Acute depletion of CTCF directly affects MYC regulation through loss of enhancer–promoter looping

Abstract

Numerous pieces of evidence support the complex, 3D spatial organization of the genome dictates gene expression. CTCF is essential to define topologically associated domain boundaries and to facilitate the formation of insulated chromatin loop structures. To understand CTCF’s direct role in global transcriptional regulation, we integrated the miniAID-mClover3 cassette to the endogenous CTCF locus in a human pediatric B-ALL cell line, SEM, and an immortal erythroid precursor cell line, HUDEP-2, to allow for acute depletion of CTCF protein by the auxin-inducible degron system. In SEM cells, CTCF loss notably disrupted intra-TAD loops and TAD integrity in concurrence with a reduction in CTCF-binding affinity, while showing no perturbation to nuclear compartment integrity. Strikingly, the overall effect of CTCF’s loss on transcription was minimal. Whole transcriptome analysis showed hundreds of genes differentially expressed in CTCF-depleted cells, among which MYC and a number of MYC target genes were specifically downregulated. Mechanically, acute depletion of CTCF disrupted the direct interaction between the MYC promoter and its distal enhancer cluster residing ∼1.8 Mb downstream. Notably, MYC expression was not profoundly affected upon CTCF loss in HUDEP-2 cells suggesting that CTCF could play a B-ALL cell line specific role in maintaining MYC expression.

BET inhibition disrupts transcription but retains enhancer-promoter contact

“…This long-distance interaction has also been observed and characterized in several other cell types 22,23,45,46 . Reciprocal Capture-C from the more proximal of the two enhancer regions demonstrated contact exclusively with the MYC promoter, avoiding intervening regions, as well as a relatively weak interaction with the more distal enhancer domain (Fig 1c).…”

“…Indeed, loss of Rad21 results in decreased enhancer-promoter contacts and transcriptional downregulation at Sik1 and Elf3 in mESCs 72 . Similarly, enhancer-promoter interactions at MYC are dependent on CTCF 22,23 . However, it is possible that this gene is unusual, as long distance enhancer-promoter interactions over 1 Mb are not common.…”

“…Whilst loop extrusion mediated by CTCF and cohesin is the most common explanation for controlling large scale chromatin structure, less is known about what stabilizes more localized enhancer-promoter loops. Possible models include CTCF/cohesin-stabilized enhancer-promoter interactions [21][22][23] and protein/RNA complexes bound to both the enhancer and promoter that interact with one another [24][25][26] . Binding of these complexes is likely initiated by key sequencespecific TFs.…”

“…In SEM cells, CTCF and RAD21 show a strong positive correlation at ATAC peaks (Fig 1b), suggesting that all or most of these CTCF binding sites are competent to enrich or stabilize RAD21 association with chromatin. It is unclear whether loop extrusion may contribute to the increased local interactions between promoters and enhancers, although recent work has suggested this possibility 21,22 . In support of this idea, CTCF and RAD21…”

“…Our data suggest that at a subset of enhancers, CTCF and cohesin may be partly responsible for facilitating enhancer-promoter interactions. Recent work in SEM cells, the cell line studied here, used Capture-C following CTCF degradation to test the effect of CTCF loss on the interaction between the MYC promoter and enhancer 22 . Consistent with the model that CTCF/RAD21 binding stabilizes enhancer-promoter interactions, loss of CTCF was found to reduce both MYC expression and interactions between the MYC enhancer and promoter 22 .…”