Retinoblastoma tumor suppressor protein-dependent methylation of histone H3 lysine 27 is associated with irreversible cell cycle exit

Abstract

The retinoblastoma tumor suppressor protein (pRb) is involved in mitotic exit, promoting the arrest of myoblasts, and myogenic differentiation. However, it is unclear how permanent cell cycle exit is maintained in differentiated muscle. Using RNA interference, expression profiling, and chromatin immunoprecipitations, we show that pRb is essential for cell cycle exit and the differentiation of myoblasts and is also uniquely required to maintain this arrest in myotubes. Remarkably, we also uncover a function for the pRb-related proteins p107 and p130 as enforcers of a G2/M phase checkpoint that prevents progression into mitosis in cells that have lost pRb. We further demonstrate that pRb effects permanent cell cycle exit in part by maintaining trimethylation of histone H3 lysine 27 (H3K27) on cell cycle genes. H3K27 trimethylation silences other genes, including Cyclin D1, in a pRb-independent but polycomb-dependent manner. Thus, our data distinguish two distinct chromatin-based regulatory mechanisms that lead to terminal differentiation.

Figure 1.

pRb depletion leads to cell cycle reentry in terminally differentiated muscle cells. (A) RT-PCR detection of pocket protein mRNA expression 48 h after transfection of C2C12 myotubes with siRNA duplexes. (B) Western blot detection of each pocket protein indicates the extent of knockdown achieved in a typical experiment. (C) Immunofluorescent detection of BrdU incorporation (green) and MHC expression (red) in differentiated C2C12 myotubes after transfection with a nonspecific siRNA duplex or duplexes targeting various pocket protein combinations. (D) Histogram representing the proportion of C2C12 myotubes with at least one BrdU⁺ nucleus after transfection with the indicated siRNA. Error bars indicate SD of three independent experiments. (E) Immunofluorescent detection of BrdU incorporation (red) and MHC expression (green) in primary myotubes after transfection with a nonspecific siRNA duplex or a duplex targeting pRb. Bars, 100 μm.

Figure 2.

Loss of pRb leads to derepression of cell cycle control genes. (A) RT-PCR detection of pocket protein, cell cycle, and muscle function gene expression 48 h after suppression of pocket protein expression in C2C12 myotubes. (B) Scatter plot representing the change in gene expression in C2C12 myotubes upon knockdown of pRb as a function of the gene expression changes during the normal course of myogenic differentiation. Each spot represents one measurement with one microarray probe. MT, untransfected myotubes; GM, untransfected growing myoblasts; NS, myotubes transfected with a nonspecific siRNA duplex; pRb, myotubes transfected with the pRb-specific duplex. All data points represent the combination of three independent experiments. (C) Scatter plot representing the change in gene expression in myotubes after pRb knockdown as a function of the degree of binding of E2F4 in a ChIP-on-chip microarray experiment performed in C2C12 myotubes. The binding ratio serves as an indirect measure of the strength of binding of E2F4 to its target genes; a cut-off corresponding to a twofold enrichment was used.

Figure 3.

H3K27 trimethylation of cell cycle gene promoters during myogenic differentiation. (A) qChIP on chromatin from either C2C12 myoblasts or myotubes using antibodies that recognize H3K27Me2/3, H3K9Me3, and H3Ac. A scatter plot indicates the fraction of input DNA immunoprecipitated from each state. Values obtained with the H3Ac antibody were divided by 10 to fit on the same plot as the other two datasets. Results represent the mean of at least three independent experiments. (B) Histogram representing the H3K27Me3 signal from A. The Hoxa5 and Mef2c genes served as specificity controls. Error bars indicate SD of three independent experiments. (C) ChIP assay performed in C2C12 myoblasts and myotubes using antibodies against histone H3, H3K27Me2 or H3K27Me3, or rabbit IgG. (D) ChIP assay performed on cells at different times after induction of differentiation. The 96-h Mt sample represents pure myotubes, whereas the samples labeled 96 h and earlier time points represent the mixed cell population before separation of myotubes from reserve cells. (E) Quantitative ChIP assays for H3K27Me2/3 performed in primary myoblasts and matched myotubes. The mean of four ChIP assays and standard errors are shown. The ratio of H3K27Me2/3 signal in myotubes over that in myoblasts is indicated in red below each gene name. Asterisks indicate an H3K27Me2/3 signal significantly higher in myotubes than in myoblasts; P < 0.03 by t test. The double asterisk indicates an H3K27Me2/3 signal significantly lower in myotubes than in myoblasts; P < 0.001 by t test.

Figure 4.

Trimethylation of H3K27 is insensitive to mitogenic stimulation and does not occur during reversible, mitogen starvation–induced quiescence. (A) RT-PCR assay of gene expression in C2C12 myoblasts and myotubes or myotubes treated with high serum concentrations for 24 h (Mt + GM). (B) ChIP assay performed on samples from cells analyzed in A. The input control corresponds to 0.4% of the amount used per immunoprecipitation. The Hoxa5 gene is shown as a control for efficiency of immunoprecipitation by the anti-H3K9Me3 antibody. (C) MEFs were harvested during asynchronous growth or after 3 d of starvation in serum-free medium. RNA was extracted and cell cycle gene expression was assessed by RT-PCR. (D) ChIP assays performed on cross-linked cells treated and processed in parallel with those indicated in C.

Figure 5.

pRb-dependent trimethylation of H3K27 at cell cycle gene promoters. (A) Histogram indicating H3K27Me2/3 levels in C2C12 myotubes transfected with a nonspecific control or pRb-silencing siRNA. The ChIP signal intensity is reported as a fraction of the signal obtained in cells transfected with the nonspecific siRNA. (B and C) Histograms depicting H3K27Me2/3 (B) and H3Ac (C) levels in C2C12 myotubes transfected with siRNAs targeting p130 or pRb or with a nonspecific control siRNA as indicated in A. Error bars represent SD of three independent experiments. (D) ChIP assay performed in C2C12 myoblasts and myotubes using antibodies directed against Suz12, Ezh2, and Bmi-1. Two regions of the Hoxd10 gene were surveyed to evaluate PRC binding. The results of two independent experiments are shown.

Figure 6.

Model detailing pRb function in differentiated muscle. See text for details.