SET Domain-containing Lysine Methyltransferase Research Articles

Methyltransferase Set7/9 as a Multifaceted Regulator of ROS Response.

Reactive oxygen species (ROS) induce multiple signaling cascades in the cell and hence play an important role in the regulation of the cell's fate. ROS can cause irreversible damage to DNA and proteins resulting in cell death. Therefore, finely tuned regulatory mechanisms exist in evolutionarily diverse organisms that are aimed at the neutralization of ROS and its consequences with respect to cellular damage. The SET domain-containing lysine methyltransferase Set7/9 (KMT7, SETD7, SET7, SET9) post-translationally modifies several histones and non-histone proteins via monomethylation of the target lysines in a sequence-specific manner. In cellulo, the Set7/9-directed covalent modification of its substrates affects gene expression, cell cycle, energy metabolism, apoptosis, ROS, and DNA damage response. However, the in vivo role of Set7/9 remains enigmatic. In this review, we summarize the currently available information regarding the role of methyltransferase Set7/9 in the regulation of ROS-inducible molecular cascades in response to oxidative stress. We also highlight the in vivo importance of Set7/9 in ROS-related diseases.

Lead discovery, chemical optimization, and biological evaluation studies of novel histone methyltransferase SET7 small-molecule inhibitors

The post-translational modifications of histones, including histone methylation and demethylation, control the expression switch of multiple genes. SET domain-containing lysine methyltransferase 7 (SET7) is the only methyltransferase, which can specifically monomethylate lysine-4 of histone H3 (H3K4me1) and play critical roles in various diseases, including breast cancer, hepatitis C virus (HCV), atherosclerotic vascular disease, diabetes, prostate cancer, hepatocellular carcinoma, and obesity. However, several known SET7 inhibitors exhibit weak activity or poor selectivity. Therefore, the development of novel SET7 inhibitors is highly desirable and of great clinical value. In this study, we identified 2–79 as a new hit compound by structure-based virtual screening and further AlphaLISA-based biochemical evaluation. Via chemical optimization, the synthesized compound DC21 was confirmed as a potent SET7 inhibitor with an IC50 value of 15.93 μM. The interaction between DC21 and SET7 was also validated through SPR experiment. Especially, DC21 retarded proliferation of MCF7 cells with an IC50 value of 25.84 μM in cellular level. In addition, DC21 has good selectivity for several other epigenetic targets, such as SUV39H1, G9a, NSD1, DOT1L and MOF. DC21 can serve as a lead compound to develop more potential SET7 inhibitors and as a chemical probe for SET7 biological function studies.

DDB1 Regulates Sertoli Cell Proliferation and Testis Cord Remodeling by TGFβ Pathway.

Testis cords are the embryonic precursors of the seminiferous tubules. Development of testis cords is a key event during embryonic testicular morphogenesis and is regulated by multiple signaling molecules produced by Sertoli cells. However, the exact nature and the cascade of molecular events underlying testis cord development remain to be uncovered. In the current study, we explored the role of DNA damage binding protein 1 (DDB1) in Sertoli cells during mouse testis cord development. The genetic ablation of Ddb1 specifically in Sertoli cells resulted in the compromised Sertoli cell proliferation and disruption of testis cord remodeling in neonatal mice. This testicular dysgenesis persisted through adulthood, resulting in smaller testis and low sperm production. Mechanistically, we observed that the DDB1 degradation can stabilize SET domain-containing lysine methyltransferase 8 (SET8), which subsequently decreases the phosphorylation of SMAD2, an essential intracellular component of transforming growth factor beta (TGFβ) signaling. Taken together, our results suggest an essential role of Ddb1 in Sertoli cell proliferation and normal remodeling of testis cords via TGFβ pathway. To our knowledge, this is the first upstream regulators of TGFβ pathway in Sertoli cells, and therefore it furthers our understanding of testis cord development.

