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An optogenetic switch for the Set2 methyltransferase provides evidence for transcription-dependent and -independent dynamics of H3K36 methylation.

ABSTRACT: Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light-activated nuclear shuttle (LANS) domain. Light activation resulted in efficient Set2-LANS nuclear localization followed by H3K36me3 deposition in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes showed disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting that H3K36me3 deposition occurs in a directed fashion on all transcribed genes regardless of their overall transcription frequency. Removal of H3K36me3 was highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggests transcription-dependent and -independent mechanisms for H3K36me deposition and removal, respectively.

SUBMITTER: Lerner AM

PROVIDER: S-EPMC7605256 | biostudies-literature |

REPOSITORIES: biostudies-literature

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An optogenetic switch for the Set2 methyltransferase provides evidence for transcription-dependent and independent dynamics of H3K36 methylation

Project description:Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification associated with transcription and DNA repair. Although the effects of H3K36 methylation have been studied, the genome-wide dynamics of H3K36me deposition and removal are not known. We established rapid and reversible optogenetic control for Set2, the sole H3K36 methyltransferase in yeast, by fusing the enzyme with the light activated nuclear shuttle (LANS) domain. Early H3K36me3 dynamics identified methylation in vivo, with total H3K36me3 levels correlating with RNA abundance. Although genes exhibited disparate levels of H3K36 methylation, relative rates of H3K36me3 accumulation were largely linear and consistent across genes, suggesting that H3K36me3 deposition occurs uniformly across all genes in a directed fashion regardless of transcription frequency. Removal of H3K36me3 was highly dependent on the demethylase Rph1. However, the per-gene rate of H3K36me3 loss weakly correlated with RNA abundance and followed exponential decay, suggesting H3K36 demethylases act in a global, stochastic manner. Altogether, these data provide a detailed temporal view of H3K36 methylation and demethylation that suggest transcription-dependent and independent mechanisms for H3K36me deposition and removal, respectively.

2020-07-01 | GSE153539 | GEO

An optogenetic switch for the Set2 methyltransferase provides evidence for transcription-dependent and independent dynamics of H3K36 methylation

Project description:An optogenetic switch for the Set2 methyltransferase provides evidence for transcription-dependent and independent dynamics of H3K36 methylation

| PRJNA643154 | ENA

Selective suppression of antisense transcription by Set2-mediated H3K36 methylation.

Project description:Maintenance of a regular chromatin structure over the coding regions of genes occurs co-transcriptionally via the 'chromatin resetting' pathway. One of the central players in this pathway is the histone methyltransferase Set2. Here we show that the loss of Set2 in yeast, Saccharomyces cerevisiae, results in transcription initiation of antisense RNAs embedded within body of protein-coding genes. These RNAs are distinct from the previously identified non-coding RNAs and cover 11% of the yeast genome. These RNA species have been named Set2-repressed antisense transcripts (SRATs) since the co-transcriptional addition of the H3K36 methyl mark by Set2 over their start sites results in their suppression. Interestingly, loss of chromatin resetting factor Set2 or the subsequent production of SRATs does not affect the abundance of the sense transcripts. This difference in transcriptional outcomes of overlapping transcripts due to a strand-independent addition of H3K36 methylation is a key regulatory feature of interleaved transcriptomes.

| S-EPMC5133703 | biostudies-literature

Set2-mediated H3K36 methylation states redundantly repress the production of antisense transcripts: role in transcription regulation.

Project description:Methyltransferase Set2-mediated methylation of histone H3 lysine 36 (H3K36), which involves the addition of up to three methyl groups at this site, has been demonstrated to function in many chromatin-coupled events. The methylation of H3K36 is known to recruit different chromatin effector proteins, affecting transcription, mRNA splicing and DNA repair. In this study, we engineered two yeast set2 mutants that lack H3K36 mono/dimethylation (H3K36me1/2) and trimethylation (H3K36me3), respectively, and characterized their roles in the production of antisense transcripts under nutrient-rich conditions. Using our new bioinformatics identification pipeline analysis, we are able to identify a larger number of antisense transcripts in set2∆ cells than has been published previously. We further show that H3K36me1/2 or H3K36me3 redundantly repressed the production of antisense transcripts. Moreover, gene ontology (GO) analysis implies that H3K36me3-mediated antisense transcription might play a role in DNA replication and DNA damage repair, which is independent of regulation of the corresponding sense gene expression. Overall, our results validate a coregulatory mechanism of different H3K36 methylation states, particularly in the repression of antisense transcription.

