High-throughput identification of chromatin-opening transcription factors

Cell fate transitions involve major changes to the chromatin landscape, some of which are thought to deepen the basin around a newly adopted cell state, stabilizing gene expression programs that might otherwise be transient. Transcription factors (TFs) are central to this process, binding a subset of their genome-wide motif occurrences to create accessible regions that can act as cell type-specific enhancers. However, apart from a few well-studied cases, it is not clear which of the ~1,600 mammalian TFs, when newly expressed, can directly or indirectly initiate newly accessible sites, nor why only some motif occurrences gain accessibility. Here, we applied pooled CRISPR activation (CRISPRa) to upregulate endogenous TFs to physiological levels in mouse embryonic stem cells (mESCs). After screening guides targeting 240 developmental TFs for transcriptional response, we selected 60 and jointly profiled gene expression and chromatin accessibility in ~140,000 single cells. Some TFs (e.g. Cdx2, Sox10) appeared to initiate new accessibility on their own, predominantly at distal sites that are closed in mESCs but coincide with cell type-specific enhancers later in development. Others appeared to partner with "co-TFs", either by leveraging preexisting mESC heterogeneity (e.g. {uparrow}Grhl1 in Tfap2c+ mESCs [->] opening of sites bearing motifs for both), or by hierarchical activation (e.g. {uparrow}Sox7 [->] {uparrow}Gata6 [->] opening of sites bearing either or both motifs). Such dependencies shape which sites are used and which fates are reachable, and may explain the differential behavior of TFs with nearly identical consensus motifs. Thus, the capacity of a newly expressed TF to open chromatin strongly depends on the cell type, cell state and gene regulatory network it lands in. Charting these dependencies will inform how cell fate transitions are initiated in development, homeostasis and disease, and how to engineer them in silico, in vitro and in vivo.