Abstract
Histone methylation plays important roles in chromatin structure, transcription, and epigenetic state of the cell. Tremendous discoveries recently demonstrated that methylation mark is not static but is dynamically regulated by both histone methyltransferases and the histone demethylases. Two families of histone demethylases have been identified to remove methyl groups from lysine side chain through different reaction mechanisms in presence of distinct cofactors. Amine oxidase LSD1 family requires flavin adenine dinucleotide (FAD) whereas dioxygenase Jmjc domain-containing proteins family relies on Fe(II) and alpha-ketoglutarate. Identification of these enzymes opened a new era in understanding how chromatin dynamic is regulated and further understanding the regulation of these enzymes will provide significant insights into fundamental mechanisms of many biological processes and human diseases. This chapter describes different assay conditions and detection methods for different family of histone demethylases. We also summarize step-by-step protocols for purification and preparation of various histone substrates for histone demethylase assays.
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References
Kouzarides, T. 2007. Chromatin modifications and their function. Cell 128:693–705.
Peterson, C.L., and Laniel, M.A. 2004. Histones and histone modifications. Curr Biol 14:R546–551.
Martin, C., and Zhang, Y. 2005. The diverse functions of histone lysine methylation. Nat Rev Mol Cell Biol 6:838–849.
Bannister, A.J., and Kouzarides, T. 2005. Reversing histone methylation. Nature 436:1103–1106.
Kim, S., Benoiton, L., and Paik, W.K. 1964. Epsilon-Alkyllysinase. Purification and Properties of the Enzyme. J Biol Chem 239:3790–3796.
Cuthbert, G.L., Daujat, S., Snowden, A.W., Erdjument-Bromage, H., Hagiwara, T., Yamada, M., Schneider, R., Gregory, P.D., Tempst, P., Bannister, A.J., et al. 2004. Histone deimination antagonizes arginine methylation. Cell 118:545–553.
Wang, Y., Wysocka, J., Sayegh, J., Lee, Y.H., Perlin, J.R., Leonelli, L., Sonbuchner, L.S., McDonald, C.H., Cook, R.G., Dou, Y., et al. 2004. Human PAD4 regulates histone arginine methylation levels via demethylimination. Science 306:279–283.
Shi, Y., Lan, F., Matson, C., Mulligan, P., Whetstine, J.R., Cole, P.A., Casero, R.A., and Shi, Y. 2004. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119:941–953.
Falnes, P.O., Johansen, R.F., and Seeberg, E. 2002. AlkB-mediated oxidative demethylation reverses DNA damage in Escherichia coli. Nature 419:178–182.
Trewick, S.C., Henshaw, T.F., Hausinger, R.P., Lindahl, T., and Sedgwick, B. 2002. Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage. Nature 419:174–178.
Tsukada, Y., Fang, J., Erdjument-Bromage, H., Warren, M.E., Borchers, C.H., Tempst, P., and Zhang, Y. 2006. Histone demethylation by a family of JmjC domain-containing proteins. Nature 439:811–816.
Tsukada, Y., and Zhang, Y. 2006. Purification of histone demethylases from HeLa cells. Methods 40:318–326.
Klose, R.J., and Zhang, Y. 2007. Regulation of histone methylation by demethylimination and demethylation. Nat Rev Mol Cell Biol 8:307–318.
Shi, Y., and Whetstine, J.R. 2007. Dynamic regulation of histone lysine methylation by demethylases. Mol Cell 25:1–14.
Fang, J., Hogan, G.J., Liang, G., Lieb, J.D., and Zhang, Y. 2007. The Saccharomyces cerevisiae histone demethylase Jhd1 fine-tunes the distribution of H3K36me2. Mol Cell Biol 27:5055–5065.
Kleeberg, U., and Klinger, W. 1982. Sensitive formaldehyde determination with Nash's reagent and a ‘tryptophan reaction’. J Pharmacol Methods 8:19–31.
Ausio, J., and van Holde, K.E. 1986. Histone hyperacetylation: its effects on nucleosome conformation and stability. Biochemistry 25:1421–1428.
Fang, J., Wang, H., and Zhang, Y. 2004. Purification of histone methyltransferases from HeLa cells. Methods Enzymol 377:213–226.
Mizzen, C.A., Brownell, J.E., Cook, R.G., and Allis, C.D. 1999. Histone acetyltransferases: preparation of substrates and assay procedures. Methods Enzymol 304:675–696.
Strahl, B.D., Ohba, R., Cook, R.G., and Allis, C.D. 1999. Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena. Proc Natl Acad Sci U S A 96:14967–14972.
Wang, H., Cao, R., Xia, L., Erdjument-Bromage, H., Borchers, C., Tempst, P., and Zhang, Y. 2001. Purification and functional characterization of a histone H3-lysine 4-specific methyltransferase. Mol Cell 8:1207–1217.
