Reduced Chromatin Accessibility Research Articles

Systematic assessment of ISWI subunits shows that NURF creates local accessibility for CTCF

Catalytic activity of the imitation switch (ISWI) family of remodelers is critical for nucleosomal organization and DNA binding of certain transcription factors, including the insulator protein CTCF. Here we define the contribution of individual subcomplexes by deriving a panel of isogenic mouse stem cell lines, each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to a strong reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. This affects adjacent nucleosome occupancy and CTCF binding. At a group of sites with reduced chromatin accessibility, CTCF binding persists but cohesin occupancy is reduced, resulting in decreased insulation. These results suggest that CTCF binding can be separated from its function as an insulator in nuclear organization and identify a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites.

Exploring the epigenetic profile of ID4 in breast cancer: bioinformatic insights into methylation patterns and chromatin accessibility dynamics.

Inhibitor of differentiation 4 (ID4) is a dominant-negative regulator of basic helix-loop-helix (bHLH) transcription factors. The expression of ID4 is dysregulated in various breast cancer subtypes, indicating a potential role for ID4 in subtype-specific breast cancer development. This study aims to elucidate the epigenetic regulation of ID4 within breast cancer subtypes, with a particular focus on DNA methylation and chromatin accessibility. Bioinformatic analyses were conducted to assess DNA methylation and chromatin accessibility in ID4 regulatory regions across breast cancer subtypes. Gene Set Enrichment Analysis (GSEA) was conducted to identify related gene sets. Transcription factor binding within ID4 enhancer and promoter regions was explored. In vitro experiments involved ER+ breast cancer cell lines treated with estradiol (E2) and Tamoxifen. Distinct epigenetic profiles of ID4 were observed, revealing increased methylation and reduced chromatin accessibility in luminal subtypes compared to the basal subtype. Gene Set Enrichment Analysis (GSEA) implicated estrogen-related pathways, suggesting a potential link between estrogen signaling and the regulation of ID4 expression. Transcription factor analysis identified ER and FOXA1 as regulators of ID4 enhancer regions. In vitro experiments confirmed the role of ER, demonstrating reduced ID4 expression and increased methylation with estradiol treatment. Conversely, Tamoxifen treatment increased ID4 expression, indicating the potential involvement of ER signaling through ERα in the epigenetic regulation of ID4 in breast cancer cells. This study shows the intricate epigenetic regulation of ID4 in breast cancer, highlighting subtype-specific differences in DNA methylation and chromatin accessibility.

ARID1B controls transcriptional programs of axon projection in an organoid model of the human corpus callosum

Mutations in ARID1B, a member of the mSWI/SNF complex, cause severe neurodevelopmental phenotypes with elusive mechanisms in humans. The most common structural abnormality in the brain of ARID1B patients is agenesis of the corpus callosum (ACC), characterized by the absence of an interhemispheric white matter tract that connects distant cortical regions. Here, we find that neurons expressing SATB2, a determinant of callosal projection neuron (CPN) identity, show impaired maturation in ARID1B+/- neural organoids. Molecularly, a reduction in chromatin accessibility of genomic regions targeted by TCF-like, NFI-like, and ARID-like transcription factors drives the differential expression of genes required for corpus callosum (CC) development. Through an invitro model of the CC tract, we demonstrate that this transcriptional dysregulation impairs the formation of long-range axonal projections, causing structural underconnectivity. Our study uncovers new functions of the mSWI/SNF during human corticogenesis, identifying cell-autonomous axonogenesis defects in SATB2+ neurons as a cause of ACC in ARID1B patients.

Abstract 3304: Modulation of enhancer accessibility by SMARCA2 PROTACs synergize with TEAD inhibitors to suppress growth of SMARCA4 mutant lung cancers

