Reporter CRISPR screens decipher cis- and trans-regulatory principles at the Xist locus

Developmental genes are controlled by an ensemble of cis-acting regulatory elements (REs), which in turn respond to multiple trans-acting transcription factors (TFs). Understanding how a cis-regulatory landscape integrates information from many dynamically expressed TFs has remained a challenge. We develop a combined CRISPR-screening approach using endogenous RNA and RE-reporters as readouts. Applied to the Xist locus, crucial for X-chromosome inactivation in females, this method allows us to comprehensively identify Xist-controlling TFs and map their TF-RE wiring. We find a group of transiently expressed TFs that regulate proximal REs, driving the binary activation of Xist expression. These basal activators are more highly expressed in cells with two X chromosomes, potentially driving female-specific Xist upregulation. A second set of developmental TFs is upregulated later during differentiation and targets distal REs. This regulatory axis is crucial to achieve high levels of Xist RNA, which is necessary for X-chromosome inactivation. Our findings support a model for developmental gene regulation in which factors targeting proximal REs drive binary ON-OFF decisions, while factors interacting with distal REs control the transcription output.