Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6
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ABSTRACT:
PROVIDER: PRJNA901941 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22301100_1.fastq.gz | Fastqsanger.gz | |||
| SRR22301100_2.fastq.gz | Fastqsanger.gz | |||
| SRR22301101_1.fastq.gz | Fastqsanger.gz | |||
| SRR22301101_2.fastq.gz | Fastqsanger.gz | |||
| SRR22301102_1.fastq.gz | Fastqsanger.gz |
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Project description:Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The mono-ubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae and K120 in humans) occurs co-transcriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its Histone Modification Domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that specifically target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro crosslinking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic and biochemical experiments, we identified separation-of-function mutations in RAD6 that greatly impair H2BK123 ubiquitylation but not other Rad6 functions. Finally, by employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.
2023-05-01 | GSE218023 | GEO
Project description:Transcription by RNA polymerase II is regulated by epigenetic modifications to the chromatin template. The mono-ubiquitylation of H2B is established during transcription elongation and broadly impacts chromatin architecture and gene expression. The Polymerase Associated Factor 1 complex (Paf1C) is required for H2B ubiquitylation through an unknown mechanism. Here, we find that a 66-amino acid histone modification domain (HMD) within the Rtf1 subunit of Paf1C promotes H2B ubiquitylation in cells lacking all Paf1C members and present the crystal structure of this domain. Using site-specific in vivo crosslinking, we show that Rtf1 directly interacts with the ubiquitin conjugase Rad6 through a conserved surface on the HMD. Through ChIP-exo analysis, we observe that enrichment of Paf1C correlates with H2B ubiquitylation, and that the HMD, Rad6 and Bre1 localize to H2B. Finally, we demonstrate that the HMD directly stimulates H2B ubiquitylation in a reconstituted system, arguing that Paf1C functions as a cofactor for Rad6-Bre1 mediated catalysis.
2016-11-07 | GSE83348 | GEO
Project description:The five-subunit yeast Paf1 complex (Paf1C) regulates all stages of transcription and is critical for the monoubiquitylation of histone H2B (H2Bub), a modification that broadly influences chromatin structure and eukaryotic transcription. Here, we show that the histone modification domain (HMD) of Paf1C subunit Rtf1 directly interacts with the ubiquitin conjugase Rad6 and stimulates H2Bub independently of transcription. We present the crystal structure of the Rtf1 HMD and use site-specific, in vivo crosslinking to identify a conserved Rad6 interaction surface. Utilizing ChIP-exo analysis, we define the localization patterns of the H2Bub machinery at high resolution and demonstrate the importance of Paf1C in targeting the Rtf1 HMD, and thereby H2Bub, to its appropriate genomic locations. Finally, we observe HMD-dependent stimulation of H2Bub in a transcription-free, reconstituted in vitro system. Taken together, our results argue for an active role for Paf1C in promoting H2Bub and ensuring its proper localization in vivo.
| S-EPMC5131541 | biostudies-literature
Project description:Histone modifications regulate transcription by RNA polymerase II and maintain a balance between active and repressed chromatin states. The conserved Paf1 complex (Paf1C) promotes specific histone modifications during transcription elongation, but the mechanisms by which it facilitates these marks are undefined. We previously identified a 90-amino acid region within the Rtf1 subunit of Paf1C that is necessary for Paf1C-dependent histone modifications in Saccharomyces cerevisiae. Here we show that this histone modification domain (HMD), when expressed as the only source of Rtf1, can promote H3 K4 and K79 methylation and H2B K123 ubiquitylation in yeast. The HMD can restore histone modifications in rtf1Δ cells whether or not it is directed to DNA by a fusion to a DNA binding domain. The HMD can facilitate histone modifications independently of other Paf1C subunits and does not bypass the requirement for Rad6-Bre1. The isolated HMD localizes to chromatin, and this interaction requires residues important for histone modification. When expressed outside the context of full-length Rtf1, the HMD associates with and causes Paf1C-dependent histone modifications to appear at transcriptionally inactive loci, suggesting that its function has become deregulated. Finally, the Rtf1 HMDs from other species can function in yeast. Our findings suggest a direct and conserved role for Paf1C in coupling histone modifications to transcription elongation.
| S-EPMC3390850 | biostudies-literature
Project description:Recent yeast genetic studies have implicated the ubiquitin-conjugating enzyme and ubiquitin ligase functions of yRad6 and yBre1, respectively, in H2B ubiquitylation. However, there have been no corresponding biochemical analyses demonstrating intrinsic enzyme activities of yRad6 and yBre1 or related mechanistic details. Here, we describe a robust in vitro chromatin ubiquitylation assay that involves purified H2B ubiquitylation factors and natural nucleosomes. Our results indicate that yRad6 has an in vitro ability to nonspecifically ubiquitylate all core histones in the absence of an ubiquitin ligase but that yBre1 functions, through direct interactions with yRad6, to direct the ubiquitin conjugating activity of yRad6 toward the physiological H2B ubiquitylation site. Moreover, a yRad6 domain mapping analysis shows that an intact UBC domain is required for binding to yBre1, whereas the C-terminal acidic tail domain that is not required for a stable yBre1-yRad6 interaction is necessary for full enzyme activity of yRad6. We also find that, analogous to heteromeric complex formation by BRE1 paralogues in other organisms, yBre1 forms a homo-multimeric complex. Of special significance, our detailed biochemical analyses further show that the yBre1 RING finger domain is essential for H2B ubiquitylation but, surprisingly, dispensable for interaction of yBre1 with yRad6. In further support of the genetically identified requirement of the RNA polymerase II-associated yPaf1 complex for H2B ubiquitylation, protein interaction studies reveal that a purified yPaf1 complex directly and selectively interacts with yBre1 and thus serves to link the H2B ubiquitylation and general transcription machineries. These studies provide a more detailed mechanistic basis for H2B ubiquitylation in yeast.