Abstract The SWI/SNF chromatin remodeling complex is frequently mutated in non-small cell lung cancer with a frequency of 33% in advanced stage disease, making it the most commonly altered complex in lung cancer. Among the various subunits of the SWI/SNF complex, SMARCA4 and ARID1A are by far the most frequently mutated. Importantly, multiple clinical reports have shown that SMARCA4 mutant lung cancers have one of the worst prognosis among genetically defined lung cancer subtypes and lack response to both immunotherapy and KRAS G12C inhibitors. Recent reports, and our own data, have identified the paralogue SMARCA2 to be synthetic lethal to SMARCA4 suggesting SMARCA2 could be a high value therapeutic target. Unfortunately, the discovery of selective inhibitors of SMARCA2 has so far been challenging. To overcome this hurdle, we have recently developed novel, potent and selective SMARCA2 degrading small molecules based on proteolysis targeting chimera (PROTAC) technology. We demonstrated that YD23, our lead SMARCA2 PROTAC, potently and selectively induced degradation of SMARCA2. Importantly, we showed that YD23 selectively inhibits growth of SMARCA4 mutant lung cancer cells. Mechanistically, we demonstrated that YD23 decreased chromatin accessibility only in SMARCA4 deficient cells. In particular, SMARCA2 degradation profoundly decreased chromatin accessibility at enhancers of a number of genes with cell cycle and cell growth regulatory roles. Gene expression profiling and pathway analysis indicated that cell cycle genes were downregulated by YD23 consistent with the reduced chromatin accessibility at their cis-regulatory regions. We further showed that YD23 robustly inhibited growth of SMARCA4-mutant xenograft tumors. While this is promising, SMARCA2 degradation by itself does not induce regression of tumors highlighting the need for identification of efficacious combination strategies. Using integrative gene expression, ATAC-Seq and CUT&RUN assays, we uncover a critical role for YAP/TEAD transcription factor signaling axis in mediating pro-growth phenotypes in SMARCA4 mutant tumors. Finally, we show that the combination of TEAD inhibitors synergizes with YD23 to induce profound tumor growth inhibition. In conclusion, our study provides a mechanistic basis for the anti-tumor action of SMARCA2 degraders and introduces a novel combination strategy for future clinical development against a genetically defined subtype of lung cancer with dismal prognosis. Citation Format: Sasi Kotagiri, Nicholas Blazanin, Mohammad Qudrattulah, Yuanxin Xi, Jing Wang, Yonathan Lissanu. Modulation of enhancer accessibility by SMARCA2 PROTACs synergize with TEAD inhibitors to suppress growth of SMARCA4 mutant lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3304.

Abstract 465: Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors

Abstract Most humans infected with Epstein-Barr virus (EBV) remain asymptomatic throughout the whole life. However, EBV is linked to several human cancers, including Burkitt's lymphoma, Hodgkin's disease, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC), and gastric carcinoma. Among these EBV-associated tumors, EBV genome is present in 100% of NPC tumor cells. In NPC cells, EBV establishes a latent infection, expressing limited number of viral proteins but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, also known as BamHI-A rightward transcripts (BARTs). The family of EBV BART RNAs includes BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). Although various functions of BART miRNAs have been identified, the role of EBV lnc-BARTs remains largely unknown. In this study, we provide evidence demonstrating that lnc-BARTs functions as a regulatory long non-coding RNA, referred to as lnc-BARTs. These lnc-BARTs play keys role in modulating the core networks that maintains EBV latency and promotes NPC oncogenesis through epigenetic mechanisms. Our study found that the apoptosis rate of NPC cells is significantly increased following the knockdown of EBV lnc-BARTs, indicating their anti-apoptotic role in EBV-associated tumors. Mass spectrometry analysis identified several host factors, including transcriptional regulators BRD4 and SC35’ interact with EBV lnc-BARTs in NPC cells. RNA-FISH assays showed lnc-BARTs co-localized with BRD4 and SC35 complexes in nuclear s peckles and such interaction were disrupted by JQ1, a BRD4 competitive inhibitor. Binding of lnc-BARTs to BRD4 RNA was confirmed using immunoprecipitation and pulldown assays. ATAC sequencing and transcriptome analyses showed that BART knockdown in EBV-harboring NPC cell line C666-1 led to reduced chromatin accessibility, downregulation of oncogenes, and decreased EBV episome replication and maintenance. Quantitative qPCR and DNA FISH analyses of cell samples treated with shRNA targeting lnc-BARTs or BRD4 reduced EBV copy numbers and EBNA1 RNA expression levels in EBV-infected NPC cells. Finally, ChIP sequencing analysis revealed that lnc-BARTs is critical for BRD4 binding to the oriP region of the EBV genome. These findings support a mechanism by which EBV lnc-BARTs epigenetically regulate host gene expression through functional interactions with elongation regulatory machinery and contribute to the maintenance of EBV latent genome by interaction with BRD4 complex. Citation Format: Jiayan Liu, Bobo Wing-Yee Mok, Honglin Chen. Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 465.

Sall4 regulates posterior trunk mesoderm development by promoting mesodermal gene expression and repressing neural genes in the mesoderm.

