Abstract
Histone lysine and arginine methylation involved in gene activation and silencing is dynamically regulated. However, partly limited to the research technologies previously available, the dynamics of global histone methylation on a site-specific basis have not been fully pursued. Heavy methyl-SILAC (Stable Isotope Labeling of Amino Acids in Cell Culture) labeling provides a remarkable signpost to distinguish the preexisting and newly generated methyl marks on histones. Using this technology coupled with quantitative LC-MS analysis make it possible to monitor changes in the dynamics of histone site-specific methylation. In this chapter, we comprehensively describe the experimental strategy to determine the dynamics of multiple histone methylated residues including SILAC labeling, histone extraction/purification and mass spectrometry analysis.
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Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705
Strahl BD, Allis CD (2000) The language of covalent histone modifications. Nature 403:41–45
Jenuwein T, Allis CD (2001) Translating the histone code. Science 293:1074–1080
Rice JC, Briggs SD, Ueberheide B, Barber CM, Shabanowitz J, Hunt DF, Shinkai Y, Allis CD (2003) Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains. Mol Cell 12:1591–1598
Yamane K, Toumazou C, Tsukada Y, Erdjument-Bromage H, Tempst P, Wong J, Zhang Y (2006) JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor. Cell 125:483–495
Klose RJ, Yamane K, Bae Y, Zhang D, Erdjument-Bromage H, Tempst P, Wong J, Zhang Y (2006) The transcriptional repressor JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine 36. Nature 442:312–316
Bernstein BE, Humphrey EL, Erlich RL, Schneider R, Bouman P, Liu JS, Kouzarides T, Schreiber SL (2002) Methylation of histone H3 Lys 4 in coding regions of active genes. Proc Natl Acad Sci USA 99:8695–8700
Kizer KO, Phatnani HP, Shibata Y, Hall H, Greenleaf AL, Strahl BD (2005) A novel domain in Set2 mediates RNA polymerase II interaction and couples histone H3 K36 methylation with transcript elongation. Mol Cell Biol 25:3305–3316
Steger DJ, Lefterova MI, Ying L, Stonestrom AJ, Schupp M, Zhuo D, Vakoc AL, Kim JE, Chen J, Lazar MA, Blobel GA, Vakoc CR (2008) DOT1L/KMT4 recruitment and H3K79 methylation are ubiquitously coupled with gene transcription in mammalian cells. Mol Cell Biol 28:2825–2839
Tschiersch B, Hofmann A, Krauss V, Dorn R, Korge G, Reuter G (1994) The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J 13:3822–3831
Cao R, Wang L, Wang H, Xia L, Erdjument-Bromage H, Tempst P, Jones RS, Zhang Y (2002) Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science 298:1039–1043
Schotta G, Lachner M, Sarma K, Ebert A, Sengupta R, Reuter G, Reinberg D, Jenuwein T (2004) A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. Genes Dev 18:1251–1262
Kirmizis A, Santos-Rosa H, Penkett CJ, Singer MA, Green RD, Kouzarides T (2009) Distinct transcriptional outputs associated with mono- and dimethylated histone H3 arginine 2. Nat Struct Mol Biol 16:449–451
Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA (2004) Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119:941–953
Thomas G, Lange HW, Hempel K (1975) Kinetics of histone methylation in vivo and its relation to the cell cycle in Ehrlich ascites tumor cells. Eur J Biochem 51:609–615
Pesavento JJ, Yang H, Kelleher NL, Mizzen CA (2008) Certain and progressive methylation of histone H4 at lysine 20 during the cell cycle. Mol Cell Biol 28:468–486
McManus KJ, Biron VL, Heit R, Underhill DA, Hendzel MJ (2006) Dynamic changes in histone H3 lysine 9 methylations: identification of a mitosis-specific function for dynamic methylation in chromosome congression and segregation. J Biol Chem 281:8888–8897
Waterborg JH (1993) Dynamic methylation of alfalfa histone H3. J Biol Chem 268:4918–4921
Zee BM, Levin RS, Xu B, LeRoy G, Wingreen NS, Garcia BA (2010) In vivo residue-specific histone methylation dynamics. J Biol Chem 285:3341–3350
Plazas-Mayorca MD, Zee BM, Young NL, Fingerman IM, LeRoy G, Briggs SD, Garcia BA (2009) One-pot shotgun quantitative mass spectrometry characterization of histones. J Proteome Res 8:5367–5374
Pedrioli PG, Eng JK, Hubley R, Vogelzang M, Deutsch EW, Raught B, Pratt B, Nilsson E, Angeletti RH, Apweiler R, Cheung K, Costello CE, Hermjakob H, Huang S, Julian RK, Kapp E, McComb ME, Oliver SG, Omenn G, Paton NW, Simpson R, Smith R, Taylor CF, Zhu W, Aebersold R (2004) A common open representation of mass spectrometry data and its application to proteomics research. Nat Biotechnol 22:1459–1466
Murray K (1966) The acid extraction of histones from calf thymus deoxyribonucleoprotein. J Mol Biol 15:409–419
Shechter D, Dormann HL, Allis CD, Hake SB (2007) Extraction, purification and analysis of histones. Nat Protoc 2:1445–1457
Garcia BA, Mollah S, Ueberheide BM, Busby SA, Muratore TL, Shabanowitz J, Hunt DF (2007) Chemical derivatization of histones for facilitated analysis by mass spectrometry. Nat Protoc 2:933–938
Rappsilber J, Ishihama Y, Mann M (2003) Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal Chem 75:663–670
Acknowledgments
B.A.G. gratefully acknowledges funding from a National Science Foundation grant (CBET-0941143), an Agilent Thought Leader award, an NSF Early Faculty CAREER award, and an NIH Innovator award (DP2OD007447) from the Office Of The Director, NIH.
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Cao, XJ., Zee, B.M., Garcia, B.A. (2013). Heavy Methyl-SILAC Labeling Coupled with Liquid Chromatography and High-Resolution Mass Spectrometry to Study the Dynamics of Site-Specific Histone Methylation. In: Bina, M. (eds) Gene Regulation. Methods in Molecular Biology, vol 977. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-284-1_24
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DOI: https://doi.org/10.1007/978-1-62703-284-1_24
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