SET Domain Containing 2 Research Articles

Effects of Glycine on epigenetic modification and early embryonic development in porcine oocytes exposed to monobutyl phthalate

Monobutyl phthalate (MBP) is the primary active metabolite of dibutyl phthalate (DBP), the key plasticizer component. A substantial body of evidence from studies conducted on both animals and humans indicates that MBP exposure could result in harmful impacts on toxicity pathways. In addition, it can seriously affect human and animal reproductive health. In our present study, we showed that exposure to MBP causes abnormal epigenetic modifications in porcine oocytes and failure of early embryonic development. However, glycine (Gly) can protect oocytes and early embryos from damage caused by MBP. Our study indicated a significant decrease in the percentage of porcine oocytes that reached the metaphase II (MII) phase when exposed to MBP. SET-domain-containing 2(SETD2)-mediated H3K36me3 histone methylation was detected, and the results showed that MBP significantly decreased the protein expression of H3K36me3 and SETD2. Moreover, the expression of the DNA break markers γH2AX and the mRNA expression of Asf1a, and Asf1b increased in the MBP group. The detection of DNA methylation marker proteins showed that MBP significantly increased the fluorescence intensity of 5-methylcytosine (5mC). The results from our RT-qPCR analysis demonstrated a significant decrease in the mRNA expression of the DNA methylation-related genes Dnmt1 and Dnmt3a, as well as the embryonic developmental potential-related genes Oct4 and Nanog, in porcine oocytes following exposure to MBP. Additionally, the mRNA expression of p53 significantly increased. Subsequently, the effects of MBP on early embryonic development were examined via parthenogenesis activation (PA) and in vitro fertilization (IVF). Exposure to MBP significantly impacted the development of embryos in both PA and IVF processes. The TUNEL staining data showed that MBP significantly increased embryonic apoptosis. However, Gly can ameliorate MBP-induced defects in oocyte epigenetic modifications and early embryonic development.

16 Linking VHL and SETD2 in a common oncogenic pathway that converges on the mitoticspindle

Abstract Background Loss of chromosome 3p is a landmark event in clear cell renal cell carcinoma (ccRCC) that results in mono-allelic loss of VHL (von Hippel Lindau) and SETD2 (Set-domain containing 2) (and other tumor suppressors co-located on 3p). Second hits in VHL inactivate this key tumor suppressor initiating tumor progression. SETD2, a histone methyltransferase, was previously shown to have a dual function in methylating both histones and microtubules, thereby contributing to both the histone and tubulin codes. Methylation by SETD2 on microtubules occurs at the mitotic spindle and is essential for normal mitosis and cytokinesis, with loss of SETD2 acting as a strong driver of apoptosis. This raises a conundrum of how cancer cells survive early mono-allelic loss of SETD2, escaping cell death. Methods Using biochemical kinase assays and mass spectrometry we have identified SETD2 as a substrate for AURKA. In addition, we have used immunoblotting and immunofluorescence assays to probe phosphorylation on SETD2 and the impact of phosphorylation of SETD2 both on its chromatin and cytoskeleton targets. Results We have identified the mitotic kinase, Aurora kinase A (AURKA), as a regulator of SETD2. Our data uncover SETD2 as a unique substrate for phosphorylation by AURKA, with mass spectrometry identifying serine 2080 (S2080) as the site of phosphorylation on SETD2. We found phosphorylation of SETD2 by AURKA at S2080 contributes to its methyltransferase (i.e. enzymatic) activity on microtubules but does not impact chromatin methylation on H3K36 which remains unaltered. We show that VHL regulates SETD2 via AURKA, and loss of phosphorylation on SETD2 results in mitotic defects and genomic instability. Importantly, we demonstrate that inhibition of AURKA is synthetic lethal in the setting of VHL and SETD2 deficiency. Conclusions AURKA expression levels are high in VHL-null cells resulting from an inability of VHL to target AURKA for degradation and our data now highlight a direct link between VHL and SETD2, two tumor suppressors believed to independently drive RCC pathogenesis. In summary, our data reveal a tumor-specific vulnerability linked to mitotic fragility that can be precisely targeted to ultimately drive mitotic catastrophe. DOD CDMRP Funding: yes

Regulation of SETD2 maintains immune regulatory function in macrophages to suppress airway allergy.

