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)


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 



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).


  1. 1.
    Rando OJ (2012) Combinatorial complexity in chromatin structure and function: revisiting the histone code. Curr Opin Genet Dev 22:148–155CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Zentner GE, Henikoff S (2013) Regulation of nucleosome dynamics by histone modifications. Nat Struct Mol Biol 20:259–266CrossRefPubMedGoogle Scholar
  3. 3.
    Osley MA (2004) H2B ubiquitylation: the end is in sight. Biochim Biophys Acta 1677:74–78CrossRefPubMedGoogle Scholar
  4. 4.
    Jason LJ, Moore SC, Lewis JD, Lindsey G, Ausio J (2002) Histone ubiquitination: a tagging tail unfolds? Bioessays 24:166–174CrossRefPubMedGoogle Scholar
  5. 5.
    Vassilev AP, Rasmussen HH, Christensen EI, Nielsen S, Celis JE (1995) The levels of ubiquitinated histone H2A are highly upregulated in transformed human cells: partial colocalization of uH2A clusters and PCNA/cyclin foci in a fraction of cells in S-phase. J Cell Sci 108(Pt 3):1205–1215PubMedGoogle Scholar
  6. 6.
    Wang H, Wang L, Erdjument-Bromage H, Vidal M, Tempst P, Jones RS, Zhang Y (2004) Role of histone H2A ubiquitination in polycomb silencing. Nature 431:873–878CrossRefPubMedGoogle Scholar
  7. 7.
    Wang H, Zhai L, Xu J, Joo HY, Jackson S, Erdjument-Bromage H, Tempst P, Xiong Y, Zhang Y (2006) Histone H3 and H4 ubiquitylation by the CUL4-DDB-ROC1 ubiquitin ligase facilitates cellular response to DNA damage. Mol Cell 22:383–394CrossRefPubMedGoogle Scholar
  8. 8.
    Kao CF, Osley MA (2003) In vivo assays to study histone ubiquitylation. Methods 31:59–66CrossRefPubMedGoogle Scholar
  9. 9.
    Minsky N, Shema E, Field Y, Schuster M, Segal E, Oren M (2008) Monoubiquitinated H2B is associated with the transcribed region of highly expressed genes in human cells. Nat Cell Biol 10:483–488CrossRefPubMedGoogle Scholar
  10. 10.
    de Napoles M, Mermoud JE, Wakao R, Tang YA, Endoh M, Appanah R, Nesterova TB, Silva J, Otte AP, Vidal M et al (2004) Polycomb group proteins Ring1A/B link ubiquitylation of histone H2A to heritable gene silencing and X. Dev Cell 7:663–676CrossRefPubMedGoogle Scholar
  11. 11.
    Doil C, Mailand N, Bekker-Jensen S, Menard P, Larsen DH, Pepperkok R, Ellenberg J, Panier S, Durocher D, Bartek J et al (2009) RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins. Cell 136:435–446CrossRefPubMedGoogle Scholar
  12. 12.
    Gatti M, Pinato S, Maspero E, Soffientini P, Polo S, Penengo L (2012) A novel ubiquitin mark at the N-terminal tail of histone H2As targeted by RNF168 ubiquitin ligase. Cell Cycle 11:2538–2544CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Mattiroli F, Vissers JH, van Dijk WJ, Ikpa P, Citterio E, Vermeulen W, Marteijn JA, Sixma TK (2012) RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling. Cell 150:1182–1195CrossRefPubMedGoogle Scholar
  14. 14.
    Stewart GS, Panier S, Townsend K, Al-Hakim AK, Kolas NK, Miller ES, Nakada S, Ylanko J, Olivarius S, Mendez M et al (2009) The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage. Cell 136:420–434CrossRefPubMedGoogle Scholar
  15. 15.
    Bergink S, Salomons FA, Hoogstraten D, Groothuis TAM, de Waard H, Wu J, Yuan L, Citterio E, Houtsmuller AB, Neefjes J et al (2006) DNA damage triggers nucleotide excision repair-dependent monoubiquitylation of histone H2A. Genes Dev 20:1343–1352CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Facchino S, Abdouh M, Chatoo W, Bernier G (2010) BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery. J Neurosci 30:10096–10111CrossRefPubMedGoogle Scholar
  17. 17.
    Ginjala V, Nacerddine K, Kulkarni A, Oza J, Hill SJ, Yao M, Citterio E, van Lohuizen M, Ganesan S (2011) BMI1 is recruited to DNA breaks and contributes to DNA damage-induced H2A ubiquitination and repair. Mol Cell Biol 31:1972–1982CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Ismail IH, Andrin C, McDonald D, Hendzel MJ (2010) BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair. J Cell Biol 191:45–60CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Xiao T, Kao C-F, Krogan NJ, Sun Z-W, Greenblatt JF, Osley MA, Strahl BD (2005) Histone H2B ubiquitylation is associated with elongating RNA polymerase II. Mol Cell Biol 25:637–651CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Kao C-F, Hillyer C, Tsukuda T, Henry K, Berger S, Osley MA (2004) Rad6 plays a role in transcriptional activation through ubiquitylation of histone H2B. Genes Dev 18:184–195CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Pavri R, Zhu B, Li G, Trojer P, Mandal S, Shilatifard A, Reinberg D (2006) Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA Polymerase II. Cell 125:703–717CrossRefPubMedGoogle Scholar
  22. 22.
