Histone Modifications H3K27ac Research Articles

Induction of OCT2 contributes to regulate the gene expression program in human neutrophils activated via TLR8.

The transcription factors (TFs) that regulate inducible genes in activated neutrophils are not yet completely characterized. Herein, we show that the genomic distribution of the histone modification H3K27Ac, as well as PU.1 and C/EBPβ, two myeloid-lineage-determining TFs (LDTFs), significantly changes in human neutrophils treated with R848, a ligand of Toll-like receptor 8 (TLR8). Interestingly, differentially acetylated and LDTF-marked regions reveal an over-representation of OCT-binding motifs that are selectively bound by OCT2/POU2F2. Analysis of OCT2 genomic distribution in primary neutrophils and of OCT2-depletion in HL-60-differentiated neutrophils proves the requirement for OCT2 in contributing to promote, along with nuclear factor κB (NF-κB) and activator protein 1 (AP-1), the TLR8-induced gene expression program in neutrophils. Altogether, our data demonstrate that neutrophils, upon activation via TLR8, profoundly reprogram their chromatin status, ultimately displaying cell-specific, prolonged transcriptome changes. Data also show an unexpected role for OCT2 in amplifying the transcriptional response to TLR8-mediated activation.

Profiling of H3K27Ac Reveals the Influence of Asthma on the Epigenome of the Airway Epithelium.

BackgroundAsthma is a chronic airway disease driven by complex genetic–environmental interactions. The role of epigenetic modifications in bronchial epithelial cells (BECs) in asthma is poorly understood.MethodsWe piloted genome-wide profiling of the enhancer-associated histone modification H3K27ac in BECs from people with asthma (n = 4) and healthy controls (n = 3).ResultsWe identified n = 4,321 (FDR < 0.05) regions exhibiting differential H3K27ac enrichment between asthma and health, clustering at genes associated predominately with epithelial processes (EMT). We identified initial evidence of asthma-associated Super-Enhancers encompassing genes encoding transcription factors (TP63) and enzymes regulating lipid metabolism (PTGS1). We integrated published datasets to identify epithelium-specific transcription factors associated with H3K27ac in asthma (TP73) and identify initial relationships between asthma-associated changes in H3K27ac and transcriptional profiles. Finally, we investigated the potential of CRISPR-based approaches to functionally evaluate H3K27ac-asthma landscape in vitro by identifying guide-RNAs capable of targeting acetylation to asthma DERs and inducing gene expression (TLR3).ConclusionOur small pilot study validates genome-wide approaches for deciphering epigenetic mechanisms underlying asthma pathogenesis in the airways.

Evolution of regulatory signatures in primate cortical neurons at cell-type resolution

The human cerebral cortex contains many cell types that likely underwent independent functional changes during evolution. However, cell-type-specific regulatory landscapes in the cortex remain largely unexplored. Here we report epigenomic and transcriptomic analyses of the two main cortical neuronal subtypes, glutamatergic projection neurons and GABAergic interneurons, in human, chimpanzee, and rhesus macaque. Using genome-wide profiling of the H3K27ac histone modification, we identify neuron-subtype-specific regulatory elements that previously went undetected in bulk brain tissue samples. Human-specific regulatory changes are uncovered in multiple genes, including those associated with language, autism spectrum disorder, and drug addiction. We observe preferential evolutionary divergence in neuron subtype-specific regulatory elements and show that a substantial fraction of pan-neuronal regulatory elements undergoes subtype-specific evolutionary changes. This study sheds light on the interplay between regulatory evolution and cell-type-dependent gene-expression programs, and provides a resource for further exploration of human brain evolution and function.

