NSD1 governs H3K36me2-mediated DNA methylation and drives endo-mesodermal differentiation of human iPSCs

Epigenetic regulatory mechanisms, which include histone modifications and DNA methylation, play a central role in development and aging. Dimethylation of H3K36, deposited mainly by the histone methyltransferase NSD1, occurs predominantly in intergenic regions, and recruits the DNA methyltransferase DNMT3A to facilitate DNA methylation. Haploinsufficiency of NSD1 results in the overgrowth disorder Sotos syndrome in vivo, which is associated with aberrant DNA methylation signatures and an enhanced epigenetic age. To understand the mechanisms by which NSD1 may regulate development, differentiation, and diseases, we generated human iPSC lines deficient in functional NSD1 (NSD1-KO). NSD1-KO cells exhibit a substrate-specific decrease in proliferation, reduced H3K36me2 levels, and extensive DNA hypomethylation. Notably, the loss of functional NSD1 altered the differentiation potential of iPSCs, with aberrant endodermal and mesodermal lineage commitment. Further analysis revealed that NSD1 might drive endodermal differentiation by regulating the expression of an endodermal lncRNA 'HIDEN' by mechanisms independent of the regulation of DNA methylation. Our NSD1-KO iPSC lines partially recapitulate the DNA methylation defects associated with NSD1-related developmental disorders. Additionally, we uncovered a novel mechanism by which NSD1 may regulate endodermal differentiation of human iPSCs.