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ATAC-seq analysis of Jurkat leukemia cell lines

ABSTRACT: Tissue-specific gene expression requires coordinated control of gene-proximal and distal cis-regulatory elements (CREs), yet functional analysis of putative gene-distal CREs such as enhancers remains challenging. Here we describe enhanced CRISPR/dCas9-based epigenetic editing systems, enCRISPRa and enCRISPRi, for multiplexed analysis of enhancer function in situ and in vivo. Using dual effector proteins capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, our systems display more robust and specific perturbations of gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Furthermore, multiplexed perturbations of lineage-specific enhancers in an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted requirement of developmentally regulated enhancers during hematopoietic lineage specification. Hence, enhanced CRSIPR epigenetic editing provides opportunities for interrogating enhancer function in development and disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE132209 | GEO | 2020/01/30

REPOSITORIES: GEO

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RNA-seq analysis of transcriptomic changes in HS2 enhancer perturbed cells

Project description:Tissue-specific gene expression requires coordinated control of gene-proximal and distal cis-regulatory elements (CREs), yet functional analysis of putative gene-distal CREs such as enhancers remains challenging. Here we describe enhanced CRISPR/dCas9-based epigenetic editing systems, enCRISPRa and enCRISPRi, for multiplexed analysis of enhancer function in situ and in vivo. Using dual effector proteins capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, our systems display more robust and specific perturbations of gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Furthermore, multiplexed perturbations of lineage-specific enhancers in an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted requirement of developmentally regulated enhancers during hematopoietic lineage specification. Hence, enhanced CRSIPR epigenetic editing provides opportunities for interrogating enhancer function in development and disease.

2020-01-30 | GSE132213 | GEO

ChIP-seq analysis of cas9, HA, H3K4me, H3K4me2, H3K9me3, H3K27ac, CTCF, GATA1 and TAL1 in HS2 enhancer perturbed K562 cells or other leukemia cell lines

2020-01-30 | GSE132212 | GEO

ChIP-seq analysis of dCas9 chromatin occupancy in K562 cells by CAPTURE2.0

Project description:The spatiotemporal control of 3D chromatin structure is fundamental for gene regulation, yet it remains challenging to obtain high-resolution chromatin interacting profiles at cis-regulatory elements (CREs) by chromatin conformation capture (3C)-based methods. Here, we describe the redesigned dCas9-based CAPTURE method for multiplexed, high-throughput and high-resolution analysis of locus-specific chromatin interactions. Using C-terminally biotinylated dCas9, endogenous biotin ligase and pooled sgRNAs, the new system enables quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling systematic comparisons across CREs within and between gene clusters. Multiplexed analyses of erythroid super-enhancers (SEs) reveal SE hierarchical structure and distinct modes of SE-gene interactions. Multiplexed capture of temporal dynamics of promoter-centric interactions establishes the instructive function of enhancer-promoter looping in transcriptional regulation during lineage differentiation. These applications illustrate the ability of multiplexed CAPTURE for decoding the organizational principles of genome structure and function.

2020-02-20 | GSE139112 | GEO

RNA-seq transcriptonal profiling in human K562 cells and G1E-ER4 cells with or without dCas9 and sgRNAs

Project description:The spatiotemporal control of 3D chromatin structure is fundamental for gene regulation, yet it remains challenging to obtain high-resolution chromatin interacting profiles at cis-regulatory elements (CREs) by chromatin conformation capture (3C)-based methods. Here, we describe the redesigned dCas9-based CAPTURE method for multiplexed, high-throughput and high-resolution analysis of locus-specific chromatin interactions. Using C-terminally biotinylated dCas9, endogenous biotin ligase and pooled sgRNAs, the new system enables quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling systematic comparisons across CREs within and between gene clusters. We reveal the hierarchical structure of super-enhancers (SEs) and distinct modes of SE-gene interactions. Multiplexed capture of temporal dynamics of promoter-centric interactions establishes the instructive function of enhancer-promoter looping in transcriptional regulation during lineage differentiation. These applications illustrate the ability of multiplexed CAPTURE for decoding the organizational principles of genome structure and function.

2020-02-20 | GSE139116 | GEO

CAPTURE-3C-seq analysis of cis-element-mediated long-range DNA interactions in K562 and G1E-ER4 cells

Project description:The spatiotemporal control of 3D chromatin structure is fundamental for gene regulation, yet it remains challenging to obtain high-resolution chromatin interacting profiles at cis-regulatory elements (CREs) by chromatin conformation capture (3C)-based methods. Here, we describe the redesigned dCas9-based CAPTURE method for multiplexed, high-throughput and high-resolution analysis of locus-specific chromatin interactions. Using C-terminally biotinylated dCas9, endogenous biotin ligase and pooled sgRNAs, the new system enables quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling systematic comparisons across CREs within and between gene clusters. We reveal the hierarchical structure of super-enhancers (SEs) and distinct modes of SE-gene interactions. Multiplexed capture of temporal dynamics of promoter-centric interactions establishes the instructive function of enhancer-promoter looping in transcriptional regulation during lineage differentiation. These applications illustrate the ability of multiplexed CAPTURE for decoding the organizational principles of genome structure and function.