SET domain-containing 2 (SETD2) is a histone methyltransferase. It regulates the activity of H3K36me3 to enhance gene transcription. Macrophages (Mϕs) are one of the cell types involved in immune response. The purpose of this study is to clarify the role of SETD2 in regulating the immune property of Mϕ. The Mφs were isolated from the bronchoalveolar lavage fluid (BALF) and analysed through flow cytometry and RNA sequencing. A mouse strain carrying Mφs deficient in SETD2 was used. A mouse model of airway allergy was established with the ovalbumin/alum protocol. Less expression of SETD2 was observed in airway Mϕs in patients with allergic asthma. SETD2 of M2 cells was associated with the asthmatic clinical response. Sensitization reduced the expression of SETD2 in mouse respiratory tract M2 cells, which is associated with the allergic reaction. Depletion of SETD2 in Mφs resulted in Th2 pattern inflammation in the lungs. SETD2 maintained the immune regulatory ability in airway M2 cells. SETD2 plays an important role in the maintenance of immune regulatory property of airway Mφs.

Exploring The Prognostic Significance of SET-Domain Containing 2 (SETD2) Expression in Advanced and Castrate-Resistant Prostate Cancer.

SET-domain containing 2 (SETD2) is a histone methyltransferase and an epigenetic modifier with oncogenic functionality. In the current study, we investigated the potential prognostic role of SETD2 in prostate cancer. A cohort of 202 patients' samples was assembled on tissue microarrays (TMAs) containing incidental, advanced, and castrate-resistant CRPCa cases. Our data showed significant elevated SETD2 expression in advanced and castrate-resistant disease (CRPCa) compared to incidental cases (2.53 ± 0.58 and 2.21 ± 0.63 vs. 1.9 ± 0.68; p < 0.001, respectively). Interestingly, the mean intensity of SETD2 expression in deceased vs. alive patients was also significantly different (2.31 ± 0.66 vs. 2 ± 0.68; p = 0.003, respectively). Overall, high SETD2 expression was found to be considered high risk and was significantly associated with poor prognosis and worse overall survival (OS) (HR 1.80; 95% CI: 1.28-2.53, p = 0.001) and lower cause specific survival (CSS) (HR 3.14; 95% CI: 1.94-5.08, p < 0.0001). Moreover, combining high-intensity SETD2 with PTEN loss resulted in lower OS (HR 2.12; 95% CI: 1.22-3.69, p = 0.008) and unfavorable CSS (HR 3.74; 95% CI: 1.67-8.34, p = 0.001). Additionally, high SETD2 intensity with ERG positive expression showed worse prognosis for both OS (HR 1.99, 95% CI 0.87-4.59; p = 0.015) and CSS (HR 2.14, 95% CI 0.98-4.68, p = 0.058). We also investigated the protein expression database TCPA, and our results showed that high SETD2 expression is associated with a poor prognosis. Finally, we performed TCGA PRAD gene set enrichment analysis (GSEA) data for SETD2 overexpression, and our data revealed a potential association with pathways involved in tumor progression such as the AMPK signaling pathway, the cAMP signaling pathway, and the PI3K-Akt signaling pathway, which are potentially associated with tumor progression, chemoresistance, and a poor prognosis.

SET domain containing 2 promotes megakaryocyte polyploidization and platelet generation through methylation of α-tubulin

BackgroundMegakaryocytes (MKs) are polyploid cells responsible for producing ∼1011 platelets daily in humans. Unraveling the mechanisms regulating megakaryopoiesis holds the promise for the production of clinical-grade platelets from stem cells, overcoming significant current limitations in platelet transfusion medicine. Previous work identified that loss of the epigenetic regulator SET domain containing 2 (SETD2) was associated with an increased platelet count in mice. However, the role of SETD2 in megakaryopoiesis remains unknown. ObjectivesHere, we examined how SETD2 regulated MK development and platelet production using complementary murine and human systems. MethodsWe manipulated the expression of SETD2 in multiple in vitro and ex vivo models to assess the ploidy of MKs and the function of platelets. ResultsThe genetic ablation of Setd2 increased the number of high-ploidy bone marrow MKs. Peripheral platelet counts in Setd2 knockout mice were significantly increased ∼2-fold, and platelets exhibited normal size, morphology, and function. By knocking down and overexpressing SETD2 in ex vivo human cell systems, we demonstrated that SETD2 negatively regulated MK polyploidization by controlling methylation of α-tubulin, microtubule polymerization, and MK nuclear division. Small-molecule inactivation of SETD2 significantly increased the production of high-ploidy MKs and platelets from human-induced pluripotent stem cells and cord blood CD34+ cells. ConclusionThese findings identify a previously unrecognized role for SETD2 in regulating megakaryopoiesis and highlight the potential of targeting SETD2 to increase platelet production from human cells for transfusion practices.

