Dr. P. Heun, MPI for Immunology, Freiburg, Germany) and SF4 (kind gift of D. Arndt-Jovin, MPI for Biophysical Chemistry, Gottingen, Germany), we found that Sf4 cells showed the best response and remained healthy under the various treatment conditions. (B) FACS analysis of asynchronously growing SF4 cells (in green, labelled As), SF4 cells arrested at in G1/S after treatment for 16 hours with aphidicholin (10 mM) and hydroxy- urea (1.5 mM) (in red, labelled A/H), and SF4 cells treated with the aurora kinase inhibitor ZM44739 (50 mM) for 16 hours (in blue, labelled ZM). (C) Western blot quantification of H3S10ph in SF4 cells arrested in G1/S (A/H) and treated with aurora kinase inhibitor (ZM). (D) Western blot quantification of H3 S10phK14ac after JIL-1 RNAi in asynchronous and G1/S arrested L2.4 cells. (E) Comparison of the high resolution ChIP on chip profile of H3S10phK14ac presented in Figure 3 with H3S10ph profiles obtained from A/H treated cells and from ZM treated cells. A 250 kb portion of the X chromosome is shown. Found at: doi:10.1371/journal.pgen.1001327.s006 (1.44 MB TIF) Figure S7 Specificities of the anti-histone H3 tail antibodies used in this study. Peptide chips (JPT Peptide Technology) consisting of three replicate arrays of 158 peptides, mostly histone peptides furnished with known posttranslational modifications or combi- nation of modifications have been used to test the selectivity of (A) the anti-H3S10phK14ac serum [40], (B) the anti-H3S10ph serum and (C) the same anti-H3S10ph antibodies after affinity purifica- tion [39]. All of them were incubated in PBS, 3% BSA at 1/ 10,000 dilution. A secondary antibody coupled to an IR dye was then used for detection with the Odyssey system (LI-COR Biosciences). The strongest reactivity seen for the H4(86-98) peptide in panel C is due to the secondary antibody. The insert in each panel shows the mean signal for each spotted peptide in the three replicated arrays. Found at: doi:10.1371/journal.pgen.1001327.s007 (1.64 MB TIF) Figure S8 Comparison of the gene-wise distribution of various features for genes of increasing ePol density. (A) Same represen- tation as in Figure 5 with 6 groups of genes of increasing ePol density for both autosomes and X chromosome. (B) Average distribution of H3K36me3 for the groups of genes defined in A. (C) Average distribution of MSL1. (D) Average distribution of H3K4me2. Found at: doi:10.1371/journal.pgen.1001327.s008 (0.96 MB TIF) Figure S9 JIL-1 and H3K36me3 distribution on long genes. (A) Five active autosomal genes and (B) 5 active X-linked genes are depicted using the same colour code and scales as in Figure 1. The strong similarity of the distribution of JIL-1 (green) and H3K36me3 (pink) on long genes in SL2 cells is illustrated. Those genes often have a long 59 intron, which is decorated by ePol (orange) and H4K16ac (blue) but depleted in H3K36me3 and JIL- 1. Found at: doi:10.1371/journal.pgen.1001327.s009 (2.30 MB TIF) Figure S10 ePol and JIL-1 distribution in SL2 cells upon heat shock treatment. SL2 cells were subjected to 29, 59 and 109 heat shock treatment as previously described [68] and processed for ChIP according to our standard protocol. Chromatin from uninduced (NHS) cells was used as a reference. The primers for References JIL-1 and Transcriptionn qPCR analysis are as described [68]. (A) ePol ChIP. (B) JIL-1 ChIP. (C) Western blot quantification of ePol, JIL-1 and H3K36me3 in the different chromatin preparation. Found at: doi:10.1371/journal.pgen.1001327.s010 (1.16 MB TIF) Figure S11 Cortactin expression upon Gal4-mof recruitment in the reporter lines. Total RNA was prepared from sorted males and females of the reporter line and of the line with additional expression of Gal4-mof. Quantitative RT-PCR was done to evaluate the expression of the Cortactin gene relative to that of the housekeeping gene Tub97E. The mean of 3 measurements on different cDNA dilutions is represented and error bars are the standard error of the mean. The corresponding relative enrich- ment of H4K16ac at the Cortactin gene in ChIP experiments is mentioned below the graph. Found at: doi:10.1371/journal.pgen.1001327.s011 (0.49 MB TIF) Figure S12 JIL-1 distribution along genes scales with H3K36me3 and H4K16ac. Average distribution profiles of chromatin features along genes scaled for length from transcrip- tion start (TS) to termination sites (TT). The JIL-1 distribution is shown for all genes covered on both our tiling array and the tiling array used for H3K36me3 profiles [30] after grouping into six equally-sized bins (containing about 455 genes each) for both autosomal and X-linked probes separately based on (A) increasing densities of H3K36me3, (B) increasing density of H4K16ac and (C) increasing density of H3K36me3+H4K16ac. Found at: doi:10.1371/journal.pgen.1001327.s012 (0.46 MB TIF) Figure S13 ePol and JIL-1 partition differentially in various chromatin types. (A) Partitioning of ePol in the five chromatin types [45]. (B) Partitioning of JIL-1 in the five types of chromatin. (C) Relative distribution of all probes in the five types of chromatins is compared to ePol and JIL-1 enriched probes. (D) A 180 kb portion of the X chromosome is shown where ePol and JIL-1 mark a green chromatin domain together with H3K9me2. (E) A 180 kb portion of an autosome is shown to illustrate the colocalisation of JIL-1, H3K36me3 and MRG15 colocalize in yellow chromatin. Found at: doi:10.1371/journal.pgen.1001327.s013 (1.12 MB TIF) Table S1 List of qPCR primer sequences used in this work. Found at: doi:10.1371/journal.pgen.1001327.s014 (0.03 MB PDF) Acknowledgments We thank M. Prestel for the reporter fly line expressing Gal4-Mof, C. Grimaud for mapping the insertion of the transgene of the reporter fly line, H. Mitlo¨hner for fly pushing, and M. Prestel and R. Villa for critical reading of the manuscript. We are grateful to C. Seiser for the anti- H3S10phK14ac serum, to L. Mahadevan for the anti-H3S10ph serum, to M. Kuroda for the anti-MSL3 serum, and to H. Saumweber for the T40 anti-lamin antibody. Author Contributions Conceived and designed the experiments: CR PBB. Performed the experiments: CR AM IKD VF. Analyzed the data: CR TS. Wrote the paper: CR TS PBB. 1. Wang Y, Zhang W, Jin Y, Johansen J, Johansen KM (2001) The JIL-1 tandem kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila. Cell 105: 433–443. 2. Jin Y, Wang Y, Walker DL, Dong H, Conley C, et al. (1999) JIL-1: a novel chromosomal tandem kinase implicated in transcriptional regulation in Drosophila. Mol Cell 4: 129–135. 3. Bao X, Cai W, Deng H, Zhang W, Krencik R, et al. (2008) The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting to chromatin. J Biol Chem 283: 32741–32750. 4. Deng H, Zhang W, Bao X, Martin JN, Girton J, et al. (2005) The JIL-1 kinase regulates the structure of Drosophila polytene chromosomes. Chromosoma 114: 173–182. PLoS Genetics | www.plosgenetics.org 15 March 2011 | Volume 7 | Issue 3 | e1001327