Binding to PRE2 in both the “ON” and “OFF” states was the same in these experiments and those in Fig. 5. doi:10.1371/journal.pone.0048765.gand pTWF respectively, were obtained from Terence Murphy and are further described at http://www.ciwemb.edu/labs/murphy/ Gateway 20vectors.html. Clone cassettes in pENTR/dTOPO were recombined into pTWF and pTFW with LR Clonase (Invitrogen) according to manufacturers instructions. The resulting constructs were fully sequenced and checked for mutations and recombination errors prior to use.Reverse Transcription-Quantitative Polymerase Chain ReactionImaginal discs, along with the central nervous system, mouth hooks, and some anterior cuticle were dissected from 3rd instar larvae (5 per sample) from ci- and en-GAL4 AZP-531 site driven Pho-FLAG larvae and immediately placed in PBS on ice. Total RNA was collected from the resulting samples using Trizol (Invitrogen) according to the manufacturers instructions. One-step RT-qPCR was performed with the QuantiTect SYBR Green RT-PCR Kit on a Roche Lightcycler 480 according to manufacturer instructions. Relative expression levels of Pho-FLAG transcript was calculated using the DC(T) 25331948 method, and expressed as a percentage of RP49 expression level. Pho-FLAG primers amplify a fragment containing the 39 end of pho gene and a portion of the FLAGencoding sequence. Pho-FLAG primers: 59-CCGTTTGTGGTATATGCAGA-39, 59-CGTCATGGTCTTTGTAGTC-39. RP49 primers: 59-CGGATCGATATGCTAAGCTGT-39, 59-CGACGCACTCTGTTGTCG-Transgenic linesUAS-PcG-FLAG transgenic lines were generated by injections into w1118 embryos by Genetic Services (Sudbury, MA, USA).Chromosome SquashesSquashes and immunofluorescent staining of polytene chromosomes were performed as described previously [41], using anti-Pc, anti-Pho, or anti-Spps (at 1:100), and monoclonal mouse antiFLAG M2 (Sigma, St. Louis, MO) at 1:1000 dilution.PcG Proteins Bind Constitutively to the en GeneFigure 5. FLAG-tagged PcG proteins are bound to the en PRE in both the “ON” and “OFF” transcriptional states. (A ) X-ChIP (with aFLAG) was performed on third instar imaginal discs and CNS, with en-GAL4 or ci-GAL4 driven Pho-FLAG (A), Sce-FLAG (B), Esc-FLAG (C), FLAG-Scm (D). Results are shown as a percentage of the input DNA, collected prior to ChIP. ns = not significant, * P#0.05, ** P#0.01, *** P#0.001, **** P#0.0001 (un-paired, two-tailed t-tests). Results shown are from three independent biological samples with 2 replicates each. (E) Fold increase (PRE/control) using the means from the experiments shown in A . The UAS-lines are shown on the left, with the drivers en-Gal4 (en) and ci-Gal4 (ci) on top. doi:10.1371/journal.pone.0048765.gRescue CrossesFLAG-tagged constructs were driven ubiquitously with an ArmGAL4 driver in the following AZP-531 web mutant backgrounds: ScmK3/ScmK4 (unpublished pharate adult lethal alleles from James A. Kennison), pp mcp Sce1/Df(3R)BSC499, esc21 b cn/escM20 (escM20 is an unpublished esc allele obtained from Mark Mortin and James A. Kennison), pho1/pho1, using standard crossing schemes.Cross-linked Chromatin Immunoprecipitation (X-ChIP)Imaginal discs, along with the central nervous system, mouth hooks, and some anterior cuticle were dissected from 3rd instar larvae (10 larvae per sample) and immediately placed in Schneider’s medium (Invitrogen) on ice. Disc sets were fixed in 2 formaldehyde (Ted Pella Inc, Redding, CA) fixing solution (50 mM Hepes pH 7.6, 100 mM NaCl, 0.1 mM EDTA, 0.5 mMEGTA) for 15 minutes, then rinsed