Methylation of the transcription factor E2F1 by SETD6 regulates SETD6 expression via a positive feedback mechanism.

The protein lysine methyltransferase SET domain-containing protein 6 (SETD6) has been shown to influence different cellular activities and to be critically involved in the regulation of diverse developmental and pathological processes. However, the upstream signals that regulate the mRNA expression of SETD6 are not known. Bioinformatic analysis revealed that the SETD6 promoter has a binding site for the transcription factor E2F1. Using various experimental approaches, we show that E2F1 binds to the SETD6 promoter and regulates SETD6 mRNA expression. Our further observation that this phenomenon is SETD6 dependent suggested that SETD6 and E2F1 are linked. We next demonstrate that SETD6 monomethylates E2F1 specifically at K117 in vitro and in cells. Finally, we show that E2F1 methylation at K117 positively regulates the expression level of SETD6 mRNA. Depletion of SETD6 or overexpression of E2F1 K117R mutant, which cannot be methylated by SETD6, reverses the effect. Taken together, our data provide evidence for a positive feedback mechanism, which regulates the expression of SETD6 by E2F1 in a SETD6 methylation-dependent manner, and highlight the importance of protein lysine methyltransferases and lysine methylation signaling in the regulation of gene transcription.

SETD6 Regulates E2-Dependent Human Papillomavirus Transcription.

Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal domain (BET) family of proteins. Brd4 regulates human papillomavirus (HPV) transcription, genome replication, and segregation by binding to the E2 protein. The SETD6 methyltransferase binds to and methylates Brd4 at lysine 99. We investigated the interactions of SETD6 and Brd4 with E2 and their role in HPV transcription. SETD6 coimmunoprecipitated with the E2 transactivation domain, and its depletion in CIN612 episomal cells reduced human papillomavirus type 31 (HPV-31) transcription, whereas depletion of SETD6 in integrated HPV cell lines had no effect on viral gene expression. The mutant Brd4 K99R (bearing a change of K to R at position 99), which cannot be methylated by SETD6, displayed decreased binding to HPV-31 E2, suggesting that SETD6 methylation of Brd4 also influences E2 association with the Brd4 protein. Using chromatin immunoprecipitation, SETD6 was detected at the enhancer region of the HPV long control region. We propose that methylation of Brd4 at K99 by SETD6 is an important mechanism for E2-Brd4 association and HPV transcriptional activation. IMPORTANCE Human papillomaviruses (HPV) cause cervical, anogenital, and oral cancers. Brd4 plays an important role in the HPV life cycle. SETD6 was recently shown to methylate Brd4. The current study demonstrates that methylation of Brd4 by SETD6 in HPV-episomal cells is required for the activation of viral transcription. This study illustrates a novel regulatory mechanism involving E2, Brd4, and SETD6 in the HPV life cycle and provides insight into the multiple roles of Brd4 in viral pathogenesis.

The methyltransferase SETD6 regulates Mitotic progression through PLK1 methylation.

Lysine methylation, catalyzed by protein lysine methyltransferases (PKMTs), is a key player in regulating intracellular signaling pathways. However, the role of PKMTs and the methylation of nonhistone proteins during the cell cycle are largely unexplored. In a recent proteomic screen, we identified that the PKMT SETD6 methylates PLK1-a key regulator of mitosis and highly expressed in tumor cells. In this study, we provide evidence that SETD6 is involved in cell cycle regulation. SETD6-deficient cells were observed to progress faster through the different mitotic steps toward the cytokinesis stage. Mechanistically, we found that during mitosis SETD6 binds and methylates PLK1 on two lysine residues: K209 and K413. Lack of methylation of these two residues results in increased kinase activity of PLK1, leading to accelerated mitosis and faster cellular proliferation, similarly to SETD6-deficient cells. Taken together, our findings reveal a role for SETD6 in regulating mitotic progression, suggesting a pathway through which SETD6 methylation activity contributes to normal mitotic pace.

Enhanced PKMT-substrate recognition through non active-site interactions.

Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on many different cellular proteins. Despite extensive biochemical and structural studies, focusing on PKMT active site-peptide interactions, little is known regarding how PKMTs recognize globular substrates. To examine whether these enzymes recognize protein substrates through interactions that take place outside of the active site, we have measured SETD6 and SETD7 activity with both protein and peptide RelA substrate. We have utilized the MTase-Glo™ methyltransferase assay to measure the activity of SETD6 and SETD7 with the different RelA substrates and calculated the Michaelis-Menten (MM) parameters. We found an up to ∼12-fold increase in KM of the PKMTs activity with RelA peptide relative to the respective full-length protein, emphasizing the significantly higher PKMT-protein interaction affinity. Examination of SETD6 and SETD7 activity toward the same RelA substrates highlight the similarity in substrate recognition for both PKMTs. Our results show that the interaction affinity of SETD6 and SETD7 with RelA is enhanced through interactions that occur outside of the active site leading to higher catalytic efficiency and specificity. These interactions can significantly vary depending on the PKMT and the specific methylation site on RelA. Overall, our results underline that PKMTs can recognize their substrates through docking interactions that occur out of the active site-peptide region for enhancing their activity and specificity in the cellular environment.

