On a Roche Lightcycler 480 according to manufacturer instructions. Relative expression levels of HIF-2��-IN-1 site Pho-FLAG transcript was calculated using the DC(T) method, and expressed as a CP21 chemical information 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constructs were driven ubiquitously with an ArmGAL4 driver in the following mutant backgrounds: ScmK3/ScmK4 (unpublished pharate adult lethal alleles from James A. Kennison), pp mcp Sce1/Df(3R)BSC499, esc21 b cn/escM20 (escM20 is an unpublished esc allele 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 solution (PBS, 0.01 Triton X-100, 0.125 M Glycine) for 10 minutes, followed by 2610 minute washes with wash solution (50vmM Tris, 10 mM EDTA, 0.5 mM EGTA, 0.25 Triton X-100). Fixed and washed samples were stored at 280uC in storage solution (10 mM TrisHCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA). Whole discs were placed in 300 ml of action buffer with Complete Protease Inhibitor Cocktail in a 1.5 microcentrifuge tube, and sonicated in BioRuptor UCD-300 (Diagenode, Denville, NJ) for 30 seconds on/30 seconds off for 20 cycles, high power, resulting in chromatin fragments tightly concentrating at 200 base pairs, with a diminishing smear up to 1500 base pairs. Remaining insoluble material was spun down at full speed for 1 min, and chromatin supernatant was transferred to a new tube. 10 ml of chromatin was removed (3.3 of total volume) and saved from each sample forPcG Proteins Bind Constitutively to the en Geneinput reactions. ChIP was performed with monoclonal mouse antiFLAG M2 (Sigma) at 1:700 dilution, and the Milli.On a Roche Lightcycler 480 according to manufacturer instructions. Relative expression levels of Pho-FLAG transcript was calculated using the DC(T) 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 solution (PBS, 0.01 Triton X-100, 0.125 M Glycine) for 10 minutes, followed by 2610 minute washes with wash solution (50vmM Tris, 10 mM EDTA, 0.5 mM EGTA, 0.25 
Triton X-100). Fixed and washed samples were stored at 280uC in storage solution (10 mM TrisHCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA). Whole discs were placed in 300 ml of action buffer with Complete Protease Inhibitor Cocktail in a 1.5 microcentrifuge tube, and sonicated in BioRuptor UCD-300 (Diagenode, Denville, NJ) for 30 seconds on/30 seconds off for 20 cycles, high power, resulting in chromatin fragments tightly concentrating at 200 base pairs, with a diminishing smear up to 1500 base pairs. Remaining insoluble material was spun down at full speed for 1 min, and chromatin supernatant was transferred to a new tube. 10 ml of chromatin was removed (3.3 of total volume) and saved from each sample forPcG Proteins Bind Constitutively to the en Geneinput reactions. ChIP was performed with monoclonal mouse antiFLAG M2 (Sigma) at 1:700 dilution, and the Milli.