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H3K79 methyltransferase Dot1p/DOT1L is phosphorylated by proline-directed MAP kinases Hog1p and p38/MAPK11

ABSTRACT: In vitro phosphorylation assays were conducted to identify phosphorylation sites on Dot1p catalysed by Hog1p. As several other kinases were co-purified with Hog1p, dead kinase control assays (Hog1p-K52R and Hog1p-D144A) were performed to verify the identified Dot1p phospho-sites. This entry contains the data pertaining to the in vitro Hog1p dead kinase phosphorylation assays, which is in supplement to existing PRIDE entry PXD036747.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Ryan Separovich

LAB HEAD: Dr Marc R Wilkins

PROVIDER: PXD040407 | Pride | 2024-01-29

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	Dot1_from_BY4741_chymo_3ul.dat	Other
	Dot1_from_BY4741_chymo_3ul.mgf	Mgf
	Dot1_from_BY4741_chymo_3ul.raw	Raw
	Dot1_from_BY4741_tryp_3ul.dat	Other
	Dot1_from_BY4741_tryp_3ul.mgf	Mgf

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Publications

Proline-directed yeast and human MAP kinases phosphorylate the Dot1p/DOT1L histone H3K79 methyltransferase.

Separovich Ryan J RJ Karakatsanis Nicola M NM Gao Kelley K Fuh David D Hamey Joshua J JJ Wilkins Marc R MR

The FEBS journal 20240125 12

Disruptor of telomeric silencing 1 (Dot1p) is an exquisitely conserved histone methyltransferase and is the sole enzyme responsible for H3K79 methylation in the budding yeast Saccharomyces cerevisiae. It has been shown to be highly phosphorylated in vivo; however, the upstream kinases that act on Dot1p are almost entirely unknown in yeast and all other eukaryotes. Here, we used in vitro and in vivo kinase discovery approaches to show that mitogen-activated protein kinase HOG1 (Hog1p) is a bona f ...[more]

PMID: 38270553

Similar Datasets

H3K79 methyltransferase Dot1p/DOT1L is phosphorylated by proline-directed MAP kinases Hog1p and p38/MAPK11

Project description:Disruptor of telomeric silencing (Dot1p) is an exquisitely conserved histone methyltransferase, and is the sole enzyme responsible for H3K79 methylation in the budding yeast, Saccharomyces cerevisiae. It has been shown to be highly phosphorylated in vivo; however, the upstream kinases that act on Dot1p are unknown in yeast and all other eukaryotes. Here, we used in vitro and in vivo kinase discovery approaches to show that the mitogen-activated protein kinase Hog1p is a bona fide kinase of Dot1p methyltransferase. In in vitro kinase assays, Hog1p phosphorylates Dot1p at multiple sites, including at several proline-adjacent sites that are consistent with its known substrate preference. Hog1p kinase activity was specifically enhanced at these proline-adjacent sites on Dot1p upon its activation by the osmostress-responsive kinase, Pbs2p. We then show that genomic deletion of HOG1 markedly reduces phosphorylation at specific sites on Dot1p in vivo, thus providing evidence for Hog1p kinase activity on Dot1p in budding yeast cells. Phenotypic analysis of knockout and phosphosite mutant yeast strains revealed the importance of Hog1p-catalysed phosphorylation of Dot1p for cellular responses to ultraviolet-induced DNA damage. This kinase-substrate relationship is conserved in mammalian systems, where human DOT1L (ortholog of Dot1p) can be phosphorylated by the proline-directed kinase p38β/MAPK11 (ortholog of Hog1p) at multiple sites in vitro. Taken together, our findings establish Hog1p and p38β/MAPK11 as the first kinases known to act on H3K79 methyltransferase enzymes in any eukaryotic species.

2024-01-29 | PXD036747 | Pride

Post-translational modification profiling of Saccharomyces cerevisiae histone methylation enzymes uncovers regulation by upstream signalling pathways

