Establishing foundational tools and datasets for investigation of NSD1 gene function in neural development - SUMMARY Sotos syndrome, characterized by childhood overgrowth, global developmental delay, intellectual disability and behavioral deficits is driven by haploinsufficiency of Nuclear Receptor Binding SET Domain Protein 1 (NSD1) which encodes a histone H3 lysine 36 (H3K36) methyltransferase. NSD1 specifically catalyzes H3K36 dimethylation (H3K36me2), which has important roles in DNA methylation (DNAme) and transcription. Previous studies using mouse or cancer cell lines have shown that H3K36me2 is essential for maintenance of DNAme via recruitment of DNA Methyltransferase 3A (DNMT3A), that NSD1 loss leads to reduced H3K36me2 and gain of the antagonistic mark histone H3 lysine 27 trimethylation (H3K27me3), and that NSD1 may regulate gene expression by facilitating the transition of RNAPII to an elongation-competent state. Cellular phenotyping studies have further identified important roles for H3K36me2 in cellular plasticity and cancer metastasis. How these mechanisms relate to neural development and dysfunction in Sotos syndrome patients, however, remains to be elucidated. Indeed, the effects of NSD1 mutation and H3K36me dysregulation are reported to be highly context dependent, resulting in opposing impacts on basic parameters such as cell proliferation across different cell types, with little known about NSD1 function and dysfunction in human brain cell types relevant for Sotos syndrome. Our laboratory recently identified a specific and significant decrease in H3K36me2 across Down syndrome (driven by trisomy 21) patient cell lines compared to euploid controls which we have been investigating by probing the relationship between H3K36me2 / H3K27me3 chromatin binding, DNAme and transcript expression in Down syndrome. These data led us to consider the intriguing possibility that Down syndrome and Sotos syndrome could share common molecular perturbations contributing to common neurodevelopmental phenotypes. However, the dearth of tools and datasets to study NSD1 function in a context relevant for Sotos syndrome (i.e., human neural development), has presented a significant barrier to progress. In Aim I, we therefore propose to generate a set of novel isogenic human iPSC models of NSD1 haploinsufficiency. Dysregulation of H3K36me2 / H3K27me3 chromatin binding patterns upon NSD1 haploinsufficiency in human neurons will then be assessed through pilot CUT&Tag datasets. Rigorous execution of this proposal will create new flexible and sustainable resources for studying NSD1 haploinsufficiency and test the molecular impacts of NSD1 haploinsufficiency in a physiologically relevant human brain cell type. This proposal is appropriate for the R03 mechanism, as the results will develop novel human cell-based models and generate discrete pilot data to support a future R01 submission.
