The Polycomb Ezh2 methyltransferase regulates muscle gene expression and skeletal muscle differentiation
Abstract
The Ezh2 protein endows the Polycomb PRC2 and PRC3 complexes with histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression. We report that Ezh2 expression was developmentally regulated in the myotome compartment of mouse somites and that its down-regulation coincided with activation of muscle gene expression and differentiation of satellite-cell-derived myoblasts. Increased Ezh2 expression inhibited muscle differentiation, and this property was conferred by its SET domain, required for the HKMT activity. In undifferentiated myoblasts, endogenous Ezh2 was associated with the transcriptional regulator YY1. Both Ezh2 and YY1 were detected, with the deacetylase HDAC1, at genomic regions of silent muscle-specific genes. Their presence correlated with methylation of K27 of histone H3. YY1 was required for Ezh2 binding because RNA interference of YY1 abrogated chromatin recruitment of Ezh2 and prevented H3-K27 methylation. Upon gene activation, Ezh2, HDAC1, and YY1 dissociated from muscle loci, H3-K27 became hypomethylated and MyoD and SRF were recruited to the chromatin. These findings suggest the existence of a two-step activation mechanism whereby removal of H3-K27 methylation, conferred by an active Ezh2-containing protein complex, followed by recruitment of positive transcriptional regulators at discrete genomic loci are required to promote muscle gene expression and cell differentiation.
By preventing inappropriate gene activation, transcriptional repression imposes unique patterns of gene expression and is essential for the specification and maintenance of cell identity (Francis and Kingston 2001). The Polycomb group (PcG) proteins repress transcription of the Drosophila Hox genes and participate in establishing the body anteroposterior axis (Simon et al. 1992). Whereas some PcG genes exert their activities at later stages of development, the PcG Enhancer of zeste, E(z), functions early in development by regulating expression of the gap genes (Pelegri and Lehmann 1994). The PcG coding sequences—and presumably their function—have been conserved throughout evolution. Both in the plant Arabidopsis thaliana and in the nematode Caenorhabditis elegans, the counterparts of Drosophila E(z) regulate homeotic gene expression (Goodrich et al. 1997; Ross and Zarkower 2003; Zhang et al. 2003). E(z) proteins are also involved in initiating X-chromosome inactivation (Plath et al. 2003) and in maintaining the epigenetic patters of pluripotent stem cells (Erhardt et al. 2003). In mammals, two E(z)-related genes have been isolated, Ezh1 and Ezh2 (Laible et al. 1997). Ezh1 expression is prevalent in the adult, whereas Ezh2 is expressed during embryonic development (Laible et al. 1997). Consistent with its expression pattern, Ezh2 is required for early mouse development. Ezh2-null mouse embryos die during the transition from pre- to postimplantation development (O'Carroll et al. 2001).
Among the PcG family, the E(z) proteins are unique in that they are chromatin-modifying enzymes with histone lysine methyltransferase (HKMT) activity (Cao et al. 2002; Czermin et al. 2002; Kuzmichev et al. 2002; Muller et al. 2002). Their catalytic activity resides in the evolutionarily conserved SET domain (Sims et al. 2003). Binding of Drosophila E(z) to a DNA Polycomb response element of the Ultrabithorax (Ubx) gene correlates with H3-K27 methylation and Ubx repression (Cao et al. 2002). Ezh2-mediated methylation of H3-K27 creates a docking site for the subsequent recruitment on the chromatin of the PRC1 (Polycomb repressive complex 1) complex containing additional PcG proteins (Czermin et al. 2002). The interaction of Ezh2 with the histone deacetylase HDAC1 suggests that both histone deacetylation and methylation converge to ensure transcriptional repression (van der Vlag and Otte 1999). The Ezh2 requirement for early mouse development has hampered the study of its role in regulating developmental and postnatal processes. However, a role for Ezh2 in cell cycle progression and cell differentiation has emerged from the analysis of several forms of aggressive tumors. Overexpression of Ezh2 has been reported in hormone-refractory, metastatic prostate cancers (Varambally et al. 2002) and in poorly differentiated and particularly aggressive breast carcinomas (Kleer et al. 2003). Resting cells derived from human lymphomas do not express Ezh2, but Ezh2 is strongly expressed in proliferating lymphoma cells (Visser et al. 2001). Pertinent to its putative role in cell differentiation are the findings that conditional inactivation of Ezh2 results in selectively impaired formation of pre-B and immature B cells but an unaltered development of pro-B cells (Su et al. 2003). Collectively, these and other (Bracken et al. 2003) findings suggest that Ezh2 may regulate cell growth and certain differentiation processes.
Because Ezh2 expression is developmentally regulated in skeletal muscle (Laible et al. 1997), we have tested the hypothesis that Ezh2 may be involved in controlling muscle gene expression and differentiation. Our results indicate that mouse skeletal muscle cells transduced with an Ezh2 retrovirus failed to undergo terminal differentiation and that this differentiation block was mediated by the SET domain, a region responsible for the HKMT activity. Ezh2 interacts with the DNA-binding protein YY1, and both proteins are found—along with the deacetylase HDAC1—on the regulatory regions of transcriptionally inactive muscle specific genes. Their presence correlated with H3-K27 methylation. Upon transcriptional activation, chromatin interaction of Ezh2, HDAC1, and YY1 was lost and replaced by the positive regulators of muscle transcription, SRF and MyoD. This molecular switch was accompanied by H3-K27 hypomethylation and histone hyperacetylation. Thus, our results indicate that the removal of an actively suppressing HKMT protein complex containing Polycomb Ezh2 and the subsequent engagement of positive transcriptional regulators characterize activation of muscle gene expression.
Acknowledgments
We thank Marcella Fulco in the Sartorelli Lab for helpful suggestions and stimulating discussion, S. Lnu and A.M Chinnaiyan for providing the Ezh2 constructs, and S. Opravil and T. Jenuwein for the H3-K27 methylation-specific antibodies. The help of S. McCroskery with the isolation and culture of mouse satellite cells is kindly acknowledged.
Notes
Supplemental material is available at http://www.genesdev.org.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.1241904.