Histone H3K36me3 mediates the genomic instability of Benzo[a]pyrene in human bronchial epithelial cells

Histone modifications maintain genomic stability and orchestrate gene expression at the chromatin level. Benzo [a]pyrene (BaP) is the ubiquitous carcinogen widely spread in the environment, but the role and regulatory mechanism of histone modification in its toxic effects remain largely undefined. In this study, we found a dose-dependent reduction of histone H3 methylations at lysine4, lysine9, lysine27, lysine36 in HBE cells treated with BaP. We observed that inhibiting H3K27 and H3K36 methylation impaired cell proliferation, whereas the loss of H3K4, H3K9, H3K27, and H3K36 methylation led to increased genomic instability and delayed DNA repair. H3K36 mutation at both H3.1 and H3.3 exhibited the most significant impacts. In addition, we found that the expression of SET domain containing 2 (SETD2), the unique methyltransferase catalyzed H3K36me3, was downregulated by BaP dose-dependently in vitro and in vivo. Knockdown of SETD2 aggravated DNA damage of BaP exposure, which was consistent with the effects of H3K36 mutation. With the aid of chromatin immunoprecipitation (ChIP) -seq and RNA-seq, we found that H3K36me3 was responsible for transcriptional regulation of genes involved in pathways related to cell survival, lung cancer, metabolism and inflammation. The enhanced enrichment of H3K36me3 in genes (CYP1A1, ALDH1A3, ACOXL, WNT5A, WNT7A, RUNX2, IL1R2) was positively correlated with their expression levels, while the reduction of H3K36me3 distribution in genes (PPARGC1A, PDE4D, GAS1, RNF19A, KSR1) were in accordance with the downregulation of gene expression. Taken together, our findings emphasize the critical roles and mechanisms of histone lysine methylation in mediating cellular homeostasis during BaP exposure.

Paternal high-fat diet altered H3K36me3 pattern of pre-implantation embryos.

The global transition towards diets high in calories has contributed to 2.1 billion people becoming overweight, or obese, which damages male reproduction and harms offspring. Recently, more and more studies have shown that paternal exposure to stress closely affects the health of offspring in an intergenerational and transgenerational way. SET Domain Containing 2 (SETD2), a key epigenetic gene, is highly conserved among species, is a crucial methyltransferase for converting histone 3 lysine 36 dimethylation (H3K36me2) into histone 3 lysine 36 trimethylation (H3K36me3), and plays an important regulator in the response to stress. In this study, we compared patterns of SETD2 expression and the H3K36me3 pattern in pre-implantation embryos derived from normal or obese mice induced by high diet. The results showed that SETD2 mRNA was significantly higher in the high-fat diet (HFD) group than the control diet (CD) group at the 2-cell, 4-cell, 8-cell, and 16-cell stages, and at the morula and blastocyst stages. The relative levels of H3K36me3 in the HFD group at the 2-cell, 4-cell, 8-cell, 16-cell, morula stage, and blastocyst stage were significantly higher than in the CD group. These results indicated that dietary changes in parental generation (F0) male mice fed a HFD were traceable in SETD2/H3K36me3 in embryos, and that a paternal high-fat diet brings about adverse effects for offspring that might be related to SETD2/H3K36me3, which throws new light on the effect of paternal obesity on offspring from an epigenetic perspective.

