Other kinases have also been shown to modulate adult EZH2 activity including AKT, CDK1, CDK2 and p38 kinase15,25,40,41
Other kinases have also been shown to modulate adult EZH2 activity including AKT, CDK1, CDK2 and p38 kinase15,25,40,41. EZH2 reduces protein stability. Thus, PRC2 formation is elevated along with H3K27me3in ATM deficiency. ChIP-sequencing shows a significant increase in H3K27me3 marks and a dramatic shift in their location. The change of H3K27me3 chromatin-binding pattern is directly related to cell cycle re-entry and cell death of ATM-deficient neurons. Lentiviral knockdown of EZH2 rescues Purkinje cell degeneration and behavioral abnormalities in mice. We propose a model of A-T in which EZH2-mediated hypermethylation of H3K27me3 is a crucial step leading to the degeneration of the mouse model10,31. We speculated that if EZH2 were involved, the disease process in the A-T nervous system might derive in part from the over-activation of PRC2. As H3K27 is the main target of EZH2, first, we examined H3K27me3 in human cerebellar cortex. Purkinje and granule cell nuclei contained low levels of H3K27me3 in healthy control cerebellar sections (Fig. 1a, top and Fig. 1b). Yet, as predicted by our hypothesis, both neuronal types in sections from A-T patients showed enhanced nuclear H3K27me3 staining; H3K9me3 was unaffected (Fig. 1a, bottom and Fig. 1b). Western blots of tissue extracts validated the immunostaining C elevated levels of H3K27me3 in A-T compared to control (Fig. 1c). We next repeated these observations in mice. Immunostaining for H3K27me3 (Fig. 1d, upper and Fig. 1e), but not H3K9me3 (Fig. 1d, lower and Fig. 1e), was substantially increased in Purkinje cell nuclei. We also found increased H3K27me3 in mouse neocortex and hippocampus (Fig. 1f; Supplementary Fig. 1a). As expected, an increased interaction of H3K27me3 with EZH2 was also found in ATM-deficiency (Supplementary Fig. 1bCc). Thus, there is a close association between elevated H3K27me3 and (right panels) mouse cerebellum were immunostained with H3K27me3 and H3k9me3 (red). Aldolase C immunostaining (green) was used as a marker of Purkinje cells. The white arrows indicate labeled Purkinje neurons. Scale bar, 50 m. e) The relative intensity of H3K27me3 immunostaining illustrated in panel d). * = p 0.05 by Students t-test. Error bar represents SEM. f) The protein level of H3K27me3 increases dramatically in mouse cerebellum. Nuclear protein extracts from adult mouse cerebellar Iodoacetyl-LC-Biotin cortex were probed on western blots with H3K27me3 and H3K9me3 antibodies. H3 Iodoacetyl-LC-Biotin was used as a loading control. Full-length blots are presented in Supplementary Figure 12. EZH2 is an ATM target The remarkable change in the levels of H3K27me3 in A-T brain suggested that EZH2 might be a novel ATM target. Analysis of the EZH2 protein sequence predicts that two serines, S652 and S734, are highly probable sites of ATM phosphorylation. We could find no comparably favorable sites in Iodoacetyl-LC-Biotin the EZH1 histone methyltransferase, or in other members (e.g., Suz12) of the polycomb repressor complex 2 (PRC2) to which EZH2 belongs. To determine whether these two EZH2 sites are used as ATM targets in vivo, we prepared cerebellar extracts from control and A-T patients as well as from wild type and mice and performed immunoprecipitation with EZH2 or EZH1 antibodies. Western blots of the immunoprecipitates were then probed with an antibody against a phosphorylated serine or threonine in the context of a following glutamine residue (P[S/T]Q), the canonical ATM/ATR target site32,33. A strong P[S/T]Q signal was found on the EZH2 band in control samples but not in A-T or extracts (Fig. 2a). We found no P[S/T]Q signal in either genotype using EZH1 immunoprecipitates (Fig. 2a). We verified that S734 is the predominant ATM site on EZH2 by performing ATM kinase assays using GST-EZH2 fusion proteins as substrates (Fig. 2b). The phosphorylation signal was nearly lost when the EZH2-S734A mutant was used as substrate; the 2SA (S652A/S734A) double mutation blocked the Iodoacetyl-LC-Biotin phosphorylation signal entirely. This indicates S734 is the major ATM phosphorylation site, but S652 may serve as an alternate kinase target. To confirm whether ATM-mediated EZH2 phosphorylation responds to DNA damage in vivo, GFP-EZH2 wild type or the non-phosphorylatable 2SA mutant was overexpressed in human A-T fibroblasts with Flag-ATM wild type or the kinase dead (KD) mutant. A strong P[S/T]Q signal was found with etoposide treatment when GFP-EZH2-WT was expressed with Flag-ATM-WT (Fig. 2c). Expression of either the EZH2-2SA mutant (lane 6) or the ATM-KD mutant (lane 3) significantly reduced the response. Open in a separate window Figure 2 ATM-mediated EZH2 phosphorylation prevents PRC2 formation and H3K27 methylationa) Protein extracts from mouse JAM3 and frozen human cerebellar cortex were immunoprecipitated with the EZH2 and EZH1, and blotted.