Structural basis of EZH2 recognition by EED

The WD-repeat domain is a highly conserved recognition module in eukaryotes involved in diverse cellular processes. It is still not well understood how the bottom of a WD-repeat domain recognizes its binding partners. The WD-repeat-containing protein EED is one component of the PRC2 complex that possesses histone methyltransferase activity required for gene repression. Here we report the crystal structure of EED in complex with a 30 residue peptide from EZH2. The structure reveals that the peptide binds to the bottom of the WD-repeat domain of EED. The structural determinants of EZH2-EED interaction are present not only in EZH2 and EZH1 but also in its Drosophila homolog E(Z), suggesting that the recognition of ESC by E(Z) in Drosophila employs similar structural motifs. Structure-based mutagenesis identified critical residues from both EED and EZH2 for their interaction. The structure presented here may provide a template for understanding of how WD-repeat proteins recognize their interacting proteins.     

The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets

Polycomb group (PcG) proteins are key regulators of stem-cell and cancer biology. They mainly act as repressors of differentiation and tumor-suppressor genes. One key silencing step involves the trimethylation of histone H3 on Lys27 (H3K27) by EZH2, a core component of the Polycomb Repressive Complex 2 (PRC2). The mechanism underlying the initial recruitment of mammalian PRC2 complexes is not well understood. Here, we show that NIPP1, a regulator of protein Ser/Thr phosphatase-1 (PP1), forms a complex with PP1 and PRC2 components on chromatin. The knockdown of NIPP1 or PP1 reduced the association of EZH2 with a subset of its target genes, whereas the overexpression of NIPP1 resulted in a retargeting of EZH2 from fully repressed to partially active PcG targets. However, the expression of a PP1-binding mutant of NIPP1 (NIPP1m) did not cause a redistribution of EZH2. Moreover, mapping of the chromatin binding sites with the DamID technique revealed that NIPP1 was associated with multiple PcG target genes, including the Homeobox A cluster, whereas NIPP1m showed a deficient binding at these loci. We propose that NIPP1 associates with a subset of PcG targets in a PP1-dependent manner and thereby contributes to the recruitment of the PRC2 complex.     

Association of BMI1 with polycomb bodies is dynamic and requires PRC2/EZH2 and the maintenance DNA methyltransferase DNMT1

Polycomb group (PcG) proteins are epigenetic chromatin modifiers involved in heritable gene repression. Two main PcG complexes have been characterized. Polycomb repressive complex 2 (PRC2) is thought to be involved in the initiation of gene silencing, whereas Polycomb repressive complex 1 (PRC1) is implicated in the stable maintenance of gene repression. Here, we investigate the kinetic properties of the binding of one of the PRC1 core components, BMI1, with PcG bodies. PcG bodies are unique nuclear structures located on regions of pericentric heterochromatin, found to be the site of accumulation of PcG complexes in different cell lines. We report the presence of at least two kinetically different pools of BMI1, a highly dynamic and a less dynamic fraction, which may reflect BMI1 pools with different binding capacities to these stable heterochromatin domains. Interestingly, PRC2 members EED and EZH2 appear to be essential for BMI1 recruitment to the PcG bodies. Furthermore, we demonstrate that the maintenance DNA methyltransferase DNMT1 is necessary for proper PcG body assembly independent of DNMT-associated histone deacetylase activity. Together, these results provide new insights in the mechanism for regulation of chromatin silencing by PcG proteins and suggest a highly regulated recruitment of PRC1 to chromatin.     

NIPP1 maintains EZH2 phosphorylation and promoter occupancy at proliferation-related target genes

The histone methyltransferase EZH2 regulates cell proliferation and differentiation by silencing Polycomb group target genes. NIPP1, a nuclear regulator of serine/threonine protein phosphatase 1 (PP1), has been implicated in the regulation of EZH2 occupancy at target loci, but the underlying mechanism is not understood. Here, we demonstrate that the phosphorylation of EZH2 by cyclin-dependent kinases at Thr416 creates a docking site for the ForkHead-associated domain of NIPP1. Recruited NIPP1 enables the net phosphorylation of EZH2 by inhibiting its dephosphorylation by PP1. Accordingly, a NIPP1-binding mutant of EZH2 is hypophosphorylated, and the knockdown of NIPP1 results in a reduced phosphorylation of endogenous EZH2. Conversely, the loss of PP1 is associated with a hyperphosphorylation of EZH2. A genome-wide promoter-binding profiling in HeLa cells revealed that the NIPP1-binding mutant shows a deficient association with about a third of the Polycomb target genes, and these are enriched for functions in proliferation. Our data identify PP1 as an EZH2 phosphatase and demonstrate that the phosphorylation-regulated association of EZH2 with proliferation-related targets depends on associated NIPP1.     

Structural Context of Disease-Associated Mutations and Putative Mechanism of Autoinhibition Revealed by X-Ray Crystallographic Analysis of the EZH2-SET Domain

The enhancer-of-zeste homolog 2 (EZH2) gene product is an 87 kDa polycomb group (PcG) protein containing a C-terminal methyltransferase SET domain. EZH2, along with binding partners, i.e., EED and SUZ12, upon which it is dependent for activity forms the core of the polycomb repressive complex 2 (PRC2). PRC2 regulates gene silencing by catalyzing the methylation of histone H3 at lysine 27. Both overexpression and mutation of EZH2 are associated with the incidence and aggressiveness of various cancers. The novel crystal structure of the SET domain was determined in order to understand disease-associated EZH2 mutations and derive an explanation for its inactivity independent of complex formation. The 2.00 Å crystal structure reveals that, in its uncomplexed form, the EZH2 C-terminus folds back into the active site blocking engagement with substrate. Furthermore, the S-adenosyl-L-methionine (SAM) binding pocket observed in the crystal structure of homologous SET domains is notably absent. This suggests that a conformational change in the EZH2 SET domain, dependent upon complex formation, must take place for cofactor and substrate binding activities to be recapitulated. In addition, the data provide a structural context for clinically significant mutations found in the EZH2 SET domain.