The trunk axial skeleton develops from paraxial mesoderm cells. Our recent study demonstrated that conditional knockout of the stem cell factor Sall4 in mice by TCre caused tail truncation and a disorganized axial skeleton posterior to the lumbar level. Based on this phenotype, we hypothesized that, in addition to the previously reported role of Sall4 in neuromesodermal progenitors, Sall4 is involved in the development of the paraxial mesoderm tissue. Analysis of gene expression and SALL4 binding suggests that Sall4 directly or indirectly regulates genes involved in presomitic mesoderm differentiation, somite formation and somite differentiation. Furthermore, ATAC-seq in TCre; Sall4 mutant posterior trunk mesoderm shows that Sall4 knockout reduces chromatin accessibility. We found that Sall4-dependent open chromatin status drives activation and repression of WNT signaling activators and repressors, respectively, to promote WNT signaling. Moreover, footprinting analysis of ATAC-seq data suggests that Sall4-dependent chromatin accessibility facilitates CTCF binding, which contributes to the repression of neural genes within the mesoderm. This study unveils multiple mechanisms by which Sall4 regulates paraxial mesoderm development by directing activation of mesodermal genes and repression of neural genes.

The ncBAF Complex Regulates Transcription in AML Through H3K27ac Sensing by BRD9.

The bromodomain-containing protein BRD9 is essential for AML cell viability, but it is unclear whether this requirement is due to the protein's role as an epigenetic reader. We inhibited this activity and identified altered gene-distal chromatin regulation and transcription consistent with a more mature myeloid cell state.

Inhibition of demethylase by IOX1 modulates chromatin accessibility to enhance NSCLC radiation sensitivity through attenuated PIF1

Chromatin accessibility is a critical determinant of gene transcriptional expression and regulated by histones modification. However, the potential for manipulating chromatin accessibility to regulate radiation sensitivity remains unclear. Our findings demonstrated that the histone demethylase inhibitor, 5-carboxy-8-hydroxyquinoline (IOX1), could enhance the radiosensitivity of non-small cell lung cancer (NSCLC) in vitro and in vivo. Mechanistically, IOX1 treatment reduced chromatin accessibility in the promoter region of DNA damage repair genes, leading to decreased DNA repair efficiency and elevated DNA damage induced by γ irradiation. Notably, IOX1 treatment significantly reduced both chromatin accessibility and the transcription of phytochrome interacting factor 1 (PIF1), a key player in telomere maintenance. Inhibition of PIF1 delayed radiation-induced DNA and telomeric DNA damage repair, as well as increased radiosensitivity of NSCLC in vitro and in vivo. Further study indicated that the above process was regulated by a reduction of transcription factor myc-associated zinc finger protein (MAZ) binding to the distal intergenic region of the PIF1. Taken together, IOX1-mediated demethylase inactivation reduced chromatin accessibility, leading to elevated telomere damage which is partly due to PIF1 inhibition, thereby enhancing NSCLC radiosensitivity.

Epigenetic and Metabolic Reprogramming of Fibroblasts in Crohn's Disease Strictures Reveals Histone Deacetylases as Therapeutic Targets.

No effective therapeutic intervention exists for intestinal fibrosis in Crohn's disease [CD]. We characterized fibroblast subtypes, epigenetic and metabolic changes, and signalling pathways in CD fibrosis to inform future therapeutic strategies. We undertook immunohistochemistry, metabolic, signalling pathway and epigenetic [Transposase-Accessible Chromatin using sequencing] analyses associated with collagen production in CCD-18Co intestinal fibroblasts and primary fibroblasts isolated from stricturing [SCD] and non-stricturing [NSCD] CD small intestine. SCD/NSCD fibroblasts were cultured with TGFβ and valproic acid [VPA]. Stricturing CD was characterized by distinct histone deacetylase [HDAC] expression profiles, particularly HDAC1, HDAC2, and HDAC7. As a proxy for HDAC activity, reduced numbers of H3K27ac+ cells were found in SCD compared to NSCD sections. Primary fibroblasts had increased extracellular lactate [increased glycolytic activity] and intracellular hydroxyproline [increased collagen production] in SCD compared to NSCD cultures. The metabolic effect of TGFβ stimulation was reversed by the HDAC inhibitor VPA. SCD fibroblasts appeared 'metabolically primed' and responded more strongly to both TGFβ and VPA. Treatment with VPA revealed TGFβ-dependent and TGFβ-independent Collagen-I production in CCD-18Co cells and primary fibroblasts. VPA altered the epigenetic landscape with reduced chromatin accessibility at the COL1A1 and COL1A2 promoters. Increased HDAC expression profiles, H3K27ac hypoacetylation, a significant glycolytic phenotype and metabolic priming characterize SCD-derived as compared to NSCD fibroblasts. Our results reveal a novel epigenetic component to Collagen-I regulation and TGFβ-mediated CD fibrosis. HDAC inhibitor therapy may 'reset' the epigenetic changes associated with fibrosis.

