Defining the basal-like A-specific enhancer landscape in PDAC

Defining the specificity and P63-dependence of B-STEP

Supplementary Table S3 contains list of genes associated with Gö4Pdx4 super enhancers overlapping TP63.

Verification of P63-dependence and B-STEP enhancer-gene regulations.

Supplementary Table S4 contains L3.6pl TP63-dependent enhancer regions.

Supplementary Table S5 contains list of genes associated with L3.6pl TP63 dependent enhancers.

The file contains additional supplementary methods

Supplementary Table S2 contains GöPdx4 super enhancers regions overlapping TP63

Supplementary Table S6 contains list of qPCR primers used in the study.

Background: The pathogenesis of human Inflammatory Bowel Disease (IBD) is a multifactorial process including interaction between immune, epithelial, and environmental factors. Growing evidence suggests that epithelial cell defects play major role in the pathogenesis of IBD. Epigenetic memory involves lasting, cell-type specific modifications to chromatin at multiple scales, which impacts transcriptional programs required for normal cell functions. A major gap in knowledge are the mechanisms involved in establishing and maintaining memory in cell types and diseases of the intestine. The objective of this study was to determine if intestinal epithelial cells (IECs) retain a memory of altered chromatin architectures and resultant regulation following inflammation. The hypothesis was that the IECs would show lasting altered chromatin structure landscapes (epigenetic memory) in response to inflammation. Methods: We have developed a multicellular model system of IBD using adult stem cell derived organoids from humans with IBD, from regions of the colon with and without active inflammation. We performed ATAC-seq, bulk RNA-seq and single cell RNA-seq on these organoids derived from the uninflamed and inflamed tissues of IBD patients, specifically Ulcerative Colitis (UC). Results: Regions of increased chromatin accessibility are enriched in organoids derived from areas of inflammation in UC patients compared to organoids from patient-matched, uninflamed regions. These open chromatin regions are associated with proinflammatory genes, with modest transcriptional alterations, pointing to an epigenetic inflammatory memory. Motif analysis for accessibly peaks in UC showed an enrichment of the AP-1 family of transcription factors and chromatin remodelers (SMARCA4). This data is consistent with other studies of inflammatory memory from other epithelial cell types, but we show this for the first time in IECs in the context of IBD. Conclusions: This study presents a novel line of investigation and development of model systems to examine epigenetic memory of IECs in human IBD. We expect that our studies will identify new chromatin-based mechanisms of IEC function that are impacted by inflammation. This research was funded by American Gastroenterological Association Research Scholar Award and KL2 Scholar Career Development Award (KL2 TR002379, National Center for Advancing Translational Science). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BACKGROUND & AIMS: The incidence of Crohn ' s disease (CD) continues to increase worldwide. The contribution of CD4 + cell populations remains to be elucidated. We aimed to provide an indepth transcriptional assessment of CD4 + T cells driving chronic in flammation in CD. METHODS: We performed single -cell RNAsequencing in CD4 + T cells isolated from ileal biopsies of patients with CD compared with healthy individuals. Cells underwent clustering analysis, followed by analysis of gene signaling networks. We overlapped our differentially expressed genes with publicly available microarray data sets and performed functional in vitro studies, including an in vitro suppression assay and organoid systems, to model gene expression changes observed in CD regulatory T (Treg) cells and to test predicted therapeutics. RESULTS: We identi fied 5 distinct FOXP3 + regulatory Treg subpopulations. Tregs isolated from healthy controls represent the origin of pseudotemporal development into in flammation - associated subtypes. These proin flammatory Tregs displayed a unique responsiveness to tumor necrosis factor -a signaling with impaired suppressive activity in vitro and an elevated cytokine response in an organoid coculture system. As predicted in silico, the histone deacetylase inhibitor vorinostat normalized gene expression patterns, rescuing the suppressive function of FOXP3 + cells in vitro. CONCLUSIONS: We identified a novel, proinflammatory FOXP3 + T cell subpopulation in patients with CD and developed a pipeline to specifically target these cells using the US Food and Drug Administration - approved drug vorinostat. samples, the contribution of individual cell populations remains largely unknown. To identify these cell types, as well as gene expression changes relevant to CD, recent investigations used single -cell RNA sequencing (scRNA-seq) approaches. Interestingly, Jaeger et al 2 detected an increased T helper 17 (Th17) over regulatory T (Treg) cell to T follicular helper cell ratio from single -cell suspensions of lamina propria isolated from CD lesions. 2 In general, CD4 + T cells develop in the thymus and na & iuml;ve CD4 + T cells encounter polarizing cytokines in the periphery, inducing the expression of specific transcription factors underlying the differentiation into specialized functional subsets. 3 The inflamed mucosa of patients with IBD displays a substantial infiltration of Th17 cells. 4 In agreement with this finding, the levels of Th17excreted cytokines were elevated in inflamed lesions, suggesting a proinflammatory function of these cells. 5,6 The X -chromosome -encoded FOXP3 transcription factor represents the master Treg lineage factor and is critical for Treg development, maintenance, and function. 7 -10 We and others have also documented a predominant infiltration of CD4 + FOXP3 + cells in the Crohn 's inflammatory lesion. 11,12 FOXP3 + Treg cells are critical to the maintenance of immune self -tolerance and are key players in the regulation of inflammatory signals in many immune -mediated disorders; yet, emerging evidence suggests altered function within the inflammatory milieu. The notion of a proinflammatory Treg is supported by single -cell sequencing technology suggesting distinct proinflammatory phenotypes resembling those

