Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the <i>CFTR</i> gene

Phylactides, Marios; Rowntree, Rebecca K.; Nuthall, Hugh; Ussery, David W.; Wheeler, Ann; Harris, Ann

doi:10.1046/j.0014-2956.2001.02679.x

Cited by 38 publications

(47 citation statements)

References 22 publications

(49 reference statements)

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“…A comparison of the power of these techniques with classic methods is warranted, particularly with respect to DHS mapping. We previously used a Southern blot-based method to screen for DHS across 400 kb spanning the CFTR locus in a number of cell lines, and identified a number of important regulatory elements within the locus (18)(19)(20)(21)(22)(23). While our experiments using DNase-chip were able to reproducibly and reliably detect DHS of the locus that functionally associate with the CFTR promoter, we were unable to detect several DHS that we observed in earlier studies.…”

Section: Discussioncontrasting

confidence: 44%

“…Additional cell-line specific DHS are evident in intron 10 (1716 ϩ 13.2 kb) in Caco2 cells and intron 18 (3600 ϩ 1.6 kb) in HT29. The intron 10 site encompasses two closely spaced DHS at 1716 ϩ 13.2 kb and ϩ13.7 kb, that were characterized in our previous work (22,23,28). Also of interest are the DHS located -35 kb and -44 kb with respect to the CFTR translational start site in primary epididymis cells.…”

Section: Resultsmentioning

confidence: 99%

“…Because we previously demonstrated enhancer function associated with several intronic DHS in the CFTR gene (19,23,26,27), we next tested whether the intron 11 DHS exhibited similar properties. The entire region flanking this site was cloned, in forward and reverse orientations, into the enhancer site of a luciferase reporter vector (23) in which 787 bp of the CFTR minimal promoter drives luciferase expression. These constructs were evaluated for firefly luciferase expression following transient transfection into Caco2 cells (Fig.…”

Section: An Element Within Cftr Intron 11 Is a Strong Transcriptionalmentioning

confidence: 99%

“…3A). Positive controls included the intron 1 DHS region enhancer in both orientations and negative controls were provided by the intron 10a,b DHS that we demonstrated previously to have no enhancer activity (23). The fragment encompassing the DHS in intron 11 enhanced CFTR promoter activity almost 20-fold in the forward and reverse orientation.…”

Section: An Element Within Cftr Intron 11 Is a Strong Transcriptionalmentioning

confidence: 99%

“…Thus, it is likely that elements outside the basal promoter region contribute to its diverse expression profile. We previously used classical methods of chromatin analysis to map potential regulatory elements in a number of cell lines that express the CFTR gene and identified several functionally important elements (18)(19)(20)(21)(22)(23). In this study we aimed to use recent technologies to comprehensively map potential regulatory elements of the CFTR locus and to establish their mechanism of action.…”

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confidence: 99%

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Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus

Ott

Blackledge

Kerschner

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The regulated expression of large human genes can depend on long-range interactions to establish appropriate three-dimensional structures across the locus. The cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encompasses 189 kb of genomic DNA, shows a complex pattern of expression with both spatial and temporal regulation. The flanking loci, ASZ1 and CTTNBP2, show very different tissue-specific expression. The mechanisms governing control of CFTR expression remain poorly understood, although they are known to involve intronic regulatory elements. Here, we show a complex looped structure of the CFTR locus in cells that express the gene, which is absent from cells in which the gene is inactive. By using chromatin conformation capture (3C) with a bait probe at the CFTR promoter, we demonstrate close interaction of this region with sequences in the middle of the gene about 100 kb from the promoter and with regions 3 to the locus that are about 200 kb away. We show that these interacting regions correspond to prominent DNase I hypersensitive sites within the locus. Moreover, these sequences act cooperatively in reporter gene constructs and recruit proteins that modify chromatin structure. The model for CFTR gene expression that is revealed by our data provides a paradigm for other large genes with multiple regulatory elements lying within both introns and intergenic regions. We anticipate that these observations will enable original approaches to designing regulated transgenes for tissue-specific gene therapy protocols.cis-acting elements ͉ enhancer:promoter interactions ͉ regulation of expression ͉ cystic fibrosis transmembrane conductance regulator U nderstanding the three-dimensional organization of individual loci within the human genome and how this relates to the regulation of gene expression is the focus of intense study. Many new technologies are being developed to locate and classify the functional elements of the human genome (1). These elements may contribute to the transcription of ubiquitously expressed genes and are also likely responsible for regulating genes with expression that is temporally and spatially controlled. Cis-acting regulatory elements located within noncoding regions of genomic DNA can influence the organization of chromosomes and the transcriptional activity of genes. These cis sequences include distal enhancers that may reside large distances from the gene promoters they control. Variations in these enhancer/promoter interactions and the nuclear localization of the genes they regulate are thought to be contributing factors in the diversity of transcriptional profiles between different cell types. Moreover, they are important in adjusting these profiles throughout cellular differentiation and development (2-7). These long-range associations are facilitated by the looping of chromatin, whereby regulatory elements come together with requisite nuclear factors to function within ''transcriptional hubs'' where transcriptional activity is coordinated (8).Here, we present...