Epigenetic landscape analysis reveals the significance of early reduced chromatin accessibility in osteoclastogenesis.

Recently improved techniques could provide snapshots of chromatin structure generated based on chromatin accessibility. Since chromatin accessibility determines transcriptional potential, it has been attempted in a variety of cell systems. However, there has been no genome-wide analysis of chromatin accessibility for the entire murine osteoclast (OC) differentiation process. We performed an Assay for Transposase-Accessible Chromatin (ATAC)-sequencing (seq) during RANKL-induced OC differentiation and found that global chromatin accessibility decreased, especially early in OC differentiation. The global histone H3K27Ac level, an active histone modification mark, was diminished during OC differentiation by western blot and histone extract experiments. Its genomic enrichment was also reduced based on publicly available H3K27Ac chromatin immunoprecipitation (ChIP)-seq data. ATAC-seq and H3K27Ac ChIP-seq data demonstrated that RANKL induced a less accessible chromatin state during OC differentiation. Restoration of reduced H3K27Ac, presumably representing accessible states upon acetate treatment, suppresses OC differentiation by provoking immune-related gene expression. Subsequential integrative analysis of ATAC-seq, RNA-seq after acetate treatment, and H3K27Ac ChIP-seq reveals that Irf8 and its downstream targets are the most vulnerable to chromatin accessibility changes and acetate supplementation. Taken together, our study generated chromatin accessibility maps during the whole OC differentiation and suggested perturbation of chromatin accessibility might be a potential therapeutic strategy for excessive OC diseases.

Abstract A064: Establishing the cellular and molecular impacts of the dual BRM/BRG1 inhibitor FHD-286 on pre-clinical models of non-small cell lung cancer (NSCLC)

Abstract The BRG/BRM-associated factor (BAF) complex plays critical roles in chromatin regulation. Within this complex, the two mutually exclusive ATPases BRM and BRG1 (SMARCA2 and SMARCA4, respectively) drive chromatin remodeling activity. Genetic alterations in the SMARCA4 gene, encoding BRG1, are found in approximately 10% of non-small cell lung cancer (NSCLC) patients, leaving this subset of BRG1-deficient tumors highly susceptible to BRM inhibition. In addition, BRG1 has oncogenic activity in NSCLC, rationalizing the use of a dual BRM/BRG1 ATPase inhibitor, such as the highly potent clinical stage compound FHD-286, in NSCLC patients, regardless of SMARCA4 status. Here, we demonstrate that a panel of NSCLC cell lines respond to dual BAF ATPase inhibition in both in vitro and in vivo settings, regardless of SMARCA4 mutation status. Interestingly, dual BRM/BRG1 inhibition in cell lines reduces chromatin accessibility at motifs normally bound by AP-1 transcription factors, which play tumorigenic roles in NSCLC. We also find that expression of the stemness-associated glycoprotein CD44 is decreased by FHD-286, suggesting that dual BRM/BRG1 inhibition impacts the differentiation state of NSCLC. Moreover, transcriptional analyses of NSCLC xenograft models reveal that a basal cell gene expression signature, which is associated with a less differentiated cell state, is downregulated by FHD-286 treatment. Finally, genome-wide CRISPR knockout screening with FHD-286 in 2 NSCLC lines further elucidates mechanisms of action and molecular vulnerabilities induced by dual BAF ATPase inhibition. Collectively, these results indicate a path forward for the development of FHD-286 for the treatment of NSCLC patients. Citation Format: Molly M. Wilson, Ammar Adam, Victoria Amaral, Oliver Mikse, GiNell Elliott, David L. Lahr, Ashley K. Gartin, Jessica Wan, Liv Johannessen, Kana Ichikawa, Katharine Feldman, Jessica Piel, Gabriel J. Sandoval, Richard C. Centore, Steven F. Bellon, Martin Hentemann. Establishing the cellular and molecular impacts of the dual BRM/BRG1 inhibitor FHD-286 on pre-clinical models of non-small cell lung cancer (NSCLC) [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 A064.

