A pair-rule pattern (panel 10), in regions that contain previous experimental evidence of transcripts and a pair-rule enhancer [31,32] (JAK unpublished data). Finally, still further upstream, central nervous system staining was observed in stage 17 embryos (panels 11, 12, and 13). The expression from probe 13 could be transcriptional read through from the tou gene. We also examined polyA and non-polyA RNA-seq data from the ModEncode project [29]. No RNAs of either type were observed at any embryonic (0?4 hours) or larval stage in the inven or en-tou regions. However, a robust signal spanning 1100 bp (2R:7360200..7361299) was observed upstream of the inv promoter and adjacent to one of the two known inv PREs (PRE coordinates 2R:7362423..7363955 [24]) (Fig. 1B). This signal was observed in all stages, beginning in 0? hour embryos. This signal is likely an artifact however, as this 1100 bp region shows near sequence identity to 21 other regions in the genome. Taken together, these results suggest that ncRNAs are not as abundant in the en/inv region as they are in the BX-C, and that inv and en PREs are not transcribed in embryos. We also examined whether the inv and en PREs are transcribed in imaginal discs and the larval CNS and saw no evidence of transcription (data not shown). We note that Schmitt et al. also found no evidence of en PRE transcription in larval tissues [20].PcG proteins bind to the en PRE in 
both the “ON” and “OFF” transcriptional states of enPcG protein binding to en and inv PREs has been examined in genome wide studies using embryos, Indolactam V larvae, and adults [26?8]. The samples in these studies contain a mixture of cells, some of which transcribe en and inv, and others that do not. en and inv exist in a “balanced” state in BG3 cells, with 18055761 transcription in the presence of PcG binding [15,16]. We wished to determine whether this was also the case in vivo. We used a UAS-driven FLAGMedChemExpress GSK -3203591 tagged PcG crosslinked-ChIP (X-ChIP) system to examine PcG binding in cells that express en and those that do not. en is expressed in stripes in embryos and in the posterior compartments of imaginal discs. cubitus interruptus (ci), is expressed in a complementary pattern with en, with no overlap in both embryos and imaginal discs [33]. By expressing UAS-FLAG-tagged proteins in specific cell populations with en-GAL4 and ci-GAL4 driver lines [34], it is possible to use ChIP to examine the binding profile of any PcG protein in the “ON” or “OFF” transcriptional states of en. Fly lines with 3XFLAG-tagged Pho, dRing/Sce, Esc, and Scm were generated. These proteins were chosen because they are present in different PcG protein complexes and might preferentially bind in the “OFF” versus the “ON” transcriptional state. All proteins were first tagged at the C-terminus. C-terminally tagged Scm-FLAG acted in a dominant negative fashion when ubiquitously expressed in a wild-type background, as indicated by strong PcG-type transformations (data not shown). Therefore, we generated and proceeded with an N-terminally tagged FLAGScm protein, which 
did not produce a phenotype when expressed ubiquitously in a wild type background. UAS-Pho-FLAG was crossed with en-GAL4 or ci-GAL4, and FLAG-expression was examined in whole embryos and imaginal discs from wandering 3rd instar larvae. As expected, Pho-FLAG driven by en-GAL4 was expressed in embryos (not shown) and in discs in a pattern that almost completely overlapped with endogenous en (Fig. 2A ). Pho-FLAG dri.A pair-rule pattern (panel 10), in regions that contain previous experimental evidence of transcripts and a pair-rule enhancer [31,32] (JAK unpublished data). Finally, still further upstream, central nervous system staining was observed in stage 17 embryos (panels 11, 12, and 13). The expression from probe 13 could be transcriptional read through from the tou gene. We also examined polyA and non-polyA RNA-seq data from the ModEncode project [29]. No RNAs of either type were observed at any embryonic (0?4 hours) or larval stage in the inven or en-tou regions. However, a robust signal spanning 1100 bp (2R:7360200..7361299) was observed upstream of the inv promoter and adjacent to one of the two known inv PREs (PRE coordinates 2R:7362423..7363955 [24]) (Fig. 1B). This signal was observed in all stages, beginning in 0? hour embryos. This signal is likely an artifact however, as this 1100 bp region shows near sequence identity to 21 other regions in the genome. Taken together, these results suggest that ncRNAs are not as abundant in the en/inv region as they are in the BX-C, and that inv and en PREs are not transcribed in embryos. We also examined whether the inv and en PREs are transcribed in imaginal discs and the larval CNS and saw no evidence of transcription (data not shown). We note that Schmitt et al. also found no evidence of en PRE transcription in larval tissues [20].PcG proteins bind to the en PRE in both the “ON” and “OFF” transcriptional states of enPcG protein binding to en and inv PREs has been examined in genome wide studies using embryos, larvae, and adults [26?8]. The samples in these studies contain a mixture of cells, some of which transcribe en and inv, and others that do not. en and inv exist in a “balanced” state in BG3 cells, with 18055761 transcription in the presence of PcG binding [15,16]. We wished to determine whether this was also the case in vivo. We used a UAS-driven FLAGtagged PcG crosslinked-ChIP (X-ChIP) system to examine PcG binding in cells that express en and those that do not. en is expressed in stripes in embryos and in the posterior compartments of imaginal discs. cubitus interruptus (ci), is expressed in a complementary pattern with en, with no overlap in both embryos and imaginal discs [33]. By expressing UAS-FLAG-tagged proteins in specific cell populations with en-GAL4 and ci-GAL4 driver lines [34], it is possible to use ChIP to examine the binding profile of any PcG protein in the “ON” or “OFF” transcriptional states of en. Fly lines with 3XFLAG-tagged Pho, dRing/Sce, Esc, and Scm were generated. These proteins were chosen because they are present in different PcG protein complexes and might preferentially bind in the “OFF” versus the “ON” transcriptional state. All proteins were first tagged at the C-terminus. C-terminally tagged Scm-FLAG acted in a dominant negative fashion when ubiquitously expressed in a wild-type background, as indicated by strong PcG-type transformations (data not shown). Therefore, we generated and proceeded with an N-terminally tagged FLAGScm protein, which did not produce a phenotype when expressed ubiquitously in a wild type background. UAS-Pho-FLAG was crossed with en-GAL4 or ci-GAL4, and FLAG-expression was examined in whole embryos and imaginal discs from wandering 3rd instar larvae. As expected, Pho-FLAG driven by en-GAL4 was expressed in embryos (not shown) and in discs in a pattern that almost completely overlapped with endogenous en (Fig. 2A ). Pho-FLAG dri.