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In Vitro and In Vivo Assays for Studying Histone Ubiquitination and Deubiquitination

  • Heui-Yun Joo
  • Qian Dai
  • Amanda E. Jones
  • Ling Zhai
  • Hengbin WangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1288)

Abstract

Posttranslational histone modifications play important roles in regulating chromatin structure and function (Rando, Curr Opin Genet Dev 22:148–155, 2012; Zentner and Henikoff, Nat Struct Mol Biol 20:259–266, 2013). 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 protein-mediated gene silencing and DNA damage repair (de Napoles et al., Dev Cell 7:663–676, 2004; Wang et al., Nature 431:873–878, 2004; Doil et al., Cell 136:435–446, 2009; Gatti et al., Cell Cycle 11:2538–2544, 2012; Mattiroli et al., Cell 150:1182–1195, 2012; Stewart et al., Cell 136:420–434, 2009; Bergink et al., Genes Dev 20:1343–1352, 2006; Facchino et al., J Neurosci 30:10096–10111, 2010; Ginjala et al., Mol Cell Biol 31:1972–1982, 2011; Ismail et al., J Cell Biol 191:45–60, 2010), H2B ubiquitination in transcription initiation and elongation (Xiao et al., Mol Cell Biol 25:637–651, 2005; Kao et al., Genes Dev 18:184–195, 2004; Pavri et al., Cell 125:703–717, 2006; Kim et al., Cell 137:459–471, 2009), pre-mRNA splicing (Jung et al. Genome Res 22:1026–1035, 2012; Shieh et al., BMC Genomics 12:627, 2011; Zhang et al., Genes Dev 27:1581–1595, 2013), nucleosome stabilities (Fleming et al., Mol Cell 31:57–66, 2008; Chandrasekharan et al., Proc Natl Acad Sci U S A 106:16686–16691, 2009), H3 methylation (Sun and Allis, Nature 418:104–108, 2002; Briggs et al., Nature 418:498, 2002; Dover et al., J Biol Chem 277:28368–28371, 2002; Ng et al., J Biol Chem 277:34655–34657, 2002), and DNA methylation (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 putative histone ubiquitin ligase(s) and deubiquitinase(s). These experimental procedures are largely based on our studies in mammalian cells. These methods should provide useful tools for studying this bulky histone modification.

Key words

Chromatin Histone ubiquitination Histone deubiquitination In vitro assay In vivo assay 

Notes

Acknowledgements

We thank Ms. Jessica Woolnough for critical reading of the manuscript. We apologize for the limited number of citations due to insufficient space. Work in Hengbin Wang’s laboratory is supported by the Leukemia and Lymphoma Society and NIH grant (GM081489).

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Heui-Yun Joo
    • 1
  • Qian Dai
    • 1
  • Amanda E. Jones
    • 1
  • Ling Zhai
    • 1
  • Hengbin Wang
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular GeneticsUniversity of Alabama at BirminghamBirminghamUSA

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