6102–6112 Nucleic Acids Research, 2016, Vol. 44, No. 13 Published online 25 March 2016 doi: 10.1093/nar/gkw193 Chromatin condensation and recruitment of PHD finger proteins to histone H3K4me3 are mutually exclusive Jovylyn Gatchalian1,†, Carmen Mora Gallardo2,†, Stephen A. Shinsky3, Ruben Rosas Ospina1, Andrea Mansilla Liendo2, Krzysztof Krajewski3, Brianna J. Klein1, Forest H. Andrews1, Brian D. Strahl3, Karel H. M. van Wely2,* and Tatiana G. Kutateladze1,* 1Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO 80045, USA, 2Department of Immunology and Oncology, Centro Nacional de Biotecnolog´ıa/CSIC, 28049 Madrid, Spain and 3Department of Biochemistry & Biophysics, The University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA Received January 06, 2016; Revised March 12, 2016; Accepted March 15, 2016 ABSTRACT modifications (PTMs), including acetylation and methyla- tion of lysine, methylation of arginine, and phosphorylation Histone post-translational modifications, and spe- of serine and threonine. Over 500 histone PTMs have been cific combinations they create, mediate a wide range identified by mass spectrometry analysis, and a number of of nuclear events. However, the mechanistic bases these modifications, or epigenetic marks, have been shown for recognition of these combinations have not been to modulate chromatin structure and cell cycle specific pro- elucidated. Here, we characterize crosstalk between cesses (1). H3T3 and H3T6 phosphorylation, occurring in mito- Methylation of histone H3 at lysine 4 is one of the canon- sis, and H3K4me3, a mark associated with active ical PTMs (2–4). The trimethylated species (H3K4me3), transcription. We detail the molecular mechanisms which is found primarily in active promoters, is impli- by which H3T3ph/K4me3/T6ph switches mediate ac- cated in transcriptional regulation (5). Methylation of lysine tivities of H3K4me3-binding proteins, including those residues in histones in general, and H3K4me3 in particular, is considered to be a stable mark compared to other PTMs containing plant homeodomain (PHD) and double Tu- (6,7). The H3K4me3 level remains fairly high and persists dor reader domains. Our results derived from nu- throughout the cell cycle owing to coordinating activities clear magnetic resonance chemical shift perturba- of K4-specific methyltransferases, such as the /KMT2 SET1 tion analysis, orthogonal binding assays and cell flu- family, and demethylases, such as the KDM5 family, though orescence microscopy studies reveal a strong anti- gene-specific alterations during mitosis have also been ob- correlation between histone H3T3/T6 phosphoryla- served. A set of epigenetic readers, including a plant home- tion and retention of PHD finger proteins in chro- odomain (PHD) finger, a double tudor domain (DTD) and matin during mitosis. Together, our findings uncover a double chromodomain (DCD), recognizes the H3K4me3 the mechanistic rules of chromatin engagement for mark by enclosing K4me3 in an aromatic cage-like bind- H3K4me3-specific readers during cell division. ing pocket (8). Preservation of H3K4me3 allows cells to re- cruit transcription complexes and resume normal gene ex- pression patterns soon after mitosis completion via bind- INTRODUCTION ing of epigenetic readers, present in these complexes, to H3K4me3-enriched promoters. Chromatin undergoes extensive reorganization throughout In contrast to lysine methylation, phosphorylation of ser- the cell cycle, transitioning from a relatively relaxed state ine and threonine residues in histone H3 fluctuates substan- in interphase to a highly condensed state in mitosis and tially throughout the cell cycle, rising sharply when cells en- returning back to the interphase state after cell division is ter mitosis (9). Phosphorylation of H3T3 (H3T3ph) is gen- completed. Cell cycle progression is highly regulated and erated, or written, by the kinase Haspin early in prophase correlates with alterations in the structure and dynamics of and is gradually erased by the PP1␥/Repo-Man phos- the nucleosome, the core building block of chromatin. Nu- phatase complex in anaphase (10,11). The H3T3ph mark cleosomal histone proteins are subject to post-translational *To whom correspondence should be addressed. Tel: +1 303 724 3593; Fax: +1 303 724 3663; Email: [email protected] Correspondence may also be addressed to Karel H. M. van Wely. Tel: +34 91 5854537; Fax: +34 91 3720493; Email: [email protected] †These authors contributed equally to the paper as first authors. C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2016, Vol. 