Polycomb group (PcG) and Trithorax group (TrxG) proteins are key components required for the maintenance of long-term repressive and active chromatin states, respectively (9
). They have been shown to play an important role in the regulation of homeotic box (HOX) genes and other developmental and cell cycle regulatory genes. Biochemical and genetic studies have provided considerable evidence that PcG proteins function in multiprotein complexes. At least two PcG protein complexes with distinct biochemical properties have been characterized. The 2-MDa Polycomb repressive complex 1 (PRC1) is comprised of the core subunits Polycomb (PC), Polyhomeotic (PH), and the three ring domain-containing proteins RING1, RING2, and BMI-1, which possess H2A-K119 ubiquitin E3 ligase activity (29
). The catalytic subunit has been mapped to RING2, while the presence of RING1 and BMI-1 can enhance the enzymatic activity (1
). Components from the transcriptional machinery, including TBP (TATA-binding protein) and TATA box-binding protein-associated factors (TAFs), are also observed to associate with the PRC1 complex in Drosophila melanogaster
), indicating that the interaction might provide a platform for PRC1 to function at promoters.
The EED-EZH2/PRC2 complex contains EZH2, SUZ12, EED, and RbAp48 and has been demonstrated to have intrinsic histone methyltransferase (HMTase) activity toward H3K27 (3
). Silencing of some Hox genes requires coordinated action of the two PcG complexes. The prerequisite of H3K27 methylation mediated by the PRC2 complex for the recruitment of the PRC1 complex, through the specific recognition of H3K27me3 by the chromo domain of the PC protein (8
), in some Hox genes provides evidence for the molecular basis of the coordinated action of these two PcG protein complexes (2
). With regard to the PRC2 complex, the SET domain-containing protein EZH2 is the catalytic subunit responsible for H3K27 methylation. However, each of the other components of the complex is indispensable for the overall function of the complex. For example, SUZ12 is required for the minimum activity of the complex in vitro and genome-wide H3K27 di- and trimethylation in vivo (4
). In contrast, EED is required for all states of H3K27 methylation, including monomethylation (17
). RbAp48, the mammalian homolog of Nurf55, has been reported to bind directly to helix 1 of histone H4, which is usually inaccessible within the nucleosome (27
). In Drosophila
, Nurf55 together with Su(z)12 is the minimal nucleosome-binding module of the Esc-E(z) complex to anchor the complex on the chromatin substrates (20
). The four components have been demonstrated to be the functional core of the PRC2 complex.
, an association between E(z) and another PcG protein named Polycomblike (PCL) has been observed and PCL shares the same polytene staining pattern with E(z) (21
). Indeed, a 1-MDa Esc-E(z) complex containing PCL and the histone deacetylase RPD3 is present during early embryogenesis. The different composition of the Esc-E(z) complex may be involved in the silencing of different targets. PCL contains two plant homeodomain (PHD) domains, a motif present in many proteins involved in chromatin function, such as CHD3, Mi2, TRX, ASH1, and ASH2. It has recently been shown that PHD domains link histone methylation to active chromatin remodeling (24
). Multiple PCL homologs have been identified in mammals. For example, there are three genes in the mouse genome that encode PCL homologs, namely mPcl1, mPcl2, and mPcl3.
Here we report the identification and functional characterization of a novel EED-EZH2 complex, which is distinguished from the previously characterized PRC2 complex by the presence of hPHF1, the closest human homolog of Drosophila PCL. By comparing the enzymatic activities of different EED-EZH2 complexes in the presence or absence of hPHF1, we show that hPHF1 positively regulates the H3K27 methyltransferase activity of the EED-EZH2 core complex in vitro. In addition, we demonstrate that the mouse homolog of hPHF1 is important for H3K27 methylation and Hox gene expression in vivo. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that mPcl1 directly contributes to HoxA10 silencing by facilitating the recruitment of the Eed-Ezh2 complex and subsequent H3K27 methylation at its promoter. Therefore, our study not only provides strong evidence that hPHF1 is an integral component of a novel EED-EZH2 complex, but also demonstrates its important function in H3K27 methylation and Hox gene silencing.