Loop stacking organizes genome folding from TADs to chromosomes

Abstract

While population level analyses reveal significant roles for CTCF and cohesin in mammalian genome organization, their contribution to chromatin structure and gene regulation at the single-cell level remain incompletely understood. Here, we use chromosome tracing microscopy to measure the effects of removal of CTCF or cohesin on genome folding across genomic scales. We find cohesin contracts the chromosome into loops, facilitating contacts both within and between Topologically Associating Domains (TADs), while increasing the separation along the chromosome arms through steric effects of loop stacking. CTCF organizes these loops radially, favoring interactions among CTCF-marked borders, including 3-way interactions that bridge TAD boundaries in developmentally important domains. Border-distal regions spread out radially from this axis, helping explain CTCF's previously described role in TAD separation. Together our data provide a structural understanding of how cohesin and CTCF reduce stochasticity in 3D folding across genomic scales and help minimize variability in gene expression.

Chromatin packing domains persist after RAD21 depletion in 3D

“…It is also worth noting that ChromSTEM imaging captures the statistics of single cell organizational states, whereas the ensemble measurement of Hi-C is an aggregate statistic of many organizational states. Consequently, this suggested that mature packing domains were largely maintained independent of cohesin mediated loop formation but that the principal effect of RAD21 extrusion was on the production of a subset of nascent domains, possibly consistent with findings in prior reports using oligo-based methods( 20, 22 ).…”

“…4 ). Although this study has limitations in that it measures packing domains in sub-portion of one nucleus in each group, other studies have similarly showed the presence of TAD-like structures after cohesin degradation( 17, 20–22, 25 ). While it is possible that TAD-like structures could be an observed subset of chromatin packing domains, the observed discordance between the number of lost TADs and the retention of most domains suggests otherwise.…”

“…As TADs arise in population measurements, it was proposed that TADs are an ensemble feature of cellular population measurements; as such, they may represent infrequent, but crucial structures necessary for proper collective function (17,23). In support of this, TAD-like structures have been observed with variable positions and frequencies utilizing oligo-paint based super-resolution microscopy (17,(20)(21)(22) and loss of RAD21 did not disrupt heterochromatin cores on structured illumination (25). Despite the association of TADs with cohesin-mediated loop extrusion, RAD21 depletion did not result in the loss of all the TAD-like structures in individual cells (14,17,(20)(21)(22).…”

“…From their initial description as foci of higher-than expected contact probability in ensemble population measurements, several key features of TADs have been described. TADs are predominantly associated with cohesinmediated loop extrusion in association with CTCF binding motifs and potentially act as boundary mediating elements in supra-nucleosomal chromatin organization (13,15,(18)(19)(20)(21)(22). Cohesin loops at individual loci appear to be transient in nature and relatively rare events, residing in a loop conformation <10% of the time in a single locus imaged in mouse embryonic stem cells (23).…”

Synergistic insulation of regulatory domains by developmental genes and clusters of CTCF sites

“…S5-6), we noticed that they are typically flanked by strong CTCF sites and, thus, located within, rather than next to, CTCF clusters. This organization might favour boundary bypass, TAD intermingling and communication with regulatory elements located on either side of the genes 26,27,31,78 . In contrast, for the two developmental genes that we investigated, we found no evidences indicating that CTCF clusters facilitate their expression or the communication with their cognate enhancers.…”