Homeotic gene clusters and gain of contacts with other PcDs. Despite these changed interactions, we did not find convincing evidence for global gene expression alterations on the In(3LR)sep chromosome. This raises the question: how relevant are long- range chromatin contacts between PcG-target genes for regulation of expression? The lack of detectable expression changes may indicate that long-range interactions have only quantitatively subtle effect on the regulation of gene expression. Nevertheless, such subtle effects on gene expression could be very important for long-term viability and species survival. In(3LR)sep animals do suffer from an overall reduced viability during several stages of development (data not shown), which may indicate a generally reduced fitness, possibly due to an dimly altered regulation of gene expression. Alternatively, PcG gene regulation may not be affected in strain In(3LR)sep, because Abd-B and Antp, although they no longer interact with each other, still prefer to interact with other PcDs (Figure 8B), suggesting that it is not relevant which PcDs interact. In such a model, the complement of all interactions contributes to PcG-mediated gene silencing in a population of cells. Finally, it is interesting to note that over ,100 million years of evolution of the Drosophila genus, exchange of genes between chromosome arms has been rare despite extensive rearrangements within each arm [45,46]. Chromosome arm territories ensure that genes within a single arm are relatively close compared to genes on other arms, which may have resulted in an increased chance of rearrangements within one arm. Alternatively, the importance of long-range interactions among sets of genes, which are topolog- ically limited to the same arm, may have contributed to the selective pressure that has led to this remarkable conservation of the gene complement of each chromosome arm. Materials and Methods Fly stocks and handling Flies were raised at 25uC on standard cornmeal/molasses/agar medium. The wild type (w1118), In(3LR)sep (In(3LR)sep, vvlsep, kniri-1, p p ), Scr1 (Scr1 pp cu1/TM3, Sb1 Ser1), and Scr4 (pb1 Scr4 red1 e1/TM3, Sb1) stocks were obtained from the Bloomington stock center (Bloomington, USA). Transgenic DamID stocks, M{UAS- Pc::Dam}ZH51C/CyO, P{Dfd-EYFP} and M{UAS-Dam}ZH51C/ CyO, were made by the BestGene corporation, using the phiC31- integrase system (see below). For detecting Scr gene expression we crossed wild type and In(3LR)sep to Scr1 and Scr4 and selected male adult offspring that lacked the Sb1 phenotypic marker from the balancer chromosome. 4C in larval brain We adapted a previously described 4C protocol [20] for D. melanogaster larval central brain tissue. For each experiment we dissected and pooled 40 pairs of central brain hemispheres. With gentle forceps manipulation we quickly removed the central nervous system from each larva, while leaving the mouth hook and imaginal discs attached, and placed it in PBS on ice. After 40 dissections, we replaced the PBS with 2% formaldehyde in PBS and fixed the samples for 15 minutes at room temperature while tumbling. The reaction was quenched for 5 minutes by adding 125 mM glycin final concentration, followed by three quick washes with cold PBS. Next, we removed the mouth hook, imaginal discs, and ventral nerve cord with gentle forceps manipulation. The remaining central brain hemispheres were placed into ice cold cell lysis buffer (10 mM Tris – pH 8.0; 10 mM NaCl; 0.2% Nonidet P40; 1x Complete protease inhibitors – Roche) and the tissue and cells were disrupted by repetitively Polycomb Domains Interact in Nuclear Space pipeting up and down several times to isolate nuclei. Finally, the nuclei were centrifuged for 15 minutes with 2700 g at 4uC and supernatant was removed afterwards. On this nuclei pellet, we applied the 3C procedure as described [47], yielding ligation products between EcoRI fragments. We used the original 4C protocol to obtain circularized DNA templates for the 4C PCR [20], and either DpnII or NlaIII as frequently cutting restriction enzymes. We supplemented our reverse 4C PCR primers with a T7 promoter sequence overhang (Table S5). 4C PCR reactions were performed directly on the circularized 4C templates using Taq polymerase (Invitrogen) and conditions were as follows: 94uC for 2 min; 30 cycles of 94uC for 15 s, 60uC for 1 min and 72uC for 1 min; and a final elongation step of 72uC for 5 min. We purified the PCR products using the Qiaquick PCR purification kit (Qiagen). We further amplified the 4C PCR products in a strictly linear fashion using a T7 RNA amplification step. For this we used the Superscript RNA amplification system (Invitrogen) according to the manufacturer’s guidelines. As a reference, we digested genomic DNA from w1118 third instar larvae with DpnII. To each restriction end, we added adaptors containing the T7 promoter sequence by means of ligation. We amplified 1 mg of this DNA using the Superscript RNA amplification system. The 4C and reference samples were differentially labeled with the ULS system (Kreatech), using Cy3 and Cy5 dyes (Amersham). For each biological replicate we used opposite dye orientations to correct for dye bias, we used the average signal of the replicates as output. For all microarray experiments we used the same reference sample. Microarray probes were designed in close proximity to EcoRI restriction sites across the fly genome. These probes were used on custom designed tiling microarrays (Agilent Technologies). DNA Adenine methyltransferase IDentification (DamID) and Chromatin Immunoprecipitation (ChIP) on chip We cloned either Pc::Dam or Dam open reading frames from our previous transfection plasmids (Tolhuis et al. 2006) into the pUAST-attB vector (gift from Konrad Basler, Zurich, Switzer- land). Plasmid integrity was verified using sequencing. The BestGene corporation used these vectors to create transgenic flies with the PhiC31-integrase system [48]. Vectors were injected into embryos of strain M{3xP3-RFP.attP’}ZH-51C (with M{vas- int.Dm}ZH-2A), which contains the attP-landing site at cytoge- netic location 51C. Transgenic strains were balanced over either CyO, P{Dfd-EYFP}2 or CyO. Importantly, the DamID assay depends on very low expression levels to avoid mistargeting [49,50]. We achieved this by depending solely on leaky expression of the uninduced pUAST promoter in the absence of a Gal4 driver. For a single DamID experiment, we used 10 M{UAS- Pc::Dam}ZH51C/CyO, P{Dfd-EYFP}2 central brains from 3rd instar larvae, and as reference sample for the microarray we used 10 brain from M{UAS-Dam}ZH51C/CyO. For the ChIP-chip we used the Red ChIP kit (Diagenode) according the manufacturers guidelines. We used the following antibodies, anti-H3K27me3 (Upstate 07-449), and anti-IgG (non- specific control; Abcam ab17890). We used central brains from 50 3rd instar larvae (w1118) in a single ChIP experiment. Amplification of DamID and ChIP isolated DNA fragments and microarray analysis were performed as described [6,51] using custom oligonucleotide tiling arrays (NimbleGen Systems). Discrete domains of either Pc or H3K27me3 enrichment were calculated using the same dynamic algorithm to extract DIDs from the 4C data (see below). A validation of the data is described in the Text S1 and Figure S2. PLoS Genetics | www.plosgenetics.org 11 March 2011 | Volume 7 | Issue 3 | e1001343