Populations. Note that the posterior compartment comprises only about a third of the cells of the imaginal disc [35], thus there are about twice as many cells expressing FLAG-tagged Epigenetics proteins with the ci-driver as with the endriver. Consistent with this, quantitative RT-PCR showed there is approximately twice as much Pho-FLAG mRNA in ci-driven samples versus en-driven samples (Fig. 2G). Next, we compared the polytene chromosome-binding pattern of the FLAG-tagged proteins to the binding pattern of an endogenous PcG protein. For these experiments, FLAG-tagged proteins were driven ubiquitously with arm-GAL4. Pho-FLAG was detected on chromosomes in a pattern that completely overlapped with endogenous Polycomb (Pc) Epigenetic Reader Domain protein (Fig. 3A). There were some Pc bands that did not contain Pho-FLAG. There are two reasons for this: one, the detection of the Pho-Flag is relatively weak, and two, endogenous Pho does not bind all Pc sites in polytene chromosomes. Similarly, Esc-FLAG and Sce-FLAG Epigenetic Reader Domain largely overlap with endogenous Pho bands on polytene chromosomes (Fig. 3B and data not shown). For Scm, we examined the overlap with the PRE DNA binding protein Spps [36] and again saw a nearly complete overlap (Fig. 3C).To test whether the FLAG-tagged proteins are functional, we ubiquitously expressed FLAG-tagged PcG proteins in flies with mutations or deletions for the respective genes to look for rescue. Esc-FLAG and Sce-FLAG completely rescued esc and Sce mutant flies, with no observable PcG or homeotic phenotypes. Pho-FLAG rescued 
pho flies with 10 of adult males showing moderate A4?A5 transformations. FLAG-Scm rescued Scm mutant flies, with about 70 of males exhibiting extra sex combs on the 2nd and 3rd legs. It is not surprising that minor PcG phenotypes are observed in some experiments, as the timing and level of expression of FLAG-tagged proteins, under the control of the UAS/GAL4 system, are not likely to perfectly match endogenous expression. Considering this, we conclude that the FLAG-tagged PcG proteins are functional, and that ChIP experiments carried out with these proteins would faithfully reflect results obtained with endogenous proteins. The validated FLAG-tagged proteins were used in X-ChIP experiments. FLAG-tagged PcG proteins were driven in flies with the en-GAL4 (“ON”) and ci-GAL4 drivers (“OFF”). Imaginal disc sets, along with the central nervous system, were collected from 3rd instar larvae, processed for X-ChIP, and analyzed with qPCR to determine binding signals at the en gene. The locations of the two PREs just upstream of en have been well characterized in functional studies (25?8; JLB and JAK, unpublished data) and are shown in Fig. 4A along with the en transcription unit and primer locations. The ChIP experiments were all done in flies that were wild type for all PcG genes, since these proteins must bePcG Proteins Bind Constitutively to the 1326631 en Genesignal in en “OFF” cells, compared with 2.4 fold in en “ON” cells (Fig. 5E). Similar results are observed with FLAG-Scm (4.8 vs. 2.7), Esc-FLAG (4.8 vs. 1.6), and less so with Sce-FLAG (2.6 vs. 2.0). However, it is important to note that there are more ci-cells than en-cells, so we cannot conclude from this data that the 
levels of PcG binding in the “OFF” state are higher than those in the “ON” state.DiscussionIn this study we sought to learn more about PcG protein complex-mediated regulation of en expression, focusing on mechanisms operating inhibitor through en PREs. First we investiga.Populations. Note that the posterior compartment comprises only about a third of the cells of the imaginal disc [35], thus there are about twice as many cells expressing FLAG-tagged proteins with the ci-driver as with the endriver. Consistent with this, quantitative RT-PCR showed there is approximately twice as much Pho-FLAG mRNA in ci-driven samples versus en-driven samples (Fig. 2G). Next, we compared the polytene chromosome-binding pattern of the FLAG-tagged proteins to the binding pattern of an endogenous PcG protein. For these experiments, FLAG-tagged proteins were driven ubiquitously with arm-GAL4. Pho-FLAG was detected on chromosomes in a pattern that completely overlapped with endogenous Polycomb (Pc) protein (Fig. 3A). There were some Pc bands that did not contain Pho-FLAG. There are two reasons for this: one, the detection of the Pho-Flag is relatively weak, and two, endogenous Pho does not bind all Pc sites in polytene chromosomes. Similarly, Esc-FLAG and Sce-FLAG largely overlap with endogenous Pho bands on polytene chromosomes (Fig. 3B and data not shown). For Scm, we examined the overlap