Polycomb group proteins Ring1b and Bmi1 (B-cell-specific Moloney murine leukaemia virus integration site 1) are critical components of the chromatin modulating PRC1 complex. Histone H2A ubiquitination by the PRC1 complex strongly depends on the Ring1b protein. Here we show that the E3-ligase activity of Ring1b on histone H2A is enhanced by Bmi1 in vitro. The N-terminal Ringdomains are sufficient for this activity and Ring1a can replace Ring1b. E2 enzymes UbcH5a, b, c or UbcH6 support this activity with varying processivity and selectivity. All four E2s promote autoubiquitination of Ring1b without affecting E3-ligase activity. We solved the crystal structure of the Ring-Ring heterodimeric complex of Ring1b and Bmi1. In the structure the arrangement of the Ringdomains is similar to another H2A E3 ligase, the BRCA1/ BARD1 complex, but complex formation depends on an N-terminal arm of Ring1b that embraces the Bmi1 Ringdomain. Mutation of a critical residue in the E2/E3 interface shows that catalytic activity resides in Ring1b and not in Bmi1. These data provide a foundation for understanding the critical enzymatic activity at the core of the PRC1 polycomb complex, which is implicated in stem cell maintenance and cancer.
X inactivation involves the stable silencing of one of the two X chromosomes in XX female mammals. Initiation of this process occurs during early development and involves Xist (X-inactivespecific transcript) RNA coating and the recruitment of Polycomb repressive complex (PRC) 2 and PRC1 proteins. This recruitment results in an inactive state that is initially labile but is further locked in by epigenetic marks such as DNA methylation, histone hypoacetylation, and MACROH2A deposition. Here, we report that the E3 ubiquitin ligase consisting of SPOP and CULLIN3 is able to ubiquitinate the Polycomb group protein BMI1 and the variant histone MACROH2A. We find that in addition to MACROH2A, PRC1 is recruited to the inactivated X chromosome in somatic cells in a highly dynamic, cell cycle-regulated manner. Importantly, RNAimediated knock-down of CULLIN3 or SPOP results in loss of MACROH2A1 from the inactivated X chromosome (Xi), leading to reactivation of the Xi in the presence of inhibitors of DNA methylation and histone deacetylation. Likewise, Xi reactivation is also seen on MacroH2A1 RNAi under these conditions. Hence, we propose that the PRC1 complex is involved in the maintenance of X chromosome inactivation in somatic cells. We further demonstrate that MACROH2A1 deposition is regulated by the CULLIN3͞ SPOP ligase complex and is actively involved in stable X inactivation, likely through the formation of an additional layer of epigenetic silencing.Bmi1 ͉ SPOP͞CULLIN3 E3 ligase ͉ X inactivation ͉ Polycomb silencing
BackgroundPolycomb repressive complex 1 (PRC1) core member Ring1b/Rnf2, with ubiquitin E3 ligase activity towards histone H2A at lysine 119, is essential for early embryogenesis. To obtain more insight into the role of Ring1b in early development, we studied its function in mouse embryonic stem (ES) cells.Methodology/Principal FindingsWe investigated the effects of Ring1b ablation on transcriptional regulation using Ring1b conditional knockout ES cells and large-scale gene expression analysis. The absence of Ring1b results in aberrant expression of key developmental genes and deregulation of specific differentiation-related pathways, including TGFbeta signaling, cell cycle regulation and cellular communication. Moreover, ES cell markers, including Zfp42/Rex-1 and Sox2, are downregulated. Importantly, retained expression of ES cell regulators Oct4, Nanog and alkaline phosphatase indicates that Ring1b-deficient ES cells retain important ES cell specific characteristics. Comparative analysis of our expression profiling data with previously published global binding studies shows that the genes that are bound by Ring1b in ES cells have bivalent histone marks, i.e. both active H3K4me3 and repressive H3K27me3, or the active H3K4me3 histone mark alone and are associated with CpG-‘rich’ promoters. However, deletion of Ring1b results in deregulation, mainly derepression, of only a subset of these genes, suggesting that additional silencing mechanisms are involved in repression of the other Ring1b bound genes in ES cells.ConclusionsRing1b is essential to stably maintain an undifferentiated state of mouse ES cells by repressing genes with important roles during differentiation and development. These genes are characterized by high CpG content promoters and bivalent histone marks or the active H3K4me3 histone mark alone.
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