Open Chromatin Regions Research Articles

MEDB-15. Dynamic chromatin alteration induces oncogenic hijacking by essential transcriptional factors during SHH medulloblastoma tumorigenesis

Medulloblastoma is a malignant brain tumor that occurs in the cerebellum, most frequently in children. Medulloblastoma is molecularly classified into four major groups, and therapies are now being developed according to the nature of these groups and subgroups. However, there are currently no effective molecularly targeted drugs for most of these groups. In recent years, we have been analyzing the genomes of medulloblastomas to identify genetic mutations involved in tumorigenesis. Among them, mutations in chromatin modifiers are frequently detected in medulloblastoma, suggesting the importance of alterations in the epigenome in tumor development. However, it remains unclear how epigenomic changes are involved in tumorigenesis. Here, we have used the SHH-group medulloblastoma (MB-SHH) mouse model to understand the epigenomic changes during tumor development and identify new therapeutic targets for medulloblastoma. To this end, we utilized an Atoh1-EGFP:Ptch1+/- mice that specifically label cerebellar granule cell progenitors (GNPs) that are known to be the cellular origin of MB-SHH, preneoplastic (PNCs) and tumor cells with EGFP during MB-SHH formation. Following FACS-based collection of EGFP-positive cells, comparative ATAC-seq analysis revealed that the open chromatin regions dynamically changed during transformation of GNPs into PNCs with enrichment of nuclear factor I (NFI) binding motifs. Cut & Tag analysis on these cells showed that NFI proteins bound chromatin regions that become more accessible during tumorigenesis, suggesting that NFI could play an important role in tumorigenesis after the epigenomic changes. Indeed, CRISPR-engineered in vivo somatic deletion of Nfia and/or Nfib prevented hyperplasia formation, confirming their essential role in tumor initiation. Knockdown of either NFIA or NFIB in patient-derived xenograft models also attenuated tumor growth. Thus, our study has uncovered a novel oncogenic mechanism that contributes to the development of MB-SHH tumors via alterations of accessible chromatin and aberrant DNA binding of NFI transcription factors.

229-LB: G6PC2, a GWAS Locus for Fasting Glucose and HbA1c, Regulates Glucose-Suppression of Glucagon Secretion in a-Cells

The G6PC2 locus, encoding the catalytic subunit of the islet-specific glucose-6-phosphatase enzyme, is an established risk locus for HbA1c and fasting glucose levels. A critical role of glycolytic flux in regulating glucagon secretion was established through manipulation of the activity and expression of the glycolytic enzyme glucokinase (GCK) which controls the setpoint for glucose-suppression of glucagon secretion (GSGS) . G6PC2 opposes the action of GCK by hydrolyzing glucose-6-phosphate back to glucose and creating a futile substrate cycle. Previous studies from germline and β-cell-specific G6PC2 null mice have demonstrated that G6PC2 ablation reduces glucose-6-phosphatase activity, glucose cycling, and glycolytic flux, resulting in a leftward shift of dose-response curve for glucose-stimulated insulin secretion. Through cell type specific chromatin accessibility maps of sorted human islet cells, we established that G6PC2 risk SNPs are in open chromatin regions in human α-cells, suggesting that they might not only affect β-cell but also α-cell expression of this critical gene. To test whether α-cell G6PC2 impacts glycemic control, we derived a new mouse line that induces ablation of the first three exons of G6PC2 after Cre activation – resulting in a null allele (G6pc2 loxP/loxP; Gcg-CreERT2; referred to as “α-G6PC2KO”) . Assessment of glucagonemia during glucose tolerance tests revealed a decrease in glucagon levels in α-G6PC2KO mice relative to controls. Intriguingly, fasting blood glucose levels were significantly lower in α-G6PC2KO mice. α-G6PC2KO mice recovered more slowly from hypoglycemia during an insulin tolerance test, and presented lower glucagon levels relative to control mice following the insulin bolus. α-G6PC2KO islets secreted less glucagon relative to control islets when incubated in low-glucose media, independent of alterations in insulin secretion or islet hormone content. Collectively, our data demonstrate that the G6PC2 locus impacts glycemic control via its action in α-cells. Disclosure V. Bahl: None. C. L. May: None. Human pancreas analysis program: n/a. K. H. Kaestner: None.

