Enhancer Mapping in Chronic Lymphocytic Leukemia
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ABSTRACT: In this study we profile the epigenomic enhancer landscapes of CLL B cells (CD19+/CD5+) harvested from peripheral blood of patients from our Center. Included are results of ChIPseq profiling using chromatin immunoprecipitation of the enhancer histone mark H3K27ac (acetylated lysine 27 on histone H3), and open chromatin profiles using ATAC-seq (assay for transposase accessible chromatin). These profiles are used to define the global enhancer and super enhancer landscape of CLL B cells, and to derive active transcription factor networks associated with this disease. Also included are H3K27ac ChIP-seq and ATAC-seq datasets for non-CLL B cells obtained from the peripheral blood of normal adult donors.
REPOSITORIES: dbGaP
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Project description:Enhancers are fundamental to gene regulation. Post-translational modifications by the small ubiquitin-like modifiers (SUMO) modify chromatin regulation enzymes, including histone acetylases and deacetylases. However, it remains unclear whether SUMOylation regulates enhancer marks, acetylation at the 27th lysine residue of the histone H3 protein (H3K27Ac). We hypothesize that SUMOylation regulates H3K27Ac. To test this hypothesis, we performed genome-wide ChIP-seq analyses. We discovered that knockdown (KD) of the SUMO activating enzyme catalytic subunit UBA2 reduced H3K27Ac at most enhancers. Bioinformatic analysis revealed that TFAP2C-binding sites are enriched in enhancers whose H3K27Ac was reduced by UBA2 KD. ChIP-seq analysis in combination with molecular biological methods showed that TFAP2C binding to enhancers increased upon UBA2 KD or inhibition of SUMOylation by a small molecule SUMOylation inhibitor. However, this is not due to the SUMOylation of TFAP2C itself. Proteomics analysis of TFAP2C interactome on the chromatin identified histone deacetylation (HDAC) machinery. TFAP2C KD reduced HDAC binding to chromatin and increased H3K27Ac marks at enhancer regions, suggesting that TFAP2C is involved in recruiting HDAC. Taken together, our findings provide important insights into regulation of enhancer marks by SUMOylation.
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Project description:Enhancer profiling has emerged as a powerful approach for discovering the cis-regulatory elements that define transcriptional core regulatory circuits. Characteristic biochemical and biophysical attributes of chromatin mark active enhancer elements, which can be leveraged with genome-wide assay technologies for discovery. This includes chromatin immunoprecipitation followed by sequencing (ChIP-seq) for histone H3 acetylated lysine 27 (H3K27ac). Enhancers are also characterized by regions of open chromatin, hypersensitive to digestion by transposases measurable by the assay for transposase-accessible chromatin (ATAC-seq). By integrating measures of chromatin accessibility and domains of enriched H3K27 acetylation, we have generated enhancer landscapes from chronic lymphocytic leukemia (CLL) representative cell lines including MEC1, MEC2, OSU-CLL, and CII. Targeting enhancer signaling via BET bromodomain inhibition (using the inhibitor JQ1) disrupts enhancer-dependent gene expression as measured with RNA-sequencing in CLL cell lines.
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Project description:Enhancers play key roles in gene regulation. However, comprehensive enhancer discovery is challenging because most enhancers, especially those affected in complex diseases, have weak effects on gene expression. Through gene regulatory network modeling, we identified that dynamic cell state transitions, a critical missing component in prevalent enhancer discovery strategies, can be utilized to improve the cells’ sensitivity to enhancer perturbation. Guided by the modeling results, we performed a mid-transition CRISPRi-based enhancer screen utilizing human embryonic stem cell definitive endoderm differentiation as a dynamic transition system. The screen discovered a comprehensive set of enhancers (4 to 9 per locus) for each of the core lineage-specifying transcription factors (TFs), including many enhancers with weak to moderate effects. Integrating the screening results with enhancer activity measurements (ATAC-seq, H3K27ac ChIP-seq) and three-dimensional enhancer-promoter interaction information (CTCF looping, Hi-C), we were able to develop a CTCF loop-constrained Interaction Activity (CIA) model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Together, our dynamic network-guided enhancer screen and the CIA enhancer prediction model provide generalizable strategies for sensitive and more comprehensive enhancer discovery in both normal and pathological cell state transitions.
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Project description:Aberrant chromatin features and epigenetic signaling emerge as an important part of the tumor disease state in chronic lymphocytic leukemia (CLL). However, an integrative analysis of the malignant chromatin landscape is currently missing. Here, we mapped DNA-methylation, nucleosome position, 7 histone modifications, ATAC-seq and the transcriptome in primary CD19+ sorted B-cells from the peripheral blood of CLL patients in comparison to non-malignant lymphocytes of healthy donor reference samples. On the > 50 kb scale we identified global differences with respect to DNA methylation, nucleosome positioning and histone modifications in CLL. Furthermore, a chromatin state annotation was developed to identify deregulated chromatin features of promoters and enhancers in CLL. By integrating these findings with an ATAC-seq based transcription factor binding analysis we identified a set of promoters and enhancers specific to CLL that were enriched in certain transcription factors binding motifs and genes of the BCR signaling pathway. Thus, our study provides an in-depth map of the CLL chromatin landscape and links chromatin state changes at promoters and enhancers to deregulated pathways.
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2023-09-26 | PXD044471 | Pride
Project description:We used chromatin-immunoprecipitation followed by sequencing (ChIP-Seq) with an antibody for the H3K27ac (a bona fide histone mark for regulatory element activation) in sorted CLL cells from 15 CLL, including cases from stereotyped subsets #1, #2, #4, and #8. The samples were sequenced by Illumina HiSeq 2500.
