In Vitro Histone Demethylase Assays
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.
Key wordsEpigenetic regulation histone methylation histone demethylation histone demethylase oxidative hydroxylation chromatin nucleosomes histone
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.
- 4.Bannister, A.J., and Kouzarides, T. 2005. Reversing histone methylation. Nature 436:1103–1106.Google Scholar
- 20.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.Google Scholar
- 22.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.Google Scholar
- 33.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.PubMedCrossRefGoogle Scholar