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

Abstract

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...

Osmolality/salinity-responsive enhancers (OSREs) control induction of osmoprotective genes in euryhaline fish

“…Of interest, one OSRE1 element of IMPA1.1 is located downstream of the TSS. However, this OSRE1 is still in very close proximity to the predicted TSS, and regions located 3′ of the TSS have been demonstrated to contain fully functional enhancers (108)(109)(110). The OSRE1 consensus sequence AGTGGAAAAA-TACTAAG is functional in both orientations (forward and reverse), as is consistent with the directional independence of other enhancers (111).…”

Post-Transcriptional Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Expression and Function by MicroRNAs

“…Although we did not detect a reduced abundance of CFTR mRNA in CF epithelia ( Figure 6A), we note that the regulation of CFTR expression is complex, and that counterregulatory effectors may exert an impact (21,35,(44)(45)(46). For example, NF-kB has been reported to interact directly with the CFTR promoter, positively regulating its transcription (35).…”

“…might influence the site availability or its thermodynamics, factors known to influence miRNA binding (48)(49)(50). CFTR expression and biosynthesis are subject to complex regulatory mechanisms involving multiple pathways (16,21,46,51), some interacting with the CFTR 39 UTR (19,30). These scenarios point to the possibility that the miRNA-mediated regulation of CFTR expression may be transient, dynamic, or subject to counterregulatory effectors that can increase CFTR transcription in CF airway epithelia (21,35).…”

A microRNA network regulates expression and biosynthesis of wild-type and ΔF508 mutant cystic fibrosis transmembrane conductance regulator

“…Despite more than 20 y of study (42), the mechanisms governing CFTR transcription remain incompletely understood. The CFTR promoter has features of a "housekeeping" gene (43,44), and several transactivating factors and regulatory elements have been characterized (43,(45)(46)(47). Given that SIN3A-mediated transcriptional silencing involves associated histone deacetylases (35), identifying an interaction between SIN3A and CTCF on the CFTR promoter at the −20.9 kb DHS improves our understanding of how CFTR is transcriptionally regulated.…”