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

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE139633 | GEO | 2022/10/24

REPOSITORIES: GEO

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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

Phase separation directs ubiquitination of gene body nucleosomes

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Critical role of the UBL-domain in stimulating the E3 ubiquitin ligase activity of UHRF1 towards chromatin

Project description:The RING E3 ubiquitin ligase UHRF1 controls DNA methylation through its ability to target the maintenance DNA methyltransferase DNMT1 to newly replicated chromatin. DNMT1 recruitment relies on ubiquitylation of histone H3 by UHRF1, however, how UHRF1 deposits ubiquitin onto the histone is unknown. Here, we demonstrate that the ubiquitin-like domain (UBL) of UHRF1 is essential for RING-mediated H3 ubiquitylation. Using chemical crosslinking and mass spectrometry, biochemical assays and recombinant chromatin substrates we show that the UBL participates in structural rearrangements of UHRF1 upon binding to chromatin and the E2 ubiquitin conjugating enzyme UbcH5a/UBE2D1. Similar to ubiquitin, the UBL exerts its effects through a hydrophobic patch that contacts a regulatory surface on the “backside” of the E2 to stabilise the E2-E3-chromatin complex. Our analysis of the enzymatic mechanism of UHRF1 uncovers an unexpected function of the UBL-domain and defines a new role for this domain in DNMT1-dependent inheritance of DNA methylation.

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2022-06-07 | GSE146296 | GEO

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