constantly transcribed. Highly transcribed genes experience nucleosome depletion and therefore do not provide anchoring sites for silencing factors. Moderately transcribed genes, however, may experience periods without active elongation [55-57]. These ‘gaps’ bear the risk of placement of silencing marks, most notable H3K9me2. In this context JIL-1 may safeguard active genes (marked by H3K36me3 as a sign of recent transcriptional activity). This function may be achieved by phosphorylation of H3S10, which prevents the recognition of HP1 by occlusion of H3K9me2 [58,59] but possibly also by phosphorylation of other substrates, like Su(var)3,9 [33]. In addition JIL-1 might have a scaffolding function for other factors, like lamins [60]. The observation that genes sensitive to the depletion of JIL-1 tend to have less JIL-1 bound both on the X chromosome and on autosomes also support a model where JIL-1 is associated to active chromatin domains to prevent the spreading of more repressive structures. We speculate that the role of JIL-1 in maintaining the balance between heterochromatin and euchromatin is not only true at the hetero/euchromatin boundary, but also within euchromatin at the level of active chromatin domains. Materials and Methods JIL-1 antibodies and kinase assay Polyclonal antisera were raised in two rabbits against a fragment of JIL-1 corresponding to amino acids 79–571, which was expressed in fusion with an N-terminal MBP-tag in E. coli (Eurogentec). The resulting sera R69 and R70 were either used crude or after affinity purification on a column with covalently coupled antigen according to a standard protocol. For Western blot, the sera were diluted 1/2000 and affinity-purified antibodies were used at 0.5 mg/ml. Recombinant Flag-tagged JIL-1 and kinase assay conditions were as previously described [33]. Reporter lines and b-galactosidase activity measurements The transgenic line used for the reporter assay in flies was originally published as U/l5 [42]. The P-element insertion site was mapped by PCR-rescue to the 39end of the Cortactin gene (Figure 6A). To activate the reporter by the Gal4 activator homozygote flies of the reporter line were crossed with males of the activator line, which carry a transgene driving the expression of the Gal4 activator under the control of the tubulin promoter (Bloomington Stock Center #5138). Gal4 is expressed from the cellular blastoderm onwards. The F1 flies were hand-sorted according to their genotype and sex in order to obtain 190–250 mg of flies for chromatin preparation. b-galactosidase activity in extracts prepared from 2–10 flies were measured as previously described [26]. The flies carrying the balancer were used as negative controls for the flies carrying the Gal4 activator. To activate the reporter by Gal4-MOF recruitment, we used a stable line homozygote for the reporter construct and prepared chromatin for ChIP from sorted 200–500 female flies [26]. Chromatin immunoprecipitation Chromatin from SL2 cells was prepared after crosslinking in 1% formaldehyde as previously described [29]. To preserve the phospho-epitopes and the acetylation status of the chromatin, we used 5 mM NaF, 20 mM b-glycerophosphate, 0.1 mM NaVanadate, 10 mM NaButyrate and a mixture of protease inhibitors (1mM PMSF, 1 mg/ml of each aprotinin, pepstatin and leupeptin) or the complete protease inhibitors (Roche, Cat No. 04693132011). In the particular case of the chromatin used for mapping H3S10phK14ac, 1 mM microcystin was also added. JIL-1 and Transcriptionn The chromatin from sorted male and/or female flies was done according to following references [26,61]. Chromatin shearing to 300–1000 bp fragments was assessed on a Bioanalyzer (Agilent) and DNA concentration was measured using NanoDrop (Thermo Scientific). 7.5 to 10 mg of DNA per chromatin was used for ChIP. For JIL-1 ChIPs we used either crude sera R69 and R70 (5 ml/IP) or the affinity-purified antibodies (5 mg/IP). A LiCl wash was done for ChIP-chip analysis. For ePol ChIPs, 5 ml of the anti-CTD S2ph monoclonal antibody Cone H5 (ab24758, Abcam) was first coupled to a 50/50 mix of protein A- and protein G-agarose using a bridging antibody (5 mg of Goat anti-mouse IgM, Jackson ImmunoResearch) and the LiCl wash was omitted. The following volumina of antibodies were used for ChIP: H3S10phK14ac: 3 ml; H4K16ac: 5 ml of antibody #39167 (Active Motif); H3K36me3: 5 ml of ab9050 (Abcam). MSL2 antibodies were used as control as previously published [62]. Quantitative PCR Quantitative PCR (qPCR) was carried out in an ABI PRISM 7000 Sequence detection system in a 25 ml reaction with Power SYBR Green Master Mix (Applied Biosystems) or in LightCycler (Roche) in 10 ml reactions with Fast SYBR Green Master mix (Applied Biosystems). Input DNA were diluted to 10 ng/ml and ChIPs were diluted 1/30, 5 ml and 2 ml template were used for ABI PRISM 7000 and LightCycler, respectively. The results were comparable on the two machines. Primer sequences are provided in Table S1. ChIP-chip and data analysis ChIP-chip data analysis was essentially performed as in [29]. Briefly, input and IP DNA were amplified using the WGA kit (Sigma) according to the online protocol (http://www.epigenome- noe.net/WWW/researchtools/protocol.php?protid=30). Labeling and hybridization to NimbleGen dual-color arrays was carried out at ImaGenes (Berlin, Germany). The layout of the array (approx. 1 probe/100 bases, isothermal probe design) was customized [24]. Data analysis was performed using R/Bioconductor (www. bioconductor.org). Experimental replication was as follows: JIL- 1: 4 biological replicates; H3S10phK14ac: 2 biological replicates including dye swap; elongating RNA Polymerase II: 3 biological replicates including one dye swap; H4K16ac: 2 biological replicates including dye swap. Raw signals of corresponding biological samples were log2 transformed and quantile-normal- ized. Enrichment statistics (IP versus input signals) were computed using the ‘sam’ algorithm within Bioconductor [63]. Fdr values of the sam statistic were determined using ‘locfdr’ [64] region summarization was performed using tileHMM [65] with the following parameters: fragment size of 700, maximal gap of 400. Genes were considered ‘bound’ if covered by at least 4 probes with a posterior probability above 0.5. All data correspond to Drosophila genome version dm3 and annotation version gadfly 5.2.2. RNAi in SL2 cells and purification of total RNA SL2 cells were cultured according to standard methods. RNA interference (RNAi) was carried out as published [66] except that 26106 cells were treated with 10 mg dsRNA and splitted 1/3 after 3 days of treatment. After 7 days cells were collected and counted prior to total RNA purification. For the 2 sets of expression analysis we used different sets of dsRNA for RNAi. In experiment 1, the 59 coding sequence of JIL-1 was targeted by a corresponding dsRNA (T7_JIL-159_for: TTAATACGACTCACTATAGGGA- GATGAGTCGCTTG, T7_JIL-159_rev: TTAATACGACTCA- CTATAGGGAGACATCCTCCTCCA), and the controls in- volved treating cells with dsRNA targeting GST sequences PLoS Genetics | www.plosgenetics.org 13 March 2011 | Volume 7 | Issue 3 | e1001327