Project description:Histone methylation is central to the regulation of eukaryotic transcription. In Saccharomyces cerevisiae, it is controlled by a system of four methyltransferases (Set1p, Set2p, Set5p, and Dot1p) and four demethylases (Jhd1p, Jhd2p, Rph1p, and Gis1p). While the histone targets for these enzymes are well characterised, the connection of the enzymes with the intracellular signalling network and thus their regulation is poorly understood, in yeast and in all other eukaryotes. Here we report the detailed characterisation of the eight S. cerevisiae enzymes, and show that they carry a total of 75 phosphorylation sites, 93 acetylation sites, and two ubiquitination sites. All enzymes are subject to phosphorylation, although demethylases Jhd1p and Jhd2p contained one and five sites respectively whereas other enzymes carried 14 to 36 sites. Phosphorylation was absent or under-represented on catalytic and other domains but strongly enriched for regions of disorder on methyltransferases, suggesting a role in the modulation of protein-protein interactions. We show that a phosphorylation cluster within an acidic and intrinsically disordered N-terminal region of methyltransferase Set2p regulates H3K36 methylation levels in vivo, thus supporting the functional relevance of disordered phosphosites. While most kinases upstream of the yeast histone methylation enzymes remain unknown, we model the possible connections between the signalling network and the histone-based gene regulatory system and propose an integrated regulatory structure. Our results provide a foundation for future, detailed exploration of the role of specific kinases and phosphosites in the regulation of histone methylation.

2020-12-15 | PXD021214 | Pride

Phosphorylation of methyltransferase Set2p and demethylase Jhd1p and its association with histone H3K36 methylation and stress response pathways in Saccharomyces cerevisiae

Project description:Histone lysine methylation is a key epigenetic modification that regulates eukaryotic transcription. In Saccharomyces cerevisiae, it is controlled by a reduced but evolutionarily conserved suite of methyltransferase (Set1p, Set2p, Dot1p, and Set5p) and demethylase (Jhd1p, Jhd2p, Rph1p, and Gis1p) enzymes. Many of these enzymes are extensively phosphorylated in vivo; however, the functions of specific phosphosites are poorly understood. Here, we comprehensively investigate the phosphoregulation of the yeast histone methylation network by analysing 40 phosphosites on six enzymes through mutagenesis. A total of 82 genomically-edited S. cerevisiae strains were generated and screened for changes in native H3K4, H3K36, and H3K79 methylation levels, and for sensitivity to environmental stress conditions. This demonstrated the functional relevance of phosphosites on methyltransferase Set2p (S6, S8, S10, and T127) and demethylase Jhd1p (S44) in the regulation of H3K36 methylation in vivo, and in the coordination of specific stress response pathways in budding yeast. Proteomic analysis of SET2 mutants revealed that phosphorylation site mutations lead to significant downregulation of membrane-associated proteins and processes, consistent with changes brought about by SET2 deletion. This study represents the first systematic investigation into the phosphoregulation of an entire epigenetic network in any eukaryote, and our findings establish phosphorylation as an important regulator of histone lysine methylation in S. cerevisiae.

2022-03-07 | PXD029088 | Pride

Genome-wide profiling of CTCF binding (ChIP-seq) in A549 and U2OS cells

Project description:To identify genome-wide CCCTC binding factor (CTCF)-binding in A549 (ATCC CCL-185) and U2OS cells stably expressing rat GR (Rogatsky et al. , Mol Cell Biol, 1997. 17(6): p. 3181-93.) we performed ChIP-seq experiments upon hormone treatment (1.5 h, 1 M dexamethasone).

2018-04-12 | E-MTAB-5352 | biostudies-arrayexpress

Biochemical Foundations of Health and Energy Conservation in Hibernating Free-Ranging Subadult Brown Bear Ursus arctos

Project description:Brown bears (Ursus arctos) hibernate for 5-7 months without eating, drinking, urinating and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We analyzed the physiological adaptations for hibernation by comparing the proteome, metabolome, and hematologic features of blood from hibernating and active free-ranging sub-adult brown bears with a focus on conservation of health and energy. We found that the concentrations of most plasma proteins and white blood cell types decreased during hibernation. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad-specificity proteins. A dramatic 45-fold increase in sex-hormone-binding-globulin SHBG levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms, (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6°C reduction in body temperature, decreased protease activity, and increased plasma viscosity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of few key proteins. This comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation, and have implications for our understanding of physiology in general.

2016-09-27 | PXD003946 | Pride

Characterisation of protein methyltransferases Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1 reveals extensive post-translational modification

Project description:Protein methylation is one of the major post-translational modifications (PTMs) in the cell. In Saccharomyces cerevisiae, over twenty protein methyltransferases (MTases) and their respective substrates have been identified. However, the way in which these MTases are modified, and potentially subject to regulation, remains poorly understood. Here, we investigated six S. cerevisiae protein MTases (Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1) to identify PTMs of potential functional relevance. We identified 51 PTM sites across the six MTases, including phosphorylation, acetylation and methylation. 45 sites are novel. We contextualised the PTM sites in structural models of the MTases and revealed that many fell in catalytic pockets or enzyme-substrate interfaces. These may regulate MTase activity. Finally, we compared PTMs on Hmt1 with those on its human homologs PRMT1, PRMT3, CARM1, PRMT6 and PRMT8. This revealed that several PTMs are conserved from yeast to human, whereas others are only found in Hmt1.

