Transcriptional Control of HSC Fitness
Authors: Muench David E. , Velu Chinavenmeni S. , Olsson Andre , Meyer Sara E. , Grimes H. Leighton 
Published: October 2019

Abstract
To sustain lifetime hematopoiesis, adult hematopoietic stem cells (HSCs) are required to remain quiescent. Proper control over cell cycle status underlies HSC fitness. Although the identity of cell cycle mediators which control HSC cycling are basically understood, the gene regulatory networks controlling them are largely unknown. Gfi1 encodes a transcriptional repressor protein that is required to maintain HSC quiescence. Over-cycling of stem-progenitors causes Gfi1-/- HSC exhaustion and prevents long-term engraftment in lethally-ablated recipients. Gfi1 controls myeloid progenitor maintenance through the direct regulation of HoxA9, but the transcriptional programming necessary to control HSC function is not known. Gfi1-mediated repression is critically dependent upon the recruitment of known corepressors; including the Lsd1/CoREST complex, and Eto proteins. Moreover, Gfi1 directly represses a suite of microRNA genes, including miR-21, miR-196b and miR-302b. We were intrigued by a series of papers in which miR-302b was shown to target the Lsd1 corepressor, which was subsequently shown to be required for HSC fitness. Lsd1 serves as a transcriptional co-repressor for Gfi1, Gfi1b, Scl1/Tal1, Bcl11a, Sall4, and Runx1. Knockdown and genetic ablation studies demonstrated that loss of Lsd1 results in a derepression of stem/progenitor genes, and leads to defects in HSC self-renewal and marrow failure. Given this published example, we next sought to determine whether miR-196b and miR-21 might also affect Gfi1 cofactors. First, we find that miR-196b targets CBFA2T3/Mtg16, encoding Eto2 (in human and mouse, respectively). Eto2 is the most abundant Eto family protein in hematopoietic stem and progenitors, and functions as a transcriptional co-repressor with Gfi1, Gfi1b, Scl1/Tal1, Gata-1, Lmo2, and Ldb1. In luciferase-sensor assays for the CBFA2T3-3'UTR, cotransfection with a miR-196b mimic reduces luciferase activity, dependent upon a canonical miR-196b binding site. In agreement with published data, we find that Mtg16-/- HSC are reduced in number and are impaired in competitive transplant assays, but this defect can be partially alleviated by increasing Mtg16-/- HSC numbers. We also find that the initial serial transplant of Mtg16-/- HSC leads to immediate exhaustion. Second, we find that miR-21 targets Ski which functions as a transcriptional corepressor for Smad, Gli, MAD, and thyroid hormone receptor transcription factors. In luciferase-sensor assays for the Ski 3'UTR, cotransfection with a miR-21 mimic reduces luciferase activity, dependent upon a canonical miR-21 binding site. We find that both synthetic and endogenous Ski and Gfi1 proteins directly and specifically interact with one another, implicating Ski as a new Gfi1-corepressor. Recently, overexpression of Ski was shown to enhance HSC repopulation in transplant recipients and caused myeloproliferative disease by promoting HSC gene signatures independent of Tgf-β signaling. Ski-/- is perinatal lethal due to cleft palate, so we analyzed Ski-/- hematopoiesis by transplanting E14.5 fetal liver cells. Similar to Mtg16-/- HSC, Ski-/- HSCs display impaired competitive engraftment that can be rescued by increasing Ski-/- HSC numbers. We also find that the initial serial transplant of Ski-/- HSC leads to immediate exhaustion. Finally, in Gfi1-/- lineage negative bone marrow (which expresses elevated levels of miR-21 and miR-196b) the level of Ski and Eto2 protein are significantly decreased. Our findings suggest that Gfi1 repression of individual Gfi1-target miR genes is required for the proper control of Gfi1 co-factors and subsequent HSC biology. To test this concept we generated mice with tetracycline-inducible miR-196b expression (in mice with intact Gfi1 expression). After only one month of transgene induction we find significant HSC exhaustion. Our data suggests that repression of Gfi1 target genes miR-21, miR-196b and miR-302b is critical for normal HSC function, because these miR target essential transcriptional cofactors.

Disclosures
No relevant conflicts of interest to declare.