PAK4 Methylation by SETD6 Promotes the Activation of the Wnt/ß-Catenin Pathway.

Lysine methylation of non-histone proteins has emerged as a key regulator of many cellular functions. Although less studied than other post-translational modifications such as phosphorylation and acetylation, the number of known methylated non-histone proteins is rapidly expanding. We have identified the p21-activated kinase 4 (PAK4) as a new substrate for methylation by the protein lysine methyltransferase SETD6. Our data demonstrate that SETD6 methylates PAK4 bothin vitroand at chromatin in cells. Interestingly, depletion of SETD6 in various cellular systems significantly hinders the activation of the Wnt/ß-catenin target genes. PAK4 was recently shown to regulate ß-catenin signaling, and we show that SETD6 is a key mediator of this pathway. In the presence of SETD6, the physical interaction between PAK4 and ß-catenin is dramatically increased, leading to a significant increase in the transcription of ß-catenin target genes. Taken together, our results uncover a new regulatory layer of the Wnt/ß-catenin signaling cascade and provide new insight into SETD6 biology.

SETD6 is a negative regulator of oxidative stress response.

The protein methyltransferase SETD6 is a key regulator of proliferation and inflammatory processes. However, the role of SETD6 in the regulation of additional cell signaling pathways has not been well studied. Here we show that SETD6 is a negative regulator of the oxidative stress response. Depletion of SETD6 from cells results in elevated Nrf2 levels and a significant increase in Nrf2 antioxidant target gene expression. Using proteomic tools, we uncovered a novel interaction between SETD6 and the oxidative stress sensor DJ1, a protein required for Nrf2-dependent transcription of antioxidant target genes. We show that SETD6 binds DJ1 both in-vitro and in cells but does not methylate DJ1. Under basal conditions, SETD6 and DJ1 are associated at chromatin. Through this interaction, SETD6 inhibits DJ1 activity, which in turn leads to the repression of Nrf2-dependent transcription. In response to oxidative stress, the transcription of Nrf2 antioxidant genes increases. We here show that under this condition, SETD6 mRNA and protein levels are reduced, leading to elevation in Nrf2 expression level and to a weaken interaction between SETD6 and DJ1 at chromatin. Taken together, these findings demonstrate that SETD6 negatively regulates the Nrf2-mediated oxidative stress response through a physical and catalytically independent interaction with DJ1 at chromatin.

Distinct specificities of the HEMK2 protein methyltransferase in methylation of glutamine and lysine residues.

The HEMK2 protein methyltransferase has been described as glutamine methyltransferase catalyzing ERF1-Q185me1 and lysine methyltransferase catalyzing H4K12me1. Methylation of two distinct target residues is unique for this class of enzymes. To understand the specific catalytic adaptations of HEMK2 allowing it to master this chemically challenging task, we conducted a detailed investigation of the substrate sequence specificities of HEMK2 for Q- and K-methylation. Our data show that HEMK2 prefers methylation of Q over K at peptide and protein level. Moreover, the ERF1 sequence is strongly preferred as substrate over the H4K12 sequence. With peptide SPOT array methylation experiments, we show that Q-methylation preferentially occurs in a G-Q-X3 -R context, while K-methylation prefers S/T at the first position of the motif. Based on this, we identified novel HEMK2 K-methylation peptide substrates with sequences taken from human proteins which are methylated with high activity. Since H4K12 methylation by HEMK2 was very low, other protein lysine methyltransferases were examined for their ability to methylate the H4K12 site. We show that SETD6 has a high H4K12me1 methylation activity (about 1000-times stronger than HEMK2) and this enzyme is mainly responsible for H4K12me1 in DU145 prostate cancer cells.

Structure-function conservation between the methyltransferases SETD3 and SETD6.