with the PRE DNA binding protein Spps [36] and again saw a nearly complete overlap (Fig. 3C).To test whether the FLAG-tagged proteins are functional, we ubiquitously expressed FLAG-tagged PcG proteins in flies with mutations or deletions for the respective genes to look for rescue. Esc-FLAG and Sce-FLAG completely rescued esc and Sce mutant flies, with no observable PcG or homeotic phenotypes. Pho-FLAG rescued pho flies with 10 of adult males showing moderate A4?A5 transformations. FLAG-Scm rescued Scm mutant flies, with about 70 of males exhibiting extra sex combs on the 2nd and 3rd legs. It is not surprising that minor PcG phenotypes are observed in some experiments, as the timing and level of expression of FLAG-tagged proteins, under the control of the UAS/GAL4 system, are not likely to perfectly match endogenous expression. Considering this, we conclude that the FLAG-tagged PcG proteins are functional, and that ChIP experiments carried out with these proteins would faithfully reflect results obtained with endogenous proteins. The validated FLAG-tagged proteins were used in X-ChIP experiments. FLAG-tagged PcG proteins were driven in flies with the en-GAL4 (“ON”) and ci-GAL4 drivers (“OFF”). Imaginal disc sets, along with the central nervous system, were collected from 3rd instar larvae, processed for X-ChIP, and analyzed with qPCR to determine binding signals at the en gene. The locations of the two PREs just upstream of en have been well characterized in functional studies (25?8; JLB and JAK, unpublished data) and are shown in Fig. 4A along with the en transcription unit and primer locations. The ChIP experiments were all done in flies that were wild type for all PcG genes, since these proteins must bePcG Proteins Bind Constitutively to the 1326631 en Genesignal in en “OFF” cells, compared with 2.4 fold in en “ON” cells (Fig. 5E). Similar results are observed with FLAG-Scm (4.8 vs. 2.7), Esc-FLAG (4.8 vs. 1.6), and less so with Sce-FLAG (2.6 vs. 2.0). However, it is important to note that there are more ci-cells than en-cells, so we cannot conclude from this data that the levels of PcG binding in the “OFF” state are higher than those in the “ON” state.DiscussionIn this study we sought to learn more about PcG protein complex-mediated regulation of en expression, focusing on mechanisms operating through en PREs. First we investiga.Populations. Note that the posterior compartment comprises only about a third of the cells of the imaginal disc [35], thus there are about twice as many cells expressing FLAG-tagged proteins with the ci-driver as with the endriver. Consistent with this, quantitative RT-PCR showed there is approximately twice as much Pho-FLAG mRNA in ci-driven samples versus en-driven 
samples (Fig. 2G). Next, we compared the polytene chromosome-binding pattern of the FLAG-tagged proteins to the binding pattern of an endogenous PcG protein. For these experiments, FLAG-tagged proteins were driven ubiquitously with arm-GAL4. Pho-FLAG was detected on chromosomes in a pattern that completely overlapped with endogenous Polycomb (Pc) protein (Fig. 3A). There were some Pc bands that did not contain Pho-FLAG. There are two reasons for this: one, the detection of the Pho-Flag is relatively weak, and two, endogenous Pho does not bind all Pc sites in polytene chromosomes. Similarly, Esc-FLAG and Sce-FLAG largely overlap with endogenous Pho bands on polytene chromosomes (Fig. 3B and data not shown). For Scm, we examined the overlap with the PRE DNA binding protein Spps [36] and again saw a nearly complete overlap (Fig. 3C).To test whether the FLAG-tagged proteins are functional, we ubiquitously expressed FLAG-tagged PcG proteins in flies with mutations or deletions for the respective genes to look for rescue. Esc-FLAG and Sce-FLAG completely rescued esc and Sce mutant flies, with no observable PcG or homeotic phenotypes. Pho-FLAG rescued pho flies with 10 of adult males showing moderate A4?A5 transformations. FLAG-Scm rescued Scm mutant flies, with about 70 of males exhibiting extra sex combs on the 2nd and 3rd legs. It is not surprising that minor PcG phenotypes are observed in some experiments, as the timing and level of expression of FLAG-tagged proteins, under the control of the UAS/GAL4 system, are not likely to perfectly match endogenous expression. Considering this, we conclude that the FLAG-tagged PcG proteins are functional, and that ChIP experiments carried out with these proteins would faithfully reflect results obtained with endogenous proteins. The validated FLAG-tagged proteins were used in X-ChIP experiments. FLAG-tagged PcG proteins were driven in flies with the en-GAL4 (“ON”) and