Transcription Defects in SF3B1K700E Induce Targetable Alterations in the Chromatin Landscape

Introduction: Acquired mutations, most commonly in RNA splicing factors and epigenetic regulators are thought to be disease drivers of clonal myeloid disorders such as MDS and AML. Both MDS and AML are amenable to epigenetic therapies regardless of underlying mutational subtypes. This prompted our hypothesis that alterations to epigenetic and chromatin landscape is important in pathobiology of clonal myeloid disorders driven by splicing factor mutations. In this study, we demonstrate that SF3B1 mutations induce distinct changes to epigenetic landscape and chromatin organization by altering RNA Polymerase II (Pol2) transcription kinetics. Critically, such epigenetic changes are also targetable - inhibiting components in the H3K4me/Sin3/HDAC pathway reverses Pol2 transcription defects and downstream effects on chromatin organization and genomic integrity. Methods: We generated inducible isogenic K562 cell lines that express single mutant allele of SF3B1 K700Ecombining two genome editing approaches (AAV-intron trap with CRISPR/Cas9 (Boddu et al, Comms Biol, 2021)) (Fig1A). RNAPII transcription kinetics changes were assessed using ChIP-seq for Pol2 and nascent-transcriptome assays (including GRO-seq and transient transcriptome-time lapse sequencing (TT-TL-seq). Chromatin accessibility/nucleosome density and histone marks (H3K4me3, H3K27ac, H3K27me3) were assessed using ATAC-seq and low-input CUT&RUN, respectively. Key results were replicated in patient samples with SF3B1 mutations and in a Sf3b1K700E mouse model. Results: Combining genome-wide approaches (ChIP-seq (Fig1B), GRO-seq, and TT-TL seq (Fig1C)), we found that Pol2 in SF3B1 K700E redistributes into the gene body due to a primary gene-body elongation defect. This transcriptional dysregulation was reflected in downstream effects such as excess R-loops (S9.6 microscopy; Fig1D), transcription-replication conflicts (proximity ligation assay for PCNA-Pol2; Fig1E), and S-phase arrest (flow cytometry; Fig1F). Importantly, normalized Pol2 density at proximal-promoter (p-p) region was reduced in SF3B1 K700E (Fig1G). This correlated with a reduction in p-p H3K4me3 in SF3B1 K700E determined by Cut and Run (Fig1H). ATAC-seq showed a reduction in promoter accessibility (Fig1I) and increased nucleosome promoter density (Fig1J). ChIP-seq for CDK9, a component of p-TEFb pause release complex, showed higher occupancy at promoters in SF3B1 K700E (Fig1K), suggesting that premature promoter pause release of Pol2 is the major driver for the observed reduction in p-p Pol2 density. Consistent with the importance of p-p Pol2 in maintaining accessible chromatin states, our findings suggest that depletion of p-p Pol2 leads to nucleosome repositioning at promoters and loss of chromatin accessibility (Gilchrist et al, Cell, 2010). Given the extensive cross talk between chromatin and transcription, we speculated that modulating epigenetic regulators might improve Pol2 elongation defects and rescue cells from downstream effects. An unbiased shRNA rescue screen, targeting epigenetic regulators and chromatin modifier protein classes, enriched for proteins in the Sin3/HDAC pathway (ING2, HDAC2) (Fig1L). Conversely, knocking down (KD) for WDR5 (H3K4me3 writer complex) further worsened survival. HDAC2 and ING2 KD cells showed reduced R-loops and DNA damage, lower gene-body Pol2 ChIP densities (Fig1M), normalization of p-p H3K4me3 densities (Fig1N), and rescue from S-phase arrest. These effects of WDR5 and HDAC2/ING2 KD on survival in SF3B1-mutant isogenic cells was confirmed in both MDS patient samples (Fig1O)- and Sf3b1K700E murine models (Fig1P)- contexts, using in vitro colony assays. Conclusion: Our findings show that SF3B1 K700E-induced disruption of Pol2 elongation kinetics is reflected in reduced Pol2 density at proximal promoter sites and results in a corresponding reduction in chromatin accessibility and promoter H3K4me3. Through an unbiased shRNA screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, improved the transcription defects and their downstream effects. Our findings shed light on the mechanisms by which oncogenic mutant spliceosomes affect chromatin organization through their effects on Pol II transcription elongation and present a rationale for targeting the Sin3/HDAC complex as a potential therapeutic strategy.

Loss of SWI/SNF Complex Subunit Arid1a in B Cells Promotes Inflammation and Perturbs Germinal Center B Cell Responses

