Phosphorylation of EZH2 by CDK1 and CDK2

Histone H3 lysine 27 trimethylation (H3K27me3) catalyzed by the enzymatic subunit EZH2 in the Polycomb repressive complex 2 (PRC2) is essential for cells to ‘memorize’ gene expression patterns through cell divisions and plays an important role in establishing and maintaining cell identity during development. However, how the epigenetic mark is inherited through cell generations remains poorly understood. Recently, we and others demonstrate that CDK1 and CDK2 phosphorylate EZH2 at threonine 350 (T350) and that T350 phosphorylation is important for the binding of EZH2 to PRC2 recruiters, such as noncoding RNAs (ncRNAs) HOTAIR and XIST, and for the effective recruitment of PRC2 to EZH2 target loci in cells. These findings imply that phosphorylation of EZH2 by CDK1 and CDK2 may provide cells a mechanism that enhances EZH2 function during S and G2 phases of the cell cycle, thereby ensuring K27me3 on de novo synthesized H3 incorporated in nascent nucleosomes before sister chromosomes are divided into two daughter cells. Additionally, a potential role of T350 phosphorylation of EZH2 in differing EZH2 from its homolog EZH1 in catalyzing H3K27me3 as well as the interplay between phosphorylation at T350 and other residues (e.g. phosphorylation by p38 at threonine 372 (T372)) in governing EZH2 activity in proliferating versus non-dividing cells are also discussed. Together, CDK phosphorylation of EZH2 at T350 may represent a key regulatory mechanism of EZH2 function that is essential for the maintenance of H3K27me3 marks through cell divisions.     

Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2

The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression. Here, we demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing.     

PCAF-primed EZH2 acetylation regulates its stability and promotes lung adenocarcinoma progression

Enhancer of zeste homolog 2 (EZH2) is a key epigenetic regulator that catalyzes the trimethylation of H3K27 and is modulated by post-translational modifications (PTMs). However, the precise regulation of EZH2 PTMs remains elusive. We, herein, report that EZH2 is acetylated by acetyltransferase P300/CBP-associated factor (PCAF) and is deacetylated by deacetylase SIRT1. We identified that PCAF interacts with and acetylates EZH2 mainly at lysine 348 (K348). Mechanistically, K348 acetylation decreases EZH2 phosphorylation at T345 and T487 and increases EZH2 stability without disrupting the formation of polycomb repressive complex 2 (PRC2). Functionally, EZH2 K348 acetylation enhances its capacity in suppression of the target genes and promotes lung cancer cell migration and invasion. Further, elevated EZH2 K348 acetylation in lung adenocarcinoma patients predicts a poor prognosis. Our findings define a new mechanism underlying EZH2 modulation by linking EZH2 acetylation to its phosphorylation that stabilizes EZH2 and promotes lung adenocarcinoma progression.     

Epigenetic Memory Meets G2/M: To Remember or To Forget?

The histone H3 lysine 27 (H3K27) methyltransferase EZH2 is essential for stem cell maintenance and proliferation. Recent insights suggest that the cyclin-dependent kinase CDK1 phosphorylates EZH2 at specific threonine residues by sensing developmental cues to mediate self-renewal or differentiation during G2/M phase.     

Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of enhancer of zeste 2 (Ezh2) regulates its stability

The H3K27 histone methyltransferase, Ezh2 (enhancer of zeste 2), is a Polycomb group protein that plays important roles in many biological processes including cellular differentiation, stem cell biology, and cancer development. Up-regulation of Ezh2 is observed in various human cancers consistent with its role in cell proliferation. Thus, understanding the regulation of Ezh2 may reveal how it contributes to the cellular proliferation process. Here, we demonstrate that Ezh2 can be regulated by the cyclin-dependent kinase, CDK1, which phosphorylates Ezh2 at threonines 345 and 487. Consistent with the cell cycle phase during which CDK1 exhibits peak activity, Ezh2 phosphorylation is enriched in cells arrested in mitosis when compared with S-phase. Phosphorylation of Thr-345 and Thr-487 promotes Ezh2 ubiquitination and subsequent degradation by the proteasome. Furthermore, expression of T345A/T487A confers a proliferative disadvantage when compared with cells expressing wild-type Ezh2, which suggests that phosphorylation of Ezh2 is important for cell proliferation. Collectively, these results establish a novel function for CDK1-mediated Ezh2 phosphorylation and provide a mechanism by which Ezh2 protein levels can be regulated in cells.     

Xenopus phospho-CDK7/cyclin H expressed in baculoviral-infected insect cells.