ci-GAL4 drivers (“OFF”). Imaginal disc sets, along with the central nervous system, were collected from 3rd instar larvae, processed for X-ChIP, and analyzed with qPCR to determine binding signals at the en gene. The locations of the two PREs just upstream of en have been well characterized in functional studies (25?8; JLB and JAK, unpublished data) and are shown in Fig. 4A along with the en transcription unit and primer locations. The ChIP experiments were all done in flies that were wild type for all PcG genes, since these proteins must bePcG Proteins Bind Constitutively to the 1326631 en Genesignal in en “OFF” cells, compared with 2.4 fold in en “ON” cells (Fig. 5E). Similar results are observed with FLAG-Scm (4.8 vs. 2.7), Esc-FLAG (4.8 vs. 1.6), and less so with Sce-FLAG (2.6 vs. 2.0). However, it is important to note that there are more ci-cells than en-cells, so we cannot conclude from this data that the levels of PcG binding in the “OFF” state are higher than those in the “ON” state.DiscussionIn this study we sought to learn more about PcG protein complex-mediated regulation of en expression, focusing on mechanisms operating through en PREs. First we investiga.Populations. Note that the posterior compartment comprises only about a third of the cells of the imaginal disc [35], thus there are about twice as many cells expressing FLAG-tagged proteins with the ci-driver as with the endriver. Consistent with this, quantitative RT-PCR showed there is approximately twice as much Pho-FLAG mRNA in ci-driven samples versus en-driven samples (Fig. 2G). Next, we compared the polytene chromosome-binding pattern of the FLAG-tagged proteins to the binding pattern of an endogenous PcG protein. For these experiments, FLAG-tagged proteins were driven ubiquitously with arm-GAL4. Pho-FLAG was detected on chromosomes in a pattern that completely overlapped with endogenous Polycomb (Pc) protein (Fig. 3A). There were some Pc bands that did not contain Pho-FLAG. There are two reasons for this: one, the detection of the Pho-Flag is relatively weak, and two, endogenous Pho does not bind all Pc sites in polytene chromosomes. Similarly, Esc-FLAG and Sce-FLAG largely overlap with endogenous Pho bands on polytene chromosomes (Fig. 3B and data not shown). For Scm, we examined the overlap with the PRE DNA binding protein Spps [36] and again saw a nearly complete overlap (Fig. 3C).To test whether the 
FLAG-tagged proteins are functional, we ubiquitously expressed FLAG-tagged PcG proteins in flies with mutations or deletions for the respective genes to look for rescue. Esc-FLAG and Sce-FLAG completely rescued esc and Sce mutant flies, with no observable PcG or homeotic phenotypes. Pho-FLAG rescued pho flies with 10 of adult males showing moderate A4?A5 transformations. FLAG-Scm rescued Scm mutant flies, with about 70 of males exhibiting extra sex combs on the 2nd and 3rd legs. It is not surprising that minor PcG phenotypes are observed in some experiments, as the timing and level of expression of FLAG-tagged proteins, under the control of the UAS/GAL4 system, are not likely to perfectly match endogenous expression. Considering this, we conclude that the FLAG-tagged PcG proteins are functional, and that ChIP experiments carried out with these proteins would faithfully reflect results obtained with endogenous proteins. The validated FLAG-tagged proteins were used in X-ChIP experiments. FLAG-tagged PcG proteins were driven in flies with the en-GAL4 (“ON”) and ci-GAL4 drivers (“OFF”). Imaginal disc sets, along with the central nervous system, were collected from 3rd instar larvae, processed for X-ChIP, and analyzed with qPCR to determine binding signals at the en gene. The locations of the two PREs just upstream of en have been well characterized in functional studies (25?8; JLB and JAK, unpublished data) and are shown in Fig. 4A along with the en transcription unit and primer locations. The ChIP experiments were all done in flies that were wild type for all PcG genes, since these proteins must bePcG Proteins Bind Constitutively to the 1326631 en Genesignal in en “OFF” cells, compared with 2.4 fold in en “ON” cells (Fig. 5E). Similar results are observed with FLAG-Scm (4.8 vs. 2.7), Esc-FLAG (4.8 vs. 1.6), and less so with Sce-FLAG (2.6 vs. 2.0). However, it is important to note that there are more ci-cells than en-cells, so we cannot conclude from this data that the levels of PcG binding in the “OFF” state are higher than those in the “ON” state.DiscussionIn this study we sought to learn more about PcG protein complex-mediated regulation of en expression, focusing on mechanisms operating through en PREs. First we investiga.