Authors Summary Active chromatin is characterised by a wealth of histone modifications that collectively define a decondensed form of chromatin that is accessible to regulators and more easily transcribed. The kinase JIL-1 associates with active chromatin and contributes to chromatin decondensation by phosphorylation of a key serine residue (S10) on the N- terminus of histone H3. We mapped the binding sites for JIL-1 on chromosomes in cells and found that the enzyme associates all along transcribed genes. A basal level of association can be found at most genes and correlates best with the presence of active chromatin exemplified by methylation of H3K36. A double dose of JIL-1 can be observed at active genes of the hyperactive male X chromosome that also bear an acetylation of histone H4 at lysine 16. Phosphorylation of H3 by JIL-1 creates a mark that characterises active chromatin and could play a role in preventing the formation of repressive chromatin. Surpris- ingly, depletion of the kinase has clear, but relatively modest, effects on transcription. We hypothesise that JIL-1 may help to reinforce the active state of chromatin that is primarily established by other factors. [18]. Johansen and co-workers by contrast argued that JIL-1 and JIL-1-dependent Ser10 phosphorylation did not have a direct function in heat shock gene activation [19]. We are interested in the potential involvement of JIL-1 in the process of X chromosome dosage compensation in D. melanogaster, which is hypothesized in light of a number of intriguing links between JIL-1 and the compensated male X chromosome. The process of dosage compensation serves to increase the transcription of genes on the single male X chromosome approximately twofold to match the combined transcription of the two alleles on the female X chromosomes [20,21]. JIL-1 is enriched on the male X chromosome and this enrichment depends on a functional DCC [2,22]. Severe reduction of JIL-1 levels leads to a global change in chromatin structure that is best appreciated on polytene chromosomes [1]. The euchromatic interbands appear much reduced in the absence of JIL-1 and replaced by atypical chromatin, in agreement with the above-mentioned role of JIL-1 in restricting the spread of heterochromatin. Interestingly, the dosage-compensated X chromosome shows an enhanced sensitiv- ity to reduced JIL-1 levels, and appears diffuse and lacking polytene banding pattern [1,4]. The Dosage Compensation Complex (DCC, also known as Male-Specific-Lethal [MSL] complex) associates along the transcribed regions of target genes and may therefore boost transcription elongation [23,24]. The activation of genes on the male X chromosome involves the DCC subunit MOF on the X chromosome, an acetyltransferase, which selectively acetylates histone H4 at K16 (H4K16ac) [25,26]. H4K16ac opposes the folding of the nucleosomal fibre in vitro and should render chromatin more accessible [27]. Accordingly, chromatin from the male X chromosome is released more efficiently from nuclei after shearing [28]. However, the precise twofold enhancement of transcription cannot be explained by H4K16ac alone [26]. We now mapped for the first time at high resolution the chromosomal distribution of JIL-1 kinase in SL2 cells using chromatin immunoprecipitation (ChIP) and hybridisation of associated DNA to oligonucleotide tiling microarrays. JIL-1 binding was compared to the distribution of elongating RNA polymerase II and related to steady-state mRNA levels. We also compared them to profiles of a number of histone modifications, JIL-1 and Transcriptionn and of DCC subunits. The vast majority of active genes is marked by a basal level of JIL-1 on gene bodies, independently of the actual transcription rates. The presence of H4K16ac on X-linked genes correlates with roughly twofold elevated levels of JIL-1. JIL- 1 thus marks transcribed genes, but it also senses dosage- compensated chromatin. The phosphorylation of H3S10 is enriched on active genes in the same manner as JIL-1 itself. Perhaps surprisingly, the depletion of JIL-1 by RNA interference (RNAi) has only mild effects of transcription, which suggests that JIL-1 is not absolutely required for transcription in general or for dosage compensation in particular. In the light of the previous genetic analysis our data suggest that JIL contributes to a composite histone mark that may serve to reinforce the active state of chromatin by preventing the inappropriate association of silencing factors. Results JIL-1 binds most transcribed genes and is enriched on dosage-compensated genes We raised two polyclonal antisera (R69 and R70) against a fragment (amino acids 79–571) of JIL-1 and established their specificity. In agreement with previous results [22], we confirmed that JIL-1 is a nuclear protein that associates with active, decondensed chromatin represented by interbands on larval polytenized chromosomes (Figure S1). We also visualized the clear enrichment of JIL-1 on the dosage-compensated, hyperactive male X chromosome. This enrichment on the X chromosomal territory can also be seen in nuclei of the Drosophila SL2 cells, which have a male genotype. By contrast, nuclei of female KC cells display a homogenous staining (Figure S1). The enrichment of JIL-1 on the male X chromosome and the conflicting reports about the role of JIL-1 in the control of early transcription elongation of heat shock genes [17–19] prompted us to generate a high-resolution in vivo chromosomal interaction profile of the kinase and to compare it to elongating RNA polymerase II (ePol). We combined chromatin immunoprecipita- tion (ChIP) with probing oligonucleotide tiling arrays as before [24,26,29], using the JIL-1 antibodies and a well-established monoclonal antibody specific for ePol (H5). This antibody recognises phosphorylated serine 2 in the heptad repeat of the C-terminal domain (CTD) of the largest RNA polymerase II subunit. Data from four biological replicates starting with independent chromatin preparations were combined for each profile. The two JIL-1 antibodies gave very coherent results (Figure S2). We also generated a profile for histone H4 acetylated at K16 (H4K16ac). Figure 1 displays the profiles of JIL-1, ePol and H4K16ac along representative 250 kb portions of the chromo- somes X and 3R (Figure 1A, 1B, respectively). The profiles resemble the distribution of histone H3 methylated at lysine 36 (H3K36me3), a modification that is placed co-transcriptionally by the CTD-associated methylase Set2 and therefore serves as a hallmark of transcribed chromatin [30,31]. Qualitatively, the distribution of JIL-1 and ePol were similar on the X chromosome and on autosomes with a preference for coding sequences. In general, binding of JIL-1 to introns is lower than binding of ePol (Figure 1C). As expected, ePol is mainly found at active genes as assessed by Affymetrix expression profiling (Figure 1D) and there is a good correlation between the density of ePol along genes and the corresponding steady-state mRNA level (Figure S3). The distribution of JIL-1 correlates well with several subunits of the DCC on the X chromosome: at the probe level, the best correlations are observed with the targeting subunit MSL1 [24] and with the spreading subunit MOF, which is the PLoS Genetics | www.plosgenetics.org 2 March 2011 | Volume 7 | Issue 3 | e1001327