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.

Genome-wide remodeling of the epigenetic landscape during myogenic differentiation

“…S5B and Dataset S1). A subset of these genes, including Myog (see below), showed a significant reduction in the density of this mark, consistent with previous studies performed on a small number of genes (7,8). Unexpectedly, other genes in these clusters were transcriptionally up-regulated in myotubes, although they retained H3K27me3 and displayed very low levels of H3K36me3 in both conditions.…”

“…H3K27me3 is known to regulate myogenic differentiation through silencing of muscle-specific genes and cell cycle genes in myoblasts and myotubes, respectively (7,8). In other studies, H3K9me3 was implicated in shutting off cell cycle genes during muscle differentiation (16).…”

“…ChIP was performed as described (8), and sonication was performed to obtain chromatin fragments of approximately 250 bp whereas MNase digestion produced approximately 150 bp fragments. Antibodies used for ChIP are listed in SI Materials and Methods.…”

“…These transcription factors collaborate with sequencespecific factors, such as MEF2c, cofactors, and histone modifying enzymes to generate transcriptional regulatory networks that promote skeletal muscle differentiation (5). MyoD1 binding coincides with histone acetylation and gene activation (6), whereas H3K27 methylation has been shown to play critical roles in selective repression of muscle-specific genes in growing cells (7)(8)(9). Whereas global changes in the epigenetic landscape have been primarily studied during ES cell differentiation, myogenic differentiation has not been comprehensively studied on a genomewide scale, and the interplay between factors and histone modifications has been studied only on a handful of genes.…”

Brahma is required for cell cycle arrest and late muscle gene expression during skeletal myogenesis

“…This phenotype coincided with the upregulation of Ccnd1, which was independently annotated as an upregulated gene by microarray analysis in Brm-depleted C2C12 myoblasts (Figs 2, EV1 and EV2). Importantly, Brm, but not Brg1, was found bound to Ccnd1 promoter by ChIP analysis, with an increased binding during the differentiation process that correlated with a proportional enrichment in H3K27 tri-methylation (H3K27me3) (Fig 3)-a marker of the repressive activity of the Polycomb Repressive Complex 2 (PRC2) for gene silencing in muscle cells [49,50]. The coincidental increase in H3K27me3 (Fig 3) suggests that the recruitment of Polycomb group complex (PcG) might contribute to establish marks of repressive chromatin, in cooperation with Brm-based SWI/SNF, as proposed by Ho & Crabtree [51].…”