Among the protein lysine methyltransferases family members, it appears that SETD6 is highly similar and closely related to SETD3. The two methyltransferases show high similarity in their structure, which raised the hypothesis that they share cellular functions. Using a proteomic screen, we identified 52 shared interacting-proteins. Gene Ontology (GO) analysis of the shared proteins revealed significant enrichment of proteins involved in transcription. Our RNA-seq data of SETD6 KO and SETD3 KO HeLa cells identified ∼100 up-regulated and down-regulated shared genes. We have also identified a substantial number of genes that changed dramatically in the double KO cells but did not significantly change in the single KO cells. GO analysis of these genes revealed enrichment of apoptotic genes. Accordingly, we show that the double KO cells displayed high apoptotic levels, suggesting that SETD6 and SETD3 inhibit apoptosis. Collectively, our data strongly suggest a functional link between SETD6 and SETD3 in the regulation of apoptosis.

Lysine methyltransferase SETD6 modifies histones on a glycine-lysine motif.

Although central to regulating the access to genetic information, most lysine methyltransferases remain poorly characterised relative to other family of enzymes. Herein, I report new substrates for the lysine methyltransferase SETD6. Based on the SETD6-catalysed site on the histone variant H2AZ, I identified similar sequences in the canonical histones H2A, H3, and H4 that are modified by SETD6 in vitro, and putative non-histone substrates. I herein expend the repertoire of substrates for methylation by SETD6.

The SETD6 Methyltransferase Plays an Essential Role in Hippocampus-Dependent Memory Formation.

BACKGROUND: Epigenetic mechanisms are critical for hippocampus-dependent memory formation. Building on previous studies that implicate the N-lysine methyltransferase SETD6 in the activation of nuclear factor-κB RELA (also known as transcription factor p65) as an epigenetic recruiter, we hypothesized that SETD6 is a key player in the epigenetic control of long-term memory. METHODS: Using a series of molecular, biochemical, imaging, electrophysiological, and behavioral experiments, we interrogated the effects of short interfering RNA-mediated knockdown of Setd6 in the rat dorsal hippocampus during memory consolidation. RESULTS: Our findings demonstrate that SETD6 is necessary for memory-related nuclear factor-κB RELA methylation at lysine 310 and associated increases in H3K9me2 (histone H3 lysine 9 dimethylation) in the dorsal hippocampus and that SETD6 knockdown interferes with memory consolidation, alters gene expression patterns, and disrupts spine morphology. CONCLUSIONS: Together, these findings suggest that SETD6 plays a critical role in memory formation and may act as an upstream initiator of H3K9me2 changes in the hippocampus during memory consolidation.

Peptide inhibition of the SETD6 methyltransferase catalytic activity.

A large body of evidence accumulating in the past few years indicates the physiological significance of non-histone proteins lysine methylation, catalyzed by protein lysine methyl transferases (PKMTs). Dysregulation of these enzymes was shown to contribute to the development and progression of numerous diseases. SETD6 lysine methylatransferase was recently shown to participate in essential cellular processes, such as the NFkB pathway, oxidative stress and also the Wnt signaling cascade. In order to test the effect of blocking SETD6 catalytic activity, we used the peptide inhibition method, which is considered highly specific and can potentially target almost any protein. We designed a 15 amino acids peptide based on the sequence of the RelA protein (residues 302-316), containing the lysine that is methylated by SETD6. To enable cellular intake, the designed peptide was fused to a cell penetrating peptide (CPP) vp22. The vp22-RelA302-316 peptide showed direct and specific interaction with SETD6 in vitro. This interaction was shown to inhibit SETD6 methyltransferase activity. SETD6 catalytic blockage by the peptide was also observed in cells upon treatment with the vp22-RelA302-316, resulting in induced cellular migration and proliferation. This new insight into the activity of a methylation inhibitory peptide could represent a milestone in the development of therapeutic tools, which can be of use in physiological cases where administration of cell proliferation is required.

Oligomerization and Auto-methylation of the Human Lysine Methyltransferase SETD6.

Signaling via lysine methylation by protein lysine methyltransferases (PKMTs), has been linked to diverse biological and disease processes. The mono-methyltransferase SETD6 (SET-domain-containing protein 6) is a member of the PKMT family and was previously shown to regulate essential cellular processes such as the NF-κB, WNT and the oxidative stress pathways. However, on the biochemical level, little is known about the enzymatic mode of action of SETD6. Here we provide evidence that SETD6 forms high-molecular-weight structures. Specifically, we demonstrate that SETD6 monomeric, dimeric and trimeric forms are stabilized by the methyl donor, S-adenosyl-l-methionine. We then show that SETD6 has auto-methylation activity at K39 and K179, which serves as the major auto-methylation sites with a moderate auto-methylation activity toward K372. A point mutation at K179 but not at K39 and K372, located at the SET domain of SETD6, impaired SETD6 ability to form a trimer, strongly implying a link between the auto-methylation and the oligomerization state. Finally, by radioactive in vitro methylation experiments and biochemical kinetics analysis, we show that the auto-methylation at K39 and K179 increases the catalytic rate of SETD6. Collectively, our data support a model by which SETD6 auto-methylation and self-interaction positively regulate its enzymatic activity in vitro and may suggest that other PKMTs are regulated in the same manner.