The Switch/Sucrose Non-Fermentable (SWI/SNF) complex is a major mediator of nucleosomal remodeling in eukaryotic genomes and plays a crucial role in the regulation of chromatin accessibility and establishment of gene expression programs. SWI/SNF complex is comprised of ~15 protein subunits, many of which are frequently mutated in several solid tumors and hematological malignancies, including B cell lymphomas. Among SWI/SNF subunits, ARID1a, ARID1B, and SMARCA4 are each mutated in ~10% of Diffuse Large B-Cell Lymphomas, which is a germinal center B cell-derived malignancy representing 95% of all adult lymphomas. Germinal Centers (GC) are microanatomical sites in secondary lymphoid tissues where B cells undergo activation and differentiation, however the role of SWI/SNF complex in GC B cell biology and development of GC-derived malignancies has not been studied. To address this, we investigated the role of Arid1a, the core subunit of SWI/SNF complex, in GC B cell differentiation and function. Using genetic mouse models with constitutive ( CD19 Cre) and activation induced ( Cg1 Cre) B cell-specific deletion of Arid1a, our studies revealed that Arid1a is indispensable for the formation of germinal centers . Arid1a-deficient B cells show profound defects in affinity maturation and fail to mount high-affinity antibody responses. However, in contrast, under in vitro conditions mimicking germinal centers, Arid1a-deficient B cells can efficiently undergo activation and class switch recombination, suggesting that Arid1a-deficient B cells are not inherently defective in their ability to proliferate and differentiate in response to stimulation. Interestingly, while Arid1a-deficient activated B cells display a global reduction in chromatin accessibility, this was also accompanied by induction of inflammatory gene and accessibility signatures. A careful examination of B cell-specific Arid1a-deficient mice demonstrated no apparent defects in the initiation of GC response, though, the sustenance of B cells in the GCs was severely compromised. In addition, Arid1a-deficient GC precursors effectively promoted the differentiation of T follicular helper cells, but also enhanced recruitment of inflammatory monocytes and neutrophils at both early and late stages post-immunization, compared to control mice. These studies suggest that Arid1a loss in B cells instigates an inflammatory milieu which may contribute to a pre-mature termination of GC program. Overall, our studies establish a critical role of SWI/SNF complex in GC B cell biology and provides insights for how loss of Arid1a could contribute to pathogenesis of B cell lymphomas. Our work is consistent with recent paradigms highlighting detrimental effects of an inflammatory micro-environment on germinal centers, which is commonly observed in patients with severe bacterial and viral infections.

Impact of Gamma-Secretase Inhibition on the Multiple Myeloma Immune Microenvironment

Background: Therapy with monoclonal antibodies, chimeric antigen receptor (CAR) T-cells, and bispecific T-cell engagers (BiTE) has significantly changed the treatment landscape for multiple myeloma (MM). B-cell maturation antigen (BCMA), a cell surface protein highly expressed in malignant plasma cells, has been established as an effective MM cell target, however, achieving long-term responses in relapsed or refractory patients remains a challenge. Resistance to BCMA targeting may result from the enzymatic cleavage of BCMA by surface membrane gamma-secretase resulting in reduced target antigen density on MM cells, and from the soluble BCMA ectodomain that may hinder tumor cell recognition through binding to circulating therapeutic antibodies or to CAR T-cells. Methods: We have previously demonstrated that gamma-secretase inhibitors (GSIs) abrogate enzymatic function and increase BCMA surface density on plasma cells with a concomitant decrease in soluble BCMA concentrations [Pont, MJ. et al. Blood, 134(2019)]. In a phase I, first-in-human clinical trial (n=18; relapsed/refractory MM) combining the GSI, crenigacestat, with anti-BCMA CAR T-cell therapy (FCARH143), we recently demonstrated that plasma cell BCMA antibody-binding capacity increased a median of 12-fold among 17/18 (94%) of participants after they received a 5-day GSI “run-in” (25 mg orally administered QOD for 3 doses) [Cowan AJ, et al. Lancet Oncology. 24(7) 2023]. Overall, 78% of participants experienced a very good partial response or better to GSI with FCAR143, but the duration of response was significantly longer in patients without prior anti-BCMA exposure. Here, we report results from an analysis of the bone marrow aspirates collected at enrollment and again within 12 hours of the third “run-in” dose. We leveraged single-cell RNA sequencing (scRNAseq) coupled with the single-cell assay for transposase-accessible chromatin sequencing (scATACseq) to profile the transcriptome and chromatin accessibility of individual cells within the bone marrow microenvironment. Results: Single-cell multi-omic analysis was performed on 32 bone marrow mononuclear cell samples from 16 patients collected before and within 12 hours after the third oral dose of GSI. Six patients had previously been treated with BCMA-directed therapy, including antibody-drug conjugates, CAR T-cells, or BiTEs. After quality filtering, including compensation for ambient RNA, 142,498 cells were analyzed (median 6,373 cells/sample, range 613-17,150 cells/sample). Cells were automatically classified into 92 immune cell types using Celltypist and a reference dataset trained on immune sub-populations from 20 tissues in 18 studies. Comparing samples pre- and post-GSI exposure revealed a significant decrease in the abundance of plasmablasts, non-classical monocytes, germinal center B cells, mid-erythroid cells, and central memory/naïve cytotoxic T cells after GSI treatment. Additionally, gene expression analysis detected increased expression of Notch signaling genes, NEURL1 and HES1, in non-classical monocytes following GSI treatment. Although RNA expression of TNFRSF17 ( BCMA) did not significantly change, chromatin accessibility was considerably higher in patients without a history of prior BCMA-directed therapy following GSI treatment. In non-plasma cells, we observed increased chromatin accessibility after GSI treatment involving genes encoding proteins known to be cleaved by gamma-secretase, such as ERBB4, NOTCH1, NOTCH2, and TNFRSF17. Accessibility of CD38, the target of daratumumab, was significantly increased in B cells, and SLAMF7, the target of elotuzumab, was significantly increased in plasma cells. Conclusions: BCMA cleavage from myeloma cells' surface is a putative resistance mechanism to BCMA-targeting immunotherapy. This study assessed the single-cell transcriptome and chromatin accessibility in the bone marrow environment of 16 patients given GSI monotherapy to ultimately enhance the efficacy of subsequent anti-BCMA CAR T-cell therapy. We found that prior BCMA-targeted therapy resulted in reduced chromatin accessibility within the BCMA epigenome. Surprisingly, GSI administration also modified the epigenome of other gamma-secretase sensitive genes. These data provide insight into the molecular consequences of prior BCMA-targeted therapy and GSI exposure that may inform future studies.