BACKGROUND:The development of Crohn's disease [CD] involves immune cell signalling pathways regulated by epigenetic modifications. Aberrant DNA methylation has been identified in peripheral blood and bulk intestinal tissue from CD patients. However, the DNA methylome of disease-associated intestinal CD4+ lymphocytes has not been evaluated.MATERIALS AND METHODS:Genome-wide DNA methylation sequencing was performed from terminal ileum CD4+ cells from 21 CD patients and 12 age- and sex-matched controls. Data were analysed for differentially methylated CpGs [DMCs] and methylated regions [DMRs]. Integration was performed with RNA-sequencing data to evaluate the functional impact of DNA methylation changes on gene expression. DMRs were overlapped with regions of differentially open chromatin [by ATAC-seq] and CCCTC-binding factor [CTCF] binding sites [by ChIP-seq] between peripherally derived Th17 and Treg cells.RESULTS:CD4+ cells in CD patients had significantly increased DNA methylation compared to those from the controls. A total of 119 051 DMCs and 8113 DMRs were detected. While hypermethylated genes were mostly related to cell metabolism and homeostasis, hypomethylated genes were significantly enriched within the Th17 signalling pathway. The differentially enriched ATAC regions in Th17 cells [compared to Tregs] were hypomethylated in CD patients, suggesting heightened Th17 activity. There was significant overlap between hypomethylated DNA regions and CTCF-associated binding sites.CONCLUSIONS:The methylome of CD patients shows an overall dominant hypermethylation yet hypomethylation is more concentrated in proinflammatory pathways, including Th17 differentiation. Hypomethylation of Th17-related genes associated with areas of open chromatin and CTCF binding sites constitutes a hallmark of CD-associated intestinal CD4+ cells.

BACKGROUND & AIMS: Loss of AT-rich interactive domain -containing protein 1A (ARID1A) fosters acinar-to-ductal metaplasia (ADM) and pancreatic carcinogenesis by down -regulating transcription programs controlling acinar cell identity. However, how ARID1A reacts to metaplasia-triggering environmental cues remains elusive. Here, we aimed to eluci-date the role of ARID1A in controlling ductal pancreatic gene signatures and deciphering hierarchical signaling cues deter-mining ARID1A-dependent chromatin regulation during acinar cell reprogramming. METHODS: Acinar cell explants with differential ARID1A status were subjected to genome-wide expression analyses. The impact of epidermal growth factor receptor (EGFR) signaling, NFATc1 activity, and ARID1A status on acinar reprogramming processes were characterized by ex vivo ADM assays and transgenic mouse models. EGFR-dependent ARID1A chromatin binding was studied by chromatin immunoprecipitation sequencing analysis and cellular fractionation. RESULTS: EGFR signaling interferes with ARID1A-dependent transcription by inducing genome-wide ARID1A displacement, thereby phenocopying ARID1A loss-of-function mutations and inducing a shift toward ADM permissive ductal transcription programs. Moreover, we show that EGFR signaling is required to push ARID1A-deficient acinar cells toward a metaplastic phenotype. Mechanistically, we identified the transcription factor nuclear factor of activated T cells 1 (NFATc1) as the central regulatory hub mediating both EGFR signaling-induced genomic ARID1A displacement and the induction of ADM -promoting gene signatures in the absence of ARID1A. Conse-quently, pharmacologic inhibition of NFATc1 or its depletion in transgenic mice not only preserves genome-wide ARID1A oc-cupancy, but also attenuates acinar metaplasia led by ARID1A loss. CONCLUSIONS: Our data describe an intimate relationship be-tween environmental signaling and chromatin remodeling in orchestrating cell fate decisions in the pancreas, and illustrate how ARID1A loss influences transcriptional regulation in acinar cell reprogramming. (Cell Mol Gastroenterol Hepatol 2023;15:1219-1246; https://doi.org/10.1016/j.jcmgh.2023.01.015)