298-OR: Variant-to-Gene Mapping at the Childhood Obesity Locus on chr12q13 and Subsequent Luciferase Assay Analyses Implicate rs71329as a Causal Variant within the 3’ UTR of FAIM2

A key limitation of genome-wide association studies (GWAS) is that they do not specify either the underlying causal variant (s) or the effector gene (s) at a given locus. The widely reproduced obesity FAIM2 locus, named arbitrarily after the nearest gene, is relatively understudied given it is more strongly associated in children than in adults. Our previously reported trans-ancestral GWAS meta-analysis of childhood obesity enabled fine-mapping of the FAIM2 locus; in the ‘credible set’ of 6 SNPs, rs71329 had the highest probability of being causal. This non-coding variant resides in the 3’ untranslated region (UTR) of FAIM2. Given that obesity-related GWAS signals have been reported to be enriched in genes relevant to neuronal function, we generated ATAC-seq and high-resolution promoter-focused Capture C data in both primary astrocytes and ESC-derived hypothalamic neurons to carry out variant-to-gene mapping at this locus. We observed that rs71329 occupied a region of open chromatin, therefore a putatively functional element, and made physical contact with the FAIM2 promoter. These findings were also supported by both GTEx testes eQTL data and a transcriptome-wide association study in brain tissue. We sought to determine in vitro if the rs71329alleles influence enhancer activity in primary astrocytes. Using technical replicates repeated 8 times, the non-risk allele region increased luciferase reporter expression relative to the FAIM2 promoter alone by a fold change of 1.87, while the risk allele decreased expression by a fold change of 0.68 (non-risk allele+FAIM2 promoter vs. risk allele+FAIM2 promoter P<0.0001) . Our results show that rs71329resides in an enhancer element and suggest that the risk allele within the 3’ UTR of FAIM2 decreases the expression of this putative effector gene, which then confers risk of childhood obesity and in turn type 2 diabetes later in life. Disclosure S.H.Littleton: None. J.Bradfield: None. J.A.Pippin: None. C.Su: None. A.Chesi: None. A.D.Wells: Stock/Shareholder; Johnson & Johnson. R.I.Berkowitz: Research Support; Eisai Inc., Novo Nordisk. M.C.Pahl: None. S.F.Grant: None. Funding National Institutes of Health (HD056465)

1396-P: Critical Cellular Functions That Mediate the Early Stages of Adipogenesis Are Directly Influenced by 14-3-3zeta and Its Interactome

Aim: We previously discovered that 14-3-3-zeta (14-3-3ζ) was essential for adipocyte differentiation. Although 14-3-3ζ scaffold is not a transcription factor (TF) , it undergoes nuclear translocation during adipogenesis. Considering its diverse interactome, we hypothesized that 14-3-3ζ anchors, nucleates and regulates essential transcriptional complexes required for adipogenesis. Methods: 3T3-L1 pre-adipocytes were modified by CRISPR-Cas9 to express a TAP (Flag-HA) -tagged 14-3-3ζ protein. At 48 first hours of adipogenesis, nuclear TAP-14-3-3-ζ complexes were purified to identify and annotate the 14-3-3ζ interactome by MS and Gene Ontology. Highly enriched proteins functions were assessed for their roles in adipogenesis using pharmacological inhibitors or siRNA. In parallel, chromatin accessibility was assessed by ATAC-Seq from control or 14-3-3ζ-depleted 3T3-L1 cells during early adipogenesis. Results: We found that the 14-3-3ζ nuclear interactome during the early stages of adipogenesis was significantly enriched with 133 proteins, including CEBP-β, an early adipogenic TF. According to GO analysis, proteins within the 14-3-3ζ interactome were primarily associated (FDR ≤ 0.01) with histone H2B ubiquitination, DNA unwinding for replication, DNA hypermethylation and ribosome assembly. Pharmacological inhibition or depletion by siRNA of these proteins significantly dampened adipocyte differentiation. Principal component analysis of ATAC-seq results revealed that samples from 14-3-3ζ-depleted cells differed from controls in term of whole chromatin accessibility, and DESeq2 identified nearly 3400 (p<0.05) differentially accessible regions of open chromatin following 14-3-3ζ depletion. Conclusion: These results reveal that via critical protein interactions, 14-3-3ζ has essential roles in chromatin accessibility, DNA replication, and mRNA translation, which are crucial events for the early adipogenesis. Disclosure S.A.Rial: None. A.Alkhoury: None. G.Lavoie: None. P.Roux: None. T.M.Durcan: None. A.Martinez-sanchez: None. G.E.Lim: None. Funding Natural Sciences and Engineering Research Council of Canada (NSERC) ; Canada Research Chairs (CRC) ; Fonds de recherche du Québec-Nature and Technologies (FRQNT)

