A loss-of-function variant in HSD17B13 was associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.).

The DiscovEHR collaboration between the Regeneron Genetics Center and Geisinger Health System couples high-throughput sequencing to an integrated health care system using longitudinal electronic health records (EHRs). We sequenced the exomes of 50,726 adult participants in the DiscovEHR study to identify ~4.2 million rare single-nucleotide variants and insertion/deletion events, of which ~176,000 are predicted to result in a loss of gene function. Linking these data to EHR-derived clinical phenotypes, we find clinical associations supporting therapeutic targets, including genes encoding drug targets for lipid lowering, and identify previously unidentified rare alleles associated with lipid levels and other blood level traits. About 3.5% of individuals harbor deleterious variants in 76 clinically actionable genes. The DiscovEHR data set provides a blueprint for large-scale precision medicine initiatives and genomics-guided therapeutic discovery.

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex temporal and spatial pattern of expression that is controlled by multiple cis-acting elements interacting with the basal promoter. Although significant progress has been made towards understanding these genomic elements, there have been no reports of post-transcriptional regulation of CFTR by miRNAs (microRNAs). In the present study, we identify two miRNAs, hsa-miR-145 and hsa-miR-494, which regulate CFTR expression by directly targeting discrete sites in the CFTR 3′ UTR (untranslated region). We show that at least 12 miRNAs are capable of repressing endogenous CFTR mRNA expression in the Caco-2 cell line. Ten of these also inhibit expression of a reporter construct containing the CFTR 3′ UTR in one or more cell lines, and five repress endogenous CFTR protein expression in Caco-2 cells. Moreover, at least three are expressed in primary human airway epithelial cells, where CFTR expression is maintained at low levels in comparison with intestinal cell lines. Three of the miRNAs that target CFTR, hsa-miR-384, hsa-miR-494 and hsamiR-1246, also inhibit expression of a reporter carrying the Na+ – K+ –Cl− co-transporter SLC12A2 [solute carrier family 12 (Na+ – K+ –Cl− transporters), member 2] 3′ UTR, suggesting that these miRNAs may play a more general role in regulating chloride transport in epithelial cells.

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.

Higher order chromatin structures across the genome are maintained in part by the architectural proteins CCCTC binding factor (CTCF) and the cohesin complex, which co-localize at many sites across the genome. Here, we examine the role of these proteins in mediating chromatin structure at the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encompasses nearly 200 kb flanked by CTCF-binding enhancer-blocking insulator elements and is regulated by cell-type-specific intronic enhancers, which loop to the promoter in the active locus. SiRNA-mediated depletion of CTCF or the cohesin component, RAD21, showed that these two factors have distinct roles in regulating the higher order organization of CFTR. CTCF mediates the interactions between CTCF/cohesin binding sites, some of which have enhancer-blocking insulator activity. Cohesin shares this tethering role, but in addition stabilizes interactions between the promoter and cis-acting intronic elements including enhancers, which are also dependent on the forkhead box A1/A2 (FOXA1/A2) transcription factors (TFs). Disruption of the three-dimensional structure of the CFTR gene by depletion of CTCF or RAD21 increases gene expression, which is accompanied by alterations in histone modifications and TF occupancy across the locus, and causes internalization of the gene from the nuclear periphery.

The forkhead box A transcription factors, FOXA1 and FOXA2, function as pioneer factors to open condensed chromatin and facilitate binding of other proteins. We showed previously that these factors are key components of a transcriptional network that drives enhancer function at the cystic fibrosis transmembrane conductance regulator (CFTR) locus in intestinal epithelial cells. The CFTR promoter apparently lacks tissue-specific regulatory elements and expression of the gene is controlled by multiple cis-acting elements, which coordinate gene expression in different cell types. Here we show that concurrent depletion of FOXA1 and FOXA2 represses CFTR expression and alters the three-dimensional architecture of the active locus by diminishing interactions between the promoter and intronic cis-acting elements. Reduction of FOXA1/A2 also modifies the enrichment profile of the active enhancer marks H3K27ac and H3K4me2 across the CFTR locus and alters chromatin accessibility at individual cis-elements. Moreover, loss of FOXA1/A2 suppresses the recruitment of other members of the transcriptional network including HNF1 and CDX2, to multiple cis-elements. These data reveal a complex molecular mechanism underlying the role of FOXA1/A2 in achieving high levels of CFTR expression in intestinal epithelial cells.

Background Distal cell-type-specific regulatory elements may be located at very large distances from the genes that they control and are often hidden within intergenic regions or in introns of other genes. The development of methods that enable mapping of regions of open chromatin genome wide has greatly advanced the identification and characterisation of these elements. Methods Here we use DNase I hypersensitivity mapping followed by deep sequencing (DNase-seq) to generate a map of open chromatin in primary human tracheal epithelial (HTE) cells and use bioinformatic approaches to characterise the distribution of these sites within the genome and with respect to gene promoters, intronic and intergenic regions. Results Genes with HTE-selective open chromatin at their promoters were associated with multiple pathways of epithelial function and differentiation. The data predict novel cell-type-specific regulatory elements for genes involved in HTE cell function, such as structural proteins and ion channels, and the transcription factors that may interact with them to control gene expression. Moreover, the map of open chromatin can identify the location of potentially critical regulatory elements in genome-wide association studies (GWAS) in which the strongest association is with single nucleotide polymorphisms in non-coding regions of the genome. We demonstrate its relevance to a recent GWAS that identifies modifiers of cystic fibrosis lung disease severity. Conclusion Since HTE cells have many functional similarities with bronchial epithelial cells and other differentiated cells in the respiratory epithelium, these data are of direct relevance to elucidating the molecular basis of normal lung function and lung disease.

The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters, and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells, we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis, including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were overrepresented in epididymis-selective DHS identified epithelial transcription factors, including ELF5 and ELF3, the androgen receptor, Pax2, and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium.