Abstract
Diverse histone modifications play important roles in transcriptional regulation throughout eukaryotes, and recent studies have implicated histone H2B ubiquitylation in active transcription. The necessity of at least three enzymes (E1-E3), as well as ongoing transcription events, for efficient H2B ubiquitylation complicates mechanistic studies of H2B ubiquitylation relative to other histone modifications. Here we describe experimental protocols for preparation of human H2B ubiquitylation factors, ubiquitylation substrates and transcription factors, as well as the use of these factors to establish H2B ubiquitylation mechanisms during transcription. The methods include reliable protein interaction and E3 ubiquitylation assays that can be widely applied to confirm cognate E2-E3 pairs in other protein ubiquitylation systems, optimized in vitro ubiquitylation assays for various histone substrates, and a transcription-coupled H2B ubiquitylation assay in a highly purified transcription system. These comprehensive analyses have revealed (i) that RAD6 serves as the cognate E2 for the BRE1 complex in human cells, as previously established in yeast, (ii) that RAD6, through direct interaction with the BRE1 complex, ubiquitylates chromatinized H2B at lysine 120 and (iii) that PAF1 complex-mediated transcription is required for efficient H2B ubiquitylation. This experimental system permits detailed mechanistic analyses of H2B ubiquitylation during transcription by providing information concerning both precise enzyme functions and physical interactions between the transcription and histone modification machineries.