P5378The methyltransferase SETD7 promotes myocardial ischemic injury by activating Hippo signalling

Abstract Introduction Despite significant advances in coronary revascularization, acute myocardial infarction remains the leading cause of heart failure and death worldwide. The Hippo pathway is a master regulator of cell survival during myocardial ischemia. Upon cellular stress, activation of Hippo signaling leads to cytosolic retention and degradation of the pro-survival transcription factor YAP. Post-translational modifications, namely methylation, critically affect protein functionality in conditions of cellular stress. The SET domain-containing lysine methyltransferase 7 (SETD7) - which induces a specific mono-methylation of both histone and non-histone proteins - has recently emerged as key player in the pathogenesis of vascular disease. However, the role of SETD7 in the heart is largely unknown. Purpose The present study investigates whether SETD7 regulates the Hippo pathway during myocardial ischemia. Methods Neonatal rat ventricular myocytes (NRVM) were exposed to normal glucose levels or glucose deprivation (GD) for 15 h, in the presence of the selective SETD7 inhibitor [(R)-PFI-2] or its inactive enantiomer [(S)-PFI-2]. Western blot and real time PCR were employed to investigate the effects of energy stress on SETD7 and the Hippo pathway, while apoptosis was assessed by Caspase-3 activity assay. YAP localization was examined by confocal microscopy while its mono-methylation was assessed by immunoblotting. SETD7 knockout (SETD7−/−) mice and wild-type (WT) littermates (male, 8–12 weeks old) underwent 1 h of left anterior descending (LAD) coronary artery ligation followed by 24 h of reperfusion. Infarct size was assessed by TTC staining and shown as infarct size per ventricle surface (I/V). Cardiac function was investigated at 24h by conventional and Tissue Doppler Imaging echocardiography (Vevo 3100, Visualsonics). Results GD for 15h in NRVMs led to both YAP phosphorylation and mono-methylation, and subsequent cytosolic retention, as assessed by confocal microscopy. Reduced nuclear content of YAP was confirmed by downregulation of YAP-dependent pro-survival genes, namely Ctgf and Fgf2. GD-induced YAP inactivation was associated with an increase in SETD7 expression. Interestingly, pharmacological inhibition of SETD7 by (R)-PFI-2 blunted YAP mono-methylation, thus restoring nuclear retention of YAP and transcription of pro-survival genes in GD-treated NRVMs. Moreover, (R)-PFI-2 prevented NRVMs apoptosis. In line with our in vitro findings, SETD7−/− mice showed decreased infarct size as compared to WT littermates (I/V: 16.27%±2 vs. 20.54%±3, p<0.005, respectively). Consistently, cardiac function, as assessed by ejection fraction (EF: 46%±2 vs. 38%±5, p<0.001), fractional shortening (FS: 22%±1 vs. 18%±3, p<0.001) as well as by TDI, was preserved in mice lacking SETD7 as compared to WT animals. Conclusions Pharmacological modulation of SETD7 by (R)-PFI-2 may represent a novel therapeutic approach to prevent myocardial ischemic damage.

SETD7 promotes TNF-α-induced proliferation and migration of airway smooth muscle cells in vitro through enhancing NF-κB/CD38 signaling

The inflammation-induced the excessive proliferation and migration of airway smooth muscle (ASM) cells in the airway wall contribute to airway remodeling in asthma pathogenesis. SET domain-containing lysine methyltransferase 7 (SETD7) has emerged as one of the key regulators of inflammation. Yet, the function of SETD7 in regulating inflammation-induced ASM cell proliferation and invasion remains unclear. In the present study, we aimed to investigate the function of SETD7 in regulating ASM cell proliferation and invasion induced by tumor necrosis factor (TNF)-α in vitro. Our results showed that SETD7 expression was upregulated in ASM cells stimulated with TNF-α. Silencing SETD7 significantly decreased TNF-α-induced ASM cell proliferation and migration, while SETD7 overexpression exhibited the opposite effect. Notably, silencing SETD7 decreased the activation of nuclear factor (NF)-κB and reduced the expression of CD38 induced by TNF-α. Blocking NF-κB activation significantly abrogated the promotional effect of SETD7 overexpression on CD38 expression. Moreover, overexpression of CD38 partially reversed the inhibitory effect of SETD7 silencing on TNF-α-induced ASM cell proliferation and migration. Overall, these results demonstrate that SETD7 regulates TNF-α-induced ASM cell proliferation and migration through modulation of NF-κB/CD38 signaling, suggesting a potential role of SETD7 in asthma airway remodeling.

