title: Purification of histone ubiquitin ligases from HeLa cells.
abstract: Posttranslational histone modifications play an important role in regulating chromatin based nuclear processes including transcription. Of these modifications, histone ubiquitination is among the least understood. Histone ubiquitination predominately targets histones H2A and H2B. While ubiquitination of H2B is evolutionarily conserved from budding yeast to mammals, ubiquitination of H2A has not been detected in budding yeast, worms, or plants. Until recently, studies of histone ubiquitination lagged far behind the study of other histone modifications, largely because antibodies specific for ubiquitinated histones are difficult to generate. Despite this obstacle, the identification of the enzymatic machineries involved in histone ubiquitination, together with the successful use of a combination of genetic and immunoblot approaches to detect ubiquitinated histones, have helped to reveal important regulatory roles for this modification in transcriptional initiation and elongation, cell cycle progression, and DNA damage response. With the aid of the recently developed ubiquitinated histone-specific antibodies, an intriguing link between histone ubiquitination and cancer development has been established. While the enzymes involved in H2B ubiquitination were identified first in budding yeast and subsequently in higher organisms based on gene homology, the identification of the enzymatic machineries involved in H2A ubiquitination largely depended on a biochemical purification approach. The unbiased search for ubiquitin ligases targeting histones also led to the identification of a H3 and H4 ubiquitin ligase. Here we detail a protocol for the biochemical approach to identify histone ubiquitin ligase(s) from HeLa cells. Similar approaches have been successfully used to identify histone methyltransferases, histone demethylases, chromatin remodeling factors, and general transcription factors. So long as an in vitro enzymatic assay can be established, the approach we describe can be easily adapted to identify other histone and non-histone modifying enzymes.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Kaul Human Genetics Building 402A, 720 South 20th Street, Birmingham, AL 35294, United States.
authors.forename: Amanda
authors.initials: A
authors.lastname: Jones
authors.forename: Heui-Yun
authors.initials: HY
authors.lastname: Joo
authors.forename: Woody
authors.initials: W
authors.lastname: Robbins
authors.forename: Hengbin
authors.initials: H
authors.lastname: Wang
mesh_terms: D002458:Cell Fractionation / Q000379:methods; D002467:Cell Nucleus / Q000737:chemistry; D002850:Chromatography, Gel; D057075:Enzyme Assays; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008836:Micrococcal Nuclease / Q000737:chemistry; D009707:Nucleosomes / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000302:isolation & purification*; D014461:Ultracentrifugation
chemical_list: D006657:Histones; D009707:Nucleosomes; D044767:Ubiquitin-Protein Ligases; D008836:Micrococcal Nuclease