2017-12-18 | PXD006767 | Pride

Impact of short photoperiod length on smoltification (RNAseq)

Project description:This experiment was set up to evaluate the effect of the duration of a short photoperiod (winter-like conditions, 8 hours light, 14 hours dark) on the smolt transcriptome in liver and gill. Fish were kept at constant light before fish were split between treatment tank or control tanks. Treatment tanks either experienced 2 weeks of short photoperiod or 8 weeks of short photoperiod. The control fish were kept in constant winter-like conditions. After winter, the fish were brought back to constant light for 4 weeks. Sampling was done before transition to short photoperiod, at the end of winter period and four weeks after transition back to constant light. Control fish were sampled at the same time points as the treatment group.

2022-05-20 | E-MTAB-10526 | biostudies-arrayexpress

Two contrasting genotypes of Theobroma cacao differ in their transcriptional response and in accumulation of reactive oxygen species in response to treatment with salicylic acid

Project description:In order to identify variety-specific differences in defense response induction, Scavina 6 (Sca6) and Imperial College Selection 1 (ICS1) cacao plantlets were treated with 2mM SA or water as control and leaves of three developmental stages (A,C, E) were collected. This process was repeated 3 times yielding 32 samples, (Stage E ICS1 and Stage A Sca6 were collected only twice.) Averaging across developmental stages within each genotype, ICS1 had 436 up- and 601 down-regulated genes and Sca6 had 490 up- and 447 down-regulated genes (Benjamini-Hochberg p-value < 0.05). Analysis of gene annotations and Gene Ontology terms revealed that more PR genes and genes associated with defense response were up-regulated in ICS1, the more pathogen-susceptible genotype, while Sca6 had more genes associated with photosynthesis and energy generation. Same 17k unigene array as described in Mejía LC, Herre EA, Sparks JP, Winter K, García MN, Van Bael SA, Stitt J, Shi Z, Zhang Y, Guiltinan MJ, Maximova SN. 2014. Pervasive effects of a dominant foliar endophytic fungus on host genetic and phenotypic expression in a tropical tree. Frontiers in Microbiology 5.

2015-03-31 | E-GEOD-64022 | biostudies-arrayexpress

Proteomic analysis of the apoplast reveals a differential cold acclimation response among winter wheat crown tissues

Project description:The crown is the critical region for survival of winter wheat exposed to low temperature stresses. When wheat is exposed to non-freezing low temperatures, they can increase their freezing tolerance (cold acclimation, ACC). Changes within the apoplast are thought to be crucial for acquisition of freezing tolerance. However, how individual tissues within the ccrown, namely the shoot apical meristem (SAM, responsible for new shoot growth) and vascular transition zone (VTZ, located at the base of the crown)enhance tolerance to freezing has not yet been characterized. In the present study, we conducted shotgun proteomic analysis of the apoplast fluid to investigate ACC-induced proteins in the SAM and VTZ.

2018-10-23 | PXD007796 | Pride

ChIP-seq of E2f, Max, Myb and Myc in Drosophila melanogaster S2 cells for mapping their genome-wide binding sites

Project description:ChIP-seq for the strongest cell cycle regulator transcription factors in Drosophila Melanogaster S2 cells. These assays have been used to validate the direct transcriptional targets of the same transcription factors investigated in RNA-seq (E-MTAB-1364) and Affymetrix microarray experiments (E-MTAB-453). ChIP-seq assays have been done with tagged fusion proteins (for example, since we dont have functional E2f antibodies against endogenous E2f , we are transfecting v5-tagged-E2f-ORF to S2 cells and then use antibodies against v5 to detect the signal from E2f binding). If the ChIP-seq has been done with tagged fusion proteins (such as v5-tagged-E2f-ORF), the protein expression has been induced with CuSO4 treatment 48h prior to cell crosslinking & lysis. Our fusion protein constructs are driven by metallothionein promoter, which is induced by CuSO4. E-MTAB-1648, E-MTAB-1364 and E-MTAB-453 are all data from: Bonke M, et al. (2013) Transcriptional networks controlling the cell cycle. G3 (Bethesda) 3, 75-90, PMID: 23316440.

2013-08-01 | E-MTAB-1648 | biostudies-arrayexpress

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