PROJECT SUMMARY In this proposal, we will define the effects of epigenetic dysregulation via Stag2 loss on three-dimensional chromatin structure and transcriptional regulation. We will define key lineage-specific chromatin features and validate their hierarchy in differentiation in contrast to stem cell self-renewal. In Aim 1, we will perform low-input Hi-ChIP assay on Ctcf (loop/TAD-boundary), Rad21 (pan-cohesin loop), ChIP assay for H3K27Ac (enhancer) and RNA-seq (gene expression) on phenotypically defined mouse and human stem and progenitors to define population-specific looping events. Using our novel ‘on-off-on’ dual recombinase (Stag2GL) allele, we will first determine if the restoration of Stag2 can reverse the effects on self-renewal/differentiation and define chromatin loops 1) perturbed during Stag2 loss and 2) restored after Stag2 reversion. Locus-specific functional assays will determine the key events required for self-renewal through in vitro studies to phenocopy in ‘Stag2-on' or prevent reversion in ‘Stag2-off’. In Aim 2, using our low cell input Hi-C and Hi-ChIP techniques to assess chromatin loops alterations identified in our fully penetrant Stag2/Npm1c and Stag2/Flt3 co-mutant AML models in combination with the Stag2GL allele. Using a multi-omics approach, we will define the chromatin landscape of Flt3 and Npm1 with and without the antecedent Stag2 mutation. We have created a well-annotated MDS and AML patient biorepository, as well as a biorepository of healthy bone marrow from hip arthroplasty patients. Using these primary patient samples, we will map chromatin loops and transcriptional output in stem and progenitor populations and intersect lineage-specific interactions in human and mouse. Functional validation of chromatin loops lost or gained in the co-mutant models will be assessed in vitro, prioritizing loci identified in primary AML patient samples. These models will constitute the system to assess the effects of Stag2 loss on therapeutic response to standard chemotherapeutic regimens ad well as to emerging targeted therapies such as Menin inhibition in Npm1c-mutant AML (DSP5536) and FLT3 inhibition in FLT3-ITD mutant AML (gilteritinib). Finally, Stag2 reversion in this system will definitively determine whether Stag2 is required for the maintenance of AML, hereby representing a genetic dependency in Stag2-mutant AML. Taken together, the results of this proposal will elucidate the molecular pathology of Stag2, thus leading to a deeper understanding of chromatin-related transcriptional regulation, epigenetic plasticity, and the contributions of Stag2 to MDS and resultant secondary AML and the identification of key novel therapeutic targets.