(T7_GST_for: TTAATACGACTCACTATAGGGAGAATGT- CCCCTATACTAGGTTA, T7_GST_rev: TTAATACGACT- CACTATAGGGAGAACGCATCCAGGCATTG). In the sec- ond set JIL-1 was knock- down by targeting its 39 coding sequences (T7_JIL-139_for: TTAATACGACTCACTATAGGGAGACAG- CAGCGTCG, T7_JIL-139_rev:TTAATACGACTCACTATAG- GGAGATTGGAACTGAT) and the control dsRNAs represented GFP sequences (T7_GFP_for: TTAATACGACTCACTATAG- GGTGCTCAGGTAGTGGTTGTCG, T7_GFP_rev:TTAAT- ACGACTCACTATAGGGCCTGAAGTTCATCTGCACCA). Off-target effects were minimized using http://dscheck.rnai.jp/. Efficiency of the depletion was assessed on whole cell extracts and quantified with the Odyssey imaging system (LI-COR, Bioscienc- es) using anti-tubulin (T9026, clone DM1A, Sigma) or anti-lamin (Clone T40, [67]) as reference. Total RNA was purified from 20- 806106 cells using either the RNA purification kit (RNeasy mini kit, Qiagen) or the lysis reagent (Qiazol, Qiagen) according to manufacturers instructions. The quality of the RNA was determined on the Bioanalyzer (Agilent). Expression analysis after JIL-1 RNAi Total RNA was processed for hybridization at ImaGenes (Berlin, Germany). Five biological replicates for each control and JIL-1 RNAi were analysed. Microarray data were processed using R/Bioconductor (www.bioconductor.org). Unless indicated other- wise we used standard parameters in all function calls. Expression values were calculated using ‘gcrma’. Probe sets were kept for differential expression analysis if there were more ‘present’ calls (calculated using ‘mas5calls’) in one of the two treatment groups than non-‘present’ calls and if their expression level variance was higher than 0 across all arrays. ‘‘One gene to many probe set‘‘ relationships were resolved by retaining only the probe set with the highest variance across all arrays. Differential expression statistics were obtained using a linear model (library ’limma) including a batch effect coefficient as two sets of arrays were processed at different times. A significant response was defined if the local false discovery (‘locfdr’ package) rate calculated on the moderated t statistic was smaller than 0.2. Microscopy Polytene chromosome staining was according to [44]. The affinity-purified antibodies R70 or R69 were used diluted 1/150 and the H5 monoclonal antibody at 1/50. Staining of SL2 cells was according to a standard protocol. In brief, after fixation with 3.7% (w/v) formaldehyde in PBS buffer, the cells were permeabilised with 0.3% triton X-100 (v/v) in PBS in the presence of 1% formaldehyde. The affinity-purified antibodies of R70 or R69 were diluted at 0.4 mg/ml, the anti-H4K16ac antibody #39167 (ACTIVE MOTIF) was diluted 1/1000 for counterstaining of the X-chromosome territory, and the anti-lamin clone T40 [67] was diluted at 1/100 to delimitate the nuclear territory. Epifluorescence microscopy was done on the Axiovert 200 inverted microscope (Zeiss) and confocal images were taken using LSM510 Meta system (Zeiss). Accession numbers Microarray data has been deposited at GEO (accession GSE22620). The Nimblegen tiling data have been deposited at GEO (accession GSE22618). Supporting Information Figure S1 Quality control of the JIL-1 antibodies in western blot and immunostaining. (A) Equivalent loading of SL2 whole cell JIL-1 and Transcriptionn extract (lane 1), Soluble fraction (lane 2) and Chromatin fraction (lane 3) were probed in western blot detection for JIL-1 (R70), tubulin (Tub) and histone H3. JIL-1 is enriched in the chromatin fraction. The asterisk marks a cross-reacting protein detected by R70 affinity purified antibodies, which is mostly in the cytoplasmic fraction. Coomassie staining of the fractions is shown below. M: Molecular weight markers. (B) Staining of male and female wt polytene chromosomes with anti-JIL-1 serum R70 and with the H5 mAb against ePol. For accurate comparison a male and a female salivary gland were processed together on one slide. On the left are the chromosomes of a female nucleus and on the right the chromosomes of a male nucleus. (C) Staining of Drosophila cells. In female KC cells JIL-1 (probed with R69) is evenly distributed in the nucleus. The hyperactivated X chromosome is detected by MSL3 staining in the male SL2 cells. JIL-1 enrichment on the X chromosome territory is observed with the two different affinity- purified antibodies R69 and R70 both used at 0.4 mg/ml. Found at: doi:10.1371/journal.pgen.1001327.s001 (3.55 MB TIF) Figure S2 Quality control of JIL-1 antibodies in ChIP. (A) The two affinity-purified antibodies raised against JIL-1 were used for ChIP-on-Chip from one batch of SL2 chromatin. A 250 kb portion of the X chromosome around phl for the two antibodies R69 and R70 is shown. (B) Corresponding correlation plot for the two independent profiles gives a Pearson correlation coefficient of R = 0.89. Found at: doi:10.1371/journal.pgen.1001327.s002 (1.17 MB TIF) Figure S3 JIL-1, H3K36me3, and MSL1 densities on genes are not proportional to transcript level. (A) Scatter plot representation illustrating the correlation of the steady-state mRNA levels determined on Affymetrix expression arrays (x axis) and the density of various features on genes (y axis). The Pearson correlation coefficients are given for each plot. Average density of ePol per gene. (B) Average density of JIL-1 per gene. (C) Average density of H3K36me3 per gene [30]. (D) Average density of MSL1 per gene [24]. Found at: doi:10.1371/journal.pgen.1001327.s003 (3.32 MB TIF) Figure S4 Comparison of ePol, JIL-1, and DCC subunits distributions on the X Chromosome. (A) Distributions of JIL-1, ePol, MSL1, MSL2, MSL3 [23] and MOF are shown on a 250 kb portion of the X chromosome. (B) Correlation plots of the different data sets. Found at: doi:10.1371/journal.pgen.1001327.s004 (2.67 MB TIF) Figure S5 JIL-1 kinase activity on histone H3 peptides, histone octamers, and oligo-nucleosomes. (A) Kinase assays of recombi- nant Flag-JIL-1 on recombinant histone H3 and H3 peptides harbouring various modifications have been analyzed by SDS- PAGE and autoradiography. The quantification on 3 independent replicate assays is represented on the left and one of the autoradiogram on the right. (B) Autoradiogram of a kinase assay with 2 mg of reconstituted recombinant histone octamers on the left and 2 mg of the same octamers assembled on a plasmid DNA to oligo-nucleosomes. Titration of increasing concentration of NaCl in the assays showed that the activity of JIL-1 (autopho- sphorylation and H3 phosphorylation in octamers) slightly drops but does not favour the phosphorylation of H3 within oligo- nucleosomes. Found at: doi:10.1371/journal.pgen.1001327.s005 (1.21 MB TIF) Figure S6 Contribution of mitotic and interphase H3S10ph in SL2 cells. (A) Western blot quantification of H3S10ph after JIL-1 (J1, J2, J3) and control GST RNAi (G1, G2, G3) in SL2 cells in 3 independent replicate experiments. After titration experiments with SL2 cells as well as with the derived clones L2.4 (kind gift of PLoS Genetics | www.plosgenetics.org 14 March 2011 | Volume 7 | Issue 3 | e1001327