The Sotos syndrome gene Nsd1 safeguards developmental gene enhancers poised for transcription by maintaining the precise deposition of histone methylation.

Germline haploinsufficiency of NSD1 is implicated as the etiology of Sotos syndrome; however, the underlying mechanism remains far from being clear. Here, we use mouse embryonic stem cell (mESC) differentiation as a model system to address this question. We found Nsd1 to be indispensable for the faithful differentiation of mESCs into three primary germ layers, particularly, various meso-endodermal cell lineages related to development of the heart and the skeletal system. Time-course transcriptomic profiling following the mESC differentiation revealed that Nsd1 not only facilitates the basal expression but also permits the differentiation-accompanied rapid induction of a suite of meso-endoderm lineage-specifying transcription factor (TF) genes such as T and Gata4. Mechanistically, Nsd1 directly occupies putative distal enhancers of the lineage TF genes under the pluripotent cell state, where it deposits H3K36me2 to antagonizes the excessive H3K27me3 and maintain the basal H3K27ac level, thereby safeguarding these gene enhancers at a primed state that responds readily to differentiation cues. In agreement, gene rescue assays using the Nsd1 knockout mESCs showed that the H3K36me2 catalysis by Nsd1 requires several functional modules within Nsd1 (namely, PHD1-4, PWWP2 and SET) to a similar degree. Disruption of either one of these Nsd1 modules severely abrogated H3K36me2 in mESCs and significantly impaired appropriate induction of developmental genes upon mESC differentiation. Altogether, our study provides novel molecular insight into how the NSD1 perturbation derails normal development and causes the disease.