Ith 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, GSM286607, GSM286611, E12_V082, E02_V081, YAF01_V084, YAM01_V083, GSM360256, GSM360257, GSM322208, GSM322245, GSM360260, GSM360262, GSM322219, GSM322338, YA_GSM280086, O_V063. RNA-seq mRNA read samples: 3358, 3317, E0.2884, E2.2885, E6.2887, E8.2888, E10.2889, E12.2890, E14.2891, E16.2892, E18.2893, E20.2894, E22.2895, L1.2872, L2.2873, L312.2874, L313.2875, L314.2876, L315.2877, P0.2878, P12.2879, P24.2880, P48.2881, P72.2882, P96.2883, YF1.2866, YF5.2868, YF30.2867, YM1.2869, YM5.2871, YM30.2870, Dm_SOLiD.Whole-mount in situ hybridization of embryosDigoxigenin (DIG)-labeled RNA antisense probe GSK0660 synthesis and whole mount in situ hybridization was carried out as previously described [40], except that fragments ranging in size from 500 to 3500 bp were cloned from genomic DNA for use as templates for probe synthesis. Probes were not fragmented with carbonate buffer. Probe template primer sequences are located in Table S1.Construction of FLAG plasmidsFLAG-tagged PcG transformation constructs were generated with the Gateway Cloning System (Invitrogen, Carlsbad, CA). Sce, esc, pho, and Scm cDNA clones were obtained from the Drosophila Genomics Resource Center (BGDP Gold cDNAs: LD23953 (Sce), SD03549 (esc), RE17954 (pho), RE16782 (Scm)). To generate Gateway entry clones, cDNAs were GGTI298 site amplified using Phusion HighFidelity DNA Polymerase and cloned into pENTR/dTOPO (Invitrogen) (for primer sequences see Table S1). Destination vectors containing N-terminal or C-terminal 3XFLAG, pTFWMaterials and Methods RNA data analysisThe following ModEncode mRNA and ncRNA reads in inv-en genomic regions were examined: Small ncRNA read samples: GSM286604, GSM364902, GSM286613, GSE24540,PcG Proteins Bind Constitutively to the en GeneFigure 4. Pho-FLAG and Sce-FLAG binding peaks at PRE2. (A) A map of the en gene showing the location of the PREs and the probes used for the qPCR (#1?). (B,C) Results of X-ChIP experiment with Sce-FLAG (B) and Pho-FLAG (C) driven by en-Gal4 (open bars) or ci-Gal4 (closed bars). Pho binding was also done on all chromatin preparations. The results of a representative experiment are shown. These experiments were done with a different batch of FLAG antibody and different ChIP reagents than those done in Fig. 5. Further, 20 larvae were used for each sample instead of 10. Under these conditions, we did not see a difference in the level of binding to the PREs between the “ON” and the “OFF” states; however, the qualitative result, PcG proteins.Ith 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, GSM286607, GSM286611, E12_V082, E02_V081, YAF01_V084, YAM01_V083, GSM360256, GSM360257, GSM322208, GSM322245, GSM360260, GSM360262, GSM322219, GSM322338, YA_GSM280086, O_V063. RNA-seq mRNA read samples: 3358, 3317, E0.2884, E2.2885, E6.2887, E8.2888, E10.2889, E12.2890, E14.2891, E16.2892, E18.2893, E20.2894, E22.2895, L1.2872, L2.2873, L312.2874, L313.2875, L314.2876, L315.2877, P0.2878, P12.2879, P24.2880, P48.2881, P72.2882, P96.2883, YF1.2866, YF5.2868, YF30.2867, YM1.2869, YM5.2871, YM30.2870, Dm_SOLiD.Whole-mount in situ hybridization of embryosDigoxigenin (DIG)-labeled RNA antisense probe synthesis and whole mount in situ hybridization was carried out as previously described [40], except that fragments ranging in size from 500 to 3500 bp were cloned from genomic DNA for use as templates for probe synthesis. Probes were not fragmented with carbonate buffer. Probe template primer sequences are located in Table S1.Construction of FLAG plasmidsFLAG-tagged PcG transformation constructs were generated with the Gateway Cloning System (Invitrogen, Carlsbad, CA). Sce, esc, pho, and Scm cDNA clones were obtained from the Drosophila Genomics Resource Center (BGDP Gold cDNAs: LD23953 (Sce), SD03549 (esc), RE17954 (pho), RE16782 (Scm)). To generate Gateway entry clones, cDNAs were amplified using Phusion HighFidelity DNA Polymerase and cloned into pENTR/dTOPO (Invitrogen) (for primer sequences see Table S1). Destination vectors containing N-terminal or C-terminal 3XFLAG, pTFWMaterials and Methods RNA data analysisThe following ModEncode mRNA and ncRNA reads in inv-en genomic regions were examined: Small ncRNA read samples: GSM286604, GSM364902, GSM286613, GSE24540,PcG Proteins Bind Constitutively to the en GeneFigure 4. Pho-FLAG and Sce-FLAG binding peaks at PRE2. (A) A map of the en gene showing the location of the PREs and the probes used for the qPCR (#1?). (B,C) Results of X-ChIP experiment with Sce-FLAG (B) and Pho-FLAG (C) driven by en-Gal4 (open bars) or ci-Gal4 (closed bars). Pho binding was also done on all chromatin preparations. The results of a representative experiment are shown. These experiments were done with a different batch of FLAG antibody and different ChIP reagents than those done in Fig. 5. Further, 20 larvae were used for each sample instead of 10. Under these conditions, we did not see a difference in the level of binding to the PREs between the “ON” and the “OFF” states; however, the qualitative result, PcG proteins.
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