High Content Cell‐Based Screening for New Classes of Histone demethylases

Histone methylation can be removed by demethylases. Up to now, two classes of about 20 histone demethylases specific for different lysine residues were identified, but demethylases for several other lysine residues have not been found. Also, a demethylase activity against the arginine residue has not been convincingly demonstrated. To identify new histone demethylases, we performed high content cell‐based screening. In this screening, we transfected each cDNA encoding 2500 nuclear proteins with more than 300 amine‐acids in length into cells and obtained hits that could reduce the levels of several specific methylations in lysine and arginine residues in transfected cells by immunocytochemistry. Through western blot analysis on transfected and non‐transfected cells as the secondary screening, we narrowed down the number of hits as the candidates. The candidates were expressed and purified from bacteria or insect cells, and reacted with bulk histones with various methylations or histone peptides with specific methylations in the presence or absence of various set of cofactors. The reaction was subjected to western blot analysis or mass spectrometry analysis to examine if the candidates could remove the specific methylation from bulk histones or histone peptides. We are now in the process of confirming the demethylase activity by demethylation product characterization, domain mapping, and crossing out of cofactors.