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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Rando OJ (2012) Combinatorial complexity in chromatin structure and function: revisiting the histone code. Curr Opin Genet Dev 22:148–155
Zentner GE, Henikoff S (2013) Regulation of nucleosome dynamics by histone modifications. Nat Struct Mol Biol 20:259–266
Osley MA (2004) H2B ubiquitylation: the end is in sight. Biochim Biophys Acta 1677:74–78
Jason LJ, Moore SC, Lewis JD, Lindsey G, Ausio J (2002) Histone ubiquitination: a tagging tail unfolds? Bioessays 24:166–174
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):1205–1215
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 431:873–878
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 22:383–394
Kao CF, Osley MA (2003) In vivo assays to study histone ubiquitylation. Methods 31:59–66
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 10:483–488
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 7:663–676
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 136:435–446
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 11:2538–2544
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 150:1182–1195
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 136:420–434
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 20:1343–1352
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 30:10096–10111
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 31:1972–1982
Ismail IH, Andrin C, McDonald D, Hendzel MJ (2010) BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair. J Cell Biol 191:45–60
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 25:637–651
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 18:184–195
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 125:703–717
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 137:459–471
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 22:1026–1035
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 12:627
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 27:1581–1595
Fleming AB, Kao CF, Hillyer C, Pikaart M, Osley MA (2008) H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation. Mol Cell 31:57–66
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 106:16686–16691
Sun Z-W, Allis CD (2002) Ubiquitination of histone H2B regulates H3 methylation and gene silencing in yeast. Nature 418:104–108
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 418:498
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 277:28368–28371
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 277:34655–34657
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 447:735–738
Robzyk K, Recht J, Osley MA (2000) Rad6-dependent ubiquitination of histone H2B in yeast. Science 287:501–504
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 11:261–266
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 11:267–274
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 49:1121–1133
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 49:1108–1120
McGinty RK, Kim J, Chatterjee C, Roeder RG, Muir TW (2008) Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453:812–816
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 20:1853–1859
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 155:817–829
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 502:249–253
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 5:1690–1703
Pham AD, Sauer F (2000) Ubiquitin-activating/conjugating activity of TAFII250, a mediator of activation of gene expression in Drosophila. Science 289:2357–2360
Pickart CM (2004) Back to the future with ubiquitin. Cell 116:181–190
Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–533
Wilkinson KD (2000) Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Semin Cell Dev Biol 11:141–148
Wilkinson KD (1997) Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J 11:1245–1256
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 25:6123–6139
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 279:1867–1871
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 17:2648–2663
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 17:585–594
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 286:7190–7201
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 449:1068–1072
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 54:296–303
Fang J, Wang H, Zhang Y (2004) Purification of histone methyltransferases from HeLa cells. Methods Enzymol 377:213–226
Luger K, Rechsteiner TJ, Richmond TJ (1999) Expression and purification of recombinant histones and nucleosome reconstitution. Methods Mol Biol 119:1–16
Jones A, Joo HY, Robbins W, Wang H (2011) Purification of histone ubiquitin ligases from HeLa cells. Methods 54:315–325
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Joo, HY., Dai, Q., Jones, A.E., Zhai, L., Wang, H. (2015). In Vitro and In Vivo Assays for Studying Histone Ubiquitination and Deubiquitination. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 1288. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2474-5_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2474-5_12
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2473-8
Online ISBN: 978-1-4939-2474-5
eBook Packages: Springer Protocols