Epigenetic response to hyperoxia in the neonatal lung is sexually dimorphic

Sex as a biological variable plays a critical role both during lung development and in modulating postnatal hyperoxic lung injury and repair. The molecular mechanisms behind these sex-specific differences need to be elucidated. Our objective was to determine if the neonatal lung epigenomic landscape reconfiguration has profound effects on gene expression and could underlie sex-biased differences in protection from or susceptibility to diseases. Neonatal male and female mice (C57BL/6) were exposed to hyperoxia (95% FiO, PND 1–5: saccular stage) or room air and euthanized on PND 7 and 21. Pulmonary gene expression was studied using RNA-seq on Illumina HiSeq 2500 platform and quantified. Epigenomic landscape was assessed using Chromatin Immunoprecipitation (ChIP-Seq) of the H3K27ac histone modification mark, associated with active genes, enhancers, and super-enhancers. These data were then integrated, pathways identified and validated. Sex-biased epigenetic modulation of gene expression leads to differential regulation of biological processes in the developing lung at baseline and after exposure to hyperoxia. The female lung exhibits a more robust epigenomic response for the H3K27ac mark in response to hyperoxia. Epigenomic changes distribute over genomic and epigenomic domains in a sex-specific manner. The differential epigenomic responses also enrich for key transcription regulators crucial for lung development. In addition, by utilizing H3K27ac as the target epigenomic change we were also able to identify new epigenomic reprogramming at super-enhancers. Finally, we report for the first time that the upregulation of p21 (Cdkn1a) in the injured neonatal lung could be mediated through gain of H3K27ac. These data demonstrate that modulation of transcription via epigenomic landscape alterations may contribute to the sex-specific differences in preterm neonatal hyperoxic lung injury and repair.

Expression of H3K4me3 and H3K9ac in breast cancer

PurposeBreast cancer is the leading cause of cancer death in females. Histone modifications have been shown to have an influence on the gene expression. This study focusses on the histone modifications H3K9ac and H3K4me3 in breast cancer and their impact on survivalMethodsH3K4me3 and H3K9ac expression was immunohistochemically examined in 235 tissue samples.ResultsPositive estrogen receptor status was correlated with a higher IRS of the nuclear (p = 0.033), and of the cytoplasmic H3K4me3 staining (p = 0.009). H3K9ac intensity was associated to the Her2 status (p = 0.045) and to poor prognosis in cells with positive Ki67 status (p = 0.013). A high intensity of nuclear H3K4me3 staining was found to be correlated with a lower 10-year-survival (p = 0.026) and with lower breast cancer-specific survival (p = 0.004). High percentage score (> 190) of H3K9ac expression was correlated to worse breast cancer-specific survival (p = 0.005). Shorter progression-free survival was found in patients with nuclear (p = 0.013) and cytoplasmic H3K4me3expression (p = 0.024) and H3K9ac expression (p = 0.023).ConclusionThis analysis provides new evidence of histone modifications in breast cancer. High H3K4me3 and H3K9ac expression was correlated with survival rates. Further investigation of histone modifications in breast cancer could lead to a more profound understanding of the molecular mechanisms of cancer development and could result in new therapeutic strategies.

KMT2D Haploinsufficiency in Kabuki Syndrome Impairs Differentiation of Neural Crest Cells

ObjectiveKabuki Syndrome (KS), caused by haploinsufficiency in lysine methyltransferase KMT2D, is a multi‐system disease characterized by distinct facial dysmorphisms, mild‐to‐moderate intellectual disability, renal and cardiac abnormalities, short stature and humoral immune deficiency. Many of the tissues in which these abnormalities manifest are derived from or developmentally modulated by the neural crest cell (NCC) lineage. This study’s objective was to determine which features of the KS phenotype can be directly linked to genetic and epigenetic dysregulation in KMT2D haploinsufficient NCCs.MethodsPeripheral blood mononuclear cells from a human donor haploinsufficient for KMT2D (KS patient, KMT2D+/−) or wild type (WT) control were used to generate induced pluripotent stem (iPS) cells. WT and KS NCCs were generated from WT and KS iPS cells, respectively. Whole transcriptome RNA sequencing (RNAseq) was performed on iPS cells (WT and KS) and NCCs (WT and KS) to characterize differential gene expression. Chromatin immunoprecipitation sequencing (ChIPseq) of histone 3 lysine 4 trimethylation (H3K4Me3) and histone 3 lysine 27 acetylation (H3K27Ac) was performed on NCCs (WT and KS) to characterize differential histone modifications at gene regulatory elements. Analysis of RNAseq and ChIPseq data was performed using BioWardrobe platform.ResultsGene set enrichment analysis of transcriptome data indicates that, compared to WT NCCs, KMT2D haploinsufficient NCCs have ectopic expression of several genes critical to normal NCC organization, morphogenesis and tissue differentiation, including NCC transcriptional repressor Msh homeobox 2 (MSX2). Analysis also identified several genes with decreased expression in KMT2D+/− NCCs, including transcription factor AP‐2 alpha (TFAP2A), which serves as a master regulator of NCC lineage progression. ChIPseq data indicate differential enrichment of H3K4Me3 and H3K27Ac histone modifications at the TFAP2A locus in WT compared to KS NCCs, which suggests that TFAP2A may have a direct role in the impaired differentiation of NCCs in KS. Further studies to explore the roles of TFAP2A and other genes identified by these data are underway.ConclusionThese results show that KMT2D haploinsufficiency impairs differentiation of the NCC lineage cells through epigenetic and transcriptomic dysregulation, thus supporting the classification of KS as a neurocristopathy.Support or Funding InformationNIH T32 Training Grant 5T32AI060515‐14 (WJL), R35 DE027557 (SB), Center for Pediatric Genomics Cincinnati Research Foundation (AWL).

