Figure 5. JIL-1 distributes differently than elongating poly- merase on active chromatin. Average distribution profiles of chromatin features along genes scaled for length from transcription start (TS) to termination sites (TT). (A) All genes covered by the tiling array were grouped into 6 equally-sized bins based on increasing densities of ePol. Average profiles of ePol (top), JIL-1 (centre) and H3S10phK14ac (bottom) were calculated for each group. The increasing darkness of the red lines indicate an increasing density of ePol on the JIL-1 and Transcriptionn genes between the groups. (B) Genes were binned based on their length. The length distribution within the bins is the following: 287- 1204, 1204-1780, 1780-2477, 2477-3778, 3778-7400 and 7400- 162904 bp. Average profiles on the grouped genes are displayed for ePol (top) and JIL-1 (bottom). doi:10.1371/journal.pgen.1001327.g005 situation akin to the heat-induced transcription mentioned earlier. In order to trigger a more modest induction of the reporter gene we expressed the acetyltransferase MOF fused to the Gal4DBD (Gal4-MOF) at a low level from its endogenous promoter [26]. We limited this analysis to females in order to avoid the complications associated with all other members of the DCC. We had shown earlier that recruitment of Gal4-MOF to the reporter locus in females leads to H4K16ac and to a substantial transcriptional activation [26]. In the current experiment the activation was determined to be about 20-fold in the b-galactosidase assay. This activation was accompanied by an enrichment of JIL-1 at the 39 end of the lacZ reporter (13-fold over the intergenic locus ptr_IG), which was about twofold higher than the enrichment at the endogenous X-linked locus phl (Figure 6C). In this case of a mild activation, we found an enrichment of H3K36me3 at the 39 of the LacZ reporter, which was comparable to the endogenous loci phl and Cortactin. Upon induction with MOF, a 7-fold enrichment of H4K16ac is detected at the 59 end of the LacZ reporter, which decreased towards the 39 end. The Cortactin gene, localised about 2-3 kb downstream of the UASGAL as mapped by PCR-rescue of the transgene, also showed an increased level of H4K16ac upon Gal4-MOF targeting. Interestingly, the presence of H4K16ac in addition to H3K36me3 on the reporter gene LacZ correlated with an enrichment of JIL-1 comparable to that of X-linked genes in males. On the neighbouring Cortactin gene the enrichment was also increased. We found that the enrichment of both H4K16ac and JIL-1 at the Cortactin gene only increased the expression of the gene by a factor 1.2 in females. In accordance with previous data [26], in males H4K16ac spreads out more broadly from the recruitment sites due to the presence of the DCC and the relative expression of the Cortactin gene was increased by a factor 1.6 (Figure S11). Taken together the recruitment experiments suggest that the association of JIL-1 with active genes rather correlates with the presence of H3K36me3 and H4K16ac than with transcription as such. This conclusion can be substantiated by visual inspection of the genome-wide ChIP-chip profiles from SL2 cells, where numerous instances can be found where JIL-1 binding closely parallels the presence of H3K36me3, whereas the presence of high levels of ePol alone and sometimes also significant enrichment of H4K16ac alone do not coincide with JIL-1 binding (see Figure S9 for a selection of views). JIL-1 recruitment and dosage compensation In order to further explore the relationship between JIL-1, transcription, H3K36me3 and H4K16ac, we looked in more detail at the relative twofold enrichment of JIL-1 on the male X chromosome. Johansen and colleagues [22] had documented earlier that the enrichment of JIL-1 on the male X chromosome depends on the DCC. Their key experiment was the ectopic expression of MSL2 in female larvae, which leads to inappropriate assembly of the DCC. The association of the DCC with the female X chromosome led to an enrichment of JIL-1, just like in males [22]. In agreement with these data, in similar experiments we found that if high levels of MSL2 are expressed from two NOPU alleles [43,44] the X chromosome is decorated with DCC and JIL- 1 is enriched concomitantly at sites of DCC binding. However, PLoS Genetics | www.plosgenetics.org 8 March 2011 | Volume 7 | Issue 3 | e1001327