SETD2 deficiency accelerates sphingomyelin accumulation and promotes the development of renal cancer

Patients with polycystic kidney disease (PKD) encounter a high risk of clear cell renal cell carcinoma (ccRCC), a malignant tumor with dysregulated lipid metabolism. SET domain–containing 2 (SETD2) has been identified as an important tumor suppressor and an immunosuppressor in ccRCC. However, the role of SETD2 in ccRCC generation in PKD remains largely unexplored. Herein, we perform metabolomics, lipidomics, transcriptomics and proteomics within SETD2 loss induced PKD-ccRCC transition mouse model. Our analyses show that SETD2 loss causes extensive metabolic reprogramming events that eventually results in enhanced sphingomyelin biosynthesis and tumorigenesis. Clinical ccRCC patient specimens further confirm the abnormal metabolic reprogramming and sphingomyelin accumulation. Tumor symptom caused by Setd2 knockout is relieved by myriocin, a selective inhibitor of serine-palmitoyl-transferase and sphingomyelin biosynthesis. Our results reveal that SETD2 deficiency promotes large-scale metabolic reprogramming and sphingomyelin biosynthesis during PKD-ccRCC transition. This study introduces high-quality multi-omics resources and uncovers a regulatory mechanism of SETD2 on lipid metabolism during tumorigenesis.

Morphological characteristics of SETD2-mutated locally advanced clear cell renal cell carcinoma: Comparison with BAP1-mutated clear cell renal cell carcinoma

SET-domain containing 2 (SETD2) and BRCA1-associated protein 1 (BAP1), both chromatin remodeling genes, are frequently mutated in clear cell renal cell carcinoma (ccRCC) and involved in tumor progression and metastasis. Herein, we studied clinicopathologic features of 7 cases of locally advanced ccRCC with single SETD2 mutation, and compared to 7 cases of locally advanced ccRCC with single BAP1 mutation. SETD2-mutated ccRCC showed high-grade transformation, comprising of enlarged tumor cells with voluminous clear cytoplasm, enlarged irregular nuclei with prominent nucleoli, eosinophilic cytoplasmic granules, arranged in various architectural patterns such as large nested, tubular, tubulopapillary and solid. 71 % (5 of 7 cases) of SETD2-mutated ccRCC showed a rhabdoid morphology. SETD2-mutated ccRCC have striking propensity for invasive growth; all cases have vascular invasion and perirenal (extracapsular) adipose tissue invasion. After nephrectomy, distant metastasis was found in 67 % (4 of 7 cases) of patients with SETD2-mutated ccRCC. The most common metastatic site was the lung (3 cases), followed by precaval lymph nodes (1 case). BAP1-mutated ccRCC also showed a similar high-grade morphology, with rhabdoid and/or sarcomatoid features. Their high-grade features mostly overlapped with those of SETD2-mutated ccRCC, which makes difficult to predict the presence of BAP1 or SETD2 mutation solely from morphology. These findings justify the use of molecular testing to detect these mutations, especially when we encounter high-grade ccRCC. Detecting SETD2 and BAP1 mutation in ccRCC is useful for risk stratification and proper therapeutic strategy.

SETD2 deficiency promotes renal fibrosis through the TGF-β/Smad signalling pathway in the absence of VHL.

Renal fibrosis is the final development pathway and the most common pathological manifestation of chronic kidney disease. Epigenetic alteration is a significant intrinsic factor contributing to the development of renal fibrosis. SET domain-containing 2 (SETD2) is the sole histone H3K36 trimethyltransferase, catalysing H3K36 trimethylation. There is evidence that SETD2-mediated epigenetic alterations are implicated in many diseases. However, it is unclear what role SETD2 plays in the development of renal fibrosis. Kidney tissues from mice as well as HK2 cells were used as research subjects. Clinical databases of patients with renal fibrosis were analysed to investigate whether SETD2 expression is reduced in the occurrence of renal fibrosis. SETD2 and Von Hippel-Lindau (VHL) double-knockout mice were used to further investigate the role of SETD2 in renal fibrosis. Renal tubular epithelial cells isolated from mice were used for RNA sequencing and chromatin immunoprecipitation sequencing to search for molecular signalling pathways and key molecules leading to renal fibrosis in mice. Molecular and cell biology experiments were conducted to analyse and validate the role of SETD2 in the development of renal fibrosis. Finally, rescue experiments were performed to determine the molecular mechanism of SETD2 deficiency in the development of renal fibrosis. SETD2 deficiency leads to severe renal fibrosis in VHL-deficient mice. Mechanically, SETD2 maintains the transcriptional level of Smad7, a negative feedback factor of the transforming growth factor-β (TGF-β)/Smad signalling pathway, thereby preventing the activation of the TGF-β/Smad signalling pathway. Deletion of SETD2 leads to reduced Smad7 expression, which results in activation of the TGF-β/Smad signalling pathway and ultimately renal fibrosis in the absence of VHL. Our findings reveal the role of SETD2-mediated H3K36me3 of Smad7 in regulating the TGF-β/Smad signalling pathway in renal fibrogenesis and provide an innovative insight into SETD2 as a potential therapeutic target for the treatment of renal fibrosis.