Epigenetic and phenotypic characterization of iPSCs-derived smooth muscle cells: Towards a cellular model for complex arterial diseases

Smooth muscle cells (SMCs) capacity to switch between proliferative (synthetic) and quiescent (contractile) phenotypes is a widely studied mechanism in cardiovascular disease. Primary SMCs tend to lose many physiological features in culture, which makes the study of their contractile function challenging. Recently, an optimized protocol of induced pluripotent stem cells (iPSCs) differentiation into contractile SMCs was described. We aimed at obtaining a deep phenotyping of SMCs derived from iPSCs and evaluating these cellular models in the context of complex cardiovascular diseases. We differentiated 3 human iPSCs lines towards SMC phenotypes using two alternative 24-day protocols, involving either RepSox (R-SMCs) or TGF-β/PDGF-BB (TP-SMCs) during late differentiation. We analyzed gene expression and open chromatin profiles at 6 time points of differentiation and compared them to primary human SMCs and artery tissue. We used CRISPR-Cas9 system to induce mutations at LRP1 cardiovascular disease locus. Both differentiation protocols provided SMCs with abundant expression of typical SMC markers. TP-SMCs exhibited greater capacity of proliferation, migration and lower calcium release in response to contractile signals compared to R-SMCs. RNA-Seq results showed that most genes involved in the contractile function of arteries were highly expressed in R-SMCs, while TP-SMCs were more similar to cultured primary human SMCs. Open chromatin regions of R-SMCs were highly enriched for variants associated with vascular diseases such as hypertension and intracranial aneurysm, whereas TP-SMCs were more enriched for variants associated to peripheral artery disease and aortic aneurysm. Deletion of a cardiovascular disease associated enhancer region led to a dysregulation of LRP1 expression in TP-SMCs, but not in R-SMCs. LRP1 inactivation in TP-SMCs led to a major decrease in SMC proliferation, migration, and deregulation of multiple genes involved in SMC function and extracellular matrix maintenance. Differentiation of SMCs from iPSCs using two complementary protocols provides cellular models suitable for the study of a variety of vascular diseases. We confirmed LRP1 as a regulatory target gene of a common variant associated to multiple cardiovascular diseases and identified multiple genes involved in SMC function downstream of LRP1.

The Dynamics of Chromatin Accessibility Prompted by Butyrate-Induced Chromatin Modification in Bovine Cells

Butyrate is produced by microbe fermentation in the rumen, and its supplementation results in rumen development. Butyrate-induced experiments are valuable in studying nutrient metabolism, cell growth, and functional genomics. This study aimed to characterize accessible chromatin regions and the dynamics of genomic accessibility prompted by butyrate-induced histone modifications in bovine cells. An average of 117,569 accessible chromatin regions were identified for all samples, and 21,347 differentially accessible regions (DARs) for butyrate. Most of the DARs were in distal intergenic regions, introns, and promoters. Gene ontology enrichment resulted in important terms related to the digestive system, regulation of epithelial cells, and cell adhesion. Ingenuity Pathway Analysis (IPA) identified critical networks (e.g., digestive system development, cell morphology and assembly, and cell cycle); canonical pathways (e.g., TGFβ, Integrin-linked kinase, and epithelial adherens junction); and upstream regulators (e.g., TGFβ1, FOS, JUNB, ATF3, and KLFs). Co-expression analysis further showcased the TGFβ and Integrin-linked kinase (ILK)-signaling pathways, which play roles in rumen development through cellular adhesions. This study is the first to provide a genome-wide characterization of differential, open chromatin regions for bovine cells by butyrate-induced treatment. These results provide valuable information for future studies of the butyrate functions in cattle gastrointestinal tract development.

Epigenetic and transcriptional dysregulation in CD4+ T cells in patients with atopic dermatitis.