Drought-inducible changes in the histone modification H3K9ac are associated with drought-responsive gene expression in Brachypodium distachyon.

H3K9ac, an epigenetic marker, is widely distributed in plant genomes. H3K9ac enhances gene expression, which is highly conserved in eukaryotes. However, genome-wide studies of H3K9ac in monocot species are limited, and the changes in H3K9ac under drought stress for individual genes are still not clear. We analysed changes in the H3K9ac level of Brachypodium distachyon under 20% PEG-6000-simulated drought stress conditions. We also performed chromatin immunoprecipitation, followed by next generation sequencing (ChIP-seq) on H3K9ac to reveal changes in H3K9ac for individual genes at the genome-wide level. Our study showed that H3K9ac was mainly enriched in gene exon regions. Drought increased or decreased the H3K9ac level at specific genomic loci. We identified 40 genes associated with increased H3K9ac levels and 36 genes associated with decreased H3K9ac levels under drought stress. Further, RT-qPCR analyses showed that H3K9ac was positively associated with gene expression of those drought-responsive genes. We conclude that H3K9ac enhances the expression level of a large number of drought-responsive genes under drought stress in B.distachyon. The data presented here will help to reveal the correlation of some specific drought-responsive genes and their enriched H3K9ac levels in the model plant B.distachyon.

Identification of key genes by integrating DNA methylation and next-generation transcriptome sequencing for esophageal squamous cell carcinoma.

Aberrant DNA methylation leads to abnormal gene expression, making it a significant regulator in the progression of cancer and leading to the requirement for integration of gene expression with DNA methylation. Here, we identified 120 genes demonstrating an inverse correlation between DNA methylation and mRNA expression in esophageal squamous cell carcinoma (ESCC). Sixteen key genes, such as SIX4, CRABP2, and EHD3, were obtained by filtering 10 datasets and verified in paired ESCC samples by qRT-PCR. 5-Aza-dC as a DNA methyltransferase (DNMT) inhibitor could recover their expression and inhibit clonal growth of cancer cells in seven ESCC cell lines. Furthermore, 11 of the 16 genes were correlated with OS (overall survival) and DFS (disease-free survival) in 125 ESCC patients. ChIP-Seq data and WGBS data showed that DNA methylation and H3K27ac histone modification of these key genes displayed inverse trends, suggesting that there was collaboration between DNA methylation and histone modification in ESCC. Our findings illustrate that the integrated multi-omics data (transcriptome and epigenomics) can accurately obtain potential prognostic biomarkers, which may provide important insight for the effective treatment of cancers.

Split hand/foot malformation associated with 20p12.1 deletion: A case report

Split hand/foot malformation (SHFM) or ectrodactyly is a rare congenital disorder affecting limb development characterized by clinical and genetic heterogeneity. SHFM is usually inherited as an autosomal dominant trait with incomplete penetrance. Isolated and syndromic forms are described. The extent of associated malformations is highly variable and multiple syndromes with clinical and genetic overlap have been described. We report here a 28 year-old man presenting with SHFM, sparse hair and widespread freckles. Array-CGH identified a 450 kb de novo 20p12.1 microdeletion encompassing three exons (exon 6 to 8) of MACROD2. Although MACROD2 mutations have not been associated with limb malformation until now, it is located next to KIF16B, which is involved in fibroblast growth factor receptor (FGFR) signaling. Additionally, the deletion encompassed a histone modification H3K27ac mark, known as a provider of quantitative readout of promoter and enhancer activity during human limb development. Altogether, these findings suggest that the 20p12.1 CNV is causative of SHFM in the present case through disturbance of regulatory elements functioning.

