Figure 4. Effects of JIL-1 RNAi on gene expression and H4K16ac localization in SL2 cells. (A) The knock-down of JIL-1 was assessed JIL-1 and Transcriptionn by western blotting on whole cell extracts after 7 days of RNA interference (RNAi). RNAi against GST and MSL2 were used as controls. The affinity-purified serum of R69 was used to detect JIL-1. A cross- reacting protein of smaller size (*) was not affected. Two and three serial dilutions of the extract were loaded for controls and JIL-1 RNAi samples, respectively. (B) Levels of ePol detected with the H5 antibody were unaffected upon JIL-1 RNAi. The ATPase ISWI served as a loading control. (C) SL2 cells were immunostained for JIL-1, H4K16ac (in green) and lamin (in white) and analyzed by confocal imaging. (D) Log2 fold changes in gene expression after JIL-1 RNAi as compared to GST or GFP RNAi (ctr RNAi) summarized by chromosomes in a box plot. The dashed horizontal line indicates the median change of all active genes. (E) The average binding of JIL-1 per gene was plotted for autosomal genes on the left (Auto) and X-linked genes on the right (X) distinguishing genes with no expression change over JIL-1 depletion (‘no’), up-regulated genes (‘up’) and down-regulated genes (‘down’). The numbers on top indicate the number of genes in each category. doi:10.1371/journal.pgen.1001327.g004 chromatin. By contrast, the relative distribution of JIL-1 along genes of various lengths is comparable on autosomes and the X chromosome, except for the twofold enrichment on the latter (Figure 5B). JIL-1 recruitment upon gene activation A recent suggestion that JIL-1-dependent H3S10 phosphoryla- tion was crucial to regulate the release of RNA polymerase into productive elongation in Drosophila in particular for the transcrip- tion of heat shock genes has remained controversial [17–19]. In our hands, heat shock treatment for 2, 5 and 10 min in SL2 cells triggered a clear enrichment of ePol at the Hsp70 locus as monitored by ChIP without strikingly affecting the distribution of JIL-1. In particular, we found that there was already some ePol and some JIL-1 at that locus in control cells as evidenced by the comparison with the active X-linked locus sta (Figure S10). Western blotting showed that the overall level of ePol drops upon heat shock, whereas the levels of JIL-1 varied only slightly. Because heat shock had been shown to trigger a massive loss of nucleosomes over a large domain [41], and because the genome-wide localization of JIL-1 appeared to correlate best with histone modifications (see below), we speculate that the lack of recruitment might be a consequence of nucleosome depletion. We next made use of an inducible reporter system in flies ([26,42]; Figure 6A), which harbours an insertion of a reporter gene cassette consisting of the lacZ gene downstream of five binding sites for the yeast transcription factor Gal4 (‘upstream activating sequences’; UASGal). The reporter was activated by crossing the reporter fly line with a line expressing either Gal4 alone or MOF fused to the Gal4 DNA binding domain (Gal4DBD). The basal level of b-galactosidase expression in the absence of activation was very low and could be induced about 1000-fold in male and female flies by expression of Gal4 under the control of a tubulin promoter. We monitored the levels of JIL-1, H3K36me3 and H4K16ac at the 59 and 39 ends of the lacZ gene by ChIP. As reference for the normalisation of the ChIP signals from different chromatin preparations we used the intergenic locus close to ptr (ptr_IG). In accordance with the results presented in Figure 2C, the X-chromosomal gene phl showed robust enrichment of JIL-1 in female flies and approximately twofold elevated levels in males. Very little recruitment of JIL-1 was observed at the reporter gene upon activation via Gal4 (Figure 6B). However, we did also not detect robust levels of H3K36me3 as compared to the endogenous loci. Since the tubulin-Gal4 driver leads to strong activation, which can be visualized by clear puffing at the level of polytene chromosomes, it is possible that these histone marks have been depleted along with nucleosomes due to vigorous transcription, a PLoS Genetics | www.plosgenetics.org 7 March 2011 | Volume 7 | Issue 3 | e1001327