Atopic dermatitis (AD) is one of the most common skin disorders among children. Disease etiology involves genetic and environmental factors, with 29 independent AD risk loci enriched for risk allele-dependent gene expression in the skin and CD4+ T cell compartments. We investigated the potential epigenetic mechanisms responsible for the genetic susceptibility of CD4+ T cells. To understand the differences in gene regulatory activity in peripheral blood T cells in AD, we measured chromatin accessibility (an assay based on transposase-accessible chromatin sequencing, ATAC-seq), nuclear factor kappa B subunit 1 (NFKB1) binding (chromatin immunoprecipitation with sequencing, ChIP-seq), and gene expression levels (RNA-seq) in stimulated CD4+ T cells from subjects with active moderate-to-severe AD, as well as in age-matched non-allergic controls. Open chromatin regions in stimulated CD4+ T cells were highly enriched for AD genetic risk variants, with almost half of the AD risk loci overlapping AD-dependent ATAC-seq peaks. AD-specific open chromatin regions were strongly enriched for NF-κB DNA-binding motifs. ChIP-seq identified hundreds of NFKB1-occupied genomic loci that were AD- or control-specific. As expected, the AD-specific ChIP-seq peaks were strongly enriched for NF-κB DNA-binding motifs. Surprisingly, control-specific NFKB1 ChIP-seq peaks were not enriched for NFKB1 motifs, but instead contained motifs for other classes of human transcription factors, suggesting a mechanism involving altered indirect NFKB1 binding. Using DNA sequencing data, we identified 63 instances of altered genotype-dependent chromatin accessibility at 36 AD risk variant loci (30% of AD risk loci) that might lead to genotype-dependent gene expression. Based on these findings, we propose that CD4+ T cells respond to stimulation in an AD-specific manner, resulting in disease- and genotype-dependent chromatin accessibility alterations involving NFKB1 binding.

Abstract B018: Targeting Notch4 and HIF2α signaling reduces the ability of cells to survive chemotherapy by undergoing the polyaneuploid transition

Abstract Giant cancer cells have been ignored for 200 years, but recently coupled to a role in metastasis and therapeutic resistance. Some cancer cells appear to survive environmental stress, such as chemotherapy or radiotherapy, via a polyaneuploid transition. This results in a PolyAneuploid Cancer Cell (PACC) state with up to 10-fold larger cell sizes. It is proposed that PACCs utilize a conserved evolutionarily or developmental program for polyploidization, which is commonly observed in other organisms and tissues. After a stress-induced formation, the PACC state first undergoes a period of quiescence and eventually undergoes reductive cell division to give rise to proliferative progeny. We hypothesize that PACCs play a role in tumor recurrence and therapy resistance by surviving the initial treatment and subsequently reseeding the tumor via pseudohypoxic HIF signaling. Dosing with LD50 cisplatin for 72h results in the induction of the PACC state in cancer cell lines from breast (HCC1806), renal (786-0), colon (HCT116), and lung (U1690) tumors. Cells in the PACC state had 3-fold larger nuclei after 3 days of treatment and 6-fold larger nuclei after 10 days. Transcriptomic data showed an overall decrease in gene expression across all 4 cell lines. However, there was an increased expression of HIF2 (2.7-fold), Notch4 (2-fold), Plekhs1 (2.3-fold) in PACCs relative to untreated control. Moreover, there was an increase in the expression of replication dependent histones. Overall, ATACseq indicated a reduction in open chromatin in PACCs, as measured by reduced levels of histone 4 and histone 4 modifications (H4k5ac, h4k16ac, H4R3me2). However, H3k27me3 was increased 20% and AP1 binding sites were enriched in open chromatin regions. HIF2 and AP1 cooperate to regulate gene expression and confer therapy resistance. We hypothesize that AP1 is activated in response to treatment and stabilizes HIF2 under oxygenated conditions. HIFs regulate regulators of the cell cycle to induce polyploidy and downregulate β-catenin, a known inducer of cytokinesis. Small molecule inhibition of Notch and HIF-2α reduced PACC formation by 50% and 65% respectively at day 3 and 60% and 75% at day 10. The surviving PACCs were smaller than those untreated by Notch and HIF-2α inhibitors. Large metabolic changes must be undertaken to generate and sustain the larger cellular mass accompanied by polyploidy. HIF signaling metabolically rewires cells to a glycolytic phenotype that provides a carbon source for increased de novo generation of nucleotides, lipids, and proteins. Indicative of this rewiring is that PACCs have increased expression of Hexokinase (2.5-fold), CPT1 (2.3-fold), and GP6D (2-fold). In conclusion, tumor cells that survive chemotherapy are polyaneuploid, larger, and in an altered metabolic state. This process appears driven by Notch4 and HIF-2α signaling. Combining conventional chemotherapy with Notch or HIF2α inhibitors presents an opportunity to reduce tumor recurrence by inhibiting a survival mechanism. Citation Format: Christopher P. Carroll, Emma Hammarlund, Kenneth J. Pienta, Sarah R. Amend. Targeting Notch4 and HIF2α signaling reduces the ability of cells to survive chemotherapy by undergoing the polyaneuploid transition [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B018.

