The histone variant H3.3 is enriched at enhancers and active genes, as well as repeat regions such as telomeres and retroelements, in mouse embryonic stem cells (mESCs) 1 – 3 . While recent studies demonstrate a role for H3.3 and its chaperones in establishing heterochromatin at repeat regions 4 – 8 , the function of H3.3 in transcription regulation has been less clear 9 – 16 . Here, we find that H3.3-specific phosphorylation 17 – 19 stimulates activity of the acetyltransferase p300 in trans , suggesting that H3.3 acts as a nucleosomal cofactor for p300. Depletion of H3.3 from mESCs reduces acetylation on histone H3 at lysine 27 (H3K27ac) at enhancers. Cells lacking H3.3 demonstrate reduced capacity to acetylate enhancers that are activated upon differentiation, along with reduced ability to reprogram cell fate. Our study demonstrates that a single amino acid in a histone variant can integrate signaling information and globally impact genome regulation, which may help better understand how mutations in these proteins contribute to human cancers 20 , 21 .

SummaryTherapeutic application of human embryonic stem cells (hESCs) requires precise control over their differentiation. However, spontaneous differentiation is prevalent, and growth factors induce multiple cell types; e.g., the mesoderm inducer BMP4 generates both mesoderm and trophoblast. Here we identify endogenous WNT signals as BMP targets that are required and sufficient for mesoderm induction, while trophoblast induction is WNT independent, enabling the exclusive differentiation toward either lineage. Furthermore, endogenous WNT signals induce loss of pluripotency in hESCs and their murine counterparts, epiblast stem cells (EpiSCs). WNT inhibition obviates the need to manually remove differentiated cells to maintain cultures and improves the efficiency of directed differentiation. In EpiSCs, WNT inhibition stabilizes a pregastrula epiblast state with novel characteristics, including the ability to contribute to blastocyst chimeras. Our findings show that endogenous WNT signals function as hidden mediators of growth factor-induced differentiation and play critical roles in the self-renewal of hESCs and EpiSCs.

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