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Following implantation, mouse epiblast cells transit from a naive to a primed state in which they are competent for both somatic and primordial germ cell (PGC) specification. Using mouse embryonic stem cells as an in vitro model to study the transcriptional regulatory principles orchestrating peri-implantation development, here we show that the transcription factor Foxd3 is necessary for exit from naive pluripotency and progression to a primed pluripotent state. During this transition, Foxd3 acts as a repressor that dismantles a significant fraction of the naive pluripotency expression program through decommissioning of active enhancers associated with key naive pluripotency and early germline genes. Subsequently, Foxd3 needs to be silenced in primed pluripotent cells to allow re-activation of relevant genes required for proper PGC specification. Our findings therefore uncover a cycle of activation and deactivation of Foxd3 required for exit from naive pluripotency and subsequent PGC specification.

The pathological consequences of structural variants disrupting 3D genome organization can be difficult to elucidate in vivodue to differences in gene dosage sensitivity between mice and humans. This is illustrated by branchiooculofacial syndrome (BOFS), a rare congenital disorder caused by heterozygous mutations within TFAP2A,a neural crest regulator for which humans, but not mice, are haploinsufficient. Here, we present a BOFS patient carrying a heterozygous inversion with one breakpoint located within a topologically associating domain (TAD) containing enhancers essential for TFAP2Aexpression in human neural crest cells (hNCCs). Using patient-specific hiPSCs, we show that, although the inversion shuffles the TFAP2AhNCC enhancers with novel genes within the same TAD, this does not result in enhancer adoption. Instead, the inversion disconnects one TFAP2Aallele from its cognate enhancers, leading to monoallelic and haploinsufficient TFAP2Aexpression in patient hNCCs. Our work illustrates the power of hiPSC differentiation to unveil long-range pathomechanisms.

Poised enhancers marked by H3K27me3 in pluripotent stem cells have been implicated in the establishment of somatic expression programs during embryonic stem cell (ESC) differentiation. However, the functional relevance and mechanism of action of poised enhancers remain unknown. Using CRISPR/Cas9 technology to engineer precise genetic deletions, we demonstrate that poised enhancers are necessary for the induction of major anterior neural regulators. Interestingly, circularized chromosome conformation capture sequencing (4C-seq) shows that poised enhancers already establish physical interactions with their target genes in ESCs in a polycomb repressive complex 2 (PRC2)-dependent manner. Loss of PRC2 does not activate poised enhancers or induce their putative target genes in undifferentiated ESCs; however, loss of PRC2 in differentiating ESCs severely and specifically compromises the induction of major anterior neural genes representing poised enhancer targets. Overall, our work illuminates an unexpected function for polycomb proteins in facilitating neural induction by endowing major anterior neural loci with a permissive regulatory topology.