PKCδ Regulates Chromatin Remodeling and DNA Repair through SIRT6.

PKCδ controls sensitivity to irradiation by regulating DNA repair.

The BAF chromatin complex component SMARCC1 does not mediate GLI transcriptional repression of Hedgehog target genes in limb buds

Transcriptional responses to the Hedgehog (HH) signaling pathway are primarily modulated by GLI repression in the mouse limb. Previous studies suggested a role for the BAF chromatin remodeling complex in mediating GLI repression. Consistent with this possibility, the core BAF complex protein SMARCC1 is present at most active limb enhancers including the majority of GLI enhancers. However, in contrast to GLI repression which reduces chromatin accessibility, SMARCC1 maintains chromatin accessibility at most enhancers, including those bound by GLI. Moreover, SMARCC1 binding at GLI-regulated enhancers occurs independently of GLI3. Consistent with previous studies, some individual GLI target genes are mis-regulated in Smarcc1 conditional knockouts, though most GLI target genes are unaffected. Moreover, SMARCC1 is not necessary for mediating constitutive GLI repression in HH mutant limb buds. We conclude that SMARCC1 does not mediate GLI3 repression, which we propose utilizes alternative chromatin remodeling complexes.

Lipid accumulation-mediated histone hypoacetylation drives persistent NK cell dysfunction in anti-tumor immunity

Hyperlipidemia impairs anti-tumor immune responses and is closely associated with increased human cancer incidence and mortality. However, the underlying mechanisms are not well understood. In the present study, we show that natural killer (NK) cells isolated from high-fat-diet mice or treated with oleic acid (OA) invitro exhibit sustainable functional defects even after removal from hyperlipidemic milieu. This is accompanied by reduced chromatin accessibility in the promoter region of NK cell effector molecules. Mechanistically, OA exposure blunts P300-mediated c-Myc acetylation and shortens its protein half-life in NK cells, which in turn reduces P300 accumulation and H3K27 acetylation and leads to persistent NK cell dysfunction. NK cells engineered with hyperacetylated c-Myc mutants surmount the suppressive effect of hyperlipidemia and display superior anti-tumor activity. Our findings reveal the persistent dysfunction of NK cells in dyslipidemia milieu and extend engineered NK cells as a promising strategy for tumor immunotherapy.

MDIG-mediated H3K9me3 demethylation upregulates Myc by activating OTX2 and facilitates liver regeneration

The mineral dust-induced gene (MDIG) comprises a conserved JmjC domain and has the ability to demethylate histone H3 lysine 9 trimethylation (H3K9me3). Previous studies have indicated the significance of MDIG in promoting cell proliferation by modulating cell-cycle transition. However, its involvement in liver regeneration has not been extensively investigated. In this study, we generated mice with liver-specific knockout of MDIG and applied partial hepatectomy or carbon tetrachloride mouse models to investigate the biological contribution of MDIG in liver regeneration. The MDIG levels showed initial upregulation followed by downregulation as the recovery progressed. Genetic MDIG deficiency resulted in dramatically impaired liver regeneration and delayed cell cycle progression. However, the MDIG-deleted liver was eventually restored over a long latency. RNA-seq analysis revealed Myc as a crucial effector downstream of MDIG. However, ATAC-seq identified the reduced chromatin accessibility of OTX2 locus in MDIG-ablated regenerating liver, with unaltered chromatin accessibility of Myc locus. Mechanistically, MDIG altered chromatin accessibility to allow transcription by demethylating H3K9me3 at the OTX2 promoter region. As a consequence, the transcription factor OTX2 binding at the Myc promoter region was decreased in MDIG-deficient hepatocytes, which in turn repressed Myc expression. Reciprocally, Myc enhanced MDIG expression by regulating MDIG promoter activity, forming a positive feedback loop to sustain hepatocyte proliferation. Altogether, our results prove the essential role of MDIG in facilitating liver regeneration via regulating histone methylation to alter chromatin accessibility and provide valuable insights into the epi-transcriptomic regulation during liver regeneration.

