The molecular mechanisms underlying context-specific Runx factor functions in directing hematopoietic cell identity

PROPOSAL SUMMARY/ABSTRACT My long-term career goal is to broaden our understandings of molecular and cellular mechanisms governing hematopoietic cell fate decisions both in normal and pathogenic conditions as an independent investigator. A central question of my research is how broadly expressed transcription factors regulate a distinct set of genes in different developmental programs, as transcription factors’ ability to provide precisely required functional inputs in each cellular context is essential for life-long production of healthy blood cells. To address this, I propose to determine molecular mechanisms driving dynamic functions of Runx transcription factors in early thymic T cell development and megakaryocyte differentiation, two vastly different programs relying on Runx activities. Also, I aim to exploit a novel cell culture technique recapitulating the connection between bone marrow progenitor stages and early thymic progenitor stages. This will establish a new opportunity to define the roles of transcription factors in this developmental window, which was previously challenging due to lack of in vitro system. My preliminary studies suggest that Runx factors possess notable ability to switch their DNA binding sites in a context-specific fashion both within the same developmental trajectory at different stages as well as across different cell types. These dynamic Runx binding sites are closely associated with the genes that are sensitive to Runx functions. Importantly, redistribution of Runx factors occurs across large genomic domains and multiple peaks appear and disappear coordinately. Also, cell type-specific Runx binding sites harbor distinct sets of other transcription factor motifs, suggesting that a unique ensemble of collaborators may be present in each cellular context. Thus, I hypothesize that 3D chromatin reconfiguration responds to or causes context-specific Runx binding site choices, and these dynamic Runx functions are driven by distinct co-factors in each program. To address this, I will determine whether developmental changes of 3D chromatin structure require or instruct Runx functions during early T cell development (AIM 1, mentored phase). Also, I will define which functional collaborators physically interact with Runx factors in early T cell development and megakaryocyte development and test which co-factors are necessary to guide cell type-specific Runx DNA binding (AIM 2A). Additionally, I will define the impact of cell type-specific partners on Runx functions independently of chromatin state by experimentally introducing mismatched-co-factors to non-native developmental context (AIM 2B). Finally, I will establish a novel in vitro system recapitulating the developmental transition from bone marrow progenitor phases to early thymic progenitor stages. I will exploit this system to test whether the principles underlying dynamic Runx functions apply to the activities of another multilineage-expressed transcription factor, c-Myb. Together, the studies in this proposal will show how globally expressed transcription factors execute context- specific functions in different developmental pathways in normal hematopoiesis, and how malfunction of these principles can cause hematologic pathologies, such as leukemia. 1