Differential contribution ofcis-regulatory elements to higher order chromatin structure and expression of theCFTRlocus

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 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.

The retrovirus HTLV-1 inserts an ectopic CTCF-binding site into the human genome

“…Sub-TADs, typically contained within TADs, appear to regulate transcription by bringing a distant enhancer near the respective promoter (8,16,18,25). However, although CTCF plays a central role in establishing chromatin structure, epigenetic borders, and transcriptional activity, removal of CTCF does not invariably lead to changes in chromatin looping (26) and epigenetic marks flanking the border (12). Further, gene expression can be changed in cis at some distance from the abrogated CTCF-BS (12,27).…”

The 3D Genome as Moderator of Chromosomal Communication

“…Third, more recently analysis of enhancer-promoter interactions around the CFTR locus showed that the CFTR promoter engages with distinct cell-type specific distal enhancers and CTCF-bound loci in different tissues. Intriguingly, all these are contained within one tissue-invariant TAD (Smith et al, 2016; Yang et al, 2015). ChIA-PET analyses of CTCF-anchored loops between TAD boundaries and RNA polymerase-anchored chromatin loops also showed that gene regulatory interactions between gene promoters and their distal regulatory elements occur mostly within TADs (Tang et al 2015).…”

Chromatin Domains: The Unit of Chromosome Organization

“…Similarly, deletion of a CTCF motif in the homologous region in a human embryonal carcinoma cell line also resulted in alterations of chromatin organization and dramatic changes in expression of genes in the locus (Xu et al, 2014). Third, deletion of CTCF binding sites at the borders of a CFTR-containing TAD in a human epithelial colorectal adenocarcinoma cell line (Caco2) altered chromatin organization and enhanced chromatin interaction between the CFTR promoter and sequences outside of the TAD (Yang et al, 2015). Fourth, comparative analysis of chromatin organization in several mammalian species revealed evolutionarily conserved TAD boundaries that are associated with conserved CTCF binding sites across species, as well as a strong correlation between the turn over of CTCF binding sites and loss or gain of TAD boundaries in these species (Vietri Rudan et al, 2015).…”