Distal and proximal cis-regulatory elements sense X-chromosomal dosage and developmental state at theXistlocus

AbstractDevelopmental genes such asXist, the master regulator of X-chromosome inactivation (XCI), are controlled by complexcis-regulatory landscapes, which decode multiple signals to establish specific spatio-temporal expression patterns.Xistintegrates information on X-chromosomal dosage and developmental stage to trigger XCI at the primed pluripotent state in females only. Through a pooled CRISPR interference screen in differentiating mouse embryonic stem cells, we identify functional enhancer elements ofXistduring the onset of random XCI. By quantifying how enhancer activity is modulated by X-dosage and differentiation, we find that X-dosage controls the promoter-proximal region in a binary switch-like manner. By contrast, differentiation cues activate a series of distal elements and bring them into closer spatial proximity of theXistpromoter. The strongest distal element is part of an enhancer cluster ∼200 kb upstream of theXistgene which is associated with a previously unannotatedXist-enhancing regulatory transcript, we namedXert. Developmental cues and X-dosage are thus decoded by distinct regulatory regions, which cooperate to ensure female-specificXistupregulation at the correct developmental time. Our study is the first step to disentangle how multiple, functionally distinct regulatory regions interact to generate complex expression patterns in mammals.