2020-02-20 | GSE139109 | GEO

ATAC-seq analysis in G1E-ER4 cells during erythroid differentiation

Project description:The spatiotemporal control of 3D chromatin structure is fundamental for gene regulation, yet it remains challenging to obtain high-resolution chromatin interacting profiles at cis-regulatory elements (CREs) by chromatin conformation capture (3C)-based methods. Here, we describe the redesigned dCas9-based CAPTURE method for multiplexed, high-throughput and high-resolution analysis of locus-specific chromatin interactions. Using C-terminally biotinylated dCas9, endogenous biotin ligase and pooled sgRNAs, the new system enables quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling systematic comparisons across CREs within and between gene clusters. We reveal the hierarchical structure of super-enhancers (SEs) and distinct modes of SE-gene interactions. Multiplexed capture of temporal dynamics of promoter-centric interactions establishes the instructive function of enhancer-promoter looping in transcriptional regulation during lineage differentiation. These applications illustrate the ability of multiplexed CAPTURE for decoding the organizational principles of genome structure and function.

2020-02-20 | GSE139105 | GEO

CRISPRai Regulatory Element Screens [Series 2]

Project description:We develop a system for bidirectional epigenetic editing (CRISPRai), in which orthogonal activating (CRISPRa) and repressive (CRISPRi) perturbations are applied simultaneously to multiple loci the same cell. We perform CRISPR gRNA enrichment screens to investigate pairwise CRISPRai perturbations of enhancers and the promoter of IL2 and IFNG genes in order to investigate enhancer-enhancer and enhancer-promoter interactions and regulatory element heirarchies.

2024-03-14 | GSE220975 | GEO

Epigenetic and genetic features that lead to discovery of enhancer function

Project description:The ability to measure epigenetic features, such as histone modifications and occupancy by transcription factors and co-activators, on a genome-wide scale is advancing the accuracy of CRM predictions. While integration of signals from multiple features is expected to improve predictions, the contribution of each feature to prediction accuracy is not known. We began with predictions of 4,915 erythroid enhancers based on genomic occupancy by TAL1, a key hematopoietic transcription factor that is strongly associated with gene induction in erythroid cells. Seventy of these DNA segments occupied by TAL1 (TAL1 OSs) were tested by transient transfections of cultured hematopoietic cells, and 56% of these were active as enhancers. Sixty-six TAL1 OSs were evaluated in transgenic mouse embryos, and 65% of these were active enhancers in various tissues. Inclusion of additional epigenetic features improved the prediction accuracy, with combinations of TAL1, GATA1, EP300, H3K4me1, and H3K27ac giving high accuracy of enhancer prediction (70%-75% success depending on method of clustering) while maintaining good sensitivity and specificity. Motifs that distinguish active from inactive TAL1 OSs implicate IRFs, STATs, and FOX protein families as candidate positive co-factors with TAL1, while REST (NRSF) and HOX family proteins are implicated in inactivity. While signals for evolutionary constraint were weak over the entire TAL1-bound DNA segments regardless of activity in either assay, phylogenetic preservation of a TF-binding site motif was associated with enhancer activity. The contribution of 8 epigenetic features including H3K27ac to identification of enhancers in 24h-induced G1E-ER4 cells.

2015-04-27 | E-GEOD-61349 | biostudies-arrayexpress

Dynamic control of enhancer repertoires drives lineage and developmental stage-specific transcription during human erythropoiesis

Project description:Transcriptional enhancers are the primary determinants of tissue-specific gene expression and influence a variety of cellular phenotypes. The regulatory components controlling enhancer assembly and turnover during stem cell development remain largely unknown. Here we compared the similarities and differences in enhancer landscape, transcriptional factor (TF) occupancy and transcriptomic changes in human primary fetal and adult hematopoietic stem/progenitor cells (HSPCs) and committed erythroid progenitors. We find that enhancers are modulated dynamically and extensively, and direct lineage and developmental stage-specific transcriptional programs. GATA2-to-GATA1 switch is prevalent within transcriptionally dynamic enhancers and drives enhancer commissioning. Further examination of lineage-specific enhancers identified TFs and their combinatorial patterns with known and unknown roles as putative drivers of enhancer turnover during differentiation. Importantly, by site-directed loss-of-function analysis of individual lineage-selective enhancers within the SLC25A37 super-enhancer using CRISPR/Cas9-mediated genomic editing, we uncover unexpected functional hierarchy of constituent enhancers within the super-enhancer cluster. Despite the indistinguishable chromatin features between the GATA switch enhancers at the GATA2 gene, we reveal through genomic editing the functional diversity of GATA switch enhancers in which enhancers with opposing functions cooperate to coordinate gene expression during development. Thus, genome-wide enhancer profiling coupled with in-depth enhancer editing in situ provide critical insights into the functional hierarchy and complexity of enhancers during stem cell development.

2016-01-11 | GSE70660 | GEO

Defining super-enhancer landscape in triple-negative breast cancer by multiomic profiling

Project description:Here we apply integrated epigenomic and transcriptomic profiling to uncover super-enhancer heterogeneity between breast cancer subtypes, and provide clinically relevant biological insights towards TNBC. Using CRISPR/Cas9-mediated gene editing, we identify genes that are specifically regulated by TNBC-specific super-enhancers, including FOXC1 and MET, thereby unveiling a mechanism for specific overexpression of the key oncogenes in TNBC. We also identify ANLN as a novel TNBC-specific gene regulated by super-enhancer. Our studies reveal a TNBC-specific epigenomic landscape, contributing to the dysregulated oncogene expression in breast tumorigenesis.

2021-03-04 | PXD024505 | iProX

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