In vitro and in vivo assays for studying histone ubiquitination and deubiquitination

Posttranslational histone modifications play important roles in regulating chromatin structure and function (Martin and Zhang, Nat Rev Mol Cell Biol 6:838-849, 2005; Jenuwein and Allis, Science 293:1074-1080, 2001). One example of such modifications is histone ubiquitination, which occurs predominately on H2A and H2B. Recent studies have highlighted important regulatory roles of H2A ubiquitination in Polycomb group proteins-mediated gene silencing (Wang et al., Nature 431:873-878, 2004; Joo et al., Nature 449:1068-1072, 2007) and H2B ubiquitination in transcription, H3 methylation, and DNA methylation (Zhang, Genes Dev 17:2733-2740, 2003; Sun and Allis, Nature 418:104-108, 2002; Sridhar et al., Nature 447:735-738, 2007). Here we describe methods for in vitro histone ubiquitination and deubiquitination assays. We also describe approaches to investigate the in vivo function of a putative histone ubiquitin ligase and deubiquitinase. These experimental procedures are largely based on our studies in mammalian cells. These methods should provide useful tools for studying this bulky histone modification.