Site-specific programming characterizes dynamic post-translational acetylation of histone H2B lysine 108 in mouse embryonic stem cells.

Dynamic histone modifications are critical for regulating stem cell fate. However, generalized strategies for site-specific programming of histone modifications profiling on unperturbed chromatin in embryonic stem cells (ESCs) remain challenging. In this study, we established a genetic code expansion-based platform in mouse ESCs (mESCs) for site-specific proteomic mapping and functional analysis within native chromatin. Using this platform, we revealed Rps19bp1 promotes Sirt1-mediated deacetylation of histone H2B lysine 108 acetylation (H2B-K108ac). Moreover, Site-specific-AcK revealed that H2B-K108ac induces the formation of H2B puncta and modulates chromatin accessibility. Functionally, elevation of H2B-K108ac via genetic code expansion and the loss of Rps19bp1 cause significant alterations in the expression of genes associated with mESC differentiation. Furthermore, Rps19bp1 deficiency promotes neural differentiation in mESC-derived teratoma. Our study develops a robust platform for linking specific histone acetylation with chromatin dynamics and cell fate determination, which lays a foundation for future exploration of additional histone PTMs in ESCs.