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Section: Discussioncontrasting

confidence: 44%

Section: Resultsmentioning

confidence: 99%

Section: An Element Within Cftr Intron 11 Is a Strong Transcriptionalmentioning

confidence: 99%

Section: An Element Within Cftr Intron 11 Is a Strong Transcriptionalmentioning

confidence: 99%

mentioning

confidence: 99%

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Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus

Ott

Blackledge

Kerschner

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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103

211

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OTX2 regulates CFTR expression during endoderm differentiation and occupies 3′ cis‐regulatory elements

Kerschner

Paranjapye

NandyMazumdar

et al. 2021

Developmental Dynamics

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Background: Cell-specific and developmental mechanisms contribute to expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene; however, its developmental regulation is poorly understood. Here we use human induced pluripotent stem cells differentiated into pseudostratified airway epithelial cells to study these mechanisms. Results: Changes in gene expression and open chromatin profiles were investigated by RNA-seq and ATAC-seq, and revealed that alterations in CFTR expression are associated with differences in stage-specific open chromatin. Additionally, two novel open chromatin regions, at +19.6 kb and +22.6 kb 3 0 to the CFTR translational stop signal, were observed in definitive endoderm (DE) cells, prior to an increase in CFTR expression in anterior foregut endoderm (AFE) cells. Chromatin studies in DE and AFE cells revealed enrichment of active enhancer marks and occupancy of OTX2 at these sites in DE cells. Loss of OTX2 in DE cells alters histone modifications across the CFTR locus and results in a 2.5-fold to 5-fold increase in CFTR expression. However, deletion of the +22.6 kb site alone does not affect CFTR expression in DE or AFE cells.Conclusions: These results suggest that a network of interacting cis-regulatory elements recruit OTX2 to the locus to impact CFTR expression during early endoderm differentiation.

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The CFTR gene and regulation of its expression

McCarthy

Harris

2005

Pediatric Pulmonology

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The cystic fibrosis transmembrane conductance regulator gene (CFTR) shows clear temporal and developmental regulation of its expression. However, there are few well-defined regulatory elements that control this pattern of expression, and their mechanism of action is poorly understood. We review the structure and organization of the CFTR gene and what is known about its regulation. The CFTR gene promoter is clearly important for maintaining levels of CFTR gene expression, but apparently it does not contain any tissue-specific elements. Thus tissue-specificity is probably controlled by sequences lying elsewhere in this large gene. We discuss data from our group and others implicating additional regions of CFTR in regulatory functions, and evaluate candidate transcription factors that may be involved. Further, we summarize aspects of the regulation of the developmental expression of CFTR. Definition of CFTR gene regulatory elements could be of considerable therapeutic significance, since only a small increase in CFTR expression in the correct cell type could alleviate the disease phenotype.

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Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR gene

Cited by 38 publications

References 22 publications

Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus

Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus

OTX2 regulates CFTR expression during endoderm differentiation and occupies 3′ cis‐regulatory elements

The CFTR gene and regulation of its expression

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