Reconstitution of nucleosome demethylation and catalytic properties of a Jumonji histone demethylase

Abstract

Jumonji histone demethylases catalyze removal of methyl marks from lysine residues in histone proteins within nucleosomes. Here, we show that the catalytic domain of demethylase JMJD2A (cJMJD2A) utilizes a distributive mechanism to remove the histone H3 lysine 9 trimethyl mark. 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. 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 further regulate demethylation activity and processivity. The quantitative assays of nucleosome demethylation developed in our work provide a platform for future work with complex chromatin substrates and full-length demethylases.

Figure 1

Catalysis and domain architecture of JMJD2A. (A) The abbreviated catalytic mechanism of Jumonji histone demethylases showing the hemiaminal intermediate. (B) Domain organization in JMJD2A.

Figure 2

Kinetic analysis of cJMJD2A-mediated demethylation of peptides. (A–B) Michaelis-Menten plots of initial velocity as a function of the concentration of ARKme3STGGK (A) and ARKme2STGGK (B). Experiments were done in triplicate and are represented as mean ± SEM. (C) Demethylation of ARKme3STGGK (200 µM) by cJMJD2A (1 µM) monitored over time using LC-MSMS. Formation of dimethylated species ([ARKme2STGGK] ~ 150 µM at 15 min) in amounts that largely exceed enzyme concentration supports distributive demethylation. Lines connecting data points are introduced to facilitate visualization and do not represent kinetic fits. For experimental conditions and procedure, see Supporting Information.

Figure 3

Demethylation of biotinylated ARKme3STGGK by cJMJD2A. Peptide (150 µM) was incubated with the demethylase (5 µM), and the extent of demethylation over time analyzed by MALDI-TOF MS. See also Figure S1.

Figure 4

Kinetic analysis of cJMJD2A-mediated demethylation of methyllysine analog (MLA)-containing peptides and nucleosomes. (A) Preparation of recombinant homogeneous H3 K_C9me3 nucleosomes. Recombinant histone H3 K9C was alkylated with (2-bromoethyl)trimethylammonium bromide to form H3 K_C9me3 histones. Subsequent assembly with histone H2A, H2B, H4, and 601 sequence DNA provided homogeneous H3 K_C9me3 nucleosomes. (B) Michaelis-Menten plot for demethylation of MLA-containing peptide. (C) Western blot analysis of an experiment performed at 350 µM cJMJD2A. Data analysis and extrapolation of k_obs is described in experimental procedures. (D) Kinetics of demethylation of MLA-containing nucleosomes. Data were obtained from three independent experiments and represented as mean ± SEM. See also Figure S2.