Coordinating Self-Renewal and Proliferation via MLL1-HOX Pathways and Beyond

Abstract SCI-28 Epigenetic regulation of gene expression plays a central role in normal hematopoietic stem cell (HSC) maintenance and leukemogenesis. The histone methyltransferase, MLL1, is essential for the maintenance of HSCs and is a common target of chromosomal translocations that result in acute leukemia. To discover genetic networks regulated by MLL1 in HSCs, we identified genes that were acutely deregulated upon Mll1 loss in HSCs, using a conditional knockout approach and lineage-negative, c-Kit+, Sca-1+, CD48-negative (LSK/CD48neg) cells. The majority of genes that changed were proliferation-associated genes, upregulated in Mll1−/− LSK/CD48neg cells. This reflected the fact that Mll1-deficient HSCs exhibit increased proliferation in vivo, a phenotype previously documented using the Mx1-cre inducible model. To determine whether the increased proliferation was cell-intrinsic, we performed single cell proliferation studies in serum-free medium containing SCF, IL-11, and Flt3L. We found that Mll1−/− LSK/CD48neg single cells entered the cell cycle earlier and that each cell cycle was shorter than wild-type controls. Evidence for failure to suppress lineage-specific gene expression was also observed; specifically, five percent of the upregulated genes encoded erythroid-specific proteins. These included erythroid transcriptional regulators such as GATA1 and KLF1, but also structural proteins such as spectrin, KEL, and EpoR. The relationship between erythroid-lineage genes and Mll1 was unique, since no other lineage-specific programs were upregulated in Mll1−/− LSK/CD48neg cells. Among the genes downregulated upon Mll1 loss, the largest category was comprised of transcriptional regulators, including Mecom, Pbx1, and Prdm16, which are known to control HSC self-renewal and quiescence. As observed in many other tissues, Mll1−/− LSK/CD48neg cells also exhibited reduced Hoxa9 expression. Interestingly, not all identified MLL1 target genes required menin, a cofactor thought to participate in directing MLL1 to particular genomic loci in vivo, and not all targets were Mll1-dependent in nonhematopoietic tissues. Chromatin immunoprecipitation and functional studies suggest that the identified genes act within a series of parallel pathways as direct transcriptional targets of MLL1. Interestingly, reexpression of Prdm16 alone could rescue Mll1-deficient cells from rapid attrition in bone marrow chimeras. Furthermore, Prdm16 corrected the hyperproliferation phenotype of Mll1−/− LSK/CD48neg cells. These data demonstrate that MLL1 coordinately regulates proliferation, lineage-specific gene expression programs, and self-renewal. By elucidating the normal MLL1-dependent transcriptional network within HSCs, we show that this pathway is overlapping but distinguishable from the leukemogenic pathway, suggesting that targeted therapy with minimal side effects on hematopoiesis will be feasible. Disclosures: No relevant conflicts of interest to declare.