P2570Synergistic activation of the cardiac enhancer landscape during reprogramming

Abstract Introduction Cardiogenic transcription factors (TFs), Gata4, Mef2c, and Tbx5, can directly reprogram fibroblasts to a cardiac fate and their cardiogenic activity is enhanced by the Hand2 TF and the Akt1 kinase. Although these cardiac reprogramming factors are key regulators of heart development, their expression and biological functions are not limited to the heart, and the mechanism by which these cardiac factors orchestrate reprogramming remains unclear. Purpose We sought to study the molecular mechanisms by which cardiac reprogramming factors contribute to cell fate conversion using a genome-wide approach. Methods Here, we used chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) to explore the genomic binding sites of reprogramming TFs and the landscape of active enhancers, annotated by H3K27ac histone modification, during cardiac reprogramming. Results We found that reprogramming TFs rapidly silence fibroblast enhancers and synergistically activate cardiac enhancers which were predominantly enriched with Mef2 motifs. Addition of Hand2 and Akt1 to GMT expands TF co-occupancy and thereby activates additional cardiac enhancers, which further augments cardiac gene expression. Moreover, additional cardiac enhancers were sequentially activated during the reprogramming process, in accordance with the temporal acquisition of functional phenotypes in induced cardiac-like myocytes. We discovered that subsets of reprogramming enhancers are conserved among other cardiogenic processes, and a collection of reprogramming enhancers displayed unique spatial expression patterns in the developing heart in transgenic mouse embryos. Conclusion Our study describes the epigenomic dynamics that underlie cardiac reprogramming which is cooperatively orchestrated by reprogramming factors to convert fibroblasts toward a cardiac lineage. Acknowledgement/Funding NIH (AR-067294, HL-130253, HL-138426, HD-087351), Fondation Leducq Transatlantic Networks of Excellence in Cardiovascular Research

NGS‐Integrator: A Tool for Combining Information from Multiple Genome‐Wide NGS Data Tracks Using Minimum Bayes Factors

Genome‐wide studies that generate multiple high‐throughput next‐generation sequencing (NGS) datasets to identify genomic DNA elements for gene transcription require data integration methods to minimize complexity and false‐positive findings. This can involve integration of multiple genome‐wide data generated from same type or different types of high‐throughput NGS techniques. Since several strategies to integrate multiple genome‐wide NGS datasets based on peak calling tools have been developed, these conventional methods are typically applied to individual replicates and not the aggregate data from multiple data.NGS‐integrator, a Java‐based tool, integrates genome‐wide NGS datasets into a single data track for a genome browser based on minimum Bayes Factor (MBF) calculated from the signal‐to‐noise ratio. NGS‐integrator consists of two elements, “Calculator” and “Integrator”. The “Calculator” element calculates the complement of MBF at each genomic position, estimating the median of the noise values across a sliding window straddling the position at which signal value is being calculated. This calculation assumes that the features being detected are relatively sparsely represented along the genome, allowing the median to be representative of the true noise level. The “Integrator” element calculates the joint probability between the multiple NGS data tracks based on the complement of MBF calculated with ‘Calculator’. We used NGS‐integrator to integrate data from three types of data generated in our laboratory in cultured mpkCCD kidney cells, viz. ChIP‐Seq for the transcription factor ELF1, ATAC‐Seq, and ChIP‐Seq for the histone modification H3K27Ac and RNA polymerase II.Support or Funding InformationThe work was supported by the Division of Intramural Research, National Heart, Lung, and Blood Institute projects ZIA‐HL001285 (to MAK) and ZIA‐HL006129 (to MAK). BW was supported by the NHLBI Summer Internship Program (June–August 2018).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The impact of the vitamin D-modulated epigenome on VDR target gene regulation

The micronutrient vitamin D significantly modulates the human epigenome via enhancing genome-wide the rate of accessible chromatin and vitamin D receptor (VDR) binding. This study focuses on histone marks of active chromatin at promoter and enhancer regions and investigates, whether these genomic loci are sensitive to vitamin D. The epigenome of THP-1 human monocytes contains nearly 23,000 sites with H3K4me3 histone modifications, 550 of which sites are significantly (p < 0.05) modulated by stimulation with the VDR ligand 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). H3K27ac histone modifications mark active chromatin and 2473 of 45,500 sites are vitamin D sensitive. The two types of ligand-dependent histone marks allow to distinguish promoter and enhancer regulation by vitamin D, respectively. Transcription start site overlap is the prime attribute of ligand-dependent H3K4me3 marks, while VDR co-location is the top ranking parameter describing 1,25(OH)2D3-sensitive H3K27ac marks at enhancers. A categorization of 1,25(OH)2D3-sensitive histone marks by machine learning algorithms - using the attributes overall peak strength and ligand inducibility - highlights 260 and 287 regions with H3K4me3 and H3K27ac modifications, respectively. These loci are found at the promoter regions of 59 vitamin D target genes and their associated enhancers. In this way, ligand-dependent histone marks provide a link of the effects of 1,25(OH)2D3 on the epigenome with previously reported mRNA expression changes of vitamin D target genes. In conclusion, the human epigenome responds also on the level of histone modifications to 1,25(OH)2D3 stimulation. This allows a more detailed understanding of vitamin D target gene regulation.