Agger, K., Cloos, P.A., Christensen, J., Pasini, D., Rose, S., Rappsilber, J., Issaeva, I., Canaani, E., Salcini, A.E., and Helin, K. 2007. UTX and JMJD3 are histone H3K27 demethylases involved in HOX gene regulation and development. Nature.
Christensen, J., Agger, K., Cloos, P.A., Pasini, D., Rose, S., Sennels, L., Rappsilber, J., Hansen, K.H., Salcini, A.E., and Helin, K. 2007. RBP2 belongs to a family of demethylases, specific for tri-and dimethylated lysine 4 on histone 3. Cell 128:1063–1076.
Cloos, P.A., Christensen, J., Agger, K., Maiolica, A., Rappsilber, J., Antal, T., Hansen, K.H., and Helin, K. 2006. The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3. Nature 442:307–311.
Fodor, B.D., Kubicek, S., Yonezawa, M., O'Sullivan, R.J., Sengupta, R., Perez-Burgos, L., Opravil, S., Mechtler, K., Schotta, G., and Jenuwein, T. 2006. Jmjd2b antagonizes H3K9 trimethylation at pericentric heterochromatin in mammalian cells. Genes Dev 20:1557–1562.
Iwase, S., Lan, F., Bayliss, P., de la Torre-Ubieta, L., Huarte, M., Qi, H.H., Whetstine, J.R., Bonni, A., Roberts, T.M., and Shi, Y. 2007. The X-linked mental retardation gene SMCX/JARID1C defines a family of histone H3 lysine 4 demethylases. Cell 128:1077–1088.
Tahiliani, M., Mei, P., Fang, R., Leonor, T., Rutenberg, M., Shimizu, F., Li, J., Rao, A., and Shi, Y. 2007. The histone H3K4 demethylase SMCX links REST target genes to X-linked mental retardation. Nature 447:601–605.
Whetstine, J.R., Nottke, A., Lan, F., Huarte, M., Smolikov, S., Chen, Z., Spooner, E., Li, E., Zhang, G., Colaiacovo, M., et al. 2006. Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell 125:467–481.
Klose, R.J., Yamane, K., Bae, Y., Zhang, D., Erdjument-Bromage, H., Tempst, P., Wong, J., and Zhang, Y. 2006. The transcriptional repressor JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine 36. Nature 442:312–316.
Yamane, K., Toumazou, C., Tsukada, Y., Erdjument-Bromage, H., Tempst, P., Wong, J., and Zhang, Y. 2006. JHDM2A, a JmjC-containing H3K9 demethylase, facilitates transcription activation by androgen receptor. Cell 125:483–495.
Klose, R.J., Yan, Q., Tothova, Z., Yamane, K., Erdjument-Bromage, H., Tempst, P., Gilliland, D.G., Zhang, Y., and Kaelin, W.G., Jr. 2007. The retinoblastoma binding protein RBP2 is an H3K4 demethylase. Cell 128:889–900.
Lee, N., Zhang, J., Klose, R.J., Erdjument-Bromage, H., Tempst, P., Jones, R.S., and Zhang, Y. 2007. The trithorax-group protein Lid is a histone H3 trimethyl-Lys4 demethylase. Nat Struct Mol Biol 14:341–343.
Yamane, K., Tateishi, K., Klose, R.J., Fang, J., Fabrizio, L.A., Erdjument-Bromage, H., Taylor-Papadimitriou, J., Tempst, P., and Zhang, Y. 2007. PLU-1 is an H3K4 demethylase involved in transcriptional repression and breast cancer cell proliferation. Mol Cell 25:801–812.
Klose, R.J., Gardner, K.E., Liang, G., Erdjument-Bromage, H., Tempst, P., and Zhang, Y. 2007. Demethylation of histone H3K36 and H3K9 by Rph1: a vestige of an H3K9 methylation system in Saccharomyces cerevisiae? Mol Cell Biol 27:3951–3961.
Fang, J., Chen, T., Chadwick, B., Li, E., and Zhang, Y. 2004. Ring1b-mediated H2A ubiquitination associates with inactive X chromosomes and is involved in initiation of X inactivation. J Biol Chem 279:52812–52815.
Acknowledgments
Work in the Fang lab is supported by funds from the American Cancer Society Institutional Research Grant (ACS-IRG) Program and H. Lee Moffitt Cancer Center & Research Institute.
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Kokura, K., Fang, J. (2009). In Vitro Histone Demethylase Assays. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 523. Humana Press. https://doi.org/10.1007/978-1-59745-190-1_17
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DOI: https://doi.org/10.1007/978-1-59745-190-1_17
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