Loss of SETD2 aggravates colorectal cancer progression caused by SMAD4 deletion through the RAS/ERK signalling pathway.

Colorectal cancer (CRC) is a complex, multistep disease that arises from the interplay genetic mutations and epigenetic alterations. The histone H3K36 trimethyltransferase SET domain-containing 2 (SETD2), as an epigenetic signalling molecule, has a 5% mutation rate in CRC. SETD2 expression is decreased in the development of human CRC and mice treated with Azoxymethane /Dextran sodium sulfate (AOM/DSS). Loss of SETD2 promoted CRC development. SMAD Family member 4 (SMAD4) has a 14% mutation rate in CRC, and SMAD4 ablation leads to CRC. The co-mutation of SETD2 and SMAD4 predicted advanced CRC. However, little is known on the potential synergistic effect of SETD2 and SMAD4. CRC tissues from mice and SW620 cells were used as research subjects. Clinical databases of CRC patients were analyzed to investigate the association between SETD2 and SMAD4. SETD2 and SMAD4 double-knockout mice were established to further investigate the role of SETD2 in SMAD4-deficient CRC. The intestinal epithelial cells (IECs) were isolated for RNA sequencing and chromatin immunoprecipitation sequencing (ChIP-seq) to explore the mechanism and the key molecules resulting in CRC. Molecular and cellular experiments were conducted to analyze the role of SETD2 in SMAD4-deficient CRC. Finally, rescue experiments were performed to confirm the molecular mechanism of SETD2 in the development of SMAD4-dificient CRC. The deletion of SETD2 promotes the malignant progression of SMAD4-deficient CRC. Smad4Vil-KO ; Setd2Vil-KO mice developed a more severe CRC phenotype after AOM/DSS induction, with a larger tumour size and a more vigorous epithelial proliferation rate. Further mechanistic findings revealed that the loss of SETD2 resulted in the down-regulation of DUSP7, which is involved in the inhibition of the RAS/ERK signalling pathway. Finally, the ERK1/2 inhibitor SCH772984 significantly attenuated the progression of CRC in Smad4Vil-KO ;Setd2Vil-KO mice, and overexpression of DUSP7 significantly inhibited the proliferation rates of SETD2KO ; SMAD4KO SW620 cells. Our results demonstrated that SETD2 inhibits the RAS/ERK signaling pathway by facilitating the transcription of DUSP7 in SMAD4-deficient CRC, which could provide a potential therapeutic target for the treatment of advanced CRC.

Cellular and molecular functions of SETD2 in the central nervous system.

The covalent modification of histones is critical for many biological functions in mammals, including gene regulation and chromatin structure. Posttranslational histone modifications are added and removed by specialised 'writer' and 'eraser' enzymes, respectively. One such writer protein implicated in a wide range of cellular processes is SET domain-containing 2 (SETD2), a histone methyltransferase that catalyses the trimethylation of lysine 36 on histone H3 (H3K36me3). Recently, SETD2 has also been found to modify proteins other than histones, including actin and tubulin. The emerging roles of SETD2 in the development and function of the mammalian central nervous system (CNS) are of particular interest as several SETD2 variants have been implicated in neurodevelopmental disorders, such as autism spectrum disorder and the overgrowth disorder Luscan-Lumish syndrome. Here, we summarise the numerous roles of SETD2 in mammalian cellular functions and development, with a focus on the CNS. We also provide an overview of the consequences of SETD2 variants in human disease and discuss future directions for understanding essential cellular functions of SETD2.

Histone methyltransferase SETD2 inhibits M1 macrophage polarization and glycolysis by suppressing HIF-1α in sepsis-induced acute lung injury.

