Mel-18, a polycomb group protein, regulates cell proliferation and senescence via transcriptional repression of Bmi-1 and c-Myc oncoproteins - PubMed (original) (raw)

Mel-18, a polycomb group protein, regulates cell proliferation and senescence via transcriptional repression of Bmi-1 and c-Myc oncoproteins

Wei-Jian Guo et al. Mol Biol Cell. 2007 Feb.

Abstract

Polycomb group (PcG) protein Bmi-1 is an important regulator of cell proliferation. It regulates cellular senescence and proliferation of cells via the transcriptional repression of INK4a/ARF locus and other target genes. Here, we report that Mel-18, a PcG ring finger protein (PCGF) transcriptionally down-regulates Bmi-1. Furthermore, the expression of Bmi-1 and Mel-18 inversely correlates in proliferating and senescent human fibroblasts. Bmi-1 down-regulation by Mel-18 results in accelerated senescence and shortening of the replicative life span in normal human cells. Importantly, using promoter-reporter, chromatin immunoprecipitation, and quantitative real-time primary transcript RT-PCR assays, and an RNA interference approach, we demonstrate that Bmi-1 is a bona fide target of c-Myc oncoprotein. Finally, our data suggest that Mel-18 regulates Bmi-1 expression during senescence via down-regulation of c-Myc. These studies link c-Myc and polycomb function in cell proliferation and senescence.

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Figures

Figure 1.

Figure 1.

Mel-18 regulates cellular senescence in human fibroblasts. (A) Overexpression of Mel-18 induces premature senescence in proliferating fibroblasts. MRC-5 fibroblasts overexpressing Mel-18 or Bmi-1 and vector-infected control cells were serially passaged in culture to determine the replicative life span (top left panel). Premature induction of senescence was determined using SA-β-gal staining (Dimri et al., 1995) of Mel-18– or Bmi-1–overexpressing and control cells (bottom left panel). % senescent cells, the percentage of SA-β-gal positive cells as determined by counting 200 cells in four different fields. Western blot analysis of various regulators of senescence (right panel) was done as described (Dimri et al., 2002; Itahana et al., 2003). CPD denotes cumulative population doublings. (B) Knockdown of Mel-18 expression leads to the extension of replicative life span in MRC-5 fibroblasts. Mel-18–expressing shRNAs (RNAi Mel-18 no. 1, RNAi Mel-18 no. 2) and control cells (Ctrl i) were passaged in culture to determine replicative life span, and SA-β-gal staining was done to determine the onset of senescence as described above. Western blot analyses (right panel) of Bmi-1, p16, pRb, and Mel-18 were done as described in Materials and Methods.

Figure 2.

Figure 2.

Mel-18 is overexpressed in senescent human fibroblasts. (A) MRC-5, BJ, and WI-38 strains of human fibroblast were serially passaged in culture until senescence as determined by measuring the SA-β-gal index. Mel-18, Bmi-1, pRb, p16, and α-tubulin in total cell lysates from proliferating presenescent (Presen) and senescent (Sen) cultures were detected by Western blot analysis. (B) Mel-18 is not up-regulated during quiescence in WI-38 fibroblasts. Proliferating presenescent (Pr) cells were made quiescent (Q) by incubating cells in 0.1% serum for 5 d. Total cell lysates were prepared from proliferating (Pr) and quiescent (Q) cells, and the expression of Mel-18, c-Myc, Bmi-1, pRb, p16, and α-tubulin was determined by Western blot analysis.

Figure 3.

Figure 3.

Mel-18 regulates Bmi-1 in human cells. (A) Stable expression of Mel-18 in MCF10A and MCF7 cells leads to down-regulation of Bmi-1 and c-Myc oncoprotein. Total cell lysate from indicated cells was analyzed by Western blot analysis using antibody against Bmi-1, Mel-18, c-Myc, and α-tubulin (loading control). (B) Transient overexpression of Mel-18 in 293T cells leads to the down-regulation of c-Myc and Bmi-1 in a dose-dependent manner. 293T cells were transiently transfected with increasing amounts pLPC-Mel-18, and 48 h after transfection total cell lysate was analyzed by Western blot analysis using antibody against Mel-18, Bmi-1, GFP (transfection control), and α-tubulin (loading control). (C) Stable knockdown of Mel-18 expression using the RNAi approach in MCF10A, WI-38, and BJ cells leads to up-regulation of c-Myc and Bmi-1 expression. MCF10A (left panel), WI-38 (middle panel), and BJ (right panel) cells were infected with pRS vector expressing either Mel-18 shRNA no. 1 (#1i), Mel-18 shRNA no. 2 (#2i), or an irrelevant control shRNA (Ctrl i), selected in puromycin and analyzed for the expression of Mel-18, c-Myc, and Bmi-1 by Western blot analysis.

Figure 4.

Figure 4.

Structural analysis of Mel-18. (A) Schematic representation of mutants of Mel-18 depicting various domains. These mutants were generated by PCR and cloned in the pLPC retroviral vector. (B) Stable overexpression of wild type (WT) and the mutants of Mel-18 in MCF10A cells; WT and the PS mutant down-regulated Bmi-1 and c-Myc expression, whereas overexpression of ΔRF and ΔRFNLS mutants led to up-regulation of Bmi-1 and c-Myc. WT or mutants of Mel-18 were stably expressed using retroviral expression, and Bmi-1, c-Myc, Mel-18, and α-tubulin were detected by Western blot analysis as described in Materials and Methods.

