Transcriptional regulation and chromatin architecture maintenance are decoupled functions at the Sox2 locus

Abstract

How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter–enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter–enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

Context-dependent transcriptional remodeling of TADs during differentiation

“…While an attractive model, RNA polymerase II is absent from the inactive Bcl6 gene in ESCs, where a relatively strong but somewhat broader TAD border is nevertheless maintained at the promoter, so other mechanisms must also contribute to architectural maintenance at this locus ( Fig 3B ). Conserved CTCF binding at the promoter would be expected to be such a mechanism, although homozygous deletion of the major binding site had mild effects on locus architecture ( Fig S5 ), in line with other studies suggesting that TADs are built up cooperatively from multiple elements (Despang et al, 2019; Rodríguez-Carballo et al, 2017; Taylor et al, 2022).…”

“…The latter are dependent on underlying gene activity and epigenetic state so are developmentally plastic, as observed (Dixon et al, 2015;Bonev et al, 2017), whereas loop extrusion barriers could be genetically coded, such as by CTCF binding sites, so in this model TADs would not be expected to change significantly across cell type. However, CTCF occupancy is not identical in all cell types and in any case does not account for all TADs (Nora et al, 2017;Taylor et al, 2022). Recent studies have shown that non-encoded features as diverse as MCM complex binding (minichromosome maintenance; most famous for replication origin licensing) and DNA double-strand breaks can also be barriers to loop extrusion (Arnould et al, 2021;Dequeker et al, 2022).…”

“…4C-seq data were mapped to the mm10, processed, normalized and visualized using 4See (Ben Zouari et al, 2020), as in (Taylor et al, 2022).…”

“…4C-seq analysis 4C-seq data were mapped to the mm10, processed, normalized and visualized using 4See (Ben Zouari et al, 2020), as in (Taylor et al, 2022). Insulation scores were essentially computed as in (Crane et al, 2015).…”

“…Live imaging experiments have visualized such predicted interactions, although they are relatively infrequent and transient (Gabriele et al, 2022). Inversion of CTCF motifs disrupts chromatin interactions (Guo et al, 2015;de Wit et al, 2015;Sanborn et al, 2015), highlighting the importance of CTCF orientation in chromatin architecture, although the prevailing absence of new interactions between CTCF pairs now brought into convergent orientation, and the existence of CTCF-independent TADs (Nora et al, 2017;Taylor et al, 2022) suggests that other factors participate in TAD formation (Sikorska and Sexton, 2020).…”