Project Summary Leukemia is a rare, but often fatal, form of cancer stemming from progenitor cells in the blood that are defective in cellular differentiation. A particularly aggressive and hard to treat form called mixed-lineage leukemia (MLL) arises from aberrant chromosomal translocations of the MLL gene with various genes encoding elongation factors. These MLL-fusions improperly drive transcription of homeotic genes like those in the Hox family, thereby maintaining a highly proliferative, stem cell-like population. These “leukemic stem cells” mature into leukemic blasts that can form tumors and metastasize throughout the body via the blood stream. However, the specific mechanisms of MLL-fusion driven leukemogenesis are still not well understood and the high frequency of relapses upon treatment call for further characterization of the main drivers of this cancer. One such factor that has recently been shown to be critical for regulating MLL-fusion driven leukemogenesis is ASH1L, a histone H3K36 dimethyltransferase with a putative BRD-PHD-BAH histone reader domain module. A scaffold protein called LEDGF was shown to bind to H3K36me2 and recruit MLL to homeotic genes in an ASH1L-dependent manner, but it is unclear whether H3K36me2 and/or ASH1L are directly recruiting LEDGF-MLL complexes to these genes. Additionally, it is completely unknown how ASH1L specifically localizes to homeotic genes rather than much of the rest of the genome. To address how ASH1L functions in non-leukemic and leukemic cells, I will employ a suite of innovative histone-binding assays, structural methodologies, and cellular-based analyses. For Aim 1, I will determine the histone binding specificity and mode of the putative ASH1L reader domain module using modernized histone peptide and nucleosome binding assays in parallel with Cryo-EM of purified ASH1L bound to modified nucleosomes. For Aim 2, I will interrogate how ASH1L reads the chromatin landscape in the well-studied and easy to genetically manipulate HEK293T cell line using a knockout/complementation system. I will assess whether ASH1L colocalizes with histone modifications that we identify in Aim 1 as well as proteins thought to associate with ASH1L like LEDGF using CUT&RUN. Additionally, I will examine how loss/complementation of ASH1L in these cells affects known gene target expression. For Aim 3, I will employ a knockdown/complementation system in MLL-fusion leukemia models to 1) assess whether ASH1L is required for viability, 2) determine where ASH1L genomically localizes in MLL, and 3) characterize how ASH1L loss and complementation affects recruitment of co-localizing gene regulators to impact transcription. This work aims to further decipher the “Histone Code” and provide insights into how ASH1L functions as a driver of MLL-fusion leukemogenesis. I will also lay out a foundation for testing ASH1L as a drug target for treating these aggressive diseases. Finally, this work serves as a strong training platform ...