JIL-1 and Transcriptionn Figure 6. JIL-1 distribution upon activation of a reporter construct in flies. (A) The reporter construct encompassing five UAS in front of a LacZ reporter carries a miniwhite gene [42]. It is inserted on 3R at position 93B8 with the UAS close to the 39 end of the Cortactin gene. The cartoon is drawn to scale, except for the negligible size of the UAS in order to visualize the distances between the amplicons (coloured boxes) used for qPCR analysis in panels B and C. (B) Activation of the LacZ reporter was achieved by crossing the homozygote reporter line to the Tubulin-Gal4/TM3 driver line expressing Gal4fl under control of the tubulin promoter. Relative b-galactosidase activity as measured in the corresponding flies is provided on the bottom of the panels. The reference was set to 0.56 for the heterozygote reporter line in the absence of activation. The ChIP signals for JIL-1 (upper panel), H3K36me3 (centre panel) and H4K16a (lower panel) at the intergenic locus (ptr_IG), at the 39 of active X-linked gene (phl-D), at the 39 of the Cortactin gene and at the 59 and 39 of the LacZ reporter gene are displayed. The data is provided as relative enrichments over the intergenic locus (ptr_IG). (C) For activation of the reporter by Gal4-MOF, the homozygote reporter line was used as a reference and compared to the stable line carrying both the homozygote reporter construct and the homozygote transgene leading to Gal4-MOF expression [26]. Only females were analyzed. The activity measurements for the two different types of females are mentioned below the graphs and can be compared to the values in B. In addition to the amplicons used in (B) we also analyzed binding to the 39 end of the miniwhite gene. Because of the inherent variability of chromatin preparation one representative biological replicate is presented here. doi:10.1371/journal.pgen.1001327.g006 when limiting levels of MSL2 are expressed from the SXB1-2 allele [43,44] the DCC only associates with about 50 high affinity sites (HAS) on polytene chromosomes. In this case, the enrichment of JIL-1 can only be seen at sites of DCC binding (Figure 7A). This enrichment requires the assembly of a complete DCC, including MOF, as it is not observed in the absence of MLE or MSL3 ([22] and data not shown). The close relationship between DCC association and JIL-1 was also apparent at the high resolution achieved by ChIP-chip mapping. We had earlier identified 131 HAS that are preferen- tially bound by limiting amounts of DCC [29]. Plotting the binding of MSL1 to genes as a function of their distance to the closest HAS shows that, in general, the closer a gene resides to a HAS, the more MSL1 and H4K16ac can be found (Figure 7B). The same trend can be observed for JIL-1 as well as for the linked H3S10phK14ac mark: their densities are decreasing with increasing distance to a HAS. This is, however, not true for H3K36me3 or ePol association (Figure 7B). In summary, diverse experiments establish two hallmarks of active chromatin that correlate with the presence of JIL-1. First, there is an excellent correlation of JIL-1 binding to H3K36me3- marked chromatin. On top of this basal level of binding, the presence of the DCC leads to an approximately twofold enrichment of JIL-1. Indeed, a global comparison of ChIP-chip probe signals (Figure 8) reveals that the correlations of JIL-1 signals with the ones of H3K36me3 (R = 0.758) or H4K16ac (R = 0.76) are very good. Remarkably, the combination of both marks (sum of the intensities of the H3K36me3 and H4K16ac signals) correlated even better with JIL-1 signals (R = 0.899) and displayed an almost linear relationship between signal intensities. This is true if all probes are considered, but also if autosomal or X chromosomal probes are plotted separately (Figure 8). This correlation can be recapitulated to some extent when the distribution of those marks is considered along genes (Figure S12). In contrast, correlation of JIL-1 and ePol (R = 0.69) is less pronounced (Figure 8). This result is consistent with the earlier observation that ePol and JIL-1 are both marks for active genes but that their relative distributions along genes differ (Figure 5). We conclude that two distinct targeting principles that are represented by H3K36me3 and H4K16ac marks determine the level of JIL-1 recruitment to active genes. Discussion Targeting of JIL-1 The genome-wide mapping of JIL-1 and ePol in male SL2 cells together with the reporter assay in flies suggest that JIL-1 is part of a network of factors that collectively define the state of active chromatin. Taken together with previous genetic analyses, our data lead us to consider to a role for JIL-1 not in the establishment of active chromatin, but in the reinforcement of the active state, independently of the extend of transcription. Although JIL-1 binds the bodies of most active gene and is recruited to a reporter gene upon activation, the degree of binding does not correlate well with ePol occupancy. In this respect JIL-1 belongs to a class of several other active chromatin components whose presence in chromatin does not scale with ePol, such as H3K36me3, the DCC subunit MSL1, or H4K16ac mark. By contrast, H3K4me2 levels are proportional to ePol levels. Conceiv- ably, JIL-1 marks active chromatin independent of whether it is currently transcribed or not. A basal level of JIL-1 binding correlates strongly with H3K36me3, an elongation marker that is placed co- transcriptionally by ePol-associated dSet2 [30]. The turnover time of this modification is not known and hence it is possible that H3K36me3 remains on chromatin between pulses of transcription. Most recently, high-resolution mapping of 56 chromatin components using Dam-ID revealed five types of chromatin in Drosophila KC cells [45]. A colour code was used to illustrate those types. The two types of active chromatin, ‘red’ and ‘yellow’, differ in that active genes in red chromatin tend to be depleted in H3K36me3 and MRG15, which are abundant constituents of yellow chromatin. Monitoring the relative distributions of ePol and JIL-1 in those types of chromatin we found that whereas ePol is equally distributed in red and yellow chromatin, JIL-1 is clearly mostly found in yellow chromatin (Figure S13). This correlation again highlights the relationship between JIL-1 and H3K36me3. The precise link between H3K36me3 and JIL-1 recruitment remains to be explored (see below). A second targeting principle is evident from the fact that JIL-1 levels are approximately twofold increased on the male X chromosome, where due to the action of the DCC H4K16ac levels are high. The situation is complex since the recruitment of the DCC to transcribed genes on the X chromosome is promoted by the potential interaction of the DCC subunit MSL3 with H3K36me3 [30]. Because JIL-1 binding is insensitive to different transcription rates on autosomes, it is not plausible that a presumed twofold increase in transcription on the X chromosome is directly responsible for this elevated association. Likewise, we do not think that direct interactions between JIL-1 and the DCC can explain the targeting. Although some interactions have been observed in vitro between JIL-1, MSL1 and MSL3 [22], a quantitative co-purification of endogenous JIL-1 with the DCC was never documented. Indeed, activation of a reporter gene in female flies through recruitment of Gal4-MOF is sufficient to recruit JIL-1 to male levels on the reporter gene and on the adjacent Cortactin gene. Altogether, we favour the hypothesis that the enrichment of JIL-1 on X-linked genes is a consequence of a modulating feature of dosage-compensated chromatin. The genome-wide distribution of JIL-1 indeed correlates best with the combination of the two chromatin modifications, H3K36me3 PLoS Genetics | www.plosgenetics.org 10 March 2011 | Volume 7 | Issue 3 | e1001327