Lysine methyltransferase SETD6 modifies histones on a glycine-lysine motif.
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ABSTRACT:
SUBMITTER: Binda O
PROVIDER: S-EPMC6961689 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:P21-activated kinase 4 (PAK4), a member of serine/threonine kinases family is over-expressed in numerous cancer tumors and is associated with oncogenic cell proliferation, migration and invasion. Our recent work demonstrated that the SET-domain containing protein 6 (SETD6) interacts with and methylates PAK4 at chromatin in mammalian cells, leading to activation of the Wnt/β-catenin signaling pathway. In our current work, we identified lysine 473 (K473) on PAK4 as the primary methylation site by SETD6. Methylation of PAK4 at K473 activates β-catenin transcriptional activity and inhibits cell adhesion. Specific methylation of PAK4 at K473 also attenuates paxillin localization to focal adhesions leading to overall reduction in adhesion-related features, such as filopodia and actin structures. The altered adhesion of the PAK4 wild-type cells is accompanied with a decrease in the migrative and invasive characteristics of the cells. Taken together, our results suggest that methylation of PAK4 at K473 plays a vital role in the regulation of cell adhesion and migration.
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Project description: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.
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Project description:Histone protein post-translational modifications (PTMs) are significant for gene expression and DNA repair. Here we report the identification and validation of a new type of PTM in histones, lysine succinylation. The identified lysine succinylated histone peptides were verified by MS/MS of synthetic peptides, HPLC co-elution, and isotopic labeling. We identified 13, 7, 10, and 7 histone lysine succinylation sites in HeLa, mouse embryonic fibroblast, Drosophila S2, and Saccharomyces cerevisiae cells, respectively. We demonstrated that this histone PTM is present in all eukaryotic cells we examined. Mutagenesis of succinylation sites followed by functional assays implied that histone lysine succinylation can cause unique functional consequences. We also identified one and two histone lysine malonylation sites in HeLa and S. cerevisiae cells, respectively. Our results therefore increase potential combinatorial diversity of histone PTMs and suggest possible new connections between histone biology and metabolism.
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Project description:BackgroundEpigenetic 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.MethodsUsing 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.ResultsOur 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.ConclusionsTogether, 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.
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Project description: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.
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Project description:Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-?B subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-?B-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-? (PKC-?) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.
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Project description:The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1(bd). These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output.
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Project description:Post-translational modifications (PTMs) regulate numerous proteins and are important for many biological processes. Lysine 4-oxononanoylation (4-ONylation) is a newly discovered histone PTM that prevents nucleosome assembly under oxidative stress. Whether there are cellular enzymes that remove 4-ONyl from histones remains unknown, which hampers the further investigation of the cellular function of this PTM. Here, we report that mammalian SIRT2 can remove 4-ONyl from histones and other proteins in live cells. A crystal structure of SIRT2 in complex with a 4-ONyl peptide reveals a lone pair-? interaction between Phe119 and the ketone oxygen of the 4-ONyl group. This is the first time that a mechanism to reverse 4-ONyl lysine modification is reported and will help to understand the role of SIRT2 in oxidative stress responses and the function of 4-ONylation.
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Project description:Rice is the most consumed cereal grain in the world, but deficient in the essential amino acid lysine. Therefore, people in developing countries with limited food diversity who rely on rice as their major food source may suffer from malnutrition. Biofortification of stable crops by genetic engineering provides a fast and sustainable method to solve this problem. In this study, two endogenous rice lysine-rich histone proteins, RLRH1 and RLRH2, were over-expressed in rice seeds to achieve lysine biofortification. Their protein sequences passed an allergic sequence-based homology test. Their accumulations in rice seeds were raised to a moderate level by the use of a modified rice glutelin 1 promoter with lowered expression strength to avoid the occurrence of physiological abnormalities like unfolded protein response. The expressed proteins were further targeted to protein storage vacuoles for stable storage using a glutelin 1 signal peptide. The lysine content in the transgenic rice seeds was enhanced by up to 35 %, while other essential amino acids remained balanced, meeting the nutritional standards of the World Health Organization. No obvious unfolded protein response was detected. Different degrees of chalkiness, however, were detected in the transgenic seeds, and were positively correlated with both the levels of accumulated protein and lysine enhancement. This study offered a solution to the lysine deficiency in rice, while at the same time addressing concerns about food safety and physiological abnormalities in biofortified crops.
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