| S-EPMC2742823 | biostudies-other
Project description:RNA polymerase II (RNAPII) transcription elongation directs an intricate pattern of histone modifications. Formation of this pattern involves a highly conserved regulatory axis, key components of which are the elongation factor Rtf1, monoubiquitylation of histone H2B (H2Bub1), and methylation of histone H3 on lysine 4 (H3K4me). Direct contact between Rtf1 and the ubiquitylation complex Rad6-Bre1 stimulates H2Bub1, which in turn promotes H3K4me by stimulating the activity of the Set1C/COMPASS methyltransferase complex. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate chromatin and transcription in vivo. We found that mutations affecting multiple components of the Rtf1-H2Bub1-H3K4me axis in the model eukaryote Schizosaccharomyces pombe caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU). Resistance correlated with a reduced effect of HU on dNTP pools, bypass of the S-phase checkpoint in the presence of HU, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain (CTD) of the RNAPII large subunit Rpb1 led to a similar spectrum of phenotypes. Moreover, all the HU resistant mutants we identified also exhibited resistance to several azole class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 CTD in controlling fungal drug tolerance.
2023-11-27 | GSE248361 | GEO
Project description:RNA polymerase II (RNAPII) transcription elongation directs an intricate pattern of histone modifications. Formation of this pattern involves a highly conserved regulatory axis, key components of which are the elongation factor Rtf1, monoubiquitylation of histone H2B (H2Bub1), and methylation of histone H3 on lysine 4 (H3K4me). Direct contact between Rtf1 and the ubiquitylation complex Rad6-Bre1 stimulates H2Bub1, which in turn promotes H3K4me by stimulating the activity of the Set1C/COMPASS methyltransferase complex. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate chromatin and transcription in vivo. We found that mutations affecting multiple components of the Rtf1-H2Bub1-H3K4me axis in the model eukaryote Schizosaccharomyces pombe caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU). Resistance correlated with a reduced effect of HU on dNTP pools, bypass of the S-phase checkpoint in the presence of HU, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain (CTD) of the RNAPII large subunit Rpb1 led to a similar spectrum of phenotypes. Moreover, all the HU resistant mutants we identified also exhibited resistance to several azole class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 CTD in controlling fungal drug tolerance.
2023-11-27 | GSE248360 | GEO
Project description:Histone H2B ubiquitination plays an important role in transcription regulation. It has been shown that H2B ubiquitination is regulated by multiple upstream events associated with elongating RNA polymerase. Here we demonstrate that H2B K34 ubiquitylation by the MOF-MSL complex is part of the protein networks involved in early steps of transcription elongation. Knocking down MSL2 in the MOF-MSL complex affects not only global H2BK34ub, but also multiple cotranscriptionally regulated histone modifications. More importantly, we show that the MSL, PAF1, and RNF20/40 complexes are recruited and stabilized at active gene promoters by direct binary interactions. The stabilized complexes serve to regulate chromatin association of pTEFb through a positive feedback loop and facilitate Pol II transition during early transcription elongation. Results from our biochemical studies are underscored by genome-wide analyses that show high RNA Pol II processivity and transcription activity at MSL target genes.
| S-EPMC4065619 | biostudies-literature
Project description:Rad6 E2 ubiquitin-conjugating enzyme and Bre1 E3 ubiquitin ligase catalyze histone H2B Lysine-123 monoubiquitination (H2Bub1), which stabilizes nucleosomes and regulates the trans-histone H3K4 and K79 methylation during gene transcription and other nuclear processes. The interaction interfaces within the Rad6-Bre1-containing H2B ubiquitin-conjugating complex has remained unknown. By solving the crystal structure of Rad6 along with a non-RING domain N-terminal region of Bre1, we report a beta-turn in Rad6's so-called backside region away from catalytic pocket as a binding site for a homodimer of Bre1 E3 ligase. Using quantitative ChIP-seq or ChIP-Rx, we further demonstrate that Rad6's backside beta-turn residues also govern the chromatin binding dynamics of the Rad6-Bre1 complex.
2022-10-24 | GSE139633 | GEO
Project description:H2B ubiquitylation has been implicated in active transcription but is not well understood in mammalian cells. Beyond earlier identification of hBRE1 as the E3 ligase for H2B ubiquitylation in human cells, we now show (1) that hRAD6 serves as the cognate E2-conjugating enzyme; (2) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120; (3) that hPAF-mediated transcription is required for efficient H2B ubiquitylation as a result of hPAF-dependent recruitment of hBRE1-hRAD6 to the Pol II transcription machinery; (4) that H2B ubiquitylation per se does not affect the level of hPAF-, SII-, and p300-dependent transcription and likely functions downstream; and (5) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and trimethylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription.
| S-EPMC2678028 | biostudies-literature