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An ectopic enhancer restores CFTR expression through de novo chromatin looping [4C-seq]

ABSTRACT: Transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is regulated by a set of ubiquitous and cell-type selective cis-regulatory elements (CREs). These elements include extragenic and intronic enhancers that bind cell-type specific transcription factors, as well as architectural protein bound structural elements. We previously reported that loss of the airway-selective -35kb enhancer in 16HBE14o- airway epithelial cells disrupts normal enhancer-promoter looping at the CFTR locus and nearly abolishes its expression. Here we expand on previous work, and explore the effect of relocation of the 500bp -35kb enhancer core to an ectopic site in intron 1 of CFTR on gene expression and chromatin dynamics. We find that although genomic relocation of this element is not able to fully restore CFTR expression to wild-type levels, the -35kb sequence is able establish a de novo enhancer signature at chromatin at the site of insertion. Additionally, the relocated -35kb enhancer is able to form chromatin contacts with known CFTR CREs. These studies reveal important direct chromatin effects, as well as limitations, of CFTR enhancers, and provide important considerations when targeting the CFTR locus for novel therapeutic gene editing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE203559 | GEO | 2022/12/13

REPOSITORIES: GEO

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An ectopic enhancer restores CFTR expression through de novo chromatin looping [ATAC-seq]

Project description:Transcription of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is regulated by a set of ubiquitous and cell-type selective cis-regulatory elements (CREs). These elements include extragenic and intronic enhancers that bind cell-type specific transcription factors, as well as architectural protein bound structural elements. We previously reported that loss of the airway-selective -35kb enhancer in 16HBE14o- airway epithelial cells disrupts normal enhancer-promoter looping at the CFTR locus and nearly abolishes its expression. Here we expand on previous work, and explore the effect of relocation of the 500bp -35kb enhancer core to an ectopic site in intron 1 of CFTR on gene expression and chromatin dynamics. We find that although genomic relocation of this element is not able to fully restore CFTR expression to wild-type levels, the -35kb sequence is able establish a de novo enhancer signature at chromatin at the site of insertion. Additionally, the relocated -35kb enhancer is able to form chromatin contacts with known CFTR CREs. These studies reveal important direct chromatin effects, as well as limitations, of CFTR enhancers, and provide important considerations when targeting the CFTR locus for novel therapeutic gene editing.

2022-12-13 | GSE203558 | GEO

The impact of genomic distance on enhancer-promoter interactions at the CFTR locus.

Project description:We identified and characterized multiple cell-type selective enhancers of the CFTR gene promoter in previous work and demonstrated active looping of these elements to the promoter. Here we address the impact of genomic spacing on these enhancer:promoter interactions and on CFTR gene expression. Using CRISPR/Cas9, we generated clonal cell lines with deletions between the -35kb airway enhancer and the CFTR promoter in the 16HBE14o- airway cell line, or between the intron 1 (185 + 10kb) intestinal enhancer and the promoter in the Caco2 intestinal cell line. The effect of these deletions on CFTR transcript abundance, as well as the 3D looping structure of the locus was investigated in triplicate clones of each modification. Our results indicate that both small and larger deletions upstream of the promoter can perturb CFTR expression and -35kb enhancer:promoter interactions in the airway cells, though the larger deletions are more impactful. In contrast, the small intronic deletions have no effect on CFTR expression and intron 1 enhancer:promoter interactions in the intestinal cells, whereas larger deletions do. Clonal variation following a specific CFTR modification is a confounding factor particularly in 16HBE14o- cells.

