Cohesin and CTCF differentially affect chromatin architecture and gene expression in human cells

Abstract

Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many selfassociating topological domains. The boundary sequences between domains are enriched for binding sites of CTCC-binding factor (CTCF) and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains. To determine the role of cohesin and CTCF in higher-order chromatin architecture in human cells, we depleted the cohesin complex or CTCF and examined the consequences of loss of these factors on higher-order chromatin organization, as well as the transcriptome. We observed a general loss of local chromatin interactions upon disruption of cohesin, but the topological domains remain intact. However, we found that depletion of CTCF not only reduced intradomain interactions but also increased interdomain interactions. Furthermore, distinct groups of genes become misregulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute differentially to chromatin organization and gene regulation.Hi-C | transcriptional regulation | 4C | HOX cluster R ecent studies of the topological organization of the genome suggest that CTCC-binding factor (CTCF) and cohesin might be involved in establishment or maintenance of topological domains in the mammalian genome, as their binding sites are enriched at the boundaries of these domains (1). It was proposed that CTCF and cohesin might work together to facilitate longrange interactions in the genome (2). First, CTCF and the cohesin complex, consisting of the core subunits SMC3, SMC1, RAD21, and STAG1/SA1 or STAG2/SA2, were found to colocalize extensively throughout mammalian genomes (3-5). Second, both factors are involved in mediating long-range interactions (6-11). Finally, cohesin was shown to be important for CTCF's chromatin insulation function (3-5), whereas CTCF is necessary to recruit cohesin to the shared binding sites but not to chromatin (3). CTCF and cohesin have also been recently correlated with both interaction frequency and gene expression during differentiation (12), indicating that they may play major roles in mediating the impacts of chromatin structure on gene regulation. However, the exact mechanisms these factors use to contribute to chromatin structure and gene regulation are unclear, as depletion of these factors has not yet been systematically tested on a genome-wide basis. Whether the two factors work in concert or independently, through mechanisms, such as long-range enhancer looping (13) or chromatin insulation (2) to control chromatin structure and gene expression, is unknown. To determine the role of cohesin and CTCF in higher-order chromatin architecture in human cells, we depleted the cohesin complex or CTCF and examined the consequences of loss of these factors on domain structure and gene expression. Results Proteolytic Cleavage of RAD21 Leads to Loss of Long-Range ChromatinInteractions. To understand the contribution of cohesin to genom...

Two independent modes of chromatin organization revealed by cohesin removal

“…1f–g), contrasting with the very mild changes reported in previous cohesin depletion experiments (Extended Data Figure 4) 19–21 . Compared to WT and TAM control samples, ΔNipbl cells show genome-wide disappearance of local TAD patterns (Fig.…”

“…Our data shows the essential role of cohesin in the formation of TADs. It is possible that the cohesin depletion in previous studies that did not report such drastic effects 19–21 was insufficient to achieve substantial loss of TADs. Our simulations suggest that TADs would still be pronounced at 2-fold cohesin depletion, requiring ~8-fold depletion for loss of TADs, which is close to what we observed in ΔNipbl samples.…”

“…The same procedure was used to analyze other existing Hi-C datasets on cohesin-depleted cells 19–21 .…”

“…(b) deletion of Rad21 in thymocytes 20 . (c) proteolytic cleavage of RAD21 in HEK293T cells 19 (d–e) deletion of Rad21 in NSCs and ASTs 21 . For each dataset: left column, top panels – the average Hi-C map around 102 peaks with size range 500–600kb 12 in WT and ΔNipbl contact maps; middle panels – the average Hi-C map of TADs called in each dataset; bottom panels – the relative contact probability between pairs of peak loci vs genomic distance, compared to randomly selected pairs of loci.…”

“…CTCF sites). Recent experimental manipulation of CTCF motifs 15–17 and CTCF depletion 18 support the proposed role of CTCFs as boundary elements, but cohesin’s role in interphase chromatin organization and the process of loop extrusion remains ambiguous, as experimental depletions of cohesin have shown limited impact on chromatin organisation 19–21 …”