Human EZH2 gene maps to the long arm of chromosome 7 at position 7q35 and encodes 746 amino acids protein, which belongs to histone-lysine methyltransferase family (Cardoso et al., 2000). EZH2 contains several functional domains which are involved in mediating its interaction with other PRC2 and regulatory proteins besides histone methyltransferase activity. The major domains include CXC (cysteine-rich domain), SET (Su(var)3-9, enhancer of zeste, trithorax domain), ncRBD (non-coding RNA-binding domain) and a DNA binding domain (Margueron and Reinberg, 2011).
Although mutations in other PRC2 members have not been reported so far, recent studies in lymphoma and myeloid neoplasms have identified EZH2 mutations that may either cause gain-of-function or complete loss of histone methyltransferase activity (Chase and Cross, 2011). A heterozygous missense somatic mutation at Tyrosine (Y) 641 in the SET domain of EZH2 results in enhanced H3K27me3 levels in follicular lymphomas and diffused large B-cell lymphomas (Morin et al., 2010). Interestingly in myeloid neoplasms, inactivating EZH2 mutations were found to be distributed throughout the gene, comprising of
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missense, nonsense and premature stop codons, which lead to the loss of histone methyltransferase activity (Ernst et al., 2010; Nikoloski et al., 2010).
Figure 2.8. Polycomb dependent mechanism of EZH2 action in transcriptional repression.
Reproduced from Deb et al. (2013)
EZH2 protein is subjected to various post-translational modifications.
Phosphorylation of EZH2 by AKT1 at Serine-21 reduces its H3K27me3 activity whereas phosphorylation at Thr-345 by CDK1 and CDK2 is required for maintenance of H3K27me3 repressive marks at target gene promoters (Cha et al., 2005; Chen et al., 2010). Furthermore, a recent study demonstrated that EZH2 and SUZ12 are potential targets for sumoylation in both in vivo and in vitro conditions (Riising et al., 2008). In Drosophila PcG proteins are recruited to
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polycomb response elements (PREs) containing target genes with the help of several other DNA binding proteins such as GAF, Pipsqueak, Zeste, or PHO . In mammals, PRC2 is targeted to CpG islands which are GC rich sequences but surprisingly no consensus sequence elements for PcG recruitment have been identified to date in mammals (Margueron and Reinberg, 2011). Recent reports suggest that YY1, the mammalian orthologue of the Drosophila PRE DNA-binding protein PHO and RYBP which interacts with both PcG proteins are involved in the PRC2 recruitment process (Ku et al., 2008; Wilkinson et al., 2006; Xi et al., 2007). Long ncRNAs have also emerged as potential elements involved in PRC2 recruitment. HOTAIR ncRNA promotes PRC2 recruitment in trans and is associated with the transcriptional repression of HOXD locus (Rinn et al., 2007;
Tsai et al., 2010). Similarly XIST and KCNQ1OT1 ncRNAs have also been implicated in PRC2 gene targeting (Kohlmaier et al., 2004; Maenner et al., 2010;
Pandey et al., 2008; Plath et al., 2003).
There are two possible molecular mechanisms for EZH2 action based on its role as a transcriptional activator or repressor. As an integral component of PRC2, the canonical role of EZH2 is of a histone methyltransferase (Figure 2.8). SET domain of EZH2 catalyze methylation of lysine in succession such that each methylation event serve as a substrate for the next (H3K27me2 is mono-methylated to form H3K27me3) and each methylation mark represent functionally distinct chromatin state. H3K27me3 has been implicated in the recruitment of PRC1 complex, suggesting that both PcG protein complexes function in gene silencing. However there are PRC2 target genes that lack H2AK119ub and genes targeted by PRC1 in the absence of PRC2, highlighting the discrepancy in the exact functional relationship between both protein complexes (Eskeland et al., 2010; Ku et al., 2008; Schoeftner et al., 2006). Trimethylation of H3 may pose steric hindrance for RNA Pol II and other proteins binding to target gene promoters and repress transcription.
The second mechanism of EZH2 action highlights its lesser known role as a transcriptional inducer. Xu et al. (2012) reported that in castration resistant prostate cancer (CRPC), EZH2 functions independent of other PRC2 members and Akt-1 mediated phosphorylation of EZH2 at serine 21 allows it to interact with androgen receptor (AR) at many solo genes. They also demonstrated that depletion in EZH2 does not alter AR levels but there is reduction in AR-
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associated lysine methylation (Lys-630 and 632). AR methylation catalyzed by Set 9 enhances its transcriptional activity (Gaughan et al., 2011; Ko et al., 2011).
Therefore, methylation of AR or AR-associated proteins is a potential mechanism for EZH2 driven transcriptional activation.
In breast cancer cells, EZH2 has been reported to induce gene transcription in two different ways depending on the ER status (Figure 2.9). In luminal-like MCF- 7 breast cancer cells, EZH2 acts as a bridge to physically link ERα and Wnt signaling components β-catenin and TCF, on the cyclin B1 and c-Myc promoters (Shi et al., 2007). EZH2 also interacts with Mediator complex through its domain II independent of the SET domain involved in HMTase activity and induces transcription by its interaction with RNA polymerase II. In ER-negative, basal like MDA-MB-231 cells, EZH2 forms a ternary complex with NF-κB components RelA and RelB and activates transcription of NF-κB target genes such as TNF and IL6 (Lee et al., 2011). In both types of cancer, although EZH2 functions independent of PRC2 as a transcriptional activator, the mechanisms are different.
Figure 2.9. Polycomb independent mechanism of EZH2 action in human breast cancer cells. When over-expressed in ER-positive luminal like MCF-7 breast cancer cells, EZH2 functions as a transcriptional activator by acting as a bridge to physically link ERα and Wnt signalling components β-catenin and TCF, on the Cyclin B1 and c-Myc promoters. It was also demonstrated that EZH2 also associates with Mediator complex through its domain II independent of the SET domain involved in HMTase activity and might enhance transcription by its interaction with RNA polymerase II. In ER-negative, basal like MDA-MB-231 cells, EZH2 forms a ternary complex with NF-κB components RelA and RelB and activates transcription of NF- κB target genes such as TNF, IL6. Reproduced from Deb et al. (2013)
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