Age-Associated Changes in Endothelial Transcriptome and Epigenetic Landscapes Correlate With Elevated Risk of Cerebral Microbleeds.

Background Stroke is a leading global cause of human death and disability, with advanced aging associated with elevated incidences of stroke. Despite high mortality and morbidity of stroke, the mechanisms leading to blood-brain barrier dysfunction and development of stroke with age are poorly understood. In the vasculature of brain, endothelial cells (ECs) constitute the core component of the blood-brain barrier and provide a physical barrier composed of tight junctions, adherens junctions, and basement membrane. Methods and Results We show, in mice, the incidents of intracerebral bleeding increases with age. After isolating an enriched population of cerebral ECs from murine brains at 2, 6, 12, 18, and 24 months, we studied age-associated changes in gene expression. The study reveals age-dependent dysregulation of 1388 genes, including many involved in the maintenance of the blood-brain barrier and vascular integrity. We also investigated age-dependent changes on the levels of CpG methylationand accessible chromatin in cerebral ECs. Our study reveals correlations between age-dependent changes in chromatin structure and gene expression, whereas the dynamics of DNA methylation changes are different. Conclusions We find significant age-dependent downregulation of the Aplnr gene along with age-dependent reduction in chromatin accessibility of promoter region of the Aplnr gene in cerebral ECs. Aplnr is associated with positive regulation of vasodilation and is implicated in vascular health. Altogether, our data suggest a potential role of the apelinergic axis involving the ligand apelin and its receptor to be critical in maintenance of the blood-brain barrier and vascular integrity.

Epigenetic Induction of Smooth Muscle Cell Phenotypic Alterations in Aortic Aneurysms and Dissections.

Smooth muscle cell (SMC) phenotypic switching has been increasingly detected in aortic aneurysm and dissection (AAD) tissues. However, the diverse SMC phenotypes in AAD tissues and the mechanisms driving SMC phenotypic alterations remain to be identified. We examined the transcriptomic and epigenomic dynamics of aortic SMC phenotypic changes in mice with angiotensin II-induced AAD by using single-cell RNA sequencing and single-cell sequencing assay for transposase-accessible chromatin. SMC phenotypic alteration in aortas from patients with ascending thoracic AAD was examined by using single-cell RNA sequencing analysis. Single-cell RNA sequencing analysis revealed that aortic stress induced the transition of SMCs from a primary contractile phenotype to proliferative, extracellular matrix-producing, and inflammatory phenotypes. Lineage tracing showed the complete transformation of SMCs to fibroblasts and macrophages. Single-cell sequencing assay for transposase-accessible chromatin analysis indicated that these phenotypic alterations were controlled by chromatin remodeling marked by the reduced chromatin accessibility of contractile genes and the induced chromatin accessibility of genes involved in proliferation, extracellular matrix, and inflammation. IRF3 (interferon regulatory factor 3), a proinflammatory transcription factor activated by cytosolic DNA, was identified as a key driver of the transition of aortic SMCs from a contractile phenotype to an inflammatory phenotype. In cultured SMCs, cytosolic DNA signaled through its sensor STING (stimulator of interferon genes)-TBK1 (tank-binding kinase 1) to activate IRF3, which bound and recruited EZH2 (enhancer of zeste homolog 2) to contractile genes to induce repressive H3K27me3 modification and gene suppression. In contrast, double-stranded DNA-STING-IRF3 signaling induced inflammatory gene expression in SMCs. In Sting-/- mice, the aortic stress-induced transition of SMCs into an inflammatory phenotype was prevented, and SMC populations were preserved. Finally, profound SMC phenotypic alterations toward diverse directions were detected in human ascending thoracic AAD tissues. Our study reveals the dynamic epigenetic induction of SMC phenotypic alterations in AAD. DNA damage and cytosolic leakage drive SMCs from a contractile phenotype to an inflammatory phenotype.