SETD6 regulates NF-κB signaling in urothelial cell survival: Implications for bladder cancer.

Non-muscle invasive bladder cancer has a high recurrence rate of 45-70%, progressing to muscle invasive disease in about 15% of those patients over a 5-year period. Administration of the mycobacterium, Bacillus Calmette-Guerin (BCG) that induces local inflammation resulting in tumor remission in responsive patients is frequently used for treatment. BCG-treated patients with NF-κB del/del genotype have an increased risk of recurrence suggesting an important role of NF-κB in bladder cancer. Since protein methyltransferases play critical roles in modulating chromatin structure and gene expression, we screened a focused array of epigenetic modification genes to identify differential expression between normal urothelial and bladder cancer cells. We found and validated high expression of the SET-domain-containing protein methyltransferase, SETD6. SETD6 monomethylates NF-κB-p65 at lysine 310. Our results show that primary urothelial cells and normal bladder tissue have nearly undetectable message and protein level of SETD6 that increases in transformed urothelial cells and is further increased in bladder cancer cells and tissues. Overexpression of SETD6 in transformed urothelial cells increased cell survival and colony formation while knockdown in cancer cells decreased both parameters. Luciferase reporter assays showed that SETD6 induced the canonical NF-κB signaling pathway. Further, the use of catalytic SETD6 and IκBα mutant shows that SETD6 positively regulates survival by affecting p65 message, protein level and its function as determined by increased expression of NF-κB target genes. Our findings suggest that SETD6 plays an important role in NF-κB regulation and may have an important role in NF-κB-mediated local inflammatory response following BCG treatment.

Proteomic analysis of SETD6 interacting proteins.

SETD6 (SET-domain-containing protein 6) is a mono-methyltransferase that has been shown to methylate RelA and H2AZ. Using a proteomic approach we recently identified several new SETD6 substrates. To identify novel SETD6 interacting proteins, SETD6 was immunoprecipitated (IP) from Human erythromyeloblastoid leukemia K562 cells. SETD6 binding proteins were subjected to mass-spectrometry analysis resulting in 115 new SETD6 binding candidates. STRING database was used to map the SETD6 interactome network. Network enrichment analysis of biological processes with Gene Ontology (GO) database, identified three major groups; metabolic processes, muscle contraction and protein folding.

SETD6 controls the expression of estrogen-responsive genes and proliferation of breast carcinoma cells.

The lysine methyltransferase SETD6 modifies the histone variant H2AZ, a key component of nuclear receptor-dependent transcription. Herein, we report the identification of several factors that associate with SETD6 and are implicated in nuclear hormone receptor signaling. Specifically, SETD6 associates with the estrogen receptor α (ERα), histone deacetylase HDAC1, metastasis protein MTA2, and the transcriptional co-activator TRRAP. Luciferase reporter assays identify SETD6 as a transcriptional repressor, in agreement with its association with HDAC1 and MTA2. However, SETD6 behaves as a co-activator of several estrogen-responsive genes, such as PGR and TFF1. Consistent with these results, silencing of SETD6 in several breast carcinoma cell lines induced cellular proliferation defects accompanied by enhanced expression of the cell cycle inhibitor CDKN1A and induction of apoptosis. Herein, we have identified several chromatin proteins that associate with SETD6 and described SETD6 as an essential factor for nuclear receptor signaling and cellular proliferation.

SETD6 monomethylates H2AZ on lysine 7 and is required for the maintenance of embryonic stem cell self-renewal.

The histone H2A variant H2AZ is an essential chromatin signaling factor. Herein, we report that H2AZ is monomethylated at lysine 7 (H2AZK7me1) by the lysine methyltransferase SETD6. We observed that methylation of H2AZ increased noticeably upon cellular differentiation of mouse embryonic stem cells (mESCs). H2AZK7me1 and the repressive H3K27me3 mark were found near the transcriptional start sites of differentiation marker genes, but were removed upon retinoic acid-induced cellular differentiation. The depletion of Setd6 in mESCs led to cellular differentiation, compromised self-renewal, and poor clonogenicity. These findings demonstrate that mESCs require Setd6 for self-renewal and portray H2AZK7me1 as a marker of cellular differentiation.