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RB maintains quiescence and prevents premature senescence through upregulation of DNMT1 in mesenchymal stromal cells.

Lin SP, Chiu FY, Wang Y, Yen ML, Kao SY, Hung SC - Stem Cell Reports (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan, ROC.

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RB Induces DNMT1 Expression through Cooperation with c-JUN(A and B) Equal aliquots of lysates from early-passage MSCs were coimmunoprecipitated by using isotype IgG or antibodies against RB, c-JUN, and E2F1, followed by western blot analysis. Input: 10% of precleared lysates (20 μg total protein).(C) ChIP assay of early-passage MSCs. The chromatin was incubated with antibodies against RB, c-JUN, or E2F1, or their isotype IgG antibodies. Fragments of AP-1 (left) and E2F1 site (right) on the DNMT1 promoter were amplified by PCR (lower) and quantitative PCR (upper). Results are shown as the mean ± SD. MsIgG, mouse IgG2a is the isotype of anti-RB antibody. RtIgG, rabbit IgG is the isotype of anti-c-JUN and anti-E2F1 antibody. The results are expressed as mean ± SD of three independent experiments.(D) Early- and late-passage MSCs were infected with lentivirus carrying control (CTR) or c-JUN-specific shRNAs, sic-JUN(1), or sic-JUN(2), followed by western blot analysis.(E) Scheme of the signaling pathway involving RB in regulating the proliferation of MSCs. In early-passage MSCs, RB either binds to E2F1 and suppresses E2F1 targets or downregulates p21 and p16 by upregulating DNMT1, thereby keeping MSCs in a state of mitotic quiescence. In late-passage MSCs, loss of RB upregulates p21 and p16 and induces premature senescence.See also Figure S5.
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fig5: RB Induces DNMT1 Expression through Cooperation with c-JUN(A and B) Equal aliquots of lysates from early-passage MSCs were coimmunoprecipitated by using isotype IgG or antibodies against RB, c-JUN, and E2F1, followed by western blot analysis. Input: 10% of precleared lysates (20 μg total protein).(C) ChIP assay of early-passage MSCs. The chromatin was incubated with antibodies against RB, c-JUN, or E2F1, or their isotype IgG antibodies. Fragments of AP-1 (left) and E2F1 site (right) on the DNMT1 promoter were amplified by PCR (lower) and quantitative PCR (upper). Results are shown as the mean ± SD. MsIgG, mouse IgG2a is the isotype of anti-RB antibody. RtIgG, rabbit IgG is the isotype of anti-c-JUN and anti-E2F1 antibody. The results are expressed as mean ± SD of three independent experiments.(D) Early- and late-passage MSCs were infected with lentivirus carrying control (CTR) or c-JUN-specific shRNAs, sic-JUN(1), or sic-JUN(2), followed by western blot analysis.(E) Scheme of the signaling pathway involving RB in regulating the proliferation of MSCs. In early-passage MSCs, RB either binds to E2F1 and suppresses E2F1 targets or downregulates p21 and p16 by upregulating DNMT1, thereby keeping MSCs in a state of mitotic quiescence. In late-passage MSCs, loss of RB upregulates p21 and p16 and induces premature senescence.See also Figure S5.

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.


RB maintains quiescence and prevents premature senescence through upregulation of DNMT1 in mesenchymal stromal cells.

Lin SP, Chiu FY, Wang Y, Yen ML, Kao SY, Hung SC - Stem Cell Reports (2014)

RB Induces DNMT1 Expression through Cooperation with c-JUN(A and B) Equal aliquots of lysates from early-passage MSCs were coimmunoprecipitated by using isotype IgG or antibodies against RB, c-JUN, and E2F1, followed by western blot analysis. Input: 10% of precleared lysates (20 μg total protein).(C) ChIP assay of early-passage MSCs. The chromatin was incubated with antibodies against RB, c-JUN, or E2F1, or their isotype IgG antibodies. Fragments of AP-1 (left) and E2F1 site (right) on the DNMT1 promoter were amplified by PCR (lower) and quantitative PCR (upper). Results are shown as the mean ± SD. MsIgG, mouse IgG2a is the isotype of anti-RB antibody. RtIgG, rabbit IgG is the isotype of anti-c-JUN and anti-E2F1 antibody. The results are expressed as mean ± SD of three independent experiments.(D) Early- and late-passage MSCs were infected with lentivirus carrying control (CTR) or c-JUN-specific shRNAs, sic-JUN(1), or sic-JUN(2), followed by western blot analysis.(E) Scheme of the signaling pathway involving RB in regulating the proliferation of MSCs. In early-passage MSCs, RB either binds to E2F1 and suppresses E2F1 targets or downregulates p21 and p16 by upregulating DNMT1, thereby keeping MSCs in a state of mitotic quiescence. In late-passage MSCs, loss of RB upregulates p21 and p16 and induces premature senescence.See also Figure S5.
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fig5: RB Induces DNMT1 Expression through Cooperation with c-JUN(A and B) Equal aliquots of lysates from early-passage MSCs were coimmunoprecipitated by using isotype IgG or antibodies against RB, c-JUN, and E2F1, followed by western blot analysis. Input: 10% of precleared lysates (20 μg total protein).(C) ChIP assay of early-passage MSCs. The chromatin was incubated with antibodies against RB, c-JUN, or E2F1, or their isotype IgG antibodies. Fragments of AP-1 (left) and E2F1 site (right) on the DNMT1 promoter were amplified by PCR (lower) and quantitative PCR (upper). Results are shown as the mean ± SD. MsIgG, mouse IgG2a is the isotype of anti-RB antibody. RtIgG, rabbit IgG is the isotype of anti-c-JUN and anti-E2F1 antibody. The results are expressed as mean ± SD of three independent experiments.(D) Early- and late-passage MSCs were infected with lentivirus carrying control (CTR) or c-JUN-specific shRNAs, sic-JUN(1), or sic-JUN(2), followed by western blot analysis.(E) Scheme of the signaling pathway involving RB in regulating the proliferation of MSCs. In early-passage MSCs, RB either binds to E2F1 and suppresses E2F1 targets or downregulates p21 and p16 by upregulating DNMT1, thereby keeping MSCs in a state of mitotic quiescence. In late-passage MSCs, loss of RB upregulates p21 and p16 and induces premature senescence.See also Figure S5.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, National Yang-Ming University, Taipei 112, Taiwan, ROC.

Show MeSH
Related in: MedlinePlus