Variant-to-gene-mapping analyses reveal a role for pancreatic islet cells in conferring genetic susceptibility to sleep-related traits.

We investigated the potential role of sleep-trait associated genetic loci in conferring a degree of their effect via pancreatic α- and β-cells, given that both sleep disturbances and metabolic disorders, including type 2 diabetes and obesity, involve polygenic contributions and complex interactions. We determined genetic commonalities between sleep and metabolic disorders, conducting linkage disequilibrium genetic correlation analyses with publicly available GWAS summary statistics. Then we investigated possible enrichment of sleep-trait associated SNPs in promoter-interacting open chromatin regions within α- and β-cells, intersecting public GWAS reports with our own ATAC-seq and high-resolution promoter-focused Capture C data generated from both sorted human α-cells and an established human beta-cell line (EndoC-βH1). Finally, we identified putative effector genes physically interacting with sleep-trait associated variants in α- and EndoC-βH1cells running variant-to-gene mapping and establish pathways in which these genes are significantly involved. We observed that insomnia, short and long sleep-but not morningness-were significantly correlated with type 2 diabetes, obesity and other metabolic traits. Both the EndoC-βH1 and α-cells were enriched for insomnia loci (p = .01; p = .0076), short sleep loci (p = .017; p = .022) and morningness loci (p = 2.2 × 10-7; p = .0016), while the α-cells were also enriched for long sleep loci (p = .034). Utilizing our promoter contact data, we identified 63 putative effector genes in EndoC-βH1 and 76 putative effector genes in α-cells, with these genes showing significant enrichment for organonitrogen and organophosphate biosynthesis, phosphatidylinositol and phosphorylation, intracellular transport and signaling, stress responses and cell differentiation. Our data suggest that a subset of sleep-related loci confer their effects via cells in pancreatic islets.

The investigation of sex‐differential open chromatin in liver, spleen and gonads in Larimichthys crocea through ATAC ‐seq

Sex dimorphism is a fascinating science problem, which includes phenotypic, transcriptomic and epigenetic differences in male and female. To explore the landscape of transcriptome and its regulation in different sex in the large yellow croaker (Larimichthys crocea), we investigated the open chromatin using ATAC-seq generated from livers, spleens and gonads in males and females. First, we found a male-specific open chromatin region upstream of the dax1 gene, which contained Nr2e1 binding footprint. Second, the motif of Nfy was significantly enriched in the open chromatins of male spleen, liver and testis using Homer. Third, the motifs of RA signal transduction and Nr2f6 were in the female-specific transcription factor footprints, which were only in ovary of the large yellow croaker, but not in the liver and spleen of female. Finally, we found a subset of super enhancers that drove the identity genes of specific cells within the testes and ovaries. In this study, the sex difference of fish was discussed from the perspective of open chromatin regions to fill the gap in this field.

Twist2-driven chromatin remodeling governs the postnatal maturation of dermal fibroblasts.

Dermal fibroblasts lose stem cell potency after birth, which prevents regenerative healing. However, the underlying intracellular mechanisms are largely unknown. We uncover the postnatal maturation of papillary fibroblasts (PFs) driven by the extensive Twist2-mediated remodeling of chromatin accessibility. A loss of the regenerative ability of postnatal PFs occurs with decreased H3K27ac levels. Single-cell transcriptomics, assay for transposase-accessible chromatin sequencing (ATAC-seq), and chromatin immunoprecipitation sequencing (ChIP-seq) reveal the postnatal maturation trajectory associated with the loss of the regenerative trajectory in PFs, which is characterized by a marked decrease in chromatin accessibility and H3K27ac modifications. Histone deacetylase inhibition delays spontaneous chromatin remodeling, thus maintaining the regenerative ability of postnatal PFs. Genomic analysis identifies Twist2 as a major regulator within chromatin regions with decreased accessibility during the postnatal period. When Twist2 is genetically deleted in dermal fibroblasts, the intracellular cascade of postnatal maturation is significantly delayed. Our findings reveal the comprehensive intracellular mechanisms underlying intrinsic postnatal changes in dermal fibroblasts.

