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

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. scRNAseq was performed in TX1072 XX and TX1072 XX dFtx-Xert -/- mESCs following 4 days of 2i/LIF-withdrawal. The cells were sequenced with 10x Chromium following a MULTIseq protocol. As the libraries were prepared together with an unrelated experiment, the provided FASTQ files were filtered for reads matching assigned cell barcodes (using UMI-tools and seqtk). The setup includes 2 replicates (REP1: 187 WT, 87 DEL cells; REP2 325 WT, 79 DEL cells).