Analysis of DNase-I-hypersensitive sites at the 3′ end of the cystic fibrosis transmembrane conductance regulator gene (<i>CFTR</i>)

Nuthall, Hugh; Moulin, Danielle S.; Huxley, Clare; Harris, Ann

doi:10.1042/bj3410601

Cited by 39 publications

(48 citation statements)

References 39 publications

(47 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: Discussionmentioning

confidence: 54%

“…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. These include the site at -20.9 kb from the CFTR translational start site (18,21) that was evident in many cell types and in human YAC-transgenic mice (25), as well as a cluster of four sites ϩ6.8 to ϩ7.4 kb distal to the translational stop site of CFTR (20). The -20.9 kb and ϩ6.8 kb sites both possess enhancer-blocking insulator activity and associate with CCCTC-binding factor (CTCF) (30,31).…”

Section: Discussionmentioning

confidence: 99%

“…A major DHS was identified at the CFTR promoter (Pr) in all cells that express the gene; several specific DHS of interest were detected, including those in intron 1 (Int1) and intron 11 (Int11). A DHS at ϩ15.6 kb 3Ј to the CFTR translational stop site (20,30) was seen in several cell types, and at ϩ48.9 kb a ubiquitous DHS (Ubiq) is marked in the last intron of the CTTBP2 gene. The zero point of the x axis represents the beginning of the first CFTR exon.…”

Section: The Cftr Locus Is Organized In a Complex Looped Structure Inmentioning

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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103

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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: Discussionmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 99%

Section: The Cftr Locus Is Organized In a Complex Looped Structure Inmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Ott

Blackledge

Kerschner

et al. 2009

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

Self Cite

103

211

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show abstract

“…The CFTR gene is known to be expressed in a tightly regulated fashion (Chou et al 1991;Denamur et al 1994;Matthews et al 1996;Pittman et al 1995;Trapnell et al 1991;Yoshimura et al 1991a,b). The control and enhancer regions for CFTR are not yet fully defined, although DNase I hypersensitive sites (DHSs) have been found at ‫‬ 20.5 kb, ‫‬ 79.5 kb, 185 + 10 kb within the first intron (Smith et al 1995;Smith et al 1996), and 4574 + 15.6 kb beyond the 3Ј end of the gene (Nuthall et al 1999). Putative cAMP response elements (CREs), Y-box, Ap-1, Sp-1, major and minor transcriptional start sites, and CAAT-like sequences have been identified by sequence analysis, 5Ј RACE, mutational analysis, and electrophoretic mobility shift assays (Chou et al 1991;Denamur et al 1994;Imler et al 1996;Matthews et al 1996;Pittman et al 1995;Vuillaumier et al 1997;White et al 1998;Yoshimura et al 1991a).…”

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confidence: 99%

Genomic Sequence Analysis of Fugu rubripes CFTR and Flanking Genes in a 60 kb Region Conserving Synteny with 800 kb of Human Chromosome 7

Davidson¹,

Taylor²,

Doherty³

et al. 2000

Genome Res.

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To define control elements that regulate tissue-specific expression of the cystic fibrosis transmembrane regulator (CFTR), we have sequenced 60 kb of genomic DNA from the puffer fish Fugu rubripes (Fugu) that includes the CFTR gene. This region of the Fugu genome shows conservation of synteny with 800-kb sequence of the human genome encompassing the WNT2, CFTR, Z43555, and CBP90 genes. Additionally, the genomic structure of each gene is conserved. In a multiple sequence alignment of human, mouse, andFugu, the putative WNT2 promoter sequence is shown to contain highly conserved elements that may be transcription factor or other regulatory binding sites. We have found two putative ankyrin repeat-containing genes that flank the CFTR gene. Overall sequence analysis suggests conservation of intron/exon boundaries betweenFugu and human CFTR and revealed extensive homology between functional protein domains. However, the immediate 5′ regions of human and Fugu CFTR are highly divergent with few conserved sequences apart from those resembling diminished cAMP response elements (CRE) and CAAT box elements. Interestingly, the polymorphic polyT tract located upstream of exon 9 is present in human and Fugu but absent in mouse. Similarly, an intron 1 and intron 9 element common to human and Fugu is absent in mouse. The euryhaline killifish CFTR coding sequence is highly homologous to the Fugusequence, suggesting that upregulation of CFTR in that species in response to salinity may be regulated transcriptionally.[The sequence data described in this paper have been submitted to the GenBank data library under accession no. AJ271361, for the combined cosmids 159C9, 146H13, 6M15, and 145M20.]

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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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Analysis of DNase-I-hypersensitive sites at the 3′ end of the cystic fibrosis transmembrane conductance regulator gene (CFTR)

Cited by 39 publications

References 39 publications

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

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

Genomic Sequence Analysis of Fugu rubripes CFTR and Flanking Genes in a 60 kb Region Conserving Synteny with 800 kb of Human Chromosome 7

The CFTR gene and regulation of its expression

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