The enhancer landscape of pluripotent stem cells undergoes extensive reorganization during early mammalian development. The functions and mechanisms behind such reorganization, however, are unclear. Here, we show that the transcription factor GRHL2 is necessary and sufficient to activate an epithelial subset of enhancers as naive embryonic stem cells (ESCs) transition into formative epiblast-like cells (EpiLCs). Surprisingly, many GRHL2 target genes do not change in expression during the ESC-EpiLC transition. Instead, enhancers regulating these genes in ESCs diminish in activity in EpiLCs while GRHL2-dependent alternative enhancers become activated to maintain transcription. GRHL2 therefore assumes control over a subset of the naive network via enhancer switching to maintain expression of epithelial genes upon exit from naive pluripotency. These data evoke a model where the naive pluripotency network becomes partitioned into smaller, independent networks regulated by EpiLC-specific transcription factors, thereby priming cells for lineage specification.

Gene regulatory networks ensure that important genes are expressed at precise levels. When gene expression is sufficiently perturbed, it can lead to disease. To understand how gene expression disruptions percolate through a network, we must first map connections between regulatory genes and their downstream targets. However, we lack comprehensive knowledge of the upstream regulators of most genes. Here, we developed an approach for systematic discovery of upstream regulators of critical immune factors-IL2RA, IL-2 and CTLA4-in primary human T cells. Then, we mapped the network of the target genes of these regulators and putative cis-regulatory elements using CRISPR perturbations, RNA-seq and ATAC-seq. These regulators form densely interconnected networks with extensive feedback loops. Furthermore, this network is enriched for immune-associated disease variants and genes. These results provide insight into how immune-associated disease genes are regulated in T cells and broader principles about the structure of human gene regulatory networks.