Abstract 532: Dioxin Modifies The Transcriptomic And Epigenetic Landscape Of Smooth Muscle Cells Leading To Adverse Remodeling Of Atherosclerosis

Background: Environmental exposure to dioxin, an active component of cigarette smoke and air pollution has been linked to increased myocardial infarction. Smooth muscle cells (SMC) in the coronary vasculature play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity. We have previously found strong dioxin response element activity in modulated SMC of atherosclerotic lesion. In this study, we aimed to understand the effect of dioxin on vascular SMC and atherosclerosis phenotype. Methods: Primary human coronary artery SMC (HCASMC) treated in culture with 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) or vehicle control were used to perform RNA-Seq and ATAC-Seq. ChIP-Seq was performed with antibodies against Aryl-hydrocarbon receptor (AHR) and TCF21. Tamoxifen inducible SMC-lineage tracing reporter mice were used to assess the in-vivo effect of TCDD on aortic SMC. After 8 weeks of high fat diet (HFD), followed by 8 weeks of TCDD injection and HFD, the aortic sinus was collected for histology and single-cell sequencing (10X Genomics). Results: Analysis of the RNA-Seq from TCDD treated HCASMC showed differential enrichment of pathways related to cell migration and vascular development. Further, ATAC-Seq data showed a significant enrichment for pathways regulating vascular development, cell migration, and apoptosis. There was an overall increase in chromatin accessibility suggesting transcriptional activation with TCDD. We observed enrichment for AHR ChIP-Seq peaks in the open chromatin regions along with a significant reciprocal reduction in TCF21 occupancy. The scRNA-Seq of mice treated with TCDD exposure showed differential gene expression of SMC-lineage cells enriching for extracellular matrix (ECM) organization, inflammation, unfolded protein response, and apoptotic process, and histological analysis of the aortic sinus showed increased lesion area with increased presence of SMC-lineage cells and Cd68 + cells compared to control. Conclusion: Dioxin adversely remodels atherosclerotic plaque affecting pathways of ECM organization, inflammation and unfolded protein response in SMC, partly by modifying chromatin accessibility and occupancy of key transcription factors of phenotypic modulation.

γδ Tcells license immature B cells to produce a broad range of polyreactive antibodies.

Immature autoreactive B cells are present in all healthy individuals, but it is unclear which signals are required for their maturation into antibody-producing cells. Inducible depletion of γδ Tcells show that direct interaction between γδ Tcells and immature B cells in the spleen support an "innate" transition to mature B cells with a broad range of antigen specificities. IL-4 production of γδ Tcells and cell-to-cell contact via CD30L support B cell maturation and induce genes of the unfolded protein response and mTORC1 signaling. Eight days after invivo depletion of γδ Tcells, increased numbers of B cells are already stuck in the transitional phase and express increased levels of IgD and CD21. Absence of γδ Tcells leads also to reduced levels of serum anti-nuclear autoantibodies, making γδ Tcells an attractive target to treat autoimmunity.

YY1 safeguard multidimensional epigenetic landscape associated with extended pluripotency.

Although extended pluripotent stem cells (EPSCs) have the potential to form both embryonic and extraembryonic lineages, how their transcriptional regulatory mechanism differs from that of embryonic stem cells (ESCs) remains unclear. Here, we discovered that YY1 binds to specific open chromatin regions in EPSCs. Yy1 depletion in EPSCs leads to a gene expression pattern more similar to that of ESCs than control EPSCs. Moreover, Yy1 depletion triggers a series of epigenetic crosstalk activities, including changes in DNA methylation, histone modifications and high-order chromatin structures. Yy1 depletion in EPSCs disrupts the enhancer-promoter (EP) interactions of EPSC-specific genes, including Dnmt3l. Yy1 loss results in DNA hypomethylation and dramatically reduces the enrichment of H3K4me3 and H3K27ac on the promoters of EPSC-specific genes by upregulating the expression of Kdm5c and Hdac6 through facilitating the formation of CCCTC-binding factor (CTCF)-mediated EP interactions surrounding their loci. Furthermore, single-cell RNA sequencing (scRNA-seq) experiments revealed that YY1 is required for the derivation of extraembryonic endoderm (XEN)-like cells from EPSCs in vitro. Together, this study reveals that YY1 functions as a key regulator of multidimensional epigenetic crosstalk associated with extended pluripotency.