| S-EPMC8329787 | biostudies-literature

A conserved genetic interaction between Spt6 and Set2 regulates H3K36 methylation.

Project description:The transcription elongation factor Spt6 and the H3K36 methyltransferase Set2 are both required for H3K36 methylation and transcriptional fidelity in Saccharomyces cerevisiae. However, the nature of the requirement for Spt6 has remained elusive. By selecting for suppressors of a transcriptional defect in an spt6 mutant, we have isolated several highly clustered, dominant SET2 mutations (SET2sup mutations) in a region encoding a proposed autoinhibitory domain. SET2sup mutations suppress the H3K36 methylation defect in the spt6 mutant, as well as in other mutants that impair H3K36 methylation. We also show that SET2sup mutations overcome the requirement for certain Set2 domains for H3K36 methylation. In vivo, SET2sup mutants have elevated levels of H3K36 methylation and the purified Set2sup mutant protein has greater enzymatic activityin vitro. ChIP-seq studies demonstrate that the H3K36 methylation defect in the spt6 mutant, as well as its suppression by a SET2sup mutation, occurs at a step following the recruitment of Set2 to chromatin. Other experiments show that a similar genetic relationship between Spt6 and Set2 exists in Schizosaccharomyces pombe. Taken together, our results suggest a conserved mechanism by which the Set2 autoinhibitory domain requires multiple Set2 interactions to ensure that H3K36 methylation occurs specifically on actively transcribed chromatin.

| S-EPMC6486648 | biostudies-literature

Nucleosome and ubiquitin position Set2 to methylate H3K36.

Project description:Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification deposited by the Set2 methyltransferases. Recent findings show that over-expression or mutation of Set2 enzymes promotes cancer progression, however, mechanisms of H3K36me are poorly understood. Set2 enzymes show spurious activity on histones and histone tails, and it is unknown how they obtain specificity to methylate H3K36 on the nucleosome. In this study, we present 3.8?Å cryo-EM structure of Set2 bound to the mimic of H2B ubiquitinated nucleosome. Our structure shows that Set2 makes extensive interactions with the H3 ?N, the H3 tail, the H2A C-terminal tail and stabilizes DNA in the unwrapped conformation, which positions Set2 to specifically methylate H3K36. Moreover, we show that ubiquitin contributes to Set2 positioning on the nucleosome and stimulates the methyltransferase activity. Notably, our structure uncovers interfaces that can be targeted by small molecules for development of future cancer therapies.

| S-EPMC6706414 | biostudies-literature

Set2 histone methyltransferase regulates transcription coupled-nucleotide excision repair in yeast.

Project description:Helix-distorting DNA lesions, including ultraviolet (UV) light-induced damage, are repaired by the global genomic-nucleotide excision repair (GG-NER) and transcription coupled-nucleotide excision repair (TC-NER) pathways. Previous studies have shown that histone post-translational modifications (PTMs) such as histone acetylation and methylation can promote GG-NER in chromatin. Whether histone PTMs also regulate the repair of DNA lesions by the TC-NER pathway in transcribed DNA is unknown. Here, we report that histone H3 K36 methylation (H3K36me) by the Set2 histone methyltransferase in yeast regulates TC-NER. Mutations in Set2 or H3K36 result in UV sensitivity that is epistatic with Rad26, the primary TC-NER factor in yeast, and cause a defect in the repair of UV damage across the yeast genome. We further show that mutations in Set2 or H3K36 in a GG-NER deficient strain (i.e., rad16Δ) partially rescue its UV sensitivity. Our data indicate that deletion of SET2 rescues UV sensitivity in a GG-NER deficient strain by activating cryptic antisense transcription, so that the non-transcribed strand (NTS) of yeast genes is repaired by TC-NER. These findings indicate that Set2 methylation of H3K36 establishes transcriptional asymmetry in repair by promoting canonical TC-NER of the transcribed strand (TS) and suppressing cryptic TC-NER of the NTS.

| S-EPMC8936446 | biostudies-literature

Set2 Methyltransferase Facilitates DNA Replication and Promotes Genotoxic Stress Responses through MBF-Dependent Transcription.

