ABSTRACT
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.
ABSTRACT
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 â¼12-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.
Subject(s)
Histone-Lysine N-Methyltransferase/chemistry , Histone-Lysine N-Methyltransferase/metabolism , Proto-Oncogene Proteins c-rel/chemistry , Proto-Oncogene Proteins c-rel/metabolism , Catalytic Domain , Kinetics , Methylation , Substrate SpecificityABSTRACT
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.
Subject(s)
Glutamine , Lysine , Site-Specific DNA-Methyltransferase (Adenine-Specific) , Humans , Glutamine/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Lysine/metabolism , Methylation , Peptides/chemistry , Protein Methyltransferases/metabolism , Substrate Specificity , Site-Specific DNA-Methyltransferase (Adenine-Specific)/geneticsABSTRACT
The transcriptional coactivator BRD4 has a fundamental role in transcription regulation and thus became a promising epigenetic therapeutic candidate to target diverse pathologies. However, the regulation of BRD4 by posttranslational modifications has been largely unexplored. Here, we show that BRD4 is methylated on chromatin at lysine-99 by the protein lysine methyltransferase SETD6. BRD4 methylation negatively regulates the expression of genes that are involved in translation and inhibits total mRNA translation in cells. Mechanistically, we provide evidence that supports a model where BRD4 methylation by SETD6 does not have a direct role in the association with acetylated histone H4 at chromatin. However, this methylation specifically determines the recruitment of the transcription factor E2F1 to selected target genes that are involved in mRNA translation. Together, our findings reveal a previously unknown molecular mechanism for BRD4 methylation-dependent gene-specific targeting, which may serve as a new direction for the development of therapeutic applications.
Subject(s)
Cell Cycle Proteins , Nuclear Proteins , Protein Methyltransferases , Transcription Factors , Cell Cycle Proteins/genetics , Chromatin , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Humans , Methylation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Biosynthesis , Protein Methyltransferases/genetics , Protein Processing, Post-Translational , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
Point Mutation , Protein Methyltransferases/chemistry , Protein Methyltransferases/metabolism , S-Adenosylmethionine/metabolism , Gene Expression Regulation, Enzymologic , HEK293 Cells , Humans , Lysine/metabolism , Methylation , Models, Molecular , Molecular Weight , Oxidative Stress , Protein Conformation , Protein Methyltransferases/genetics , Protein MultimerizationABSTRACT
SETD3 is a member of the protein lysine methyltransferase (PKMT) family, which catalyzes the addition of methyl group to lysine residues. Accumulating data suggest that PKMTs are involved in the regulation of a broad spectrum of biological processes by targeting histone and non-histone proteins. Using a proteomic approach, we have identified 172 new SETD3 interacting proteins. We show that SETD3 binds and methylates the transcription factor FoxM1, which has been previously shown to be associated with the regulation of VEGF expression. We further demonstrate that under hypoxic conditions SETD3 is down-regulated. Mechanistically, we find that under basal conditions, SETD3 and FoxM1 are enriched on the VEGF promoter. Dissociation of both SETD3 and FoxM1 from the VEGF promoter under hypoxia correlates with elevated expression of VEGF. Taken together, our data reveal a new SETD3-dependent methylation-based signaling pathway at chromatin that regulates VEGF expression under normoxic and hypoxic conditions.