Sepsis is a severe syndrome caused by the imbalance of the host response to infection, accompanied by multiple organ damage, especially acute lung injury. SET Domain-Containing 2 (SETD2) is a methyltransferase catalyzing H3 lysine 36 trimethylation (H3K36me3) that regulates multiple biological processes. This study focused on explicating the action of SETD2 on macrophage function in sepsis and the precise mechanism involved. Enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction (RT-qPCR), and Western blotting were used to determine expression. Luciferase reporter assay and chromatin immunoprecipitation assay were conducted to detect the binding of SETD2 or H3K36me3 with the hypoxia-inducible factor 1, alpha subunit (Hif1a) gene. A sepsis-induced acute lung injury model was constructed via cecal ligation and puncture (CLP). SETD2 was decreased in RAW 264.7 cells stimulated by lipopolysaccharide (LPS). Besides, SETD2 suppressed M1 macrophage polarization and glycolysis caused by LPS. HIF-1α was enhanced in RAW 264.7 cells stimulated by LPS and inversely related to SETD2 expression. In addition, SETD2-catalyzed H3K36me3 bound to the Hif1a gene to modulate HIF-1α expression. Furthermore, Hif1a silencing suppressed Setd2 silencing-induced M1 macrophage polarization and glycolysis in RAW 264.7 cells. Moreover, overexpression of Setd2 inhibited CLP-induced lung injury and M1 macrophage polarization in mice. SETD2 suppressed M1 macrophage polarization and glycolysis via regulating HIF-1α through catalyzing H3K36me3 in sepsis.

Abstract 119: Exploring The Role Of Rho-related BTB Domain Containing 1(RhoBTB1) And Phosphodiesterase 5(PDE5) To Identify Potential Proteomic Targets In Cardiovascular Function

The study of macromolecular (protein:protein) interactions has significantly enhanced our comprehension of human impairments, such as cardiovascular diseases (CVDs). We previously identified that RhoBTB1 acts as a key regulator of vascular tone by associating with Cullin-3 (CUL3) to control the activity of phosphodiesterase 5 (PDE5) through the ubiquitination-proteasomal pathway. PDE5 selectively hydrolyzes cGMP thus regulating NO-mediated vasodilation. We hypothesized that the PDE5 binding region on RhoBTB1 could be responsible for binding to other unknown partners in RhoBTB1-CUL3 pathway. We truncated RhoBTB1 into its component domains individually (GTPase, BTB1, BTB2, and CT) and in combination (B1B2C, PB1B2, and B2C) and studied their interaction with PDE5 by co-immunoprecipitation (co-IP) in HEK293 cells. These analyses revealed that the B1B2C domain of RhoBTB1 is the minimal region required to bind PDE5. Interestingly, the CT region is essential as the truncations lacking CT could not participate in PDE5 binding and its subsequent proteasomal degradation. The GTPase and Proline-rich (P) domains were dispensable for binding of RhoBTB1 to PDE5. We identified that Pro 353 and Ser 363 are key amino acid residues in the B1B2C region involved in CUL3 binding to RhoBTB1. Mutation of these amino acids resulted in hampered binding to CUL3. The mutants failed to degrade PDE5 despite their preserved binding to PDE5. We employed ascorbate peroxidase (APEX2) labeling using a B1B2C-APEX2 fusion construct as a bait to identify unknown RhoBTB1 binding partners. As a contemporaneous screen, we used B1B2-APEX2 since it does not bind to PDE5, and therefore is not expected to bind to those unknown partners. We “filtered” the captured proteome for its interaction with B1B2C better than B1B2 (without the CT). SET Domain Containing 2 (SETD2) was among several B1B2C binding partners identified and validated. SETD2 and RhoBTB1 directly interacted, and the level of SETD2 increased in response to RhoBTB1-inhibition with siRNA, inhibition of proteasomal machinery either by pharmacological means or CUL3 editing. This suggests that SETD2 is regulated by the RhoBTB1-CUL3 axis. Future studies may pave a way to determine if SETD2 is important for cardiovascular function.

