Role of SIRT1 in Proliferation and Self-Renewal of Mouse Embryonic Stem Cells.

A detailed understanding of regulation of self-renewal and ex-vivo expansion of embryonic stem cells is vital to ensure their safe and efficient use for differentiation to hematopoietic cells to be used in various therapeutic methodologies. The maintenance of murine embryonic stem (mES) cell self-renewal is regulated by leukemia inhibitory factor (LIF)-dependent activation of signal transducer and activator of transcription 3 (STAT3) and LIF-independent mechanisms including Nanog, BMP2/4, and Wnt signaling. Here we describe a novel role for the NAD-dependent protein deacetylase, SIRT1, in regulation of mES cell proliferation and self-renewal. SIRT1 mRNA expression in mES cells was induced by LIF through hypoxia-independent activation of hypoxia inducing factor-1α (HIF-1α. We cloned the 2 kb 5′-flanking region of the mouse SIRT1 gene and fused it to the luciferase gene. Overexpression of HIF-1α markedly enhanced SIRT1 promoter activity and a dominant-negative isoform, HIF-1α completely suppressed it. We demonstrated that two putative HIF-1α response element in the SIRT1 gene 5′-flanking region (-216 to -146) is essential for this SIRT1 induction. The two promoter regions, containing HIF-1-binding sequences, were regulated directly by HIF-1 α. These results suggest that HIF-1 enhances transactivation of the SIRT1 gene in mES cells that maintain their undifferentiated state with LIF, implying that stimulation of the SIRT1 promoter by HIF-1 may be responsible for induction of the SIRT1 gene. HIF-1α activation was blocked by the reversible phosphatidylinositol 3-OH kinase (PI3K) inhibitor, LY294002, as well as a more specific inhibititory class I(A) PI3K via regulated expression of dominant negative Deltap85, indicating that PI3K signaling is important for the hypoxia-independent HIF activity. We also demonstrate that SIRT1 deacetylates p53 at Lys 379 and then exports p53 to the cytoplasm, which results in inhibition of p53 transcriptional activity on p21. Inhibition of SIRT1 expression by siRNA translocated p53 to the nucleus and recovered p53 transactivational activity, suggesting that the deacetylated form of p53 at Lys 379 cannot access the p21 promoter and activate p21 transcription. To our knowledge, this is the first description of an influence of SIRT1 on p53 nuclear translocation via its deacetylation activity on p53. Surprisingly, we also found that N-terminal deleted p73 (DeltaNp73), a p53 family member, binds to p53 binding sites on the nanog promoter in normal mES cells, while p53 binds to p53 binding sites on the nanog promoter only in SIRT1-deficient mES cells. We then cloned the 2 kb 5′-flanking region of the mouse nanog gene and fused it to the luciferase gene. Overexpression of DeltaNp73 enhanced nanog promoter activity, but additional overexpression of p53 with DeltaNp73 abolished DeltaNp73-induced enhancement of nanog promoter activity. These data suggest that SIRT1 may regulate binding of two transcription factors, p53 and DeltaNp73, to the p53 binding site of the nanog promoter and turn on/off the nanog gene, which may eventually determine ES cell fate to self-renewal or differentiation. Upon long term inhibition of SIRT1 by SIRT1 siRNA retroviral infection, we observed a reduction in cell proliferation rate, whereas long term overexpression of SIRT by retroviral vector containing SIRT1 gene enhanced cell proliferation. Our results suggest SIRT1 controls self-renewal and proliferation of mES cells by regulation of p53 localization through deacetylation of p53.