Mechanisms of RUNX1 transcriptional control in hematopoiesis

PROJECT SUMMARY/ABSTRACT Germline loss-of-function mutations in the RUNX1 gene cause a familial platelet disorder with a predisposition to the development of myeloid malignancy (RUNX1-FPDMM). RUNX1 encodes a sequence-specific transcription factor that regulates critical hematopoietic gene expression programs. Consistently, mouse models of Runx1 deletion have revealed critical roles in definitive hematopoiesis, platelet production, stem cell function, and the development and function of both myeloid and lymphoid cells. However, while mouse models have defined critical Runx1 knockout phenotypes, it has been difficult to define the Runx1 target genes that underlie these phenotypes. This is because, while transcription is a highly dynamic process changing within minutes, Runx1 conditional knockout models (germline deletion is embryonic lethal) allow tissues to develop in the absence of the Runx1 protein resulting in the accumulation of both direct as well as indirect and compensatory changes in gene expression. In order to overcome this limitation and define the mechanism of RUNX1 function, we have engineered the endogenous RUNX1 locus with a degron tag. This technology allows the rapid removal of the RUNX1 proteins within 1-2 hours of treatment with the small molecule, dTAG, thus collapsing the timeframe for studying RUNX1-mediated transcriptional changes from weeks to hours. Using this technology in cell lines, we have identified complex mechanisms of RUNX1-mediated transcriptional control, which suggest that RUNX1 collaborates with a number of other sequence-specific transcription factors and co-regulatory proteins to regulate hematopoietic gene networks. Importantly, some of these factors (e.g., FLI1, GATA1, GATA2) are also associated with germline mutations that cause either familial thrombocytopenia or predisposition to malignancy, suggesting that these factors may contribute to similar phenotypes by converging on an overlapping gene network. Here we propose to understand the cooperative and/or antagonistic nature of these proteins on RUNX1 gene expression networks. At the same time, in order to uncover how Runx1 transcriptional control contributes to hematopoietic development, we will complement these cell-based models with the first Runx1dTAG mouse model. This model will allow in vivo and ex vivo characterization of the direct consequences of acute Runx1 depletion on HSPC transcription and cell fate decisions. Combined, these studies will define mechanisms by which RUNX1 cooperates with other transcription factors to fine tune hematopoietic gene expression programs and will determine how Runx1 controlled transcriptional programs facilitate distinct lineage choices to maintain normal hematopoiesis. Importantly, the mechanistic insights provided by these studies will shed light on the molecular underpinnings of RUNX1-FPDMM.