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

Developmental genes are controlled by several cis-acting regulatory elements (REs), which in turn respond to multiple trans-acting transcription factors (TFs). Understanding this regulatory complexity has remained a challenge. Here, we combine pooled CRISPR screens with different phenotypic readouts to dissect how a cis-regulatory landscape integrates information from a large number of TFs. We apply our approach to the murine X-inactivation center, which harbors the Xist gene. We identify a large set of Xist-controlling TFs and map their TF-RE wiring at the Xist locus. We show that a transiently expressed group of XX-biased TFs, which includes the X-linked factor ZIC3, control Xist's promoter-proximal REs. These factors promote initial Xist upregulation in a binary fashion, potentially ensuring female-specific Xist expression. A second set of developmental TFs, which include the epiblast master regulator of the epiblast OTX2, are upregulated slightly later and activate distal REs associated with the lncRNA genes Jpx, Ftx and Xert. We show that this second regulatory axis is required to ensure sufficiently high Xist RNA levels for efficient establishment of X-chromosome inactivation. Our unbiased, systematic approach provides a framework for the comprehensive dissection of regulatory interactions across genomic loci. Overall design: scRNAseq was performed in TX1072 SP427 XX CasTuner mESCs with guides targeting the Xist TSS or the Xist-regulating REs 61, 85, 93 and 96 following 4 days of 2i/LIF-withdrawal. Gradual Xist repression was induced via the addition of different dTAG concentrations to the medium (0, 1, 2, 4, 8 and 500 nM). The cells were sequenced with 10x Chromium following a MULTIseq protocol. Sample barcodes for de-convoluting the raw .fastq files, as well as filtered counts with assigned samples are included in the supplementary files. The experiment was conducted in 2 replicates.