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

Blais A, van Oevelen CJ, Margueron R, Acosta-Alvear D, Dynlacht BD - J. Cell Biol. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Microbiology, and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa K1H 8M5, Canada.

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.

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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.
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fig2: 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.

Mentions: We have conclusively shown that myotubes reenter the cell cycle after pRb loss, prompting us to determine whether this event is associated with changes at the gene expression level. We performed genome-wide expression profiling on myotubes treated with pRb-specific siRNA and observed widespread derepression of cell cycle genes (Fig. 2 A and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200705051/DC1). As expected, because its expression is barely detectable in myotubes, p107 suppression had very mild effects on the gene expression profile. In addition, we observed remarkably few differences after suppressing p130 or p107 and p130 (Fig. 2 A; Table S1, and not depicted), despite the fact that p130 is expressed at high levels in myotubes and was suppressed to an extent comparable to pRb (Fig. 1, A and B).


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

Blais A, van Oevelen CJ, Margueron R, Acosta-Alvear D, Dynlacht BD - J. Cell Biol. (2007)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2373492&req=5

fig2: 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.
Mentions: We have conclusively shown that myotubes reenter the cell cycle after pRb loss, prompting us to determine whether this event is associated with changes at the gene expression level. We performed genome-wide expression profiling on myotubes treated with pRb-specific siRNA and observed widespread derepression of cell cycle genes (Fig. 2 A and Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200705051/DC1). As expected, because its expression is barely detectable in myotubes, p107 suppression had very mild effects on the gene expression profile. In addition, we observed remarkably few differences after suppressing p130 or p107 and p130 (Fig. 2 A; Table S1, and not depicted), despite the fact that p130 is expressed at high levels in myotubes and was suppressed to an extent comparable to pRb (Fig. 1, A and B).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Microbiology, and Immunology, Ottawa Institute of Systems Biology, University of Ottawa, Ottawa K1H 8M5, Canada.

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.

Show MeSH
Related in: MedlinePlus