Purification of histone demethylases from HeLa cells.
Posttranslational histone modifications play an important role in regulating chromatin dynamics and function. One of the modifications, methylation, occurs on both lysine and arginine residues and participates in diverse range of biological processes including heterochromatin formation, X-chromosome inactivation, and transcriptional regulation. While acetylation, phosphorylation, and ubiquitylation are dynamically regulated by enzymes that catalyze the addition and removal of a particular modification, enzymes that are capable of removing methyl groups were not known until recently. Thus far, two families of histone demethylases with distinct cofactor requirements and reaction mechanisms have been identified. One is the FAD (flavin adenine dinucleotide)-dependent amine oxidase family LSD1 (lysine-specific demethylase), the other is the Fe(II) and alpha-KG (alpha-ketoglutarate)-dependent dioxygenase family JHDM (JmjC domain-containing histone demethylase). Identification and characterization of these histone demethylases is an important step towards understanding both the function and regulation of histone methylation. Here, we describe assays currently used for measuring histone demethylase activity and chromatography strategies used in purifying histone demethylases from HeLa cells.
Tsukada
Yu-ichi
Y
Howard Hughes Medical Institute, Chapel Hill, NC 27599-7295, USA.
Zhang
Yi
Y
D002845:Chromatography / Q000379:methods; D044783:F-Box Proteins; D006367:HeLa Cells; D056466:Histone Demethylases; D006657:Histones / Q000378:metabolism*; D006801:Humans; D056484:Jumonji Domain-Containing Histone Demethylases; D008745:Methylation; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D010089:Oxidoreductases, N-Demethylating / Q000032:analysis* / Q000302:isolation & purification*
D044783:F-Box Proteins; D006657:Histones; D046912:Multiprotein Complexes; D056466:Histone Demethylases; D056484:Jumonji Domain-Containing Histone Demethylases; C534524:KDM2A protein, human; C495195:KDM1A protein, human; D010089:Oxidoreductases, N-Demethylating