Abstract
Abstract 1612
SIRT1 is a conserved NAD-dependent deacetylase capable of deacetylating a number of protein substrates including, but not limited to, p53 and FOXO transcription factors. SIRT1 plays an important role in a variety of biological processes including stress resistance, metabolism, differentiation and aging (Rodgers et al, Nature, 2005; 434:113). SIRT1 is expressed at high levels in mouse embryos. A role for SIRT1 in mouse (m) embryonic stem cell (ESC) maintenance and differentiation is only beginning to be elucidated (Han et al, Cell Stem Cell, 2008; 2:241, Calvanese V et al, PNAS, 2010; 10713736). Here we focus on a role for SIRT1 in differentiation of mESCs into hematopoietic progenitors (HPCs), and in embryonic and adult hematopoiesis. We hypothesized that SIRT1 is involved in hematopoietic commitment within the mouse. We initially assessed the ability of WT and SIRT1-/- mESC to give rise to blast colony forming cells (BL-CFC), a transient population that is present in EBs between day 2.5 and day 3.5 of differentiation and represents the in vitro equivalent of the hemangioblast and as such, the earliest commitment step in the differentiation of mesoderm to the hematopoietic and endothelial lineages. SIRT1-/- ESCs exhibited markedly delayed formation of BL-CFC. The emergence of the Flk-1+/c-Kit- cell population pattern was also delayed, consistent with the delayed pattern of BL-CFC development in SIRT1-/- EBs. This observed delay appears to result from a slower differentiation of the SIRT1-/- ESCs as the kinetics of decline in secondary EB potential, an indication of undifferentiated ES cells, is delayed compared to that of SIRT1+/+ ES cells. When analyzed for hematopoietic and endothelial potential of individual blast colony, replated SIRT1-/- BL-CFC presented limited hematopoietic potential, whereas endothelial potential was essentially unaltered. Next, the ability of SIRT1-/- ESCs to form primitive and definitive hematopoietic cells was evaluated and we found that primitive erythroid progenitors formed from SIRT1-/- R1 cells were not only delayed but greatly decreased. Moreover, after differentiation of SIRT1 -/- mESC there were also significant decreases in granulocyte-macrophage (CFU-GM), and multipotential (CFU-GEMM) progenitors. Differences in primitive and definitive erythroid progenitors were confirmed by gene analysis of βH1 globin (embryonic hemoglobin), a marker for primitive erythroid cells, and βmajor globin (adult hemoglobin). The above delay defects were associated with delayed ability to switch off Oct4, Nanog and Fgf5, decreased β-H1 Globin, β-major globin, Scl gene expression and reduced activation of the Erk1/2 pathway upon SIRT1-/- ESC commitment. Reintroduction of WT SIRT1 into SIRT1-/- cells partially rescued the primitive erythroid progenitor formation of SIRT1-/- cells and the expression of hemoglobin genes, Hbb-bh1 and Hbb-b1, suggesting that the defect of hematopoietic commitment is due to deletion of SIRT1, and not to genetic drifting of SIRT1-/- cells. To confirm SIRT1 effects, we assessed embryonic and adult hematopoiesis in SIRT1+/+, +/− and -/- mice. Yolk sacs from SIRT1 mutant embryos generated fewer primitive erythorid precursors compared to wild-type and heterozygous mice. Moreover, knockout of SIRT1 decreased primary bone marrow HPCs in 5 week and 12 month old mice, effects especially notable at lower (5%) O2 tension. Taken together, these results demonstrate that SIRT1 plays a role in mouse embryonic and adult stem cell differentiation.
Disclosures:
No relevant conflicts of interest to declare.
MLL3/MLL4 Histone Methyltranferase Activity Dependent Chromatin Organization at Enhancers during Embryonic Stem Cell Differentiation