    Kim J, Guermah M, McGinty RK, Lee J-S, Tang Z, Milne TA, Shilatifard A, Muir TW, Roeder RG (2009) RAD6-mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells. Cell 137:459–471CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Jung I, Kim SK, Kim M, Han YM, Kim YS, Kim D, Lee D (2012) H2B monoubiquitylation is a 5′-enriched active transcription mark and correlates with exon-intron structure in human cells. Genome Res 22:1026–1035CrossRefPubMedCentralPubMedGoogle Scholar
  24. 24.
    Shieh G, Pan CH, Wu JH, Sun YJ, Wang CC, Hsiao WC, Lin CY, Tung L, Chang TH, Fleming AB et al (2011) H2B ubiquitylation is part of chromatin architecture that marks exon-intron structure in budding yeast. BMC Genomics 12:627CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Zhang Z, Jones A, Joo HY, Zhou D, Cao Y, Chen S, Erdjument-Bromage H, Renfrow M, He H, Tempst P et al (2013) USP49 deubiquitinates histone H2B and regulates cotranscriptional pre-mRNA splicing. Genes Dev 27:1581–1595CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Fleming AB, Kao CF, Hillyer C, Pikaart M, Osley MA (2008) H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation. Mol Cell 31:57–66CrossRefPubMedGoogle Scholar
  27. 27.
    Chandrasekharan MB, Huang F, Sun ZW (2009) Ubiquitination of histone H2B regulates chromatin dynamics by enhancing nucleosome stability. Proc Natl Acad Sci U S A 106:16686–16691CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Sun Z-W, Allis CD (2002) Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418:104–108CrossRefPubMedGoogle Scholar
  29. 29.
    Briggs SD, Xiao T, Sun Z-W, Caldwell JA, Shabanowitz J, Hunt DF, Allis CD, Strahl BD (2002) Gene silencing: trans-histone regulatory pathway in chromatin. Nature 418:498CrossRefPubMedGoogle Scholar
  30. 30.
    Dover J, Schneider J, Tawiah-Boateng MA, Wood A, Dean K, Johnston M, Shilatifard A (2002) Methylation of histone H3 by COMPASS requires ubiquitination of histone H2B by Rad6. J Biol Chem 277:28368–28371CrossRefPubMedGoogle Scholar
  31. 31.
    Ng HH, Xu RM, Zhang Y, Struhl K (2002) Ubiquitination of histone H2B by Rad6 is required for efficient Dot1-mediated methylation of histone H3 lysine 79. J Biol Chem 277:34655–34657CrossRefPubMedGoogle Scholar
  32. 32.
    Sridhar VV, Kapoor A, Zhang K, Zhu J, Zhou T, Hasegawa PM, Bressan RA, Zhu JK (2007) Control of DNA methylation and heterochromatic silencing by histone H2B deubiquitination. Nature 447:735–738CrossRefPubMedGoogle Scholar
  33. 33.
    Robzyk K, Recht J, Osley MA (2000) Rad6-dependent ubiquitination of histone H2B in yeast. Science 287:501–504CrossRefPubMedGoogle Scholar
  34. 34.
    Hwang WW, Venkatasubrahmanyam S, Ianculescu AG, Tong A, Boone C, Madhani HD (2003) A conserved RING finger protein required for histone H2B monoubiquitination and cell size control. Mol Cell 11:261–266CrossRefPubMedGoogle Scholar
  35. 35.
    Wood A, Krogan NJ, Dover J, Schneider J, Heidt J, Boateng MA, Dean K, Golshani A, Zhang Y, Greenblatt JF (2003) Bre1, an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter. Mol Cell 11:267–274CrossRefPubMedGoogle Scholar
  36. 36.
    Kim J, Kim JA, McGinty RK, Nguyen UT, Muir TW, Allis CD, Roeder RG (2013) The n-SET domain of Set1 regulates H2B ubiquitylation-dependent H3K4 methylation. Mol Cell 49:1121–1133CrossRefPubMedCentralPubMedGoogle Scholar
  37. 37.
    Wu L, Lee SY, Zhou B, Nguyen UT, Muir TW, Tan S, Dou Y (2013) ASH2L regulates ubiquitylation signaling to MLL: trans-regulation of H3 K4 methylation in higher eukaryotes. Mol Cell 49:1108–1120CrossRefPubMedCentralPubMedGoogle Scholar
  38. 38.
