Distal and proximal cis-regulatory elements sense X chromosome dosage and developmental state at the Xist locus

Abstract

Developmental genes such as Xist, which initiates X chromosome inactivation, are controlled by complex cisregulatory landscapes, which decode multiple signals to establish specific spatiotemporal expression patterns. Xist integrates information on X chromosome dosage and developmental stage to trigger X inactivation in the epiblast specifically in female embryos. Through a pooled CRISPR screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements of Xist at the onset of random X inactivation. Chromatin profiling reveals that X-dosage controls the promoter-proximal region, while differentiation cues activate several distal enhancers. The strongest distal element lies in an enhancer cluster associated with a previously unannotated Xist-enhancing regulatory transcript, which we named Xert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specific Xist upregulation at the correct developmental time. With this study, we start to disentangle how multiple, functionally distinct regulatory elements interact to generate complex expression patterns in mammals.

Mechanisms of Choice in X-Chromosome Inactivation

“…Among the positive regulators of Xist within the same TAD as the Xist promoter, three lncRNA loci have been reported to influence Xist expression in cis and could therefore affect choice-Ftx and Xert via their transcription [62,104], and Jpx transcriptionally or posttranscriptionally [63,105]. Jpx has also been reported as a trans-acting regulator of Xist via its lncRNA [106].…”

“…Jpx has also been reported as a trans-acting regulator of Xist via its lncRNA [106]. A recent study has identified a series of proximal and distal enhancers that are also required for activation of Xist upregulation in cis [104]. Elements within the Xist locus itself have also been reported to contribute to Xist upregulation-e.g., [107], including an antisense transcript, XistAR [108].…”

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of a distal enhancer element

“…Xist expression is controlled by a large genomic region, which contains a series of lncRNA loci, thought to repress (Tsix, Linx) or activate (Jpx, Ftx, Xert) Xist transcription mostly in cis (15,16). Large (210-460 kb) single-copy Xist-containing transgenes (tg53, tg80), encompassing ~100kb genomic sequence upstream of Xist, can recapitulate initial Xist upregulation after fertilization and maintenance in extraembryonic lineages, but not rXCI in somatic tissues (17,18).…”

X Chromosome Inactivation Timing is Not eXACT: Implications for Autism Spectrum Disorders

“…Consistent with Jpx escaping XCI, female mouse ES cells express twice the expression level as males ( Sun et al, 2013 ), but whether that means that autosomal Jpx developmental gene targets are expressed at a higher level in female versus male preimplantation embryos was not determined in this study. Another recently described activator of Xist in XX mouse ES cells is an enhancer localized lncRNA called Xert ( Gjaltema et al, 2021 ). Unlike Jpx , whose expression is ubiquitous in somatic tissues, the expression of Xert appears limited to the mouse epiblast at embryonic days 5.5–6.5 ( Gjaltema et al, 2021 ), but expected sex differences in embryonic Xert expression were not directly determined in this study.…”

“…Another recently described activator of Xist in XX mouse ES cells is an enhancer localized lncRNA called Xert ( Gjaltema et al, 2021 ). Unlike Jpx , whose expression is ubiquitous in somatic tissues, the expression of Xert appears limited to the mouse epiblast at embryonic days 5.5–6.5 ( Gjaltema et al, 2021 ), but expected sex differences in embryonic Xert expression were not directly determined in this study.…”