Cancer-predicting transcriptomic and epigenetic signatures revealed for ulcerative colitis in patient-derived epithelial organoids

Ulcerative colitis (UC) is a prevalent form of inflammatory bowel disease (IBD) whose pathogenic mechanisms remain unclear. Elucidating these mechanisms is important to reduce UC symptoms and to prevent UC progression into colitis-associated colon cancer (CAC). Our goal was to develop and validate faithful, human-derived, UC models and analyze them at histologic, transcriptomic and epigenetic levels to allow mechanistic studies of UC and CAC pathogenesis.We generated patient-derived primary-organoid cultures from UC and non-IBD colonic epithelium. We phenotyped them histologically and used next-generation-sequencing approaches to profile whole transcriptomes and epigenomes of organoids and primary tissues.Tissue organization and expression of mucin 2 (MUC2) and lysozyme (LYZ) demonstrated histologic faithfulness of organoids to healthy and diseased colonic epithelium. Transcriptomic analyses showed increased expression of inflammatory pathways in UC patient-derived organoids and tissues. Profiling for active enhancers using the H3K27ac histone modification revealed UC-derived organoid enrichment for pathways indicative of gastrointestinal cancer, including S100 calcium-binding protein P (S100P), and revealed novel markers for GI cancer, including both LYZ and neuropeptide S receptor 1 (NPSR1). Immunolocalization showed increased levels of LYZ, S100P, and NPSR1 proteins in UC and CAC.In conclusion, primary colonic organoid cultures from UC and non-IBD patients can be established that faithfully represent diseased or normal colonic states. These models reveal precancerous molecular pathways that are already activated in UC. The findings demonstrate the suitability of primary organoids for dissecting UC and CAC pathogenic mechanisms and suggest new targets for therapeutic intervention.

Multiple enhancer regions govern the transcription of CCN2 during embryonic development

CCN2 is a critical matricellular protein that is expressed in several cells with major implications in physiology and different pathologies. However, the transcriptional regulation of this gene remains obscure. We used the Encyclopaedia of DNA Elements browser (ENCODE) to visualise the region spanning from 300 kb upstream to the CCN2 start site in silico in order to identify enhancer regions that regulate transcription of this gene. Selection was based on three criteria associated with enhancer regions: 1) H3K4me1 and H3K27ac histone modifications, 2) DNase I hypersensitivity of chromatin and 3) inter-species conservation. Reporter constructs were created with sequences spanning each of the regions of interest placed upstream of an Hsp68 silent proximal promoter sequence in order to drive the expression of β-galactosidase transgene. Each of these constructs was subsequently used to create transgenic mice in which reporter gene production was assessed at the E15.5 developmental stage. Four functional enhancers were identified, with each driving distinct, tissue-specific patterns of transgene expression. An enhancer located -100 kb from the CCN2 transcription start site facilitated expression within vascular tissue. An enhancer -135 kb upstream of CCN2 drove expression within the articular chondrocytes of synovial joints. The other two enhancers, located at -198 kb and -229 kb, mediated transgene expression within dermal fibroblasts, however the most prevalent activity was found within hypertrophic chondrocytes and periosteal tissue, respectively. These findings suggest that the global expression of CCN2 during development results from the activity of several tissue-specific enhancer regions in addition to proximal regulatory elements that have previously been demonstrated to drive transcription of the gene during development.

The Psychiatric Risk Gene Transcription Factor 4 (TCF4) Regulates Neurodevelopmental Pathways Associated With Schizophrenia, Autism, and Intellectual Disability.