Project description:Chromatin modification through histone H3 lysine 36 methylation by the SETD2 tumor suppressor plays a key role in maintaining genome stability. Here, we describe a role for Set2-dependent H3K36 methylation in facilitating DNA replication and the transcriptional responses to both replication stress and DNA damage through promoting MluI cell-cycle box (MCB) binding factor (MBF)-complex-dependent transcription in fission yeast. Set2 loss leads to reduced MBF-dependent ribonucleotide reductase (RNR) expression, reduced deoxyribonucleoside triphosphate (dNTP) synthesis, altered replication origin firing, and a checkpoint-dependent S-phase delay. Accordingly, prolonged S phase in the absence of Set2 is suppressed by increasing dNTP synthesis. Furthermore, H3K36 is di- and tri-methylated at these MBF gene promoters, and Set2 loss leads to reduced MBF binding and transcription in response to genotoxic stress. Together, these findings provide new insights into how H3K36 methylation facilitates DNA replication and promotes genotoxic stress responses in fission yeast.

| S-EPMC5608972 | biostudies-literature

Unique and Shared Roles for Histone H3K36 Methylation States in Transcription Regulation Functions.

Project description:Set2 co-transcriptionally methylates lysine 36 of histone H3 (H3K36), producing mono-, di-, and trimethylation (H3K36me1/2/3). These modifications recruit or repel chromatin effector proteins important for transcriptional fidelity, mRNA splicing, and DNA repair. However, it was not known whether the different methylation states of H3K36 have distinct biological functions. Here, we use engineered forms of Set2 that produce different lysine methylation states to identify unique and shared functions for H3K36 modifications. Although H3K36me1/2 and H3K36me3 are functionally redundant in many SET2 deletion phenotypes, we found that H3K36me3 has a unique function related to Bur1 kinase activity and FACT (facilitates chromatin transcription) complex function. Further, during nutrient stress, either H3K36me1/2 or H3K36me3 represses high levels of histone acetylation and cryptic transcription that arises from within genes. Our findings uncover the potential for the regulation of diverse chromatin functions by different H3K36 methylation states.

| S-EPMC7334899 | biostudies-literature

H3K36 methylation antagonizes PRC2-mediated H3K27 methylation.

Project description:H3K27 methylation mediated by the histone methyltransferase complex PRC2 is critical for transcriptional regulation, Polycomb silencing, Drosophila segmentation, mammalian X chromosome inactivation, and cancer. PRC2-mediated H3K27 methylation can spread along the chromatin and propagate the repressive chromatin environment; thus, chromatin components that antagonize the activity of PRC2 are important for restraining Polycomb silencing. Here we report that in HeLa cells, H3 histones unmethylated at Lys-36 are mostly methylated at Lys-27, with the exception of newly synthesized H3. In addition, K27me3 rarely co-exists with K36me2 or K36me3 on the same histone H3 polypeptide. Moreover, PRC2 activity is greatly inhibited on nucleosomal substrates with preinstalled H3K36 methylation. These findings collectively identify H3K36 methylation as a chromatin component that restricts the PRC2-mediated spread of H3K27 methylation. Finally, we provide evidence that the controversial histone lysine methyltransferase Ash1, a known Trithorax group protein that antagonizes Polycomb silencing in vivo, is an H3K36-specific dimethylase, not an H3K4 methylase, further supporting the role of H3K36 methylation in antagonizing PRC2-mediated H3K27 methylation.

| S-EPMC3048685 | biostudies-literature

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