Abstract
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.
Highlights
Regulation of gene expression via gene silencing is a critical and conserved mechanism for the management of cellular growth, differentiation, survival, and senescence
The crystal structure (Figure 2) was determined by SAD using the anomalous signal of bound zinc for phasing
The final model consisted of one enhancer-of-zeste homolog 2 (EZH2)-SET domain containing a total of 209 residues, 6 zinc atoms, 159 water molecules, and 1 sulfate ion. 96.6% of residues are in the most favored region of the Ramachandran plot, and 100% are in the allowed region
Summary
Regulation of gene expression via gene silencing is a critical and conserved mechanism for the management of cellular growth, differentiation, survival, and senescence. In Drosophila, the Polycomb-group complexes, PRC1 and PRC2, and the trithorax group protein assemblies act via opposing mechanisms to regulate homeobox (HOX) genes, the former being repressive and the latter being activating, generally. The PRC complexes repress gene expression through the SAM-catalyzed methylation of histone 3 (H3) proteins at lysine 27 (H3K27) [6] as well as other mechanisms [7,8]. Methylation of histone (H3K27) by PRC complexes is believed to be catalyzed by its conserved SETdomain containing member, Enhancer of zeste [E(z)] [9]. EZH2 is the human homolog of E(z), the C-terminal SET domain of which is the most conserved region of the protein by primary sequence [10]. Unlike other SET domains, i.e., SET7, SET8, and SUV39H1, EZH2 is inactive on its own and requires binding partners (SUZ12 [13,14] and EED [15,16]) for activity
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