44, No. 13 6103 recruits Survivin, a subunit of the chromosomal passenger MATERIALS AND METHODS complex (CPC) to chromatin, which is essential for proper Cloning and protein purification chromosome segregation and modulation of the Aurora B kinase activity (12–15). The protein kinase C␤I phosphory- The DIDO PHD finger construct (aa 260–325) was cloned lates H3T6 during androgen receptor-dependent gene acti- from full-length cDNA (Open Biosystems) into pDEST15 vation (16), however, a phosphatase responsible for the re- using GatewayR cloning technology. The ING1 PHD fin- moval of this mark has not yet been identified. ger construct (aa 200–279) was cloned into pGex6P1 as The close proximity of the T3ph, K4me3 and T6ph described previously (28). The PHF8 PHD finger con- residues in histone H3 suggests functional and regulatory struct (aa 37–106) in a pGex6P1 vector was a kind gift interplay between the phosphorylation and methylation from Xiaodong Cheng. Proteins were expressed in Rosetta2 modifications that can influence the binding ability ofread- (DE3) pLysS or BL21 (DE3) RIL in either Luria Broth or ers of these PTMs. In 2003, Fischle et al. introduced the M19 minimal media supplemented with 50 ␮M ZnCl2 and 15 concept of binary phospho/methyl switches and proposed NH4Cl. Cells were induced with 0.5 mM IPTG, grown for a role of these switches in the release of readers from chro- 16 h at 18◦C and harvested by centrifugation at 6k rpm. matin (17). Since then, a substantial body of work has been Cells were lysed by sonication in lysis buffer containing 25 carried out in support of this concept, which has provided a mM Tris pH 7.5 at 4◦C, 150 mM NaCl, 3 mM dithiothreitol, notable example of H3S10 phosphorylation promoting the 0.5% Triton X-100, 1 mM PMSF, 5 mM MgCl2, DNaseI dissociation of H3K9me3-binding heterochromatin protein and clarified by centrifugation at 15k rpm for 30 min. Pro- 1 (HP1) from mitotic chromatin (18,19). Co-occurrence of teins were purified on glutathione agarose beads (PierceR H3T3ph/K4me, H3K9me/S10ph, H3K27me/S28ph and cat# 16101) and the GST-tag cleaved using PreScission pro- other phospho/methyl modifications has been confirmed tease for at least overnight at 4◦C. Cleaved proteins were experimentally, and combinatorial PTM-specific antibody concentrated into 25 mM Tris pH 7.5, 150 mM NaCl and studies reveal that K4me3 and T3ph can be present on the 3 mM dithiothreitol. Unlabelled proteins were further puri- same histone H3 tail (20–22). fied by size exclusion chromatography using a HiPrep 16/60 The inhibitory effect of H3T3 phosphorylation on the Sephacryl S-100 column. interaction with H3K4me3 has been reported for DCD of the ATP-dependent chromodomain helicase DNA-binding Nuclear magnetic resonance protein 1 (CHD1) (23) and the PHD finger of the transcrip- tion initiation factor TFIID subunit 3 (TAF3) (24). In vivo NMR experiments were collected on a Varian INOVA 600 studies demonstrate that overexpression of Haspin leads to MHz spectrometer equipped with a cryogenic probe. CSP repression of TAF3-mediated gene transcription and a de- experiments were carried out at 298K using uniformly 15N- creased association of the TFIID complex with chromatin, labelled PHD finger proteins. 1H, 15N HSQC spectra were whereas this complex is retained at mitotic chromosomes as recorded in the presence of increasing concentrations of hi- a result of Haspin knockdown (24). On-bead histone pep- stone H3 peptides (aa 1–12) carrying single, dual or triple tide library screening and peptide microarrays show a con- modifications. Kd value was calculated by a nonlinear least- siderable attenuation in recognition of H3K4me3 by DTD squares analysis in Kaleidagraph using the following equa- tion: of the demethylase KDM4A and by the PHD fingers of 2 mixed lineage leukemia 5 (MLL5), death inducer obliter- ([L] + [P] + Kd) − ([L] + [P] + Kd) − 4 [P][L]) δ = δmax ator 3 (DIDO3), inhibitor of growth 2 (ING2) and recom- 2 [P] bination activating gene 2 (RAG2) when the adjacent H3T3 is phosphorylated, whereas the effect of phosphorylation where [L] is the concentration of the peptide, [P] is the con- centration of the protein, ␦ is the observed normalized of H3T6 varies (25–27). The differential reduction in bind- ␦ / chemical shift change and max is the normalized chem- ing activities of these readers suggests that phospho methyl ical shift change at saturation, calculated as switches provide a fine-tuned (possibly phase-specific) reg- ulation of the H3K4me3-recognizing