CTCF Promotes Long-range Enhancer-promoter Interactions and Lineage-specific Gene Expression in Mammalian Cells

Abstract

23 24 [All datasets have been deposited to GEO, with accession number GSE94452, and can be 25 accessed here]. 26 3 Abstract: 27 Topologically associating domains (TAD) and insulated neighborhoods (INs) have been 28 proposed to constrain enhancer-promoter communications to enable cell-type specific 29 transcription programs, but recent studies show that disruption of TADs and INs resulted in 30 relatively mild changes in gene expression profiles. To better understand the role of 31 chromatin architecture in dynamic enhancer-promoter contacts and lineage-specific gene 32 expression, we have utilized the auxin-inducible degron system to acutely deplete CTCF, a 33 key factor involved in TADs and IN formation, in mouse embryonic stem cells (mESCs) and 34 examined chromatin architecture and gene regulation during neural differentiation. We find 35 that while CTCF depletion leads to global weakening of TAD boundaries and loss of INs, only 36 a minor fraction of enhancer-promoter contacts are lost, affecting a small subset of genes. 37 The CTCF-dependent enhancer-promoter contacts tend to be long-range, spanning hundreds 38 of kilobases, and are established directly by CTCF binding to promoters. Disruption of CTCF 39 binding at the promoter reduces enhancer-promoter contacts and transcription, while 40 artificial tethering of CTCF to the promoter restores the enhancer-promoter contacts and 41 gene activation. Genome-wide analysis of CTCF binding and gene expression across multiple 42 mouse tissues suggests that CTCF-dependent promoter-enhancer contacts may regulate 43 4 expression of additional mouse genes, particularly those expressed in the brain. Our results 44 uncover both CTCF-dependent and independent enhancer-promoter contacts, and highlight a 45 distinct role for CTCF in promoting enhancer-promoter contacts and gene activation in 46 addition to its insulator function. 47 48 5 Introduction: 49Transcriptional regulation in mammalian cells is orchestrated by cis-regulatory elements that include 50 promoters, enhancers, insulators and other less well characterized sequences 1,2 . Large-scale 51 projects such as ENCODE have annotated millions of candidate cis-regulatory elements in the 52 human genome and genomes of other mammalian species 3-5 . A majority of these candidate 53 regulatory elements are located far from transcription start sites(i.e. promoters), display tissue and 54 cell-type specific chromatin accessibility, and likely act as enhancers to regulate cell-type specific 55 gene expression. Enhancers are frequently found to be positioned close to their target gene 56 promoters in 3D space at the time of gene activation, suggesting a role for the chromatin 57 architecture in gene regulation 6,7 . Indeed, artificially induced spatial proximity between enhancers 58 and promoters has been shown to lead to gene activation 8,9 . Insulators, on the other hand, act to 59 block enhancer-promoter contacts to prevent ectopic gene activation 10-12 . Clearly, in-depth 60 knowledge of the chromatin architecture in each...

CTCF knockout in zebrafish induces alterations in regulatory landscapes and developmental gene expression

“…2c-d). This indicates that CTCF is required for the expression of thousands of genes during zebrafish development, an impacting result that contrasts to previous observations in in vitro experimental setups showing alteration of a few hundred genes upon CTCF removal 5,32 . Next, we analyzed gene expression changes in the transition from 24 to 48 hpf in wild-type embryos and found that genes that get activated in this period tend to be down-regulated in ctcf −/− embryos, and vice versa, indicating that many developmental genes fail to acquire their normal expression level during this developmental period (Extended Data Fig.…”

“…The interaction profile was generally characterized by a loss of long-range contacts but retaining some contacts at shorter ranges (Fig. 4a), consistent with observations in mammalian cells 32,44 . Genomic regions showing a reduced contact frequency with the ptch2 promoter included CTCF-binding sites as well as ATAC peaks with reduced accessibility in the mutant and others not affected by CTCF absence.…”

“…However, to what extent TADs are crucial for gene regulation is currently under debate. Depletion of CTCF in mammalian in vitro systems causes only modest transcriptional alterations 5,15,32 , in agreement with some in vivo studies 33,34 . However, targeted deletion of several CTCF sites and quantitative gene expression analyses reveal loss of gene expression 35–37 .…”

“…In this work, we have established a new in vivo model to study the loss of CTCF in zebrafish and demonstrate that chromatin structure is required to maintain developmental gene regulatory landscapes during body plan formation. In the last years, the function of CTCF in chromosome folding has been clearly demonstrated in mammalian in vitro systems, including mouse embryonic stem cells, neural progenitor cells as well as human morula embryos 5,15,32 . These studies showed by different depletion mechanisms that CTCF knock-down severely reduces TAD formation and insulation.…”

Revisiting 3D chromatin architecture in cancer development and progression

“…In line with this, Bing Ren lab recently revealed that disruption of CTCF binding on the promoter led to reduction of promoter–enhancer interaction and decreased transcription, while artificial tethering of CTCF on the promoter led to restoration of enhancer–promoter interaction and transcription. Moreover, the occupancy of CTCF on the promoter is associated with cell-specific transcription ( 87 ). In addition, Keji Zhao lab uncovered that CTCF could bring distal enhancer to the vicinity to their target genes via interaction with cohesin ( 88 ).…”