Pan-cancer analysis of mutations in open chromatin regions and their possible association with cancer pathogenesis.

BackgroundOpen chromatin is associated with gene transcription. Previous studies have shown that the density of mutations in open chromatin regions is lower than that in flanking regions because of the higher accessibility of DNA repair machinery. However, in several cancer types, open chromatin regions show an increased local density of mutations in activated regulatory regions. Although the mutation distribution within open chromatin regions in cancer cells has been investigated, only few studies have focused on their functional implications in cancer. To reveal the impact of highly mutated open chromatin regions on cancer, we investigated the association between mutations in open chromatin regions and their possible functions.MethodsWhole‐genome sequencing data of 18 cancer types were downloaded from the PanCancer Analysis of Whole Genomes and Catalog of Somatic Mutations in Cancer. We quantified the mutations located in open chromatin regions defined by The Cancer Genome Atlas and classified open chromatin regions into three categories based on the number of mutations. Then, we investigated the chromatin state, amplification, and possible target genes of the open chromatin regions with a high number of mutations. We also analyzed the association between the number of mutations in open chromatin regions and patient prognosis.ResultsIn some cancer types, the proportion of promoter or enhancer chromatin state in open chromatin regions with a high number of mutations was significantly higher than that in the regions with a low number of mutations. The possible target genes of open chromatin regions with a high number of mutations were more strongly associated with cancer than those of other open chromatin regions. Moreover, a high number of mutations in open chromatin regions was significantly associated with a poor prognosis in some cancer types.ConclusionsThese results suggest that highly mutated open chromatin regions play an important role in cancer pathogenesis and can be effectively used to predict patient prognosis.

Inferring mammalian tissue-specific regulatory conservation by predicting tissue-specific differences in open chromatin

BackgroundEvolutionary conservation is an invaluable tool for inferring functional significance in the genome, including regions that are crucial across many species and those that have undergone convergent evolution. Computational methods to test for sequence conservation are dominated by algorithms that examine the ability of one or more nucleotides to align across large evolutionary distances. While these nucleotide alignment-based approaches have proven powerful for protein-coding genes and some non-coding elements, they fail to capture conservation of many enhancers, distal regulatory elements that control spatial and temporal patterns of gene expression. The function of enhancers is governed by a complex, often tissue- and cell type-specific code that links combinations of transcription factor binding sites and other regulation-related sequence patterns to regulatory activity. Thus, function of orthologous enhancer regions can be conserved across large evolutionary distances, even when nucleotide turnover is high.ResultsWe present a new machine learning-based approach for evaluating enhancer conservation that leverages the combinatorial sequence code of enhancer activity rather than relying on the alignment of individual nucleotides. We first train a convolutional neural network model that can predict tissue-specific open chromatin, a proxy for enhancer activity, across mammals. Next, we apply that model to distinguish instances where the genome sequence would predict conserved function versus a loss of regulatory activity in that tissue. We present criteria for systematically evaluating model performance for this task and use them to demonstrate that our models accurately predict tissue-specific conservation and divergence in open chromatin between primate and rodent species, vastly out-performing leading nucleotide alignment-based approaches. We then apply our models to predict open chromatin at orthologs of brain and liver open chromatin regions across hundreds of mammals and find that brain enhancers associated with neuron activity have a stronger tendency than the general population to have predicted lineage-specific open chromatin.ConclusionThe framework presented here provides a mechanism to annotate tissue-specific regulatory function across hundreds of genomes and to study enhancer evolution using predicted regulatory differences rather than nucleotide-level conservation measurements.

Distinct cytosine modification profiles define epithelial-to-mesenchymal cell-state transitions.