Inhibition of SETD7 protects cardiomyocytes against hypoxia/reoxygenation-induced injury through regulating Keap1/Nrf2 signaling

The protein SET domain-containing lysine methyltransferase 7 (SETD7) has recently been shown to regulate apoptosis in various cells. However, the role of SETD7 on cardiomyocyte apoptosis during myocardial ischemia/reperfusion injury remains unclear. This study aimed to investigate the potential role of SETD7 in hypoxia/reoxygenation (H/R)-induced apoptosis of rat cardiomyocytes and reveal the underlying mechanism. Our results demonstrated that SETD7 expression was significantly up-regulated in cardiomyocytes in response to H/R injury. The inhibition of SETD7 by siRNA-mediated gene silencing significantly suppressed H/R-induced apoptosis and decreased the production of reactive oxygen species (ROS). The overexpression of SETD7 markedly enhanced H/R-induced apoptosis and ROS production. Moreover, the knockdown of SETD7 reduced the expression of Keap1 and promoted the expression of Nrf2. In addition, the knockdown of SETD7 increased the activity of antioxidant response element and promoted the expression of heme oxygenase-1 and NADPH-quinone oxidoreductase 1. However, the knockdown of Nrf2 partially abrogated the SETD7 inhibition-mediated protective effect against H/R injury. Taken together, these results indicate that the inhibition of SETD7 attenuates H/R-induced injury of cardiomyocytes via the down-regulation of Keap1 and promotion of the Nrf2-mediated anti-oxidation signaling pathway.

Expression and clinical significance of COMPASS family of histone methyltransferases in clear cell renal cell carcinoma

The regulation of histone 3 lysine 4 (H3K4) methylation code is indispensable for the cell as any disturbance in this has been associated with many pathologic conditions, like cancer. The SET domain-containing lysine methyltransferase 2 (KMT2) family of histone methyltransferases was the first to be identified as writers of H3K4 methylation. In mammals, seven members of the KMT2 family are responsible for carrying out the bulk of H3K4 methylation. Recent studies documented alterations in various histone methylases in human cancer, which were associated with therapeutic and prognostic potential. Notwithstanding, no report has documented the role of KMT2 family of histone methyltransferases in clear cell renal cell carcinoma (ccRCC). Therefore, we examined the expression profile of all the seven KMT2 family members of histone methyltransferases in ccRCC with the aim to establish their role as prognostic or therapeutic targets. Real-time PCR was employed to study the expression of KMT2 family genes using specific primers in 50 cases of ccRCC. The mRNA levels were correlated with stage, grade, and metastasis of the tumor. Among seven genes, KMT2G was significantly up-regulated in higher stages of the tumor as compared to low stage tumors (p = 0.017). Similarly, KMT2G levels were higher in the metastatic tumor (p = 0.027). Additionally, some of the KMT2G target genes were found to be up-regulated in metastatic tumors as compared to non-metastatic. ROC curve indicated KMT2G as a good marker for discriminating metastatic tumors from non-metastatic tumors (AUC = 0.738). Thus, we conclude that KMT2G histone methyltransferase could be a good prognostic marker for ccRCC. Additionally, KMT2G can also serve as a therapeutic target for ccRCC.

Crosstalk between lysine methylation and phosphorylation of ATG16L1 dictates the apoptosis of hypoxia/reoxygenation-induced cardiomyocytes

ABSTRACTPost-translational modifications of autophagy-related (ATG) genes are necessary to modulate their functions. However, ATG protein methylation and its physiological role have not yet been elucidated. The methylation of non-histone proteins by SETD7, a SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. Here we present evidence that the precise activity of ATG16L1 protein in hypoxia/reoxygenation (H/R)-treated cardiomyocytes is regulated by a balanced methylation and phosphorylation switch. We first show that H/R promotes autophagy and decreases SETD7 expression, whereas autophagy inhibition by 3-MA increases SETD7 level in cardiomyocytes, implying a tight correlation between autophagy and SETD7. Then we demonstrate that SETD7 methylates ATG16L1 at lysine 151 while KDM1A/LSD1 (lysine demethylase 1A) removes this methyl mark. Furthermore, we validate that this methylation at lysine 151 impairs the binding of ATG16L1 to the ATG12–ATG5 conjugate, leading to inhibition of autophagy and increased apoptosis in H/R-treated cardiomyocytes. However, the cardiomyocytes with shRNA-knocked down SETD7 or inhibition of SETD7 activity by a small molecule chemical, display increased autophagy and decreased apoptosis following H/R treatment. Additionally, methylation at lysine 151 inhibits phosphorylation of ATG16L1 at S139 by CSNK2 which was previously shown to be critical for autophagy maintenance, and vice versa. Together, our findings define a novel modification of ATG16L1 and highlight the importance of an ATG16L1 phosphorylation-methylation switch in determining the fate of H/R-treated cardiomyocytes.

Histone Methyltransferase Setd7 Regulates Nrf2 Signaling Pathway by Phenethyl Isothiocyanate and Ursolic Acid in Human Prostate Cancer Cells.