BackgroundCommon genetic variants in and around the gene encoding transcription factor 4 (TCF4) are associated with an increased risk of schizophrenia. Conversely, rare damaging TCF4 mutations cause Pitt–Hopkins syndrome and have also been found in individuals with intellectual disability (ID) and autism spectrum disorder (ASD).MethodsChromatin immunoprecipitation and next generation sequencing were used to identify the genomic targets of TCF4. These data were integrated with expression, epigenetic and disease gene sets using a range of computational tools.ResultsWe identify 10604 TCF4 binding sites in the genome that were assigned to 5437 genes. De novo motif enrichment found that most TCF4 binding sites contained at least one E-box (5′-CAtcTG). Approximately 77% of TCF4 binding sites overlapped with the H3K27ac histone modification for active enhancers. Enrichment analysis on the set of TCF4 targets identified numerous, highly significant functional clusters for pathways including nervous system development, ion transport and signal transduction, and co-expression modules for genes associated with synaptic function and brain development. Importantly, we found that genes harboring de novo mutations in schizophrenia (P = 5.3 × 10−7), ASD (P = 2.5 × 10−4), and ID (P = 7.6 × 10−3) were also enriched among TCF4 targets. TCF4 binding sites were also found at other schizophrenia risk loci including the nicotinic acetylcholine receptor cluster, CHRNA5/CHRNA3/CHRNB4 and SETD1A.ConclusionsThese data demonstrate that TCF4 binding sites are found in a large number of neuronal genes that include many genetic risk factors for common neurodevelopmental disorders.

Putative enhancer sites in the bovine genome are enriched with variants affecting complex traits

BackgroundEnhancers are non-coding DNA sequences, which when they are bound by specific proteins increase the level of gene transcription. Enhancers activate unique gene expression patterns within cells of different types or under different conditions. Enhancers are key contributors to gene regulation, and causative variants that affect quantitative traits in humans and mice have been located in enhancer regions. However, in the bovine genome, enhancers as well as other regulatory elements are not yet well defined. In this paper, we sought to improve the annotation of bovine enhancer regions by using publicly available mammalian enhancer information. To test if the identified putative bovine enhancer regions are enriched with functional variants that affect milk production traits, we performed genome-wide association studies using imputed whole-genome sequence data followed by meta-analysis and enrichment analysis.ResultsWe produced a library of candidate bovine enhancer regions by using publicly available bovine ChIP-Seq enhancer data in combination with enhancer data that were identified based on sequence homology with human and mouse enhancer databases. We found that imputed whole-genome sequence variants associated with milk production traits in 16,581 dairy cattle were enriched with enhancer regions that were marked by bovine-liver H3K4me3 and H3K27ac histone modifications from both permutation tests and gene set enrichment analysis. Enhancer regions that were identified based on sequence homology with human and mouse enhancer regions were not as strongly enriched with trait-associated sequence variants as the bovine ChIP-Seq candidate enhancer regions. The bovine ChIP-Seq enriched enhancer regions were located near genes and quantitative trait loci that are associated with pregnancy, growth, disease resistance, meat quality and quantity, and milk quality and quantity traits in dairy and beef cattle.ConclusionsOur results suggest that sequence variants within enhancer regions that are located in bovine non-coding genomic regions contribute to the variation in complex traits. The level of enrichment was higher in bovine-specific enhancer regions that were identified by detecting histone modifications H3K4me3 and H3K27ac in bovine liver tissues than in enhancer regions identified by sequence homology with human and mouse data. These results highlight the need to use bovine-specific experimental data for the identification of enhancer regions.

Abstract 1446: Super-enhancer identification via Bru/BrUV-Seq and H3K27Ac ChIP-Seq

