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

Developmental genes such as Xist , the master regulator of X-chromosome inactivation (XCI), are controlled by complex cis -regulatory landscapes, which decode multiple signals to establish specific spatio-temporal expression patterns. Xist integrates 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 of Xist during 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 the Xist promoter. The strongest distal element is part of an enhancer cluster ∼200 kb upstream of the Xist gene which is associated with a previously unannotated Xist -enhancing regulatory transcript, 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. Our study is the first step to disentangle how multiple, functionally distinct regulatory regions interact to generate complex expression patterns in mammals.