The cyclin-dependent kinase-activating kinase (CAK) catalyzes the phosphorylation of the cyclin-dependent protein kinases (CDKs) on a threonine residue (Thr160 in human CDK2). The reaction is an obligatory step in the activation of the CDKs. In higher eukaryotes, the CAK complex has been characterized in two forms. The first consists of three subunits, namely CDK7, cyclin H, and an assembly factor called MAT1, while the second consists of phospho-CDK7 and cyclin H. Phosphorylation of CDK7 is essential for cyclin association and kinase activity in the absence of the assembly factor MAT1. The Xenopus laevis CDK7 phosphorylation sites are located on the activation segment of the kinase at residues Ser170 and at Thr176 (the latter residue corresponding to Thr160 in human CDK2). We report the expression and purification of X. laevis CDK7/cyclin H binary complex in insect cells through coinfection with the recombinant viruses, AcCDK7 and Accyclin H. Quantities suitable for crystallization trials have been obtained. The purified CDK7/cyclin H binary complex phosphorylated CDK2 and CDK2/cyclin A but did not phosphorylate histone H1 or peptide substrates based on the activation segments of CDK7 and CDK2. Analysis by mass spectrometry showed that coexpression of CDK7 with cyclin H in baculoviral-infected insect cells results in phosphorylation of residues Ser170 and Thr176 in CDK7. It is assumed that phosphorylation is promoted by kinase(s) in the insect cells that results in the correct, physiologically significant posttranslational modification. We discuss the occurrence of in vivo phosphorylation of proteins expressed in baculoviral-infected insect cells.     

Kaposi’s sarcoma-associated herpesvirus promotes mesenchymal-to-endothelial transition by resolving the bivalent chromatin of PROX1 gene

Increasing evidence suggests that Kaposi’s sarcoma (KS) arises from Kaposi’s sarcoma-associated herpesvirus (KSHV)-infected mesenchymal stem cells (MSCs) through mesenchymal-to-endothelial transition (MEndT). KSHV infection promotes MSC differentiation of endothelial lineage and acquisition of tumorigeneic phenotypes. To understand how KSHV induces MEndT and transforms MSCs to KS cells, we investigated the mechanism underlying KSHV-mediated MSC endothelial lineage differentiation. Like embryonic stem cells, MSC differentiation and fate determination are under epigenetic control. Prospero homeobox 1 (PROX1) is a master regulator that controls lymphatic vessel development and endothelial differentiation. We found that the PROX1 gene in MSCs harbors a distinctive bivalent epigenetic signature consisting of both active marker H3K4me3 and repressive marker H3K27me3, which poises expression of the genes, allowing timely activation upon differentiation signals or environmental stimuli. KSHV infection effectively resolves the bivalent chromatin by decreasing H3K27me3 and increasing H3K4me3 to activate the PROX1 gene. vIL-6 signaling leads to the recruitment of MLL2 and SET1 complexes to the PROX1 promoter to increase H3K4me3, and the vGPCR-VEGF-A axis is responsible for removing PRC2 from the promoter to reduce H3K27me3. Therefore, through a dual signaling process, KSHV activates PROX1 gene expression and initiates MEndT, which renders MSC tumorigenic features including angiogenesis, invasion and migration.     

Enhancer of zeste homolog 2 promotes the proliferation and invasion of epithelial ovarian cancer cells

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that includes noncatalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3 (H3K27Me3), resulting in epigenetic silencing of gene expression. Here, we investigated the expression and function of EZH2 in epithelial ovarian cancer (EOC). When compared with primary human ovarian surface epithelial (pHOSE) cells, EZH2, SUZ12, and EED were expressed at higher levels in all 8 human EOC cell lines tested. Consistently, H3K27Me3 was also overexpressed in human EOC cell lines compared with pHOSE cells. EZH2 was significantly overexpressed in primary human EOCs (n = 134) when compared with normal ovarian surface epithelium (n = 46; P < 0.001). EZH2 expression positively correlated with expression of Ki67 (P < 0.001; a marker of cell proliferation) and tumor grade (P = 0.034) but not tumor stage (P = 0.908) in EOC. There was no correlation of EZH2 expression with overall (P = 0.3) or disease-free survival (P = 0.2) in high-grade serous histotype EOC patients (n = 98). Knockdown of EZH2 expression reduced the level of H3K27Me3 and suppressed the growth of human EOC cells both in vitro and in vivo in xenograft models. EZH2 knockdown induced apoptosis of human EOC cells. Finally, we showed that EZH2 knockdown suppressed the invasion of human EOC cells. Together, these data demonstrate that EZH2 is frequently overexpressed in human EOC cells and its overexpression promotes the proliferation and invasion of human EOC cells, suggesting that EZH2 is a potential target for developing EOC therapeutics.     

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.     

TNF/p38α/polycomb signaling to Pax7 locus in satellite cells links inflammation to the epigenetic control of muscle regeneration

How regeneration cues are converted into the epigenetic information that controls gene expression in adult stem cells is currently unknown. We identified an inflammation-activated signaling in muscle stem (satellite) cells, by which the polycomb repressive complex 2 (PRC2) represses Pax7 expression during muscle regeneration. TNF-activated p38α kinase promotes the interaction between YY1 and PRC2, via threonine 372 phosphorylation of EZH2, the enzymatic subunit of the complex, leading to the formation of repressive chromatin on Pax7 promoter. TNF-α antibodies stimulate satellite cell proliferation in regenerating muscles of dystrophic or normal mice. Genetic knockdown or pharmacological inhibition of the enzymatic components of the p38/PRC2 signaling--p38α and EZH2--invariably promote Pax7 expression and expansion of satellite cells that retain their differentiation potential upon signaling resumption. Genetic knockdown of Pax7 impaired satellite cell proliferation in response to p38 inhibition, thereby establishing the biological link between p38/PRC2 signaling to Pax7 and satellite cell decision to proliferate or differentiate.     

Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.     

JNK and STAT3 signaling pathways converge on Akt-mediated phosphorylation of EZH2 in bronchial epithelial cells induced by arsenic

The molecular mechanisms by which arsenic (As³⁺) causes human cancers remain to be fully elucidated. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb-repressive complexes 2 (PRC2) that promotes trimethylation of lysine 27 of histone H3, leading to altered expression of tumor suppressors or oncogenes. In the present study, we determined the effect of As³⁺ on EZH2 phosphorylation and the signaling pathways important for As³⁺-induced EZH2 phosphorylation in human bronchial epithelial cell line BEAS-2B. The involvement of kinases in As³⁺-induced EZH2 phosphorylation was validated by siRNA-based gene silencing. The data showed that As³⁺ can induce phosphorylation of EZH2 at serine 21 in human bronchial epithelial cells and that the phosphorylation of EZH2 requires an As³⁺-activated signaling cascade from JNK and STAT3 to Akt. Transfection of the cells with siRNA specific for JNK1 revealed that JNK silencing reduced serine727 phosphorylation of STAT3, Akt activation and EZH2 phosphorylation, suggesting that JNK is the upstream kinase involved in As³⁺-induced EZH2 phosphorylation. Because As³⁺ is capable of inducing miRNA-21 (miR-21), a STAT3-regulated miRNA that represses protein translation of PTEN or Spry2, we also tested the role of STAT3 and miR-21 in As³⁺-induced EZH2 phosphorylation. Ectopic overexpression of miR-21 promoted Akt activation and phosphorylation of EZH2, whereas inhibiting miR-21 by transfecting the cells with anti-miR-21 inhibited Akt activation and EZH2 phosphorylation. Taken together, these results demonstrate a contribution of the JNK, STAT3 and Akt signaling axis to As³⁺-induced EZH2 phosphorylation. Importantly, these findings may reveal new molecular mechanisms underlying As³⁺-induced carcinogenesis.     

Regulated activating Thr172 phosphorylation of cyclin-dependent kinase 4(CDK4): its relationship with cyclins and CDK "inhibitors"

Cyclin-dependent kinase 4 (CDK4) is a master integrator of mitogenic and antimitogenic extracellular signals. It is also crucial for many oncogenic transformation processes. Various molecular features of CDK4 activation remain poorly known or debated, including the regulation of its association with D-type cyclins, its activating Thr172 phosphorylation, and the roles of Cip/Kip CDK "inhibitors" in these processes. Thr172 phosphorylation of CDK4 was reinvestigated using two-dimensional gel electrophoresis in various experimental systems, including human fibroblasts, canine thyroid epithelial cells stimulated by thyrotropin, and transfected mammalian and insect cells. Thr172 phosphorylation of CDK4 depended on prior D-type cyclin binding, but Thr172 phosphorylation was also found in p16-bound CDK4. Opposite effects of p27 on cyclin D3-CDK4 activity observed in different systems depended on its stoichiometry in this complex. Thr172-phosphorylated CDK4 was enriched in complexes containing p21 or p27, even at inhibitory levels of p27 that precluded CDK4 activity. Deletion of the p27 nuclear localization signal sequence relocalized cyclin D3-CDK4 in the cytoplasm but did not affect CDK4 phosphorylation. Within cyclin D3 complexes, T-loop phosphorylation of CDK4, but not of CDK6, was directly regulated, identifying it as a determining target for cell cycle control by extracellular factors. Collectively, these unexpected observations indicate that CDK4-activating kinase(s) should be reconsidered.     

Methylation of EZH2 by PRMT1 regulates its stability and promotes breast cancer metastasis

Enhancer of zeste homolog 2 (EZH2), a key histone methyltransferase and EMT inducer, is overexpressed in diverse carcinomas, including breast cancer. However, the molecular mechanisms of EZH2 dysregulation in cancers are still largely unknown. Here, we discover that EZH2 is asymmetrically dimethylated at R342 (meR342-EZH2) by PRMT1. meR342-EZH2 was found to inhibit the CDK1-mediated phosphorylation of EZH2 at T345 and T487, thereby attenuating EZH2 ubiquitylation mediated by the E3 ligase TRAF6. We also demonstrate that meR342-EZH2 resulted in a decrease in EZH2 target gene expression, but an increase in breast cancer cell EMT, invasion and metastasis. Moreover, we confirm the positive correlations among PRMT1, meR342-EZH2 and EZH2 expression in the breast cancer tissues. Finally, we report that high expression levels of meR342-EZH2 predict a poor clinical outcome in breast cancer patients. Our findings may provide a novel diagnostic target and promising therapeutic target for breast cancer metastasis.Subject terms: Metastasis, Tumour biomarkers, Ubiquitylation, Gene regulation