This study aims to investigate the role of the epigenetic regulator SET domain-containing lysine methyltransferase 7 (Setd7) in regulating the antioxidant Nrf2 pathway in prostate cancer (PCa) cells and examines the effects of two phytochemicals, phenethyl isothiocyanate (PEITC) and ursolic acid (UA). Lentivirus-mediated shRNA knockdown of Setd7 in LNCaP and PC-3 cells decreases the expression of downstream Nrf2 targets, such as NAD(P)H: quinone oxidoreductase 1 (Nqo1) and glutathione S-transferase theta 2 (Gstt2). Downregulation of Setd7 decreases soft agar colony formation ability of PCa cells. Knockdown of Setd7 increases reactive oxygen species (ROS) generation. Furthermore, Setd7 knockdown attenuates Nqo1 and Gstt2 expression in response to H2 O2 challenge, whereas increased DNA damage is observed in Setd7 knockdown cells in comet assay. Interestingly, Setd7 expression could be induced by the dietary phytochemicals PEITC and UA. Chromatin immunoprecipitation (ChIP) assays show that Setd7 knockdown decreased H3K4me1 enrichment in the Nrf2 and Gstt2 promoter regions, while PEITC and UA treatments elevated the enrichment. Taken together, these results indicate that Setd7 knockdown decreases Nrf2 and Nrf2-target genes expression and that PEITC and UA induce Setd7 expression, which activates the Nrf2/antioxidant response element (ARE) signaling pathway and protects DNA from oxidative damage.

Inhibition of SET Domain-Containing Lysine Methyltransferase 7/9 Ameliorates Renal Fibrosis.

TGF-β1 activity results in methylation of lysine 4 of histone H3 (H3K4) through SET domain-containing lysine methyltransferase 7/9 (SET7/9) induction, which is important for the transcriptional activation of fibrotic genes in vitro. However, in vivo studies utilizing an experimental model of renal fibrosis are required to develop therapeutic interventions that target SET7/9. In this study, we investigated the signaling pathway of TGF-β1-induced SET7/9 expression and whether inhibition of SET7/9 suppresses renal fibrosis in unilateral ureteral obstruction (UUO) mice and kidney cell lines. Among the SET family, SET7/9 was upregulated on days 3 and 7 in UUO mice, and the upregulation was suppressed by TGF-β1 neutralizing antibody. TGF-β1 induced SET7/9 expression via Smad3 in normal rat kidney (NRK)-52E cells. In human kidney biopsy specimens from patients diagnosed with IgA nephropathy and membranous nephropathy, SET7/9 expression was positively correlated with the degree of interstitial fibrosis (r=0.59, P=0.001 in patients with IgA nephropathy; and r=0.58, P<0.05 in patients with membranous nephropathy). In addition, small interfering RNA-mediated knockdown of SET7/9 expression significantly attenuated renal fibrosis in UUO mice. Sinefungin, an inhibitor of SET7/9, also suppressed the expression of mesenchymal markers and extracellular matrix proteins and inhibited H3K4 mono-methylation (H3K4me1) in kidneys of UUO mice. Moreover, sinefungin had an inhibitory effect on TGF-β1-induced α-smooth muscle actin expression and H3K4me1 in both NRK-52E and NRK-49F cells. In conclusion, sinefungin, a SET7/9 inhibitor, ameliorates renal fibrosis by inhibiting H3K4me1 and may be a candidate therapeutic agent.

Repression of hypoxia-inducible factor α signaling by Set7-mediated methylation.

Hypoxia-inducible factor (HIF)-1α and HIF-2α are the main regulators of cellular responses to hypoxia. Post-translational modifications of HIF-1α and 2α are necessary to modulate their functions. The methylation of non-histone proteins by Set7, an SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. In this study, we show that Set7 methylates HIF-1α at lysine 32 and HIF-2α at lysine K29; this methylation inhibits the expression of HIF-1α/2α targets by impairing the occupancy of HIF-α on hypoxia response element of HIF target gene promoter. Set7-null fibroblasts and the cells with shRNA-knocked down Set7 exhibit upregulated HIF target genes. Set7 inhibitor blocks HIF-1α/2α methylation to enhance HIF target gene expression. Set7-null fibroblasts and the cells with shRNA-knocked down Set7 or inhibition of Set7 by the inhibitor subjected to hypoxia display an increased glucose uptake and intracellular adenosine triphosphate levels. These findings define a novel modification of HIF-1α/2α and demonstrate that Set7-medited lysine methylation negatively regulates HIF-α transcriptional activity and HIF-1α-mediated glucose homeostasis.