A UAS-Pho-FLAG, ci-GAL4 cross. Panel F shows complementary staining of anti-FLAG and anti-En. Note that the size of the anterior compartment, where Ci is expressed is about twice the size of the posterior compartment, where En is expressed [35]. (G) qRT-PCR showing that there is about twice as much Pho-FLAG transcript when it is driven by ci-Gal4 than by en-Gal4 (*** P#0.001). doi:10.1371/journal.pone.0048765.gexpressed in all cells for proper development. ci- and en-driven Pho-FLAG and Sce-FLAG binding were measured using probes upstream and within the en transcription unit (Fig. 4). Sce-FLAG was bound to PRE2 in both the “ON” and “OFF” transcriptional states. Pho-FLAG has a similar binding profile CASIN chemical information except that binding to the non-PRE probes in the “ON” chromatin was higher than the “OFF” chromatin, and there was some binding to PRE1. For comparison, Pho binding was measured using the same chromatin used for the FLAG-samples. Pho ChIP measures binding in both the “ON” and the “OFF” cells. Note that the Pho-binding was similar in both the Pho-FLAG samples and the Sce-FLAG samples, suggesting that the Pho-FLAG accurately reflects the distribution of endogenous Pho. We compared the level of X-ChIP binding to en PRE 2 with that of a control fragment from the en intron (probe 8) for 
all of the FLAG-tagged PcG proteins. Each experiment was repeated 3 times and the results were pooled in Fig. 5. Pho-FLAG, FLAGScm, Sce-FLAG, Esc-FLAG, were present at en PRE2 in both the “ON” and “OFF” transcriptional states of en. These ChIP results suggest that PcG proteins are present in the en “OFF” transcriptional state at higher levels than in the “ON” state. For example, the Pho-FLAG signal is 4 fold higher than the controlPcG Proteins Bind Constitutively to the en GeneFigure 3. FLAG-tagged PcG proteins co-localize with endogenous PcG proteins on polytene chromosomes. FLAG-tagged proteins were driven by arm-Gal4. doi:10.1371/journal.pone.0048765.gOne unexpected result from these experiments was that FLAGSce binds to PRE2 but not to PRE1 (Fig. 4). This is an interesting result that needs to be followed up on. Recent ChIP-Seq data in our lab using imaginal disk/brain larval samples and the anti-Pho antibody show 5 additional Pho binding peaks between en and tou, which could be 5 additional PREs (S. De and JAK, unpublished data). Three of these correspond to Pho binding peaks already identified by Oktaba et al. [39]. ChIP-seq experiments with the FLAG-tagged proteins expressed in the “ON” and “OFF” transcriptional states would be necessary to ask whether the distribution of PcG-proteins is altered at any of the PREs or any other region of the en/inv domain. In conclusion, our data allows us to rule out two simple models of PcG-regulation of the en/inv genes. First, the en/inv PREs are not transcribed, so this cannot determine their activity state. Felypressin chemical information Second, PcG proteins bind to at least one of the PREs of the en/inv locus in the “ON” state, therefore a simple model of PcG-binding determining the activity state of en/inv is not correct. Perhaps the proteins that activate en expression modify the PcG-proteins or the 3D structure of the locus and interfere with PcG-silencing. While FLAG-tagged PcG proteins offer a good tool to study PcGbinding particularly in the “OFF” state, cell-sorting of en positive and negative cells will be necessary to study the 3D structure and chromatin modification of the en/inv locus.GSM286605, GSM286606, GS.A UAS-Pho-FLAG, ci-GAL4 cross. Panel F shows complementary staining of anti-FLAG and anti-En. Note that the size of the anterior compartment, where Ci is expressed is about twice the size of the posterior compartment, where En is expressed [35]. (G) qRT-PCR showing that there is about twice as much Pho-FLAG transcript when it is driven by ci-Gal4 than by en-Gal4 (*** P#0.001). doi:10.1371/journal.pone.0048765.gexpressed in all cells for proper development. ci- and en-driven Pho-FLAG and Sce-FLAG binding were measured using probes upstream and within the en transcription unit (Fig. 4). Sce-FLAG was bound to PRE2 in both the “ON” and “OFF” transcriptional states. Pho-FLAG has a similar binding profile except that binding to the non-PRE probes in the “ON” chromatin was higher than the “OFF” chromatin, and there was some binding to PRE1. For comparison, Pho binding was measured using the same chromatin used for the FLAG-samples. Pho ChIP measures binding in both the “ON” and the “OFF” cells. Note that the Pho-binding was similar in both the Pho-FLAG samples and the Sce-FLAG samples, suggesting that the Pho-FLAG accurately reflects the distribution of endogenous Pho. We compared the level of X-ChIP binding to en PRE 2 with that of a control fragment from the en intron (probe 8) for all of the FLAG-tagged PcG proteins. Each experiment was repeated 3 times and the results were pooled in Fig. 5. Pho-FLAG, FLAGScm, Sce-FLAG, Esc-FLAG, were present at en PRE2 in both the “ON” and “OFF” transcriptional states of en. These ChIP results suggest that PcG proteins are present in the en “OFF” transcriptional state at higher levels than in the “ON” state. For example, the Pho-FLAG signal is 4 fold higher than the controlPcG Proteins Bind Constitutively to the en GeneFigure 3. FLAG-tagged PcG proteins co-localize with endogenous PcG proteins on polytene chromosomes. FLAG-tagged proteins were driven by arm-Gal4. doi:10.1371/journal.pone.0048765.gOne unexpected result from these experiments was that FLAGSce binds to PRE2 but not to PRE1 (Fig. 4). This is an interesting result that needs to be followed up on. Recent ChIP-Seq data in our lab using imaginal disk/brain larval samples and the anti-Pho antibody show 5 additional Pho binding peaks between en and tou, which could be 5 additional PREs (S. De and JAK, unpublished data). Three of these correspond to Pho binding peaks already identified by Oktaba et al. [39]. ChIP-seq experiments with the FLAG-tagged proteins expressed in the “ON” and “OFF” transcriptional states would be necessary to ask whether the distribution 
of PcG-proteins is altered at any of the PREs or any other region of the en/inv domain. In conclusion, our data allows us to rule out two simple models of PcG-regulation of the en/inv genes. First, the en/inv PREs are not transcribed, so this cannot determine their activity state. Second, PcG proteins bind to at least one of the PREs of the en/inv locus in the “ON” state, therefore a simple model of PcG-binding determining the activity state of en/inv is not correct. Perhaps the proteins that activate en expression modify the PcG-proteins or the 3D structure of the locus and interfere with PcG-silencing. While FLAG-tagged PcG proteins offer a good tool to study PcGbinding particularly in the “OFF” state, cell-sorting of en positive and negative cells will be necessary to study the 3D structure and chromatin modification of the en/inv locus.GSM286605, GSM286606, GS.