Background:Epithelial-to-mesenchymal transition (EMT) is an early step in the invasion-metastasis cascade, involving progression through intermediate cell states. Due to challenges with isolating intermediate cell states, genome-wide cytosine modifications that define transition are not completely understood.Methods:The authors measured multiple DNA cytosine modification marks and chromatin accessibility across clonal populations residing in specific EMT states.Results:Clones exhibiting more intermediate EMT phenotypes demonstrated increased 5-hydroxymethylcytosine and decreased 5-methylcytosine. Open chromatin regions containing increased 5-hydroxymethylcytosine CpG loci were enriched in EMT transcription factor motifs and were associated with Rho GTPases.Conclusion:The results indicate the importance of both distinct and shared epigenetic profiles associated with EMT processes that may be targeted to prevent EMT progression.

Gene expression and chromatin conformation differs between worker bees performing different tasks

BackgroundRevealing the effect of transcriptomic regulation on behavioral differences is a fundamental goal in biology, but the relationship between gene regulatory networks and individual behavior differences remains largely unknown. Honey bees are considered as good models for studying the mechanisms underlying gene expression changes and behavioral differences since they exhibit strong and obvious differences in tasks between individuals. The cis-regulatory regions usually contain the binding sites of diverse transcription factor (TFs) influencing bee behavior. Thus, the identification of cis-regulatory elements in the brains across different behavioral states is important for understanding how genomic and transcriptomic variations affect different tasks in honeybees. MethodsIn this study, we employed transcriptome and genome-wide chromatin accessibility assays to analyze brain tissues of honey bees in different behavioral states for examining the relationship between individual behavior differences and brain gene expression changes. We also used the obtained open chromatin regions to identify cis-motifs associating differentially expressed TFs and genes in order to reveal the transcriptional regulatory mechanism related to the different tasks. ResultsWe identified genetic regulatory modules regulating different tasks that contained key TFs (CTCF, Trl and schlank) associated with open chromatin regions enriched for DNA sequence motifs belonging to the family of the corresponding TFs. The most prominent transcriptomic changes, which correlated with chromatin accessibility modifications within their proximal promoter regions, occurred in nervous system development, and were associated with behavior switch. ConclusionsOur results revealed the regulatory landscape among three behavioral stages in honeybees and identified interactive molecular networks regulating different tasks. These results provide a comprehensive insight into behavioral differences of honeybees, which offers reference for future study.

HNF4A modulates glucocorticoid action in the liver.

SummaryThe glucocorticoid receptor (GR) is a nuclear receptor critical to the regulation of energy metabolism and inflammation. The actions of GR are dependent on cell type and context. Here, we demonstrate the role of liver lineage-determining factor hepatocyte nuclear factor 4A (HNF4A) in defining liver specificity of GR action. In mouse liver, the HNF4A motif lies adjacent to the glucocorticoid response element (GRE) at GR binding sites within regions of open chromatin. In the absence of HNF4A, the liver GR cistrome is remodeled, with loss and gain of GR recruitment evident. Loss of chromatin accessibility at HNF4A-marked sites associates with loss of GR binding at weak GRE motifs. GR binding and chromatin accessibility are gained at sites characterized by strong GRE motifs, which show GR recruitment in non-liver tissues. The functional importance of these HNF4A-regulated GR sites is indicated by an altered transcriptional response to glucocorticoid treatment in the Hnf4a-null liver.

Whole-genome profiling of DNA methylation and hydroxymethylation identifies distinct regulatory programs among innate lymphocytes.

Innate lymphocytes encompass a diverse array of phenotypic identities with specialized functions. DNA methylation and hydroxymethylation are essential for epigenetic fidelity and fate commitment. The landscapes of these modifications are unknown in innate lymphocytes. Here, we characterized the whole-genome distribution of methyl-CpG and 5-hydroxymethylcytosine in mouse ILC3, ILC2, and NK cells. We identified differentially methylated and hydroxymethylated DNA regions between ILC-NK subsets and correlated them with transcriptional signatures. We associated lineage-determining transcription factors with demethylation and demonstrated unique patterns of DNA methylation/hydroxymethylation in relationship to open chromatin regions, histone modifications, and transcription factor binding sites. We further discovered a novel association between hydroxymethylation and NK cell super-enhancers. Using mice lacking DNA hydroxymethylase TET2, we showed its requirement for optimal production of hallmark cytokines by ILC3 and IL-17A by inflammatory ILC2. These findings provide a powerful resource for studying innate lymphocyte epigenetic regulation and decode the regulatory logic governing their identity.