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Project description:Chronic lymphocytic leukemia (CLL) is characterized by substantial clinical heterogeneity, despite relatively few genetic alterations. To provide a basis for studying epigenome deregulation in CLL, we established genome-wide chromatin accessibility maps for 88 CLL samples from 55 patients using the ATAC-seq assay, and we also performed ChIPmentation and RNA-seq profiling for ten representative samples. Based on the resulting dataset, we devised and applied a bioinformatic method that links chromatin profiles to clinical annotations. Our analysis identified sample-specific variation on top of a shared core of CLL regulatory regions. IGHV mutation status â which distinguishes the two major subtypes of CLL â was accurately predicted by the chromatin profiles, and gene regulatory networks inferred for IGHV-mutated vs. IGHV-unmutated samples identified characteristic differences between these two disease subtypes. In summary, we found widespread heterogeneity in the CLL chromatin landscape, established a community resource for studying epigenome deregulation in leukemia, and demonstrated the feasibility of chromatin accessibility mapping in cancer cohorts and clinical research. Genome-wide profiling of chromatin states and gene expression levels in 88 CLL samples from 55 individuals gave rise to 88 ATAC-seq profiles, 40 ChIPmentation profiles (10 samples, each with 3 different antibodies and matched immunoglobulin control), and 10 RNA-seq profiles. Raw sequence data has been deposited at the EBI's European Genome-phenome Archive (EGA) under the accession number EGAS00001001821 (controlled access to protect patient privacy).
2016-06-26 | E-GEOD-81274 | biostudies-arrayexpress
Project description:We determined changes in enhancer chromatin that occur during colonic inflammation, found that dynamic chromatin regions are enriched for HNF4A binding motirfs, and then measured HNF4A binidng by ChIP-seq in each condition. Examination of H3K27ac histone modification in normal and DSS-treated colon, examination of HNF4A binding by ChIP-seq in normal and DSS-treated colon epithelium
2014-06-30 | E-GEOD-52426 | biostudies-arrayexpress
Project description:Chronic lymphocytic leukemia (CLL) is a biologically and clinically heterogeneous disease. The somatic hypermutation status of the immunoglobulin heavy chain variable (IGHV) genes has been identified as one of the most robust prognostic markers in CLL. Patients with unmutated IGHV status (U-CLL) typically experience an inferior outcome compared to those whose clones express mutated IGHV genes (M-CLL). We conducted a genome-wide DNA methylation analysis in CD19+ B-cells from a group of 43 CLL patients using reduced representation bisulfite sequencing (RRBS). Using base-pair resolution methylation sequencing, 2323 differentially methylated regions between CLL and normal B-cells (CLL-specific DMRs) and 569 between M-CLL and U-CLL samples (IGHV-specific DMRs) were identified in the CLL genomes. The IGHV-specific DMRs are mostly unique when compared to the CLL-specific DMRs. Less than 10% of the IGHV-specific DMRs are located in promoter regions; however, more than half of these overlap with known DNase I hypersensitive sites, enhancer regions marked by histone modification (H3K4Me1 and H3K27Ac), and transcription factor binding sites in the ENCODE datasets, which indicates that these DMRs contain regulatory sequences. Distinctive DNA methylation patterns were observed in M-CLL and U-CLL samples. Overall, U-CLL was found to contain 50% more hypermethylated regions than M-CLL samples. The hypermethylated loci observed in the U-CLL samples also appear to be hypermethylated in normal naïve B-cells as compared memory B-cells, suggesting that M-CLL and U-CLL differ in differentiation status corresponding to normal B-cell differentiation stages. RNA-seq analysis performed using matched samples (n=34), in which both DNA methylation and gene expression data were available, demonstrated excellent correlation between DNA methylation and gene expression. Several genes whose expression status was previously shown to be associated with CLL prognosis such as ZAP70, CRY1, LDOC1, SEPT10, LAG3, and LPL were differentially methylated in the promoter regions between M-CLL and U-CLL samples indicating that DNA methylation plays an important role in defining the gene expression patterns of these prognostic genes. We further validated 9 genes with IGHV-specific DMRs in the promoter regions using bisulfite pyrosequencing, and the results demonstrated excellent correlation between differential methylation and IGHV mutation status. These novel differentially methylated genes could be developed into biomarkers for CLL prognosis. In addition, DNA hypomethylation was observed in a significant number of genes involved in lymphocyte activation such as PDCD1, NFAT1, and CD5. DNA hypomethylation was observed in the proximal promoter and far up-stream enhancer regions of CD5, an important cell surface marker that uniquely identifies CLL. Overall, the DNA methylation landscape in CLL patients indicates that CLL B cells possess an active B-cell phenotype; at the same time, U-CLL and M-CLL are faithfully committed to their lineage resembling either naïve or memory B-cells. In summary, this comprehensive DNA methylation analysis has identified a large number of novel epigenetic changes in CLL patients. The results from this study will further advance our understanding of the epigenetic contribution to molecular subtypes in CLL. To perform a transcriptome analysis in CLL, we generated sequencing libraries from total RNA isolated from purified B-cells of CLL patients and healthy donnors. The RNA-seq libraries were sequenced using Illumina HiSeq2000 sequencer with a read length of 100bp. 11 CLL B-cell samples, 3 normal control samples including one each of normal CD19+ B cells were studied. We generated 20-30 million Illumina sequencing reads for each sample.
2015-02-21 | E-GEOD-66117 | biostudies-arrayexpress
Project description:EZH2, H3K4me3, H3K27ac and H3K27me3 ChIP-seq data consisting of fastq single-end reads from peripheral blood CLL cells
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