Abstract Transcriptional super-enhancers (SEs) are large clusters of active enhancers that drive expression of genes critical to cell identity and function. In cancer and other human diseases, SEs can be acquired through somatic aberrations. Importantly, SEs and their target genes are particularly sensitive to perturbation, making this mechanism a promising target for both diagnosis and therapy. Histone modification H3K27Ac is a good single predicative marker of active enhancers, though additional marks would help. Accordingly, a common approach to SE identification is using H3K27Ac ChIP-Seq. The non-TSS (transcription start site) histone binding sites (e.g., principally excluding TSS±2.5kb regions) are mapped to genome and then subject to near-neighbor stitching (e.g., within 12.5kb). From the resulted relatively broad regions, SEs are identified by exceptional binding enrichment. Another potentially powerful approach is to use Bru/BrUV-Seq in combination. By directional sequencing of bromouridine(Bru)-labeled nascent RNAs, Bru-Seq has been developed to study nascent transcription. In the derivative BrUV-Seq, UV irradiation is additionally conducted to stabilize transcripts prior to labeling, thereby allowing more sensitive detection of TSSs and unstable transcripts, including enhancer RNAs (eRNAs). It has been shown that eRNAs indicate enhancer activity better than H3K27Ac binding. In this study, both approaches were used to identify SEs for three colon cancer cell lines, HCT-116, SW48, and SW620. We have demonstrated that Bru/BrUV-Seq has advantages in differentiating between mRNAs and eRNAs, and between overlapping transcripts; the reduced complexity helps prevent false identifications, which inflated our results obtained from ChIP-Seq. Citation Format: Chunlao Tang, Vipin Yadav, Hui-Rong Qian, Jason B. Cunningham, Hong Gao, Yushi Liu, Jason C. Ting, Steven M. Bray, Philip J. Ebert, Yuhao Lin, Amit Aggarwal, John N. Calley, Bharvin K. Patel. Super-enhancer identification via Bru/BrUV-Seq and H3K27Ac ChIP-Seq [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1446. doi:10.1158/1538-7445.AM2017-1446

A GWAS of 170,000 Individuals Identifies Thousands of Alleles Perturbing Blood Cell Traits, Many of Which Are in Super Enhancers Setting Cell Identity

Adults release ~1011 blood cells into circulation every day. These cells derive, by clonal expansion, from a minuscule population of hematopoietic stem cells (HSCs), which differentiate into mature cells through progressively fate-restricted progenitor cells. In order to identify the genetic loci controlling these processes, we performed a hypothesis-free genome-wide association study (GWAS) of complete blood count data measured on 170,000 subjects in the UK Biobank and INTERVAL cohorts. This identified almost 3,000 causally distinct variants (hits) modulating hematopoiesis or blood cell clearance, including several hundred low frequency variants (minor allele frequencies <5%) with very large effect sizes.Over three quarters of the GWAS hits have no coding consequence and analysis of the 700 platelet hits showed a striking enrichment in the DNAse-I hypersensitivity sites of HSC-derived megakaryocytes (MKs) compared to those of HSC-derived erythroblasts (EBs), suggesting that a substantial proportions of blood trait heritability can be explained by variants modulating cell type specific genetic regulators.To understand the nature of gene regulation in the erythroid-megakaryocytic lineage we firstly used long-range chromosomal interaction data (from genome-wide promoter-capture chromosome conformation assays) and found that the platelet trait hits are enriched in regulatory elements that interact with a gene promoter in MKs. We then scanned the genome for super-enhancers (SEs) (regions characterized by a high density of enhancer elements and a strong H3K27ac histone modification). We identified 1067 SEs in MKs and 1294 SEs in EBs, with an overlap count of 400. Platelet GWAS hits were strongly enriched in MK SEs over MK typical enhancers (TEs) relative to red cell hits and were frequently localised in SEs specific to MKs. Finally, we found that SE (but not TE) MK-interacting genes are significantly enriched for gene-ontology (GO) terms for megakaryopoiesis, platelet function and hemostasis, supporting the notion that SEs play a major role in the determination of cell identity.To corroborate this, we focused on the second (SE2) of three SEs for the CD9-VWF gene cluster, which contained the GWAS hit rs2363877. We recalled 100 individuals by genotype at rs2363877 and found that the minor allele was associated with decreased expression of CD9 and increased expression of VWF in platelets. By flowing blood over collagen and a VWF-binding collagen-mimetic we showed this allele has a significant effect on the extent of ex-vivo thrombus formation. Finally, using CRISPR/Cas9 we eliminated SE2 from IPSC derived MKs and showed an increase in VWF expression.In summary, recently generated epigenetic and long-range chromosomal interaction data suggest that a significant fraction of platelet GWAS hits perturb super-enhancer activity in MKs with downstream effect on networks of genes. In the light of this the maxim that 'a GWAS hit acts through a single gene' cannot be sustained. DisclosuresNo relevant conflicts of interest to declare.

Phenotypic Modulation of Smooth Muscle Cells in Atherosclerosis Is Associated With Downregulation of LMOD1, SYNPO2, PDLIM7, PLN, and SYNM.

