Deletion of an X-Inactivation Boundary Disrupts Adjacent Gene Silencing

Abstract

In mammalian females, genes on one X are largely silenced by X-chromosome inactivation (XCI), although some “escape” XCI and are expressed from both Xs. Escapees can closely juxtapose X-inactivated genes and provide a tractable model for assessing boundary function at epigenetically regulated loci. To delimit sequences at an XCI boundary, we examined female mouse embryonic stem cells carrying X-linked BAC transgenes derived from an endogenous escape locus. Previously we determined that large BACs carrying escapee Kdm5c and flanking X-inactivated transcripts are properly regulated. Here we identify two lines with truncated BACs that partially and completely delete the distal Kdm5c XCI boundary. This boundary is not required for escape, since despite integrating into regions that are normally X inactivated, transgenic Kdm5c escapes XCI, as determined by RNA FISH and by structurally adopting an active conformation that facilitates long-range preferential association with other escapees. Yet, XCI regulation is disrupted in the transgene fully lacking the distal boundary; integration site genes up to 350 kb downstream of the transgene now inappropriately escape XCI. Altogether, these results reveal two genetically separable XCI regulatory activities at Kdm5c. XCI escape is driven by a dominant element(s) retained in the shortest transgene that therefore lies within or upstream of the Kdm5c locus. Additionally, the distal XCI boundary normally plays an essential role in preventing nearby genes from escaping XCI.

When the Lyon(ized chromosome) roars: ongoing expression from an inactive X chromosome

“…Four different single-copy integrations showed escape from inactivation for Kdm5c while flanking genes were silenced, delimiting a 112 kb region containing such an intrinsic element [81]. Further analysis of deletions of additional BAC integrations also showed evidence for a distal boundary that when disrupted allowed spread of euchromatin (escape) into flanking genes [82]. CTCF has long been implicated as a boundary [72], and has clear roles in the structure of the inactive X (discussed above) as well as generally defining TADs; however, CTCF itself is too abundant on the X chromosome to be the sole delineator of escape genes, thus other factors must interact.…”

X chromosome regulation: diverse patterns in development, tissues and disease

“…Chromatin conformation analyses show that these genes apparently adopt a specific chromatin configuration with different domains interacting with each other, which suggests that escape genes may occupy a specific nuclear compartment 71 . Domains of silencing may be protected by insulator elements from encroachment by escape domains 72,73 . Furthermore, lncRNAs (the genes of which are often located adjacent to protein-coding genes) may facilitate their escape 74,75 .…”

X-chromosome inactivation: new insights into cis and trans regulation

“…Loci lying upstream of Xist have been proposed to participate in its activation: the X-pairing region (Xpr), 58 Genome architecture and expression …”