in stop solut.Binding to PRE2 in both the “ON” and “OFF” states was the same in these experiments and those in Fig. 5. doi:10.1371/journal.pone.0048765.gand pTWF respectively, were obtained from Terence Murphy and are further described at http://www.ciwemb.edu/labs/murphy/ Gateway 20vectors.html. Clone cassettes in pENTR/dTOPO were recombined into pTWF and pTFW with LR Clonase (Invitrogen) according to manufacturers instructions. The resulting constructs were fully sequenced and checked for mutations and recombination errors prior to use.Reverse Transcription-Quantitative Polymerase Chain ReactionImaginal discs, along with the central nervous system, mouth hooks, and some anterior cuticle were dissected from 3rd instar larvae (5 per sample) from ci- and en-GAL4 driven Pho-FLAG larvae and immediately placed in PBS on ice. Total RNA was collected from the resulting samples using Trizol (Invitrogen) according to the manufacturers instructions. One-step RT-qPCR was performed with the QuantiTect SYBR Green RT-PCR Kit on a Roche Lightcycler 480 according to manufacturer instructions. Relative expression levels of Pho-FLAG transcript was calculated using the DC(T) 25331948 method, and expressed as a percentage of RP49 expression level. Pho-FLAG primers amplify a fragment containing the 39 end of pho gene and a portion of the FLAGencoding sequence. Pho-FLAG primers: 59-CCGTTTGTGGTATATGCAGA-39, 59-CGTCATGGTCTTTGTAGTC-39. RP49 primers: 59-CGGATCGATATGCTAAGCTGT-39, 59-CGACGCACTCTGTTGTCG-Transgenic linesUAS-PcG-FLAG transgenic lines were generated by injections into w1118 embryos by Genetic Services (Sudbury, MA, USA).Chromosome SquashesSquashes and immunofluorescent staining of polytene chromosomes were performed as described previously [41], using anti-Pc, anti-Pho, or anti-Spps (at 1:100), and monoclonal mouse antiFLAG M2 (Sigma, St. Louis, MO) at 1:1000 dilution.PcG Proteins Bind Constitutively to the en GeneFigure 5. FLAG-tagged PcG proteins are bound to the en PRE in both the “ON” and “OFF” transcriptional states. (A ) X-ChIP (with aFLAG) was performed on third instar imaginal discs and CNS, with en-GAL4 or ci-GAL4 driven Pho-FLAG (A), Sce-FLAG (B), Esc-FLAG (C), FLAG-Scm (D). Results are shown as a percentage of the input DNA, collected prior to ChIP. ns = not significant, * P#0.05, ** P#0.01, *** P#0.001, **** P#0.0001 (un-paired, two-tailed t-tests). Results shown are from three independent biological samples with 2 replicates each. (E) Fold increase (PRE/control) using the means from the experiments shown in A . The UAS-lines are shown on the left, with the drivers en-Gal4 (en) and ci-Gal4 (ci) on top. doi:10.1371/journal.pone.0048765.gRescue CrossesFLAG-tagged constructs were driven ubiquitously with an ArmGAL4 driver in the following mutant backgrounds: ScmK3/ScmK4 (unpublished pharate adult lethal alleles from James A. Kennison), pp mcp Sce1/Df(3R)BSC499, esc21 b cn/escM20 (escM20 is an unpublished esc allele obtained from Mark Mortin and James A. Kennison), pho1/pho1, using standard crossing schemes.Cross-linked Chromatin Immunoprecipitation (X-ChIP)Imaginal discs, along with the central nervous system, mouth hooks, and some anterior cuticle were dissected from 3rd instar larvae (10 larvae per sample) and immediately placed in Schneider’s medium (Invitrogen) on ice. Disc sets were fixed in 2 formaldehyde (Ted Pella Inc, Redding, CA) fixing solution (50 mM Hepes pH 7.6, 100 mM NaCl, 0.1 mM EDTA, 0.5 mMEGTA) for 15 minutes, then rinsed in stop solut.