2024-03-07 | GSE239930 | GEO

Looping of upstream cis-regulatory elements is required for CFTR expression in human airway epithelial cells [4C-seq]

Project description:The CFTR gene lies within an invariant topologically associated domain (TAD) demarcated by CTCF and cohesin, but shows cell-type specific control mechanisms utilizing different cis-regulatory elements (CRE) within the TAD. Within the respiratory epithelium, more than one cell type expresses CFTR and the molecular mechanism controlling its transcription are likely divergent between them. Here we determine how two extragenic CREs that are prominent in secretory epithelial cells in the lung, regulate expression of the gene. We showed earlier that these CREs, located at -44kb and -35 kb upstream of the promoter, have strong cell-type-selective enhancer function. They are also responsive to inflammatory mediators and to oxidative stress, consistent with a key role in CF lung disease. Here we use CRISPR/Cas9 technology to remove these CREs from the endogenous locus in human bronchial epithelial cells. Loss of either site extinguished CFTR expression and abolished long-range interactions between these sites and the gene promoter. The deletions also greatly reduced promoter interactions with the 5’ TAD boundary. We show substantial recruitment of RNAPII to the -35kb element and identify CEBPβ as a key activating transcription factor for airway expression of CFTR, likely through occupancy at both the CRE and the gene promoter.

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Looping of upstream cis-regulatory elements is required for CFTR expression in human airway epithelial cells [Omni-ATAC-seq]

2020-04-22 | GSE132806 | GEO

Looping of upstream cis-regulatory elements is required for CFTR expression in human airway epithelial cells [ChIP-seq]

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Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus

Project description:Higher order chromatin structure establishes domains that organize the genome and coordinate gene expression. However, the molecular mechanisms controlling transcription of individual loci within a topological domain (TAD) are not fully understood. The cystic fibrosis transmembrane conductance regulator (CFTR) gene provides a paradigm for investigating these mechanisms. CFTR occupies a TAD bordered by CTCF/cohesin binding sites within which are cell-type-selective cis-regulatory elements for the locus. We showed previously that intronic and extragenic enhancers, when occupied by specific transcription factors, are recruited to the CFTR promoter by a looping mechanism to drive gene expression. Here we use a combination of CRISPR/Cas9 editing of cis-regulatory elements and siRNA-mediated depletion of architectural proteins to determine the relative contribution of structural elements and enhancers to the higher order structure and expression of the CFTR locus. We found the boundaries of the CFTR TAD are conserved among diverse cell types and are dependent on CTCF and cohesin complex. Removal of an upstream CTCF-binding insulator alters the interaction profile, but has little effect on CFTR expression. Within the TAD, intronic enhancers recruit cell-type selective transcription factors and deletion of a pivotal enhancer element dramatically decreases CFTR expression, but has minor effect on its 3D structure. Examination of open chromatin region in Caco2 (colorectal adenocarcinoma cells), HBE (primary human bronchial epithelial cells), and primary adult human epididymis cells

2016-01-07 | E-GEOD-74709 | biostudies-arrayexpress

BACH1, the master regulator of oxidative stress, modulates CFTR gene expression [RNA-seq]

Project description:The cystic fibrosis transmembrane conductance regulator (CFTR) gene lies within a TAD in which multiple cis-regulatory elements (CREs) and transcription factors (TFs) regulate its cell-specific expression. The CREs are recruited to the gene promoter by a looping mechanism that depends upon both architectural proteins and specific TFs. An siRNA screen to identify TFs coordinating CFTR expression in airway epithelial cells suggested an activating role for BTB Domain and CNC Homolog 1 (BACH1). BACH1 is a ubiquitous master regulator of the cellular response to oxidative stress. Here we show that BACH1 may have a dual effect on CFTR expression by direct occupancy of CREs at physiological oxygen (~8%), while indirectly modulating expression under conditions of oxidative stress. Hence BACH1, can activate or repress the same gene, to fine tune expression in response to environmental cues such as cell stress. Furthermore, our 4C-seq data suggest that BACH1 can also directly regulate CFTR gene expression by modulating locus architecture through occupancy at known enhancers and structural elements, and depletion of BACH1 alters the higher order chromatin structure.

2021-11-17 | GSE165718 | GEO

BACH1, the master regulator of oxidative stress, modulates CFTR gene expression [ChIP-seq]

2021-11-17 | GSE165717 | GEO

BACH1, the master regulator of oxidative stress, modulates CFTR gene expression [4C-seq]

2021-11-17 | GSE165716 | GEO

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

Project description:Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated endonuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human β-globin genes establishes evidence for composition-based hierarchical organization of enhancer structure. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally controlled super-enhancers reveals spatial features causally regulate gene transcription. Thus, comprehensive analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.

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