Differential contribution of cis-regulatory elements to higher order chromatin structure and expression of the CFTR locus

Abstract

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 CFTRTAD 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.

Figure 1.

Cell-type-specific chromatin structure at the CFTR locus: 4C-seq profiles with a CFTR promoter viewpoint. A schematic at the top shows the genomic location of CFTR and adjacent genes on chromosome 7 and below are known cis-regulatory elements for the CFTR locus. The nomenclature represents distance in kb (−) 5′ to the translational start site and (+) 3′ to the last coding base. Open chromatin mapped by DNase-seq and 4C-seq data are shown for Caco2, Calu3, HBE, epididymis and skin fibroblast cells. Representative data from one of the replicates are shown here. DNase-seq data are presented as histograms from screen shots of data sets uploaded to the UCSC genome browser. 4C-seq data are presented in alignment with the DNase-seq data and have two subpanels. The upper panel indicates the main trend of contact profile using a 5-kb window size. Relative interactions are normalized to the strongest point (which is set to 1) within each panel. The lower panel is a domainogram (39), which uses color coded intensity values to show relative interactions with window sizes varying from 2 to 50 kb. Here, red denotes the strongest interactions and dark blue, through turquoise, to gray represent gradually decreasing frequencies. Arrows denote important data features described in the results.

Figure 2.

Cell-type-specific chromatin structure at the CFTR locus: 4C-seq profiles with a −20.9 kb CTCF-binding insulator viewpoint. Cell types, DNase-seq data and 4C-seq data presentation as described in Figure 1.

Figure 3.

Chromatin structure at the CFTR locus in Caco2 cells analyzed from multiple viewpoints. 4C-seq data and DNase-seq data presentation in Caco2 cells as described in Figure 1. Viewpoints are CTCF-binding sites at −80.1, −20.9 and +48.9 kb and at the promoter.

Figure 4.

Depletion of CTCF and cohesin complex has a dramatic impact on chromatin structure at the CFTR locus. 4C interaction profiles of the CFTR locus in Caco2 cells after CTCF/Rad21 knockdown (KD). Top right inset. Efficacy of CTCF/Rad21 KD: western blots of cell lysates from Caco2 cells co-transfected with CTCF, RAD21-specific or negative control (NC) siRNAs, probed with antibodies to CTCF, RAD21 and β-tubulin. 4C-seq viewpoints are at the CFTR promoter and −20.9 kb in negative control or CTCF/Rad21 double knockdown (KD) Caco2 cells. CTCF ChIP-seq data combined from multiple cell types are from ENCODE (62).

Figure 5.

Deletion of the −20.9 kb CTCF site from the CFTR locus disrupts its chromatin structure but has little effect on gene expression. −20.9 kb CTCF site removed from endogenous locus in Caco2 cells by CRISPR/Cas9 (Supplementary Figure S3A, B). (A) ChIP for CTCF in wildtype (WT, gray) and del-20.9 kb (black) Caco2 clones. CTCF occupancy measured by qPCR at CTCF sites (−80.9 kb, intron 1, +48.8 kb and +83.7 kb) and at the intron 11 enhancer. Results are pooled from three independent experiments from two WT and two deletion clones. Error bars represent S.E.M. (B) 4C-seq interaction profiles of WT and del-20.9 kb Caco2 clones using viewpoints at the promoter, −80.1 kb, and +48.9 kb. (C) CFTR expression measured by RT-qPCR relative to 18s RNA in WT and del-20.9 kb Caco2 clones, showing no significant change. Results are pooled from three independent experiments from two WT and two deletion clones. Error bars represent S.E.M.

Figure 6.

Deletion of the intron 11 enhancer from the CFTR locus has little impact on chromatin structure but substantially reduces gene expression. (A) RT-qPCR analysis showing CFTR expression relative to 18s RNA in WT (gray) and del11 (black) Caco2 clones. Values are mean ± S.E.M., n = 3, ***P < 0.001. (B) 4C-seq interaction profiles of WT and del11 Caco2 clones using viewpoints at the promoter and −20.9 kb. (C) FOXA2 ChIP in WT (gray) and del11 (black) Caco2 clones, followed by qPCR with primers for the promoter and intronic cis-regulatory elements in CFTR. (D) RNA pol II ChIP in WT (gray) and del11 (black) Caco2 clones, followed by qPCR. ChIP results are pooled from three independent experiments from two WT and two deletion clones. Data present the mean ± S.E.M., *P < 0.05, **P < 0.01.