Knockdown of SETD2 promotes erastin-induced ferroptosis in ccRCC

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer and is associated with poor prognosis. The histone H3 lysine 36 methyltransferase SET-domain-containing 2 (SETD2) has been reported to be expressed at low levels and frequently mutated in ccRCC. Ferroptosis, a form of death distinct from apoptosis and necrosis, has been reported in recent years in renal cancer. However, the relationship between SETD2 and ferroptosis in renal cancer is not clear. Here, we demonstrated that SETD2 was expressed at low levels in ccRCC and was associated with poor prognosis. Moreover, we found that knockdown of SETD2 increased lipid peroxidation and Fe2+ levels in tumor cells, thereby increasing the sensitivity of erastin, a ferroptosis inducer. Mechanistically, histone H3 lysine 36 trimethylation (H3K36me3) which was catalyzed by SETD2, interacted with the promoter of ferrochelatase (FECH) to regulate its transcription and ferroptosis-related signaling pathways. In conclusion, the presesnt study revealed that knockdown of the epigenetic molecule, SETD2, significantly increases the sensitivity of ferroptosis inducers which promotes tumor cell death, thereby indicating that SETD2 may be a potential therapeutic target for ccRCC.

Loss of SETD2-mediated downregulation of intracellular and exosomal miRNA-10b determines MAPK pathway activation and multidrug resistance in renal cancer.

SET domain-containing 2 (SETD2) is the most frequently mutated gene among all the histone methyltransferases in clear cell renal cell carcinoma (ccRCC). Microarrays, RNA sequencing analysis and exosomes analysis of cellular supernatant were performed after transfection A498 cells with si-SETD2 or siRNA of negative control. Chromatin immunoprecipitation and Luciferase reporter assay were conducted to evaluate the interaction between SETD2 and miR-10b. Functional and drug experiments in vitro and in vivo were performed to verify the role of SETD2, miR-10b and MAP4K4. The results showed that loss of SETD2 mediated downregulation of intracellular and exosomal microRNA-10b. MAP4K4 were relevant to oncogenesis of ccRCC caused by loss of SETD2 and miR-10b. SETD2 could directly target miR-10b and regulate the expression of multidrug resistance (MDR)-1 (P-gp170) through JNK pathway, which was one of the downstream pathways of MAP4K4. The coordinated expression of SETD2/H3K36me3/miR-10b/MAPKs/JNK/MDR pathway was revealed to the progression of ccRCC.

Abstract PR009: Linking VHL and SETD2 in a common oncogenic pathway that converges on the mitotic spindle

Abstract Loss of chromosome 3p is a landmark event in clear cell renal cell carcinoma (ccRCC) that results in mono-allelic loss of VHL (von Hippel Lindau) and SETD2 (Set-domain containing 2) (and other tumor suppressors co-located on 3p). Second hits in VHL inactivate this key tumor suppressor initiating tumor progression. SETD2, a histone methyltransferase, was previously shown to have a dual function in methylating both histones and microtubules, thereby contributing to both the histone and tubulin codes. Methylation by SETD2 on microtubules occurs at the mitotic spindle and is essential for normal mitosis and cytokinesis, with loss of SETD2 acting as a strong driver of apoptosis. This raises a conundrum of how cancer cells survive early mono-allelic loss of SETD2, escaping cell death. We have identified the mitotic kinase, Aurora kinase A (AURKA), as a regulator of SETD2. Our data uncover SETD2 as a unique substrate for phosphorylation by AURKA, with mass spectrometry identifying serine 2080 (S2080) as the site of phosphorylation on SETD2. Phospho-SETD2 (S2080) localized at the spindle poles during mitosis, and this specific localization was modulated by AURKA enzymatic activity. Initial observations reveal a role for AURKA driven SETD2 phosphorylation at S2080 in nucleocytoplasmic shuttling of this methyltransferase. Our data suggest that phosphorylation of SETD2 by AURKA contributes to both its methyltransferase (i.e. enzymatic) activity and its geo-spatial location with the cell. Importantly, AURKA expression levels are high in VHL-null cells resulting from an inability of VHL to target AURKA for degradation and our data now highlight a direct link between VHL and SETD2, two tumor suppressors believed to independently drive RCC pathogenesis. In summary, our data reveal a tumor-specific vulnerability linked to mitotic fragility that can be precisely targeted to ultimately drive mitotic catastrophe. Citation Format: Ruhee Dere, Pratim Chowdhury, Sung Yun Jung, Rim Aboulhassane, Manga Motrapu, Xiaoli Wang, Richard Han, Cheryl Walker, Ian Frew, W. Kimryn Rathmell. Linking VHL and SETD2 in a common oncogenic pathway that converges on the mitotic spindle [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR009.

