Effect of Rad6 backside beta-turn mutations on gene expression

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. Overall design: Gene expression changes were generated from six different yeast strains representing wild type control, rad6 null and bre1 null mutants, and beta-turn mutants: rad6-P43L, rad6-P47T and rad6-E49K. Three independent biological samples were grown for each strain, total RNA was isolated, and libraries were prepared, sequenced, and analyzed separately.