microRNA Control of Hematopoietic Stem Cell Fitness and Myelopoiesis.

The Growth factor independent -1 (Gfi1) transcriptional repressor regulates both hematopoietic stem cell (HSC) self renewal and myeloid differentiation. Gfi1 null mice eventually die of HSC exhaustion, and Gfi1^−/− HSC are not competitive in transplantation assays. Here we show that Gfi1 is a master regulator of microRNAs and that transcriptional control of a microRNA gene is critical for Gfi1-directed stem cell competitiveness and myelopoiesis. First, we show that the gene encoding miR21 is a direct transcriptional target of Gfi1. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal Gfi1 binding to specific DNA sequences upstream of the miR21 stem loop. Moreover, the expression of Gfi1 and miR21 is reciprocal in 1) wild type and Gfi1^−/− marrow cells, 2) during normal differentiation from common myeloid progenitors (CMP) to granulocyte monocyte progenitors (GMP), and 3) during treatment-induced differentiation of human myeloid leukemia cell lines. Forced expression of Gfi1 lowers miR21 levels in wild type Lin⁻ bone marrow cells and human cancer cell lines. Knockdown of Gfi1 expression with shRNA in human cancer cell lines increases miR21 expression. Moreover, conditional deletion of Gfi1 induces miR21 expression in primary murine hematopoietic cells, including sorted CMP and GMP. Thus, Gfi1 transcriptionally regulates miR21 in both human and murine hematopoietic cells. Interestingly, we find that the Ski oncoprotein/transcriptional corepressor is a direct target of miR-21. Subsequently, Ski is dramatically reduced in Gfi1^−/− HSC and in wild type bone marrow Lin⁻ cells forced to express miR21. Gfi1 may repress miR21 to maintain functional competence. Specifically, we find that Ski is a previously undescribed Gfi1 corepressor. Both endogenous Ski and Gfi1 physically interact. Synthetic Ski and Gfi1 proteins reveal that the interaction is both strong and specific. Chromatin immunoprecipitation reveals Ski and Gfi1 occupy several Gfi1 target genes. However, Ski function is critical as a corepressor on only a subset of Gfi1 target genes. To determine the importance of Ski corepression to Gfi1 induced biology, we examined two well established phenotypes of Gfi1 loss of function; HSC competitiveness and myelopoiesis. When Gfi1^−/− embryonic stem cells are injected into a wild type blastocyst, they do not participate in hematopoiesis. Similarly, we find that when Ski^−/− embryonic stem cells are injected into a blastocyst, they infrequently participate in hematopoeisis. Next, because Ski^−/− animals die at or before birth, we examined the fitness of Ski^−/− fetal liver HSC. In competitive transplantation assays, Ski^−/− fetal liver HSC were significantly impaired in reconstitution compared to congenic wild-type competitor fetal-liver HSC. Moreover, Ski null HSC generated significantly less myeloid progeny. Thus, Ski^−/− HSC display a partial phenocopy of Gfi1^−/− hematopoiesis. We conclude that Gfi1 directly targets miR21 to control the expression of Ski, a corepressor for Gfi1, and that the Gfi1/Ski complex is critical to regulate a subset of Gfi1 target genes important for HSC fitness and myeloid cell production.