at newly-accessible diverging regulatory elements. Together, our findings decipher key traits of chromatin accessibility that sustain lineage integrity and point to a powerful strategy for dissecting transcriptional circuits central to cell fate commitment.

Reorganization of postmitotic neuronal chromatin accessibility for maturation of serotonergic identity.

Assembly of transcriptomes encoding unique neuronal identities requires selective accessibility of transcription factors to cis-regulatory sequences in nucleosome-embedded postmitotic chromatin. Yet, the mechanisms controlling postmitotic neuronal chromatin accessibility are poorly understood. Here, we show that unique distal enhancers define the Pet1 neuron lineage that generates serotonin (5-HT) neurons in mice. Heterogeneous single-cell chromatin landscapes are established early in postmitotic Pet1 neurons and reveal the putative regulatory programs driving Pet1 neuron subtype identities. Distal enhancer accessibility is highly dynamic as Pet1 neurons mature, suggesting the existence of regulatory factors that reorganize postmitotic neuronal chromatin. We find that Pet1 and Lmx1b control chromatin accessibility to select Pet1-lineage-specific enhancers for 5-HT neurotransmission. Additionally, these factors are required to maintain chromatin accessibility during early maturation suggesting that postmitotic neuronal open chromatin is unstable and requires continuous regulatory input. Together, our findings reveal postmitotic transcription factors that reorganize accessible chromatin for neuron specialization.

GATA6 defines endoderm fate by controlling chromatin accessibility during differentiation of human-induced pluripotent stem cells.

SUMMARYIn addition to driving specific gene expression profiles, transcriptional regulators are becoming increasingly recognized for their capacity to modulate chromatin structure. GATA6 is essential for the formation of definitive endoderm; however, the molecular basis defining the importance of GATA6 to endoderm commitment is poorly understood. The members of the GATA family of transcription factors have the capacity to bind and alter the accessibility of chromatin. Using pluripotent stem cells as a model of human development, we reveal that GATA6 is integral to the establishment of the endoderm enhancer network via the induction of chromatin accessibility and histone modifications. We additionally identify the chromatin-modifying complexes that interact with GATA6, defining the putative mechanisms by which GATA6 modulates chromatin architecture. The identified GATA6-dependent processes further our knowledge of the molecular mechanisms that underpin cell-fate decisions during formative development.

FAK regulates IL-33 expression by controlling chromatin accessibility at c-Jun motifs

Focal adhesion kinase (FAK) localizes to focal adhesions and is overexpressed in many cancers. FAK can also translocate to the nucleus, where it binds to, and regulates, several transcription factors, including MBD2, p53 and IL-33, to control gene expression by unknown mechanisms. We have used ATAC-seq to reveal that FAK controls chromatin accessibility at a subset of regulated genes. Integration of ATAC-seq and RNA-seq data showed that FAK-dependent chromatin accessibility is linked to differential gene expression, including of the FAK-regulated cytokine and transcriptional regulator interleukin-33 (Il33), which controls anti-tumor immunity. Analysis of the accessibility peaks on the Il33 gene promoter/enhancer regions revealed sequences for several transcription factors, including ETS and AP-1 motifs, and we show that c-Jun, a component of AP-1, regulates Il33 gene expression by binding to its enhancer in a FAK kinase-dependent manner. This work provides the first demonstration that FAK controls transcription via chromatin accessibility, identifying a novel mechanism by which nuclear FAK regulates biologically important gene expression.

Mapping the Effects of Genetic Variation on Chromatin State and Gene Expression Reveals Loci That Control Ground State Pluripotency.

Mouse embryonic stem cells (mESCs) cultured in the presence of LIF occupy a ground state with highly active pluripotency-associated transcriptional and epigenetic circuitry. However, ground state pluripotency in some inbred strain backgrounds is unstable in the absence of ERK1/2 and GSK3 inhibition. Using an unbiased genetic approach, we dissect the basis of this divergent response to extracellular cues by profiling gene expression and chromatin accessibility in 170 genetically heterogeneous mESCs. We map thousands of loci affecting chromatin accessibility and/or transcript abundance, including 10 QTL hotspots where genetic variation at a single locus coordinates the regulation of genes throughout the genome. For one hotspot, we identify a single enhancer variant ∼10 kb upstream of Lifr associated with chromatin accessibility and mediating a cascade of molecular events affecting pluripotency. We validate causation through reciprocal allele swaps, demonstrating the functional consequences of noncoding variation in gene regulatory networks that stabilize pluripotent states invitro.

The transcription factor GLI1 cooperates with the chromatin remodeler SMARCA2 to regulate chromatin accessibility at distal DNA regulatory elements

The transcription factor GLI1 (GLI family zinc finger 1) plays a key role in the development and progression of multiple malignancies. To date, regulation of transcriptional activity at target gene promoters is the only molecular event known to underlie the oncogenic function of GLI1. Here, we provide evidence that GLI1 controls chromatin accessibility at distal regulatory regions by modulating the recruitment of SMARCA2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 2) to these elements. We demonstrate that SMARCA2 endogenously interacts with GLI1 and enhances its transcriptional activity. Mapping experiments indicated that the C-terminal transcriptional activation domain of GLI1 and SMARCA2's central domains, including its ATPase motif, are required for this interaction. Interestingly, similar to SMARCA2, GLI1 overexpression increased chromatin accessibility, as indicated by results of the micrococcal nuclease assay. Further, results of assays for transposase-accessible chromatin with sequencing (ATAC-seq) after GLI1 knockdown supported these findings, revealing that GLI1 regulates chromatin accessibility at several regions distal to gene promoters. Integrated RNA-seq and ATAC-seq data analyses identified a subset of differentially expressed genes located in cis to these regulated chromatin sites. Finally, using the GLI1-regulated gene HHIP (Hedgehog-interacting protein) as a model, we demonstrate that GLI1 and SMARCA2 co-occupy a distal chromatin peak and that SMARCA2 recruitment to this HHIP putative enhancer requires intact GLI1. These findings provide insights into how GLI1 controls gene expression in cancer cells and may inform approaches targeting this oncogenic transcription factor to manage malignancies.

Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle.

The pioneer activity of transcription factors allows for opening of inaccessible regulatory elements and has been extensively studied in the context of cellular differentiation and reprogramming. In contrast, the function of pioneer activity in self-renewing cell divisions and across the cell cycle is poorly understood. Here we assessed the interplay between OCT4 and SOX2 in controlling chromatin accessibility of mouse embryonic stem cells. We found that OCT4 and SOX2 operate in a largely independent manner even at co-occupied sites, and that their cooperative binding is mostly mediated indirectly through regulation of chromatin accessibility. Controlled protein degradation strategies revealed that the uninterrupted presence of OCT4 is required for post-mitotic re-establishment and interphase maintenance of chromatin accessibility, and that highly OCT4-bound enhancers are particularly vulnerable to transient loss of OCT4 expression. Our study sheds light on the constant pioneer activity required to maintain the dynamic pluripotency regulatory landscape in an accessible state.