Structure and Function of RhoBTB1 Required for Substrate Specificity and Cullin-3 Ubiquitination.

We identified Rho-related BTB domain containing 1 (RhoBTB1) as a key regulator of phosphodiesterase 5 (PDE5) activity, and through PDE5, a regulator of vascular tone. We identified the binding interface for PDE5 on RhoBTB1 by truncating full-length RhoBTB1 into its component domains. Co-immunoprecipitation analyses revealed that the C-terminal half of RhoBTB1 containing its two BTB domains and the C-terminal domain (B1B2C) is the minimal region required for PDE5 recruitment and subsequent proteasomal degradation via Cullin-3 (CUL3). The C-terminal domain was essential in recruiting PDE5 as constructs lacking this region could not participate in PDE5 binding or proteasomal degradation. We also identified Pro353 and Ser363 as key amino acid residues in the B1B2C region involved in CUL3 binding to RhoBTB1. Mutation of either of these residues exhibited impaired CUL3 binding and PDE5 degradation, although the binding to PDE5 was preserved. Finally, we employed ascorbate peroxidase 2 (APEX2) proximity labeling using a B1B2C-APEX2 fusion protein as bait to capture unknown RhoBTB1 binding partners. Among several B1B2C-binding proteins identified and validated, we focused on SET domain containing 2 (SETD2). SETD2 and RhoBTB1 directly interacted, and the level of SETD2 increased in response to pharmacological inhibition of the proteasome or Cullin complex, CUL3 deletion, and RhoBTB1-inhibition with siRNA. This suggests that SETD2 is regulated by the RhoBTB1-CUL3 axis. Future studies will determine whether SETD2 plays a role in cardiovascular function.

SETD2-H3K36ME3: an important bridge between the environment and tumors.

Epigenetic regulation plays an important role in the occurrence, development and treatment of tumors. The histone methyltransferase SET-domain-containing 2 (SETD2) plays a key role in mammalian epigenetic regulation by catalyzing histone methylation and interacting with RNA polymerase II to mediate transcription elongation and mismatch repair. As an important bridge between the environment and tumors, SETD2-H3K36me3 plays an important role in the occurrence and development of tumors. Many tumors, including renal cancer, gastric cancer, lung cancer, are closely related to SETD2 gene mutations. As a key component of common tumor suppressor mechanisms, SETD2-H3K36me3is an important target for clinical disease diagnosis and treatment. Here, we reviewed the structure and function of the SETD2 and how SETD2-H3K36me3 functions as a bridge between the environment and tumors to provide an in-depth understanding of its role in the occurrence and development of various tumors, which is of great significance for future disease diagnosis and treatment.

Targeted Profiling of Epitranscriptomic Reader, Writer, and Eraser Proteins Regulated by H3K36me3.

Trimethylation of lysine 36 on histone H3 (H3K36me3), an epigenetic mark associated with actively transcribed genes, plays an important role in multiple cellular processes, including transcription elongation, DNA methylation, DNA repair, etc. Aberrant expression and mutations of the main methyltransferase for H3K36me3, i.e., SET domain-containing 2 (SETD2), were shown to be associated with various cancers. Here, we performed targeted profiling of 154 epitranscriptomic reader, writer, and eraser (RWE) proteins using a scheduled liquid chromatography-parallel-reaction monitoring (LC-PRM) method coupled with the use of stable isotope-labeled (SIL) peptides as internal standards to investigate how H3K36me3 modulates the chromatin occupancies of epitranscriptomic RWE proteins. Our results showed consistent changes in chromatin occupancies of RWE proteins upon losses of H3K36me3 and H4K16ac and a role of H3K36me3 in recruiting METTL3 to chromatin following induction of DNA double-strand breaks. In addition, protein-protein interaction network and Kaplan-Meier survival analyses revealed the importance of METTL14 and TRMT11 in kidney cancer. Taken together, our work unveiled cross-talks between histone epigenetic marks (i.e., H3K36me3 and H4K16ac) and epitranscriptomic RWE proteins and uncovered the potential roles of these RWE proteins in H3K36me3-mediated biological processes.