RB maintains quiescence and prevents premature senescence through upregulation of DNMT1 in mesenchymal stromal cells.
Bottom Line: RB knockdown induces premature senescence and reduced differentiation potentials in early-passage MSCs.Furthermore, DNMT1 knockdown in early-passage MSCs induces senescence and reduces differentiation potentials, whereas DNMT1 overexpression in late-passage MSCs has the opposite effect.These results demonstrate that RB expressed in early-passage MSCs upregulates DNMT1 expression and inhibits senescence in MSCs.
Affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan, ROC.Show MeSH
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Mentions: A previous report demonstrated that RB modulates DNMT1 expression either by binding with c-JUN to a noncanonical AP-1 site in the DNMT1 promoter and thereby activating DNMT1 promoter activity, or by binding with E2F1 and thereby blocking E2F1-induced DNMT1 promoter activity (Slack et al., 2001). To demonstrate that RB upregulates DNMT1 in early-passage MSCs through the above-mentioned mechanism, we first showed that RB bound with c-JUN rather than E2F1 in early-passage MSCs (Figures 5A and 5B). Consistently, chromatin immunoprecipitation (ChIP) analysis revealed that the AP-1 site of the DNMT1 promoter was bound by RB and c-JUN, whereas the E2F binding site of the DNMT1 promoter was only bound by E2F1, and not by RB (Figure 5C), suggesting that RB plays a coactivator role in upregulation of DNMT1 by binding c-JUN in the AP-1 site in early-passage MSCs. We also performed ChIP assays in non-MSCs. We found the E2F1 binding site, but not the AP-1 site, of the DNMT1 promoter was bound by RB in a control somatic cell line, H1299 cells (Figure S5), suggesting that RB has a differentiation role in regulating DNMT1 between early-passage MSCs and late-passage MSCs or other somatic cells. Moreover, we found that the c-JUN level was not changed between early- and late-passage MSCs or before and after RB knockdown (Figure 5D), suggesting that the increase of DNMT1 expression in early-passage MSCs was not due to upregulation of c-JUN or through RB-mediated c-JUN upregulation in early-passage MSCs. To further demonstrate that RB upregulation of DNMT1 in early-passage MSCs requires c-JUN binding, we transfected MSCs with c-JUN shRNAs. We found that c-JUN knockdown reduced DNMT1 expression in early-passage MSCs, but not late-passage MSCs (Figure 5E), although the expression of RB in early-passage MSCs was not suppressed (Figure 5E). Together, these data suggest that RB binds with c-JUN to activate DNMT1 expression in early-passage MSCs.
Affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan, ROC.