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

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. Overall design: Genome-wide occupancy maps were generated from yeast strains representing 3V5-epitope-tagged wild type Rad6 or the backside beta-turn mutants: rad6-P43L, rad6-P47T and rad6-E49K. A bre1 null strain was also used as a negative or background control. Two independent biological samples were grown for each S. cerevisiae strain. S. cerevisiae cells were mixed with spike-in or reference endogenous genome C. glabrata cells expressing cgRad6 with C-terminal protein AG tag at 2:1 ratio before chromatin preparation by sonication. Chromatin immunoprecipitation was performed using V5 or custom Bre1 antibodies, libraries were prepared from ChIP and input DNA, sequenced, and analyzed separately.