Key augmented processes in atherosclerosis have been identified, whereas less is known about downregulated pathways. Here, we applied a systems biology approach to examine suppressed molecular signatures, with the hypothesis that they may provide insight into mechanisms contributing to plaque stability. Muscle contraction, muscle development, and actin cytoskeleton were the most downregulated pathways (false discovery rate=6.99e-21, 1.66e-6, 2.54e-10, respectively) in microarrays from human carotid plaques (n=177) versus healthy arteries (n=15). In addition to typical smooth muscle cell (SMC) markers, these pathways also encompassed cytoskeleton-related genes previously not associated with atherosclerosis. SYNPO2, SYNM, LMOD1, PDLIM7, and PLN expression positively correlated to typical SMC markers in plaques (Pearson r>0.6, P<0.0001) and in rat intimal hyperplasia (r>0.8, P<0.0001). By immunohistochemistry, the proteins were expressed in SMCs in normal vessels, but largely absent in human plaques and intimal hyperplasia. Subcellularly, most proteins localized to the cytoskeleton in cultured SMCs and were regulated by active enhancer histone modification H3K27ac by chromatin immunoprecipitation-sequencing. Functionally, the genes were downregulated by PDGFB (platelet-derived growth factor beta) and IFNg (interferron gamma), exposure to shear flow stress, and oxLDL (oxidized low-density lipoprotein) loading. Genetic variants in PDLIM7, PLN, and SYNPO2 loci associated with progression of carotid intima-media thickness in high-risk subjects without symptoms of cardiovascular disease (n=3378). By eQTL (expression quantitative trait locus), rs11746443 also associated with PDLIM7 expression in plaques. Mechanistically, silencing of PDLIM7 in vitro led to downregulation of SMC markers and disruption of the actin cytoskeleton, decreased cell spreading, and increased proliferation. We identified a panel of genes that reflect the altered phenotype of SMCs in vascular disease and could be early sensitive markers of SMC dedifferentiation.

Hypomethylation coordinates antagonistically with hypermethylation in cancer development: a case study of leukemia.

BackgroundMethylation changes are frequent in cancers, but understanding how hyper- and hypomethylated region changes coordinate, associate with genomic features, and affect gene expression is needed to better understand their biological significance. The functional significance of hypermethylation is well studied, but that of hypomethylation remains limited. Here, with paired expression and methylation samples gathered from a patient/control cohort, we attempt to better characterize the gene expression and methylation changes that take place in cancer from B cell chronic lymphocyte leukemia (B-CLL) samples.ResultsAcross the dataset, we found that consistent differentially hypomethylated regions (C-DMRs) across samples were relatively few compared to the many poorly consistent hypo- and highly conserved hyper-DMRs. However, genes in the hypo-C-DMRs tended to be associated with functions antagonistic to those in the hyper-C-DMRs, like differentiation, cell-cycle regulation and proliferation, suggesting coordinated regulation of methylation changes. Hypo-C-DMRs in B-CLL were found enriched in key signaling pathways like B cell receptor and p53 pathways and genes/motifs essential for B lymphopoiesis. Hypo-C-DMRs tended to be proximal to genes with elevated expression in contrast to the transcription silencing-mechanism imposed by hypermethylation. Hypo-C-DMRs tended to be enriched in the regions of activating H4K4me1/2/3, H3K79me2, and H3K27ac histone modifications. In comparison, the polycomb repressive complex 2 (PRC2) signature, marked by EZH2, SUZ12, CTCF binding-sites, repressive H3K27me3 marks, and “repressed/poised promoter” states were associated with hyper-C-DMRs. Most hypo-C-DMRs were found in introns (36 %), 3′ untranslated regions (29 %), and intergenic regions (24 %). Many of these genic regions also overlapped with enhancers. The methylation of CpGs from 3′UTR exons was found to have weak but positive correlation with gene expression. In contrast, methylation in the 5′UTR was negatively correlated with expression. To better characterize the overlap between methylation and expression changes, we identified correlation modules that associate with “apoptosis” and “leukocyte activation”.ConclusionsDespite clinical heterogeneity in disease presentation, a number of methylation changes, both hypo and hyper, appear to be common in B-CLL. Hypomethylation appears to play an active, targeted, and complementary role in cancer progression, and it interplays with hypermethylation in a coordinated fashion in the cancer process.Electronic supplementary materialThe online version of this article (doi:10.1186/s40246-016-0071-5) contains supplementary material, which is available to authorized users.