A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions

Abstract

A variety of self-interacting domains, defined at different levels of resolution, have been described in mammalian genomes. These include Chromatin Compartments (A and B)1, Topologically Associated Domains (TADs)2,3, contact domains4,5, sub-TADs6, insulated neighbourhoods7 and frequently interacting regions (FIREs)8. Whereas many studies have found the organisation of self-interacting domains to be conserved across cell types389, some do form in a lineage-specific manner6710. However, it is not clear to what degree such tissue-specific structures result from processes related to gene activity such as enhancer-promoter interactions or whether they form earlier during lineage commitment and are therefore likely to be prerequisite for promoting gene expression. To examine these models of genome organisation in detail, we used a combination of high-resolution chromosome conformation capture, a newly-developed form of quantitative fluorescence in-situ hybridisation and super-resolution imaging to study a 70 kb self-interacting domain containing the mouse α-globin locus. To understand how this self-interacting domain is established, we studied the region when the genes are inactive and during erythroid differentiation when the genes are progressively switched on. In contrast to many current models of long-range gene regulation, we show that an erythroid-specific, decompacted self-interacting domain, delimited by convergent CTCF/cohesin binding sites, forms prior to the onset of robust gene expression. Using previously established mouse models we show that formation of the self-interacting domain does not rely on interactions between the α-globin genes and their enhancers. As there are also no tissue-specific changes in CTCF binding, then formation of the domain may simply depend on the presence of activated lineage-specific cis-elements driving a transcription-independent mechanism for opening chromatin throughout the 70 kb region to create a permissive environment for gene expression. These findings are consistent with a model of loop-extrusion in which all segments of chromatin, within a region delimited by CTCF boundary elements, can contact each other. Our findings suggest that activation of tissue-specific element(s)within such a self-interacting region is sufficient to influence all chromatin within the domain.

Single-cell chromatin interactions reveal regulatory hubs in dynamic compartmentalized domains

“…This is consistent with the observed additive effects of individual enhancers at the globin (5, 7) and many other genes (6). Importantly, we have previously shown that no single enhancer element in the a-globin locus is critical for the formation of the chromatin structures associated with active a-globin transcription (3,5). The formation of complexes in which multiple enhancers and promoters interact simultaneously therefore provides a structural basis for the observed functional cooperativity and also suggests a role for apparent redundant enhancer elements (8,9).…”

“…The globin loci, which provide ideal models to elucidate the general principles of mammalian gene regulation, have been extensively studied in this way. For example, we have previously shown that the active mouse a-globin cluster and its regulatory elements are located in a decompacted ~70 kb chromatin domain that forms early in erythroid differentiation and is flanked by CTCFbinding sites (2)(3)(4). Within this domain, the a-globin genes interact with five enhancer elements, which cooperate in an additive manner to upregulate gene expression (5).…”

Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells

“…HoxD10/Dlx2 loci and a second set for similarly interacting polycomb target genes, Nkx2-3/Pax2 ( Figure S7), and performed non-denaturing RASER-FISH (Brown et al, 2018) to measure the three-dimensional distance between these loci in individual cells ( Figures 6B, S6C, and S7A). This revealed a distribution of distances in cells where cohesin is intact, including some in close proximity ( Figures 6C, S6D, S7B, and S7D).…”

“…RASER-FISH was conducted as previously described (Brown et al, 2018) with minor changes. Briefly, cells were grown on coverslips, labeled for 24 h with BrdU/BrdC mix (3:1) at final conc.…”

H3K79me2/3 controls enhancer–promoter interactions and activation of the pan-cancer stem cell marker PROM1/CD133 in MLL-AF4 leukemia cells

“…One characteristic of an active enhancer is that it physically contacts the promoter of the gene it regulates [42][43][44][45]. To test whether this was true for these putative enhancers, we performed next generation Capture-C to identify regions interacting with the promoters of PROM1 and TAPT1.…”