    McGinty RK, Kim J, Chatterjee C, Roeder RG, Muir TW (2008) Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453:812–816CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Bratzel F, Lopez-Torrejon G, Koch M, Del Pozo JC, Calonje M (2010) Keeping cell identity in Arabidopsis requires PRC1 RING-finger homologs that catalyze H2A monoubiquitination. Curr Biol 20:1853–1859CrossRefPubMedGoogle Scholar
  40. 40.
    Han J, Zhang H, Zhang H, Wang Z, Zhou H, Zhang Z (2013) A Cul4 E3 ubiquitin ligase regulates histone hand-off during nucleosome assembly. Cell 155:817–829CrossRefPubMedCentralPubMedGoogle Scholar
  41. 41.
    Nishiyama A, Yamaguchi L, Sharif J, Johmura Y, Kawamura T, Nakanishi K, Shimamura S, Arita K, Kodama T, Ishikawa F et al (2013) Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication. Nature 502:249–253CrossRefPubMedGoogle Scholar
  42. 42.
    Kim K, Lee B, Kim J, Choi J, Kim JM, Xiong Y, Roeder RG, An W (2013) Linker Histone H1.2 cooperates with Cul4A and PAF1 to drive H4K31 ubiquitylation-mediated transactivation. Cell Rep 5:1690–1703CrossRefPubMedCentralPubMedGoogle Scholar
  43. 43.
    Pham AD, Sauer F (2000) Ubiquitin-activating/conjugating activity of TAFII250, a mediator of activation of gene expression in Drosophila. Science 289:2357–2360CrossRefPubMedGoogle Scholar
  44. 44.
    Pickart CM (2004) Back to the future with ubiquitin. Cell 116:181–190CrossRefPubMedGoogle Scholar
  45. 45.
    Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–533CrossRefPubMedGoogle Scholar
  46. 46.
    Wilkinson KD (2000) Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Semin Cell Dev Biol 11:141–148CrossRefPubMedGoogle Scholar
  47. 47.
    Wilkinson KD (1997) Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J 11:1245–1256PubMedGoogle Scholar
  48. 48.
    Gardner RG, Nelson ZW, Gottschling DE (2005) Ubp10/Dot4p regulates the persistence of ubiquitinated histone H2B: distinct roles in telomeric silencing and general chromatin. Mol Cell Biol 25:6123–6139CrossRefPubMedCentralPubMedGoogle Scholar
  49. 49.
    Daniel JA, Torok MS, Sun ZW, Schieltz D, Allis CD, Yates JR 3rd, Grant PA (2004) Deubiquitination of histone H2B by a yeast acetyltransferase complex regulates transcription. J Biol Chem 279:1867–1871CrossRefPubMedGoogle Scholar
  50. 50.
    Henry KW, Wyce A, Lo W-S, Duggan LJ, Emre NCT, Kao C-F, Pillus L, Shilatifard A, Osley MA, Berger SL (2003) Transcriptional activation via sequential histone H2B ubiquitylation and deubiquitylation, mediated by SAGA-associated Ubp8. Genes Dev 17:2648–2663CrossRefPubMedCentralPubMedGoogle Scholar
  51. 51.
    Emre NCT, Ingvarsdottir K, Wyce A, Wood A, Krogan NJ, Henry KW, Li K, Marmorstein R, Greenblatt JF, Shilatifard A et al (2005) Maintenance of low histone ubiquitylation by Ubp10 correlates with telomere-proximal Sir2 association and gene silencing. Mol Cell 17:585–594CrossRefPubMedGoogle Scholar
  52. 52.
    Joo HY, Jones A, Yang C, Zhai L, Smith AD 4th, Zhang Z, Chandrasekharan MB, Sun ZW, Renfrow MB, Wang Y et al (2011) Regulation of histone H2A and H2B deubiquitination and Xenopus development by USP12 and USP46. J Biol Chem 286:7190–7201CrossRefPubMedCentralPubMedGoogle Scholar
  53. 53.
    Joo HY, Zhai L, Yang C, Nie S, Erdjument-Bromage H, Tempst P, Chang C, Wang H (2007) Regulation of cell cycle progression and gene expression by H2A deubiquitination. Nature 449:1068–1072CrossRefPubMedGoogle Scholar
  54. 54.
    Trujillo KM, Tyler RK, Ye C, Berger SL, Osley MA (2011) A genetic and molecular toolbox for analyzing histone ubiquitylation and sumoylation in yeast. Methods 54:296–303CrossRefPubMedCentralPubMedGoogle Scholar
  55. 55.
    Fang J, Wang H, Zhang Y (2004) Purification of histone methyltransferases from HeLa cells. Methods Enzymol 377:213–226CrossRefPubMedGoogle Scholar
  56. 56.
    Luger K, Rechsteiner TJ, Richmond TJ (1999) Expression and purification of recombinant histones and nucleosome reconstitution. Methods Mol Biol 119:1–16PubMedGoogle Scholar
  57. 57.
    Jones A, Joo HY, Robbins W, Wang H (2011) Purification of histone ubiquitin ligases from HeLa cells. Methods 54:315–325CrossRefPubMedCentralPubMedGoogle Scholar

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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