An ectopic enhancer restores CFTR expression through de novo chromatin looping.
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ABSTRACT:
PROVIDER: PRJNA841375 | ENA |
REPOSITORIES: ENA
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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 | GSE203559 | GEO
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
Project description:An ectopic enhancer restores CFTR expression through de novo chromatin looping [ATAC-seq]
| PRJNA841377 | ENA
Project description:An ectopic enhancer restores CFTR expression through de novo chromatin looping [4C-seq]
| PRJNA841376 | ENA
Project description:This SuperSeries is composed of the SubSeries listed below.
2022-12-13 | GSE203560 | GEO
Project description:Three-dimensional genome structure plays an important role in gene regulation. Globally chromosomes are organized into active and inactive compartments, while at the gene level looping interactions connect promoters to regulatory elements. Topologically Associating Domains (TADs), typically several hundred kilobases in size form an intermediate level of organization. Major questions include how TADs are formed and what their relation is with looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb region on chromosome 7 surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Further, we find that these TAD boundaries are present irrespective of expression and looping of genes located between them. In contrast looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell type-specific long-range interactions and gene regulation. We investigated a 2.8 Mb region on Chromosome 7 (hg18 chr7: 115797757-118405450) containing the ENCODE region ENm001 42. The 5C experiment was designed to interrogate looping interactions between HindIII fragments containing transcription start sites (TSSs) and any other HindIII restriction fragment (distal fragments) in the target region. Libraries were generated for five cell lines: Caco2, Calu3, Capan1, GM12878 and HepG2, with two biological replicates for each line. 5C probes were designed at HindIII restriction sites (AAGCTT) using 5C primer design tools previously developed and made publicly available online at our My5C website (http://my5C.umassmed.edu). Probes were designed based on the ENCODE manual region 1 (ENM001) design 25 with additional probes placed throughout the region when appropriate. We also added probes to extend the analysis to include a 700 Kb gene desert region located directly adjacent to ENM001. Probe settings were: U-BLAST, 3; S-BLAST, 100; 15-MER, 3,000; MIN_FSIZE, 250; MAX_FSIZE, 20,000; OPT_TM, 65; OPT_PSIZE, 40. We designed 74 reverse 5C probes, and 605 forward 5C probes.
2016-01-07 | E-GEOD-75634 | biostudies-arrayexpress
Project description:Three-dimensional genome structure plays an important role in gene regulation. Globally chromosomes are organized into active and inactive compartments, while at the gene level looping interactions connect promoters to regulatory elements. Topologically Associating Domains (TADs), typically several hundred kilobases in size form an intermediate level of organization. Major questions include how TADs are formed and what their relation is with looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb region on chromosome 7 surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Further, we find that these TAD boundaries are present irrespective of expression and looping of genes located between them. In contrast looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell type-specific long-range interactions and gene regulation.
2016-01-07 | GSE75634 | GEO
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
Project description:Background. Accumulating evidence indicates that CFTR modulators can be effective for CF sufferers. However, in vivo the microRNAs that regulate CFTR are increased in the CF lung milieu and this can nullify the beneficial effects of CFTR modulators. Methods. A panel of target site blockers (TSBs) designed against the CFTR 3’untranslated region (UTR) were tested for their ability to increase CFTR expression by CFTR 3’UTR luciferase assay and western blot. The effect of two lead TSBs on CFTR activity were tested alone or in combination with selected CFTR modulators in four separate p.Phe508del/p.Phe508del in vitro and ex vivo models (CFBE41o-, Cufi-1, primary bronchial epithelial cells, iPSC-derived lung organoids). Respirable poly-lactic-co-glycolic acid (PLGA) nanoparticles encapsulating the TSBs were formulated and tested. Findings. TSBs that target the miR-145-5p or miR-223-3p binding sites at positons 298-305 and 166-173 in the CFTR 3’UTR, respectively, increased CFTR expression and CFTR anion channel activity, and enhance the effects of Lumacaftor/Ivacaftor or Lumacftor/Tezacaftor in CF BECs. PLGAs encapsulating the TSBs promote CFTR expression in primary BECs and retain their biophysical characteristics following nebulization. Interpretation. Alone or in combination with CFTR modulators, CFTR-targeting TSBs encapsulated in PLGA nanoparticles and nebulized to the lung could overcome microRNA-mediated inhibition of CFTR in CF bronchial epithelium. This strategy represents a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung.
2019-03-27 | GSE128861 | GEO
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