Dissecting contributions of catalytic and reader domains in regulation of histone demethylation

Histone demethylases catalyze the removal of methyl marks from histones, an activity associated with transcriptional regulation and DNA damage repair. As these processes are critical for normal physiology, deregulation of histone demethylases is disease causative, and their function and regulation are targets for therapeutic intervention. The larger of two histone demethylase families are Jumonji C (JmjC) demethylases. The members of the JmjC family share a conserved catalytic domain, and often contain non-catalytic domains that “read” the modification state of chromatin. By binding to specific histone modifications, reader domains assist in recruitment and promote accumulation of demethylases at their targets, as well as regulate their activity and substrate specificity. Here, we present protocols for the investigation of this functional coupling between reader and catalytic domains in human histone demethylase KDM5A. Although we use KDM5A and its PHD1 domain as our model system, the procedures presented herein can be applied for the biochemical characterization of other JmjC demethylases and chromatin readers.