Figure 5.

Figure 5.

Mel-18 does not regulate Bmi-1 protein level. (A) Treatment with MG132, a proteosome inhibitor does not restore Bmi-1 expression in Mel-18–overexpressing cells. Control, Mel-18– and Bmi-1–overexpressing cells were treated with 10 μM MG132 for the indicted time period and analyzed by Western blot analysis for the expression of Bmi-1, Mel-18, and α-tubulin as described in Figure 1. (B) Bmi-1 half-life is similar in control and Mel-18–overexpressing cells. Vector control and Mel-18–overexpressing cells were treated with 100 μg/ml cyclohexamide (CHX) for the indicated amounts of time and analyzed for the expression of Bmi-1, Mel-18, p53, and β-actin. The percent remaining Bmi-1 (bottom left panel) or p53 (bottom right panel) protein was calculated by densitometry of the Bmi-1 signal present in different lanes and by normalizing it with the β-actin control signal present in the corresponding lanes. Only the lower band of Bmi-1 Western analysis, which clearly showed time-dependent degradation, was used to calculate half-life of Bmi-1.

Figure 6.

Figure 6.

Mel-18 regulates mRNA levels of c-Myc and Bmi-1 as determined by QRT-PCR analysis. (A) The mRNA levels of Bmi-1 and c-Myc in Mel-18–overexpressing and control MCF10A and MCF7 cells were quantified by QRT-PCR and normalized to GAPDH mRNA levels as described in Materials and Methods. (B) Using QRT-PCR assay, the mRNA levels of c-Myc and Bmi-1 were quantified and normalized to GAPDH mRNA levels in control (Ctrl RNAi) and Mel-18 knockdown cells (Mel-18 i no. 1 and Mel-18 i no. 2). The QRT-PCR assays were performed in triplicates.

Figure 7.

Figure 7.

c-Myc binds to the Bmi-1 promoter and regulates its activity. (A) c-Myc binds to the E-box sequences in the Bmi-1 promoter as shown by the ChIP analysis. The ChIP analysis was performed using vector control or Mel-18–overexpressing MCF10A and MCF7 cells as indicated. The cell lysates were IPed using c-Myc antibody or control IgG and a primer set that either amplifies the c-Myc binding flanking region in the Bmi-1 promoter (c-Myc site) or a region further upstream that does not contain a c-Myc binding site (Non-Myc site). (B) Detailed analysis of Bmi-1 promoter activity. The pGL-Bmi PrWT, pGL-Bmi PrMut, and pGL-Bmi PrΔMyc reporters (described in the text) were analyzed for the luciferase activity in 293T cells by transient transfection as described in Materials and Methods.

Figure 8.

Figure 8.

Mel-18 and c-Myc regulate Bmi-1 promoter activity. (A) Overexpression of Mel-18 down-regulates only the wild-type Bmi-1 promoter. pGL3-Bmi-1 PrWT, pGL3-Bmi-1PrMut, and pGL3- Bmi-1PrΔMyc plasmids were transiently transfected into 293T cells together with an increasing amount of Mel-18–overexpressing plasmid (pLPC-Mel-18) and a plasmid expressing renilla luciferase. Forty-eight hours after transfection luciferase activity was determined as described in Materials and Methods. (B) Transient overexpression of c-Myc up-regulates wild-type Bmi-1 promoter activity through the c-Myc binding site. Different promoter-reporter constructs (as indicated) were transiently transfected into 293T cells with an increasing amount of pCMV-Myc expression plasmid together with a plasmid expressing renilla luciferase, and luciferase activity was determined as described in Materials and Methods. (C) c-Myc knockdown using transient transfection of a plasmid containing c-Myc shRNA down-regulates activity of the Bmi-1 promoter, which contains an intact c-Myc binding site. The promoter activity of various promoter-reporter constructs with the increasing amount of a plasmid expressing c-Myc shRNA was analyzed in 293T cells as described in Materials and Methods.

Figure 9.

Figure 9.

Mel-18 regulates c-Myc and Bmi-1 transcription. Quantitative PT RT-PCR analysis of primary transcripts of c-Myc and Bmi-1 in control and Mel-18–overexpressing MCF7 (left panel) and MCF10A cells (right panel). PT RT-PCR analysis was performed in triplicate as described in Materials and Methods.

Figure 10.

Figure 10.

c-Myc regulates endogenous Bmi-1 expression and transient expression of c-Myc in Mel-18–overexpressing cells restores Bmi-1 expression. (A) Stable overexpression of c-Myc leads to Bmi-1 up-regulation. MCF10A cells were infected with a c-Myc expressing retrovirus (pLNCX2-Myc), selected in G418, and the expression of c-Myc, Bmi-1, and α-tubulin was determined by Western blot analysis. (B) Knockdown of c-Myc expression by RNAi approach leads to down-regulation of endogenous Bmi-1. MCF10A cells expressing c-Myc shRNA (Myc RNAi) or a control shRNA (Ctrl. RNAi) were generated and analyzed for the expression of c-Myc, Bmi-1, and α-tubulin by Western blot analysis. (C) Restoration of c-Myc in Mel-18–overexpressing cells by its transient overexpression leads to the reversal of Bmi-1 repression by Mel-18. 293T cells were transfected with either Mel-18, c-Myc, or both and a GFP expressing plasmid. The total cell lysate from each set was analyzed for the expression of Mel-18, c-Myc, Bmi-1, GFP, and α-tubulin by Western blot analysis.

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