SUMMARY Down syndrome (DS), driven by an extra copy of chromosome 21, is associated with profound changes in genome-wide gene expression and DNA methylation, but underlying mechanisms remain incompletely resolved. Leveraging human induced pluripotent stem cell (iPSC) models to capture molecular mechanisms relevant for human development, we previously identified a significant decrease in the abundance of a specific histone modification, histone H3 lysine 36 dimethylation (H3K36me2), in DS patient cells compared with euploid controls; this finding was also highly reproducible across a panel of DS patient and euploid control lymphoblastoid cell lines, indicating the finding extends to some peripheral cell types. H3K36me2 is localized to euchromatin where it impacts transcriptional regulation and is essential for maintenance of DNA methylation via DNMT3A recruitment, particularly at intergenic regions. Notably, haploinsufficiency of the histone methyltransferase which catalyzes H3K36me2 drives a neurodevelopmental disorder called Sotos syndrome, characterized by reduced H3K36me2, transcriptional dysregulation and DNA hypomethylation. The profound developmental consequences of reduced H3K36me2 in Sotos syndrome supports the novel hypothesis that the reduced H3K36me2 we found in DS could also contribute to developmental abnormalities in this disease context. Our overall hypothesis is that reduced H3K36me2 in DS drives DNA hypomethylation and transcriptional dysregulation, and that normalization of H3K36me2 will partially rescue these phenotypes. In Aim I, we will test the hypothesis that decreased H3K36me2 drives DNA hypomethylation in DS, by generating genome-wide DNA methylation maps and integrating them with existing H3K36me2 gene occupancy and transcriptional datasets, all from the same DS patient and isogenic euploid control iPSC- derived glutamatergic neurons. Importantly, these experiments are designed to connect a well-characterized phenotype in DS (aberrant DNA methylation) with a novel molecular mechanism (reduced H3K36me2). In Aim II, we will test the hypothesis that normalizing H3K36me2 levels in DS patient iPSC-derived neurons can rescue DNA methylation phenotypes. These data would serve as proof-of-principle that inhibition or activation of specific epigenetic modifiers can reverse well-characterized phenotypes in DS, using physiologically relevant human cellular models. Collectively, our rigorous molecular analyses will elucidate novel mechanisms of epigenetic dysregulation in DS, which may ultimately inform on new therapeutic strategies for DS patients; they will also generate an essential roadmap for future studies to further investigate how reduced H3K36me2 and its normalization impacts iPSC-derived and peripheral blood cell phenotypes.