HIF-1α deletion partially rescues defects of hematopoietic stem cell quiescence caused by Cited2 deficiency

Abstract

Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence-and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1␣ gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1␣ doubleknockout mice. Additional deletion of HIF-1␣ in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1␣ restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1-dependent and HIF-1-independent pathways. (Blood. 2012;119(12):2789-2798) IntroductionHematopoietic stem cells (HSCs) are thought to be localized in the hypoxic microenvironment of the BM and remain quiescent or differentiate into multiple blood cell lineages. Several factors have been found to regulate HSC quiescence in either a cell-intrinsic (eg, p21, p57, Bmi1, Egr1, GATA2, Gfi1, Pbx1, and others) or a cell-extrinsic (eg, Tie2/Angpt1, c-Mpl/Tpo, CXCR4/CXCL12, and others) manner.CBP/p300-interacting transactivator with glutamic acid (E) and aspartic acid (D)-rich tail 2 (Cited2), a member of the Cited family of transcriptional modulators, is a cytokine-inducible gene 1 that plays various roles during mouse development. [2][3][4][5][6] In particular, Cited2 plays an important role in fetal liver hematopoiesis, which is supported by severely impaired fetal liver HSC function and decreased expression of genes known to be essential for hematopoiesis in Cited2-deficient fetal liver HSC/progenitors. 7 Cited2 has also been implicated in tumor cell invasion and progression. 8,9 Cited2 is a negative regulator of hypoxia-inducible factor 1 (HIF-1). Bhattacharya et al first demonstrated in vitro that Cited2 competes with HIF-1␣ for binding to p300-CH1 and inhibits HIF-1-mediated transactivation. 10 Bakker et al also showed that FOXO3a inhibits HIF-1-induced apoptosis by stimulating the transcription of Cited2, which reduces the expression of the pro-apoptotic HIF-1 target genes NIX (also called "Bnip3l") and RTP801 (also called "Ddit4"). 11 In our previous studies, we showed that deletion of HIF-1␣ partially rescues defects in heart and lens caused by Cited2 deficiency. 4,12 In addition, at the structural level, Freedman et al revealed that Cited2 and HIF-1␣ share a conserved...

Cited2 Gene Controls Pluripotency and Cardiomyocyte Differentiation of Murine Embryonic Stem Cells through Oct4 Gene

“…It was demonstrated that 2-3-fold overexpression of Oct4 inhibits hematopoietic differentiation of ESCs (8). Because ineffective down-regulation of Oct4 expression was observed in Cited2 KO EBs and Cited2 deletion affects fetal and adult hematopoiesis (23,26,27), we examined whether Cited2 depletion in ESCs affects hematopoietic differentiation. Cells from primary day 9 EBs were re-plated in methylcellulose media containing cytokines supporting hematopoietic colony formation and scored for CFU-E, CFU-GM, and CFU-Mix by day 7.…”

“…In particular, deletion of Cited2 at the epiblast stage using Sox2-Cre phenocopies cardiac defects in Cited2 total knock-out mouse model (28). Depletion of Cited2 in the hematopoietic tissues via Mx1-Cre affects hematopoietic stem cell quiescence and apoptosis (26,27).…”

Cited2, a Transcriptional Modulator Protein, Regulates Metabolism in Murine Embryonic Stem Cells

“…The role of p300 in ESC metabolism remains elusive. By monitoring glucose consumption and extracellular lactate production, we found that undifferentiated Cited2 ⌬/Ϫ ESCs metabolize ϳ20% more glucose via aerobic glycolysis than WT counterparts, with a concomitant regulation of glycolysis-associated genes, such as HK1 and PFK1-L. HK1 is a target gene of Oct4 and HIF-1 (41,50), two transcription factors previously shown to be modulated by Cited2 (8,22). A ChIP assay indicated that Cited2 is recruited to the HK1 gene promoter, further supporting the direct role of Cited2 in coordinating HK1 expression and glucose metabolism.…”

“…Cited2 competes with HIF-1␣ in binding to the CH1 domain of CBP/p300 and thus modulates key biological functions in heart, eye, and hematopoietic stem cells (8,(52)(53)(54). HIF-1 is responsible for controlling the rate of glycolysis in ESC lines with gain-or loss-of-function of HIF-1␣.…”

“…In adult mice, hematopoietic stem cells are metabolically inactive, as evidenced by cell quiescence, whereas the maintenance of hematopoietic stem cells is sensitive to changes in the metabolism of glucose and fatty acids (5,6). Importantly, we and others have shown that Cited2 regulates quiescence and apoptosis in adult hematopoietic stem cells via HIF-1 and p53-dependent mechanisms (7,8). Recently, by adenovirus-mediated delivery of Cited2 small hairpin RNA (shRNA) to mouse hepatocytes, it was demonstrated that Cited2 regulates gluconeogenesis by interacting with histone acetyltransferase GCN5 to affect acetylation and activity of peroxisome proliferative-activated receptor ␥ co-activator 1␣ (PGC-1␣) (9).…”