The Tudor Domain of the PHD Finger Protein 1 Is a Dual Reader of Lysine Trimethylation at Lysine 36 of Histone H3 and Lysine 27 of Histone Variant H3t Ina Kycia 1 , Srikanth Kudithipudi 1 , Raluca Tamas 1 , Goran Kungulovski 1 , Arunkumar Dhayalan 2 and Albert Jeltsch 1 1 - Institute of Biochemistry, Stuttgart University, Pfaffenwaldring 55, 70569 Stuttgart, Germany 2 - Department of Biotechnology, Pondicherry University, R. V. Nagar, Kalapet, Puducherry 605014, India Correspondence to Albert Jeltsch: albert.jeltsch@ibc.uni-stuttgart.de http://dx.doi.org/10.1016/j.jmb.2013.08.009 Edited by S. Khorasanizadeh Abstract PHF1 associates with the Polycomb repressive complex 2 and it was demonstrated to stimulate its H3K27-trimethylation activity. We studied the interaction of the PHF1 Tudor domain with modified histone peptides and found that it recognizes H3K36me3 and H3tK27me3 (on the histone variant H3t) and that it uses the same trimethyllysine binding pocket for the interaction with both peptides. Since both peptide sequences are very different, this result indicates that reading domains can have dual specificities. Sub-nuclear localization studies of full-length PHF1 in human HEK293 cells revealed that it co-localizes with K27me3, but not with K36me3, and that this co-localization depends on the trimethyllysine binding pocket indicating that K27me3 is an in vivo target for the PHF1 Tudor domain. Our data suggest that PHF1 binds to H3tK27me3 in human chromatin, and H3t has a more general role in Polycomb regulation. © 2013 Elsevier Ltd. All rights reserved. Introduction The Polycomb group proteins are key regulators of the homeotic (HOX) genes and play central roles in development [1–3]. The Polycomb repressive complex 2 (PRC2) catalyzes the trimethylation of H3K27 resulting in transcriptional repression of target genes. PRC2 contains four essential core members, viz. the catalytic subunit EZH2, SUZ12, EED and RbAp46/48 proteins [1]. Other proteins including PHF1, JARID2 and AEBP2 associate with PRC2 and regulate its activity [4–6]. One of these proteins is the human PHF1 protein, which consists of an N-terminal Tudor domain (TD) and two PHD fingers, which interact with EZH2 [7]. It co-localizes with PRC2 complexes at HOXA loci and other genes [4,8]. Knockdown of PHF1 led to a decrease in H3K27me3 levels suggesting that PHF1 stimulates the activity of EZH2 [8]. We studied histone peptide and chromatin interaction of the PHF1-TD with the aim to understand its interplay with K27 trimethyla- tion. During the process of our work, other data were published and surprisingly showed that the PHF1-TD interacts with H3K36me3 [9–11]. They reported that this interaction leads to the inhibition of PRC2-mediated H3K27 methylation [9] and spread- ing of H3K27me3 into H3K36me3-containing chromatin regions [10]. The finding that PHF1-TD interacts with H3K36me3 and its inhibition of PRC2 activity was unexpected since it was shown previ- ously to stimulate PRC2 [8] and H3K27me3 and H3K36me3 are known to be antagonistic marks [12]. We show here that PHF1-TD interacts with the H3 variant H3t trimethylated at Lys27 in addition to H3K36me3 using the same trimethyl binding pocket for the interaction with both marks. We also show that PHF1 co-localizes with the K27me3 mark in vivo, and this interaction depends on the integrity of the trimethyllysine binding pocket of the Tudor domain. Hence, our data suggest that PHF1 is a dual reader of H3K36me3 and K27me3 in histone variant H3t. 0022-2836/$ - see front matter © 2013 Elsevier Ltd. All rights reserved. J. Mol. Biol. (2014) 426, 1651–1660 Article