Mutant Genetic Background Affects the Functional Rearrangement and Kinetic Properties of JMJD2b Histone Demethylase Eva Bártová⁎, Lenka Stixová, Gabriela Galiová, Andrea Harničarová Horáková, Soňa Legartová and Stanislav Kozubek Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i., Královopolská 135, CZ-612 65 Brno, Czech Republic Received 6 August 2010; accepted 2 November 2010 Available online 10 November 2010 Edited by J. Karn Keywords: histone code; SUV39h; JMJD2b; epigenetics; HDAC inhibitors We have studied JMJD2b histone demethylase, which antagonizes H3K9me3 in the pericentromeric heterochromatin. In cells with a deficiency in the histone methyltransferase SUV39h, the level of full-length JMJD2b (JMJD2b-GFP-1086) at chromocenters was reduced, corresponding to a global decrease in JMJD2b and H3K9me3. In wild-type fibroblasts, the chromatin of ribosomal genes, which is dense with H3K9 methylation, lacked JMJD2b-GFP-1086, while mutant and truncated forms of JMJD2b densely occupied the nucleolar compartment. This implies that the PHD Zn- fingers and Tudor domains, which were removed in truncated JMJD2b, are responsible for the aberrant JMJD2b function. Intriguingly, the JMJD2b- GFP-1086 level was significantly higher in tumor cell nucleoli. The kinetic properties of JMJD2b-GFP-1086 in the nucleoli and nucleoplasm of normal and tumor cells were similar; ∼ 50% recovery of prebleached intensity was reached after b 1 s. However, the mobile fraction of JMJD2b-GFP-1086 was increased in SUV39h-deficient cells. Similarly, the mobile fractions of mutant JMJD2b(1–424)H189A-GFP and truncated JMJD2b(1–424)GFP were greater than that measured for the full-length protein. We suggest that nucleoli are the site of an aberrant function of JMJD2b, the kinetic properties of which can be influenced by a mutant genetic background. © 2010 Elsevier Ltd. All rights reserved. Introduction Histone signature is a major determinant of transcriptionally active and silent chromatin. In particular, histone acetylation and methylation are thought to be central epigenetic events that have a fundamental role in the biology of chromatin. 1–3 There are several lysine residues that can be methylated on N-terminal histone tails. Specifically, H3K4 and H3K36 methylations are related to transcriptional activation, while H3K9, H3K27, and H4K20 methylations are mostly responsible for gene silencing and heterochromatinization, similar to heterochromatin protein 1 (HP1) binding to H3K9 methylation. 3–6 A notable exception occurs in ribosomal genes, where histones, when transcrip- tionally active, can be associated with HP1β and HP1γ. 7,8 In nucleolar regions, as well as in non- nucleolar compartments, histone methylation can appear as monomethylation, dimethylation, and *Corresponding author. E-mail address: bartova@ibp.cz. Abbreviations used: HP1, heterochromatin protein 1; HDAC, histone deacetylase; rDNA, ribosomal DNA; FRAP, fluorescence recovery after photobleaching; TSA, trichostatin A; wt, wild type; MEF, mouse embryonic fibroblast; ChIP, chromatin immunoprecipitation; dn, double-null; DFC, dense fibrillar component; PcG, Polycomb group; GFP, green fluorescence protein; αJMJD2b, antibody to JMJD2b. doi:10.1016/j.jmb.2010.11.001 J. Mol. Biol. (2011) 405, 679–695 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.