Deletion of an X-Inactivation Boundary Disrupts Adjacent Gene Silencing

<div><p>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 <i>Kdm5c</i> and flanking X-inactivated transcripts are properly regulated. Here we identify two lines with truncated BACs that partially and completely delete the distal <i>Kdm5c</i> XCI boundary. This boundary is not required for escape, since despite integrating into regions that are normally X inactivated, transgenic <i>Kdm5c</i> 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 <i>Kdm5c</i>. XCI escape is driven by a dominant element(s) retained in the shortest transgene that therefore lies within or upstream of the <i>Kdm5c</i> locus. Additionally, the distal XCI boundary normally plays an essential role in preventing nearby genes from escaping XCI.</p>