acetyltransferase responsible for H4K16 acetylation (Figure S4). On the X chromosome 1413 genes are detectably transcribed according to ePol occupancy. Among those, the vast majority (93%) have both JIL-1 and MSL1 bound, a minority of 55 genes (4%) show no detectable JIL-1, 86 genes (6%) have no MSL1 bound, and 46 genes (3%) have neither JIL-1 nor MSL1 bound. Remarkably, the densities of ePol on active genes are comparable on autosomes and on the X chromosome (Figure 1D), although the latter ones benefit from dosage compensation (see discussion). By contrast, the mean density of JIL-1 on active genes is 0.92 for autosomal and 1.71 for X-linked genes, thus the calculated enrichment of JIL-1 on X chromosomal genes relative to autosomal ones is about 1.86 (Figure 1E). These results quantitatively reproduces the picture obtained from polytene chromosome staining (Figure S1), where a twofold enrichment had been estimated [22]. The enrichment of JIL-1 at various loci was confirmed by quantitative PCR (qPCR) on the non-amplified immunoprecipi- tate. Figure 2A shows as example the X chromosomal gene pole hole (phl) where JIL-1 accumulates towards the 39 end of the gene, together with H4K16ac and H3K36me3. We also confirmed the male-specific enrichment of JIL-1 on several X-linked genes by qPCR of immunoprecipitates of chromatin prepared from sorted male and female flies (Figure 2B). The X-linked genes phl, CG14804, sta, Pgd and RpII215 show elevated levels of JIL-1 in males as compared to females. As an example for autosomal genes, we chose the Cortactin gene, which is similarly bound by JIL-1 in males and females. The H3K36me3 levels at the same genes are rather similar in both sexes, whereas H4K16ac is clearly found there only in males. We conclude that JIL-1 binds active genes in both sexes and exhibits a twofold enrichment on X chromosomal genes in males. H3 phosphorylation by JIL-1 contributes to a composite phospho-acetyl mark associated with active chromatin In order to characterise the potential of JIL-1 to phosphorylate histones we carried out in vitro kinase assays using baculovirus- expressed FLAG-tagged JIL-1. Recombinant histone H3 was phosphorylated, but not histones H2A, H2B, H4 (Figure S5A). Histone H1 prepared from Drosophila embryos, which can be phosphorylated to some extent at serine 10 [32] was not a substrate for JIL-1 in vitro (not shown). We did not detect phosphorylation of H3 in nucleosomes even by increasing the salt concentration in the assay (Figure S5A). A JIL-1 derivative, in which the catalytic domain was impaired by mutating an aspartate to alanine, had no H3 kinase activity (JIL-1D392A; [33]). We used H3-derived peptides to characterise the substrate specificity further and found that JIL-1 was unable to phosphorylate S28 in the context of the H321-34 peptide but efficiently phosphorylated the peptide H31–21. The phosphorylation occurred at serine 10 (S10) since it was abolished by incorporation of phospho-serine during peptide synthesis. At saturating amounts of peptide, neighbouring modifications of the H3 tail do not impair S10 phosphorylation (Figure S5). H3S10 phosphorylation, particularly in the context of K14 acetylation (H3S10phK14ac) had previously been shown to be enriched on the male X chromosome [1]. By ChIP the enrichment of the straight H3S10ph epitope is difficult to document (a 1.09- fold enrichment in the data of Zhang and Oliver [28]), presumably due to the considerable amount of H3S10ph contributed by aurora kinase in mitotic cells (for review, see [34,35]. Consistent with this, JIL-1 depletion by RNAi in SL2 cells did not lead to reproducible decrease of global H3S10ph levels (Figure S6). In order to estimate the contribution of mitotic H3S10ph in JIL-1 and Transcriptionn asynchronously growing SL2 cells, we arrested cells in G1/S by combined aphidicholine and hydroxyurea treatment and found the level of H3S10ph 8-fold reduced as compared to asynchro- nously growing cells. In the presence of the potent aurora kinase inhibitor ZM447439 [36] the global H3S10ph signal was 20 times reduced (Figure S6C). Since the proportion of mitotic SL2 cells in a culture has been estimated to be 4% [37], we conclude that the level of interphase H3S10ph is two orders of magnitude lower than that of mitotic H3S10ph. This may be an overestimate since an H3S10ph profile from ZM44743-treated cells showed very little signal over background (Figure S6E). The detection of interphase H3S10ph in salivary glands has been shown to greatly depend on the antibody itself as well as on the procedure [19]. In particular, some antibodies may be occluded by neighbouring modifications in the H3 tail and not recognize S10ph as part of a composite epitope [38]. The two antisera used in this study, anti-H3S10ph [39] and anti- H3S10phK14ac [40] were therefore tested for their ability to recognise the S10ph mark in the presence of neighbouring modifications on the H3 tail using a custom-made peptide microarray (Figure S7). Interaction of the anti-S10ph serum, but not of the anti-S10phK14ac serum was adversely affected by the K14 acetylation. However, the latter also detected S10ph in the absence of K14 acetylation. This may explain why upon JIL-1 depletion by RNAi in SL2 cells, no decrease in H3S10phK14ac was detected by Western blotting (Figure S6D). Because the H3S10phK14ac epitope had been found enriched on the X [1] and the anti-H3S10phK14ac had been successfully used in ChIP experiments [40] we generated a global ChIP profile with the latter antibody (Figure 3A). A view of this profile along 140 kb of the X chromosome showed that H3S10phK14ac coincides with the presence of JIL-1. In addition, the H3S10phK14ac signal was enhanced on X chromosomal probes (Figure 3B) and preferen- tially mapped to the CDS of genes. Average binding profiles revealed that an enrichment of H3S10phK14ac throughout the transcription units can be observed in particular on X chromo- somal JIL-1 target genes (Figure 3C). The global depletion of H3S10phK14ac could not be assessed by Western blot (Figure S6D), given the cross-reactivity of the antibody with (mitotic) H3S10ph but, the enrichment at the 39 end of tested target genes is reduced upon JIL-1 RNAi (Figure 3D). Taken together, the data suggest that JIL-1 contributes to generating the composite S10phK14ac modification on histone H3 at active genes. JIL-1 increases the transcription efficiency of genes on the male X chromosome The H3S10ph and H3S10phK14ac marks were correlated with active transcription in various model systems. In order to assess the contribution of JIL-1 to transcriptional output we reduced the levels of the kinase by RNAi in SL2 cells. Efficient depletion (up to 95%) was reproducibly achieved while ePol remained unaffected (Figure 4). This observation is in support of the results from the Johansen laboratory on salivary glands of the JIL-1 mutant [19]. Removal of JIL-1 did not visibly affect the association of the DCC with the X chromosomal territory, which in Figure 4C is visualised by the diagnostic H4K16ac mark. Although flies with decreasing JIL-1 levels show increasing lethality [1] the prolifer- ation of SL2 and KC cells appears only slightly affected (proliferation was monitored for up to 19 days in RNAi conditions, not shown). Comparison of steady-state mRNA levels in JIL-1-depleted versus control cells were assessed by Affymetrix profiling. Two sets of experiments each consisting of three biological replicates were conducted with different sets of dsRNA. The statistical analysis of PLoS Genetics | www.plosgenetics.org 4 March 2011 | Volume 7 | Issue 3 | e1001327