Cited2 Regulates Hematopoietic Stem Cell Quiescence Through HIF-1α Dependent and Independent Pathways

Abstract 912

Hematopoietic stem cells (HSCs) are thought to be localized in hypoxic microenvironment of the bone marrow (BM) and can remain quiescent or differentiate into multiple blood cell lineages. A number of factors have been found to regulate HSC quiescence in either cell-intrinsic or cell-extrinsic manner. Cited2 (CBP/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2), a member of a newly identified transcriptional modulator, is a cytokine inducible gene and plays various roles during mouse development. In particular, Cited2 is essential for fetal liver hematopoiesis. In this study, we used conditional knockout strategy to delete Cited2 in order to further investigate its function in adult hematopoiesis. Sequential injection of poly(I)-poly(C) (pI-pC) efficiently deleted the Cited2 gene in Cited2^fl/fl;Mx1-Cre mice. In this mouse model (Cited2^−/− mice), the white blood cell (WBC) count, BM cellularity and Lin⁻Sca-1⁺c-Kit⁺ (LSK) cell number were within the normal range. However, the long-term HSC (LT-HSC; defined as Flt3⁻CD34⁻LSK or CD48⁻CD150⁺LSK) frequency was significantly decreased. Cited2^−/− mice also exhibited increased apoptosis in both LSK and CD34⁻LSK cells. In addition, Cited2 deficiency led to loss of HSC quiescence evidenced by cell cycle analysis and BrdU incorporation assay. HSC reconstitution capacity was significantly impaired assessed by 5-fluorouracil (5-FU) treatment and transplantation experiments. Transcriptional profiling revealed that multiple HSC quiescence and hypoxia related genes were affected, including p57, Hes1 and Egr1. Recent studies have shown that both HIF-1α-deficient and HIF-1α-stabilized HSCs result in impaired hematopoietic reconstitution, suggesting that precise regulation of HIF-1α level is essential for maintaining HSC quiescence and transplantation activity. Cited2 has been shown to be a negative regulator for HIF-1α through competitive binding to CBP/p300 with higher affinity. In addition, we previously showed that HIF-1α haploinsufficiency (HIF-1α^+/−) partially rescues the heart defects in Cited2^−/− embryos and HIF-1α deletion (HIF-1α^−/−) rescues aberrant vasculature in Cited2^−/− embryonic lens. These findings prompted us to explore whether defects of Cited2^−/− HSC in adult mice are mediated by dysregulated HIF-1 activity by generating Cited2^fl/flHIF-1α^fl/fl;Mx1-Cre mice. Additional deletion of HIF-1α partially rescued impaired HSC quiescence and reconstitution capacity caused by Cited2 deficiency. Cited2^−/− HIF-1α^−/− HSCs displayed comparable apoptosis to Cited2^−/− counterparts. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to a normal level. Taken together, these results suggest that Cited2 regulates HSC quiescence through HIF-1 dependent and independent pathways.