X-inactivation escapee domains are CTCF-cohesin independent chromatin compartments

Abstract

X-chromosome inactivation involves chromosome-wide gene silencing accompanied by extensive chromatin changes, as well the loss of topologically associating domains. Yet discrete regions of the inactive X chromosome retain activity within localised 3D domains, which contain active genes that variably escape from X inactivation. The transcription factor and architectural protein CTCF has been proposed to be implicated in escape by insulating escape domains or sustaining their topology via cohesin-mediated loop extrusion. Here, we test the role of CTCF and cohesin in escape using acute degron-mediated depletion of CTCF and RAD21 in neural progenitor cells with established escape profiles. Although CTCF occupancy correlates with escape status on the inactive X chromosome, its removal - together with loss of loop extrusion - does not disrupt escapee gene expression, or domain organization, nor does it result in spreading of silencing or activation of genes in cis. Rather, we show that facultative escape regions are self-sustaining compartments of active chromatin enriched in H3K27 acetylation and depleted in H3K27 methylation, with the magnitude of compartment strength scaling up with the degree of transcriptional activity on the inactive X chromosome. These active escapee compartments are propagated independently of CTCF and RAD21-dependent 3D architecture. Our findings identify chromatin compartmentalization as the primary feature of facultative escapee domains.