Global Analysis of the Relationship between JIL-1 Kinase and Transcription Catherine Regnard, Tobias Straub, Angelika Mitterweger, Ina K. Dahlsveen, Viola Fabian, Peter B. Becker* Adolf-Butenandt-Institute and Centre for Integrated Protein Science (CiPSM), Ludwig-Maximilians-University, Munich, Germany Abstract The ubiquitous tandem kinase JIL-1 is essential for Drosophila development. Its role in defining decondensed domains of larval polytene chromosomes is well established, but its involvement in transcription regulation has remained controversial. For a first comprehensive molecular characterisation of JIL-1, we generated a high-resolution, chromosome-wide interaction profile of the kinase in Drosophila cells and determined its role in transcription. JIL-1 binds active genes along their entire length. The presence of the kinase is not proportional to average transcription levels or polymerase density. Comparison of JIL-1 association with elongating RNA polymerase and a variety of histone modifications suggests two distinct targeting principles. A basal level of JIL-1 binding can be defined that correlates best with the methylation of histone H3 at lysine 36, a mark that is placed co-transcriptionally. The additional acetylation of H4K16 defines a second state characterised by approximately twofold elevated JIL-1 levels, which is particularly prominent on the dosage-compensated male X chromosome. Phosphorylation of the histone H3 N-terminus by JIL-1 in vitro is compatible with other tail modifications. In vivo, phosphorylation of H3 at serine 10, together with acetylation at lysine 14, creates a composite histone mark that is enriched at JIL-1 binding regions. Its depletion by RNA interference leads to a modest, but significant, decrease of transcription from the male X chromosome. Collectively, the results suggest that JIL-1 participates in a complex histone modification network that characterises active, decondensed chromatin. We hypothesise that one specific role of JIL-1 may be to reinforce, rather than to establish, the status of active chromatin through the phosphorylation of histone H3 at serine 10. Citation: Regnard C, Straub T, Mitterweger A, Dahlsveen IK, Fabian V, et al. (2011) Global Analysis of the Relationship between JIL-1 Kinase and Transcription. PLoS Genet 7(3): e1001327. doi:10.1371/journal.pgen.1001327 Editor: Bas van Steensel, Netherlands Cancer Institute, The Netherlands Received July 16, 2010; Accepted February 5, 2011; Published March 10, 2011 Copyright: 2011 Regnard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been supported by Grants Be1140/6-1 and SFB-TR5-A1 of the Deutsche Forschungsgemeinschaft to PBB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pbecker@med.uni-muenchen.de Introduction JIL-1 is a ubiquitously expressed, nuclear tandem kinase of Drosophila melanogaster that associates with chromatin at all stages of development. JIL-1 phosphorylates histone H3 in chromatin and the kinase is thought to be responsible for the majority of phosphorylation of histone H3 at serine 10 (H3S10ph) in interphase [1]. At the low level of resolution afforded by staining larval polytene chromosomes JIL-1 is seen to associate with active chromatin such as the decondensed interbands of chromosomes [2]. Ectopic recruitment of JIL-1 to a series of Lac operator repeats by fusion to a lacI DNA binding domain leads to local decondensation of polytene chromatin. This reorganisation of chromatin depends on JIL-1’s kinase activity [3]. The essential function of JIL-1 can be explained, at least in part, by the involvement of the kinase in chromosome organisation [4,5], where it plays a role in maintaining the balance between euchromatin and heterochromatin [6–8]. This is supported by the fact that Su(var)3-1 alleles of the JIL-1 gene are strong suppressors of position effect variegation, which provides a sensitive assay for heterochromatin propagation [9]. In the absence of JIL-1 the H3K9me2 mark and HP1 are redistributed genome-wide and tend to be enriched on the X chromosome in both males and females [6]. In addition, the lethal loss-of-function mutation of JIL-1 is rescued by reduced levels of the H3K9 methyltransferase Su(var)3-9 but not by reduced levels of the major HP1 isoform HP1a [10] suggesting that the spreading of H3K9me2 might be the cause of the lethality of the JIL-1 mutants. However, reducing the dose of Su(var)3-7, another essential component of heterochromatin, also rescues the lethality of the JIL-1 mutant. In this case the redistribution of H3K9me2 was still observed [11]. Su(var)3-7 may thus be an essential effector in the pathway of heterochromatin spreading, which is counteracted by JIL-1. In mammalian cells the H3S10ph mark has been implicated in transcriptional activation, notably in immediate early response to mitogen, stress or steroid hormones, which trigger the transient phosphorylation of H3S10 and H3S28 by the kinases Msk1/2 or PIM-1 (for review see [12]. Whereas some of the inducing effects involve the dissociation of euchromatic HP1 isoforms [13,14], the H3S10ph mark also serves as docking site for 14-3-3 proteins, which in turn recruit additional activating enzymes [13,15,16]. In D. melanogaster, the role of JIL-1 and the H3S10ph mark in transcription regulation is less clear. Corces and colleagues reported that upon heat shock gene activation, H3S10 phosphor- ylation by JIL-1 leads to recruitment of 14-3-3 and of the elongator protein 3 (Elp3) [17] that in turn would trigger the transition of an initiated RNA polymerase II (Pol II) into the elongation mode PLoS Genetics | www.plosgenetics.org 1 March 2011 | Volume 7 | Issue 3 | e1001327