UCSF

Jumonji histone demethylases catalyze removal of methyl marks from lysine residues in histone proteins within nucleosomes. Misregulation of demethylation has been correlated with diseases including various forms of cancer, obesity, and x-linked mental retardation. Our study of these enzymes is described in this dissertation. In Chapter 1, we described the preparation of histone H3 lysine 9 (H3K9) demethylases and the optimization of demethylase assay conditions. In Chapter 2, we described our work on the demethylation of homogeneously methylated nucleosomes and the investigation of the intrinsic processivity of the catalytic domains of a H3K9 Jumonji demethylase, JMJD2A. By developing a method to assess demethylation of homogeneous, site-specifically methylated nucleosomes, we determined that the kinetic parameters for demethylation of nucleosomes by cJMJD2A are comparable to those of peptide substrates, suggesting that the catalytic domain does not recognize additional features of the nucleosome. These findings imply that other domains of the demethylase or its protein partners may contribute to nucleosome recognition in vivo, and in this way, may regulate demethylation activity and processivity. Importantly, our work demonstrates that quantitative assessment of nucleosome demethylation is feasible and provides a platform for future work with complex chromatin substrates and full-length demethylases. In Chapter 3, we described our studies on the possible oligomerization of JMJD2A. Lastly, in Chapter 4, we presented our work on the synthesis and evaluation of candidate mechanism-based inhibitors of JMJD2A. Together, our work provides a better understanding of the intrinsic properties of JMJD2A mediated demethylation and serves as foundations for future studies in demethylase activity regulation.