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Effect of Rad6 backside beta-turn mutations on the chromatin occupancy of the histone H2B ubiquitin-conjugating complex subunits

ABSTRACT: Effect of Rad6 backside beta-turn mutations on the chromatin occupancy of the histone H2B ubiquitin-conjugating complex subunits

PROVIDER: PRJNA580526 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10375800.fastq.gz	Fastqsanger.gz
	SRR10375801.fastq.gz	Fastqsanger.gz
	SRR10375802.fastq.gz	Fastqsanger.gz
	SRR10375803.fastq.gz	Fastqsanger.gz
	SRR10375804.fastq.gz	Fastqsanger.gz

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Effect of Rad6 backside beta-turn mutations on the chromatin occupancy of the histone H2B ubiquitin-conjugating complex subunits

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

Effect of Rad6 backside beta-turn mutations on gene expression

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 have 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 and transcriptional regulatory functions of the Rad6-Bre1 complex.

2022-10-24 | GSE139632 | GEO

Effect of Rad6 backside beta-turn mutations

Project description:Effect of Rad6 backside beta-turn mutations

| PRJNA580524 | ENA

The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Promotes H2B Ubiquitylation Through a Direct Interaction with the Ubiquitin Conjugase Rad6

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

Structure and functional determinants of Rad6-Bre1 subunits in the histone H2B ubiquitin-conjugating complex.

Project description:The conserved complex of the Rad6 E2 ubiquitin-conjugating enzyme and the Bre1 E3 ubiquitin ligase catalyzes histone H2B monoubiquitination (H2Bub1), which regulates chromatin dynamics during transcription and other nuclear processes. Here, we report a crystal structure of Rad6 and the non-RING domain N-terminal region of Bre1, which shows an asymmetric homodimer of Bre1 contacting a conserved loop on the Rad6 'backside'. This contact is distant from the Rad6 catalytic site and is the location of mutations that impair telomeric silencing in yeast. Mutational analyses validated the importance of this contact for the Rad6-Bre1 interaction, chromatin-binding dynamics, H2Bub1 formation and gene expression. Moreover, the non-RING N-terminal region of Bre1 is sufficient to confer nucleosome binding ability to Rad6 in vitro. Interestingly, Rad6 P43L protein, an interaction interface mutant and equivalent to a cancer mutation in the human homolog, bound Bre1 5-fold more tightly than native Rad6 in vitro, but showed reduced chromatin association of Bre1 and reduced levels of H2Bub1 in vivo. These surprising observations imply conformational transitions of the Rad6-Bre1 complex during its chromatin-associated functional cycle, and reveal the differential effects of specific disease-relevant mutations on the chromatin-bound and unbound states. Overall, our study provides structural insights into Rad6-Bre1 interaction through a novel interface that is important for their biochemical and biological responses.

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Effect of Rad6 backside beta-turn mutations on gene expression

Project description:Effect of Rad6 backside beta-turn mutations on gene expression

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An E2 ubiquitin conjugating enzyme links diubiquitinated H2B to H3K27M oncohistone function

Project description:The H3K27M oncogenic histone (oncohistone) mutation drives ~80% of incurable childhood brain tumors known as diffuse midline gliomas (DMGs). The major molecular feature of H3K27M mutant DMGs is a global loss of H3K27 tri-methylation (H3K27me3), a phenotype conserved in Caenorhabditis elegans (C. elegans). Here we perform unbiased genome-wide suppressor screens in C. elegans expressing H3K27M, and isolate 20 suppressors, all of which at least partially restore H3K27me3. 19/20 suppressor mutations map to the same histone H3.3 gene in which the K27M mutation was originally introduced. Most of these create single amino acid substitutions between residues R26-Y54, which do not disrupt oncohistone expression. Rather, they are predicted to impair interactions with the Polycomb Repressive Complex 2 (PRC2) and are functionally conserved in human cells. Further, we mapped a single extragenic H3K27M suppressor to ubc-20, an E2 ubiquitin conjugating enzyme, whose loss rescued H3K27me3 to nearly 50% wild-type levels despite continued oncohistone expression and chromatin incorporation. We demonstrate that ubc-20 is the major enzyme responsible for generating di-ubiquitinated histone H2B. Our study provides in vivo support for existing models of PRC2 inhibition via direct oncohistone contact, and suggests that the effects of H3K27M may be modulated by H2B ubiquitination.

2024-11-20 | GSE275118 | GEO

An E2 ubiquitin conjugating enzyme links diubiquitinated H2B to H3K27M oncohistone function

Project description:An E2 ubiquitin conjugating enzyme links diubiquitinated H2B to H3K27M oncohistone function

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Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6

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

Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes

Project description:Supporting .raw data for "Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes", DOI: 10.15252/embj.2021108823 Related to Figure 1E and EV1A.

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