In Vitro Histone Demethylase Assays

  • Kenji Kokura
  • Lidong Sun
  • Jia FangEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1288)


Histone methylation plays pivotal roles in modulating chromatin structure and dynamics and in turn regulates genomic processes that require access to the DNA template. The methylation status at different sites is dynamically regulated by histone methyltransferases and demethylases. During the past decade, two classes of proteins have been characterized to actively remove methyl groups from lysine residues through different mechanisms. The LSD1/KDM1 family of amine oxidases require flavin adenine dinucleotide (FAD) for reaction, while the larger Jumonji C (JmjC) family of hydroxylases utilize Fe(II) and α-ketoglutarate as cofactors to demethylate histones. Since their discoveries, histone demethylases have been implicated in the precise control of gene expression program during development, cell identity, and fate decision. Several demethylases have also been linked to various human diseases such as neurological disorders and cancer. This chapter describes several in vitro assay conditions and detection methods for two classes of histone demethylases. We also discuss the protocols to prepare various substrates for different histone demethylase assays.

Key words

Epigenetic regulation Histone methylation Histone demethylation Histone demethylase Oxidative hydroxylation Chromatin Nucleosomes Histone 



Works in Fang lab are supported by grant from the James and Esther King Biomedical Research Program, Florida Department of Health (3KN02), and RO1CA172774 from the NIH.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Tumor Biology DepartmentH Lee Moffitt Cancer Center and Research InstituteTampaUSA
  2. 2.Department of Molecular OncologyH Lee Moffitt Cancer Center and Research InstituteTampaUSA

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