Drosophila Chromosome Condensation Proteins Topoisomerase II and Barren Colocalize with Polycomb and Maintain Fab-7 PRE Silencing  Rossella Lupo, Achim Breiling, Marco E Bianchi, Valerio Orlando  Molecular Cell  Volume 7, Issue 1, Pages 127-136 (January 2001) DOI: 10.1016/S1097-2765(01)00161-7

Figure 1 Southern Analysis of In Vivo Fixed Chromatin Immunoprecipitation with TOPOII and BARR Antibodies Specific BX-C genomic fragments previously identified by low resolution analysis as containing binding sites for TOPOII and BARR were digested with restriction enzymes and resolved on 1.2% agarose gels (A), transferred onto nitrocellulose filters, and hybridized with DNA probes from TOPOII (C), BARR (D), and mock (no antibody, [B]) chromatin immunoprecipitations. Loading reflects the proximal-distal position in the BX-C map (Martin et al. 1995). Lane M is a molecular weight marker (1 kb ladder, Promega). Lane 1, AbdB γ promoter (p8106), HindIII, AccI; lane 2, iab-7 (p3434), EcoRI, XhoI; lane 3, Fab-7 (p4344), EcoRI, XbaI, XhoI; lane 4, Fab-7 (p4389), PstI, HindIII; lane 5, Mcp (p5989), EcoRI, AccI, PstI; lane 6, iab-4 (p7652), EcoRI, XhoI, PstI; lane 7, iab-3 (p6741), EcoRI, SalI, XhoI; lane 8, iab-3 (p2088), EcoRI, SmaI; lane 9, abdA II promoter (p3060), EcoRI, BamHI; lane 10, bxd (p3384), EcoRI, EcoRV, ClaI; lane 11, p6807, EcoRI, ClaI, EcoRV; lane 12, Ubx promoter (p3901), EcoRI, BamHI, ClaI; lane 13, p7480, EcoRI, SmaI, XbaI; lane 14, p1617, EcoRI; lane 15 bx (p2571), EcoRI, Eco47III, HindIII; lane 16, p2333, EcoRI; and lane 17, p10074, EcoRI, AccI, HindIII, HincII. The names of all subclones (except p8106) reflect the size of the corresponding genomic fragment in bp. Molecular Cell 2001 7, 127-136DOI: (10.1016/S1097-2765(01)00161-7)

Figure 2 High-Resolution Distribution of TOPOII and BARR in the BX-C Hybridization signals in Figure 1 were quantified by Phosphorimager and plotted with ImageQuant software. Each panel provides a schematic view of the subclones and the position of TOPOII and BARR binding sites based on the quantitation values. Intensity values were normalized to an arbitrary 1 kb fragment and subtracted from the corresponding background values obtained from the mock hybridizations. The x axis represents the restriction fragments, featuring the genomic subclone structure below each panel. Numbers at the edges of the subclones indicate the position in the genetic map of the BX-C (Martin et al. 1995). The y axis represents TOPOII and BARR values after subtraction of the background, expressed in arbitrary units. Panel letters are also reported in Figure 3, permitting the identification of the position mapped for the two proteins in the locus. Restriction enzymes: AccI, A; BamHI, B; ClaI, C; EcoRI, E; EcoRV, EV; Eco47III, E47; HincII, HII; HindIII, H; PstI, P; SalI, S; SmaI, Sm; XbaI, Xb; and XhoI, Xh. Molecular Cell 2001 7, 127-136DOI: (10.1016/S1097-2765(01)00161-7)

Figure 3 Distribution of TOPOII and BARR in the BX-C The scheme represents the BX-C, including transcription units and genetically characterized regulatory regions. The numbers represent the map coordinates expressed in kilobases (0 distal, 340 proximal; Martin et al. 1995). Previously identified major PC binding sites (green squares) are shown. A highly significant colocalization is observed between these sites and the BARR (red squares) and TOPOII (blue squares) sites. The capital letters (A–N) refer to genomic subfragments shown in Figure 2. Molecular Cell 2001 7, 127-136DOI: (10.1016/S1097-2765(01)00161-7)

Figure 4 Coimmunoprecipitations of PH, BARR, and TOPOII Nuclear extracts from Drosophila embryos were immunoprecipitated with antibodies against BARR, TOPOII, PH, PC, PSC, and the preimmune serum (PI-serum) from the rabbit used for the PH antibody production (as indicated on top of the Western blots shown). After washing and SDS–PAGE (see Experimental Procedures), TOPOII (A), BARR (B), and PH (C) were detected with the respective antibodies. In each panel, an aliquot (10 μl) of the extract is shown as input. The molecular weights are 164 kDa for TOPOII, 97 kDa for BARR, and 170 kDa (PH 170p), 140 kDa (PH 140p), and 130 kDa (PH 130d) for the three PH isoforms (note the higher apparent molecular weight of TOPOII and of the PH isoforms in the blots shown). The three different isoforms and the size of the marker bands (New England BioLabs, prestained protein marker, broad range) are indicated. In (C), an additional blot of proteins immunoprecipitated with TOPOII antibodies is shown with an aliquot of nuclear extract run on the same gel for exact size comparison of the PH isoforms. Molecular Cell 2001 7, 127-136DOI: (10.1016/S1097-2765(01)00161-7)

Figure 5 Loss of Fab-7 PRE-Induced Silencing of a mini-white Reporter Gene in barren Null Heterozygotes (A) Effect of a null allele (L305) of the barren gene on the expression of a mini-white gene under the control of Fab-7 PRE (5F24 25,2 line). Heterozygous females for barren show strong derepression of the mini-white gene (red eye). (B) Female progeny resulting from the same cross as in (A) carrying the balancer chromosome CyO (wild type for barren) instead of the barrL305 allele. In this case, no effect on the mini-white gene expression is observed (white eye). (C) Effect of the barrL305 allele on the expression of a mini-white gene not placed under the control of Fab-7 PRE (UI5 1,1 line). In this case, no derepression of the mini-white gene is observed (white eye). (D) The corresponding progeny as in (C) carrying the balancer chromosome CyO. Also in this case, no effect on mini-white expression is observed (white eye). Expression of the mini-white gene (red eye) was detected as flies hatched. In all cases (white and red eyes), no changes were observed up to ten days after eclosure. In all panels, 1-week-old females are shown. Molecular Cell 2001 7, 127-136DOI: (10.1016/S1097-2765(01)00161-7)

Figure 6 Enhancement of the Fab-72 Phenotype by the barrL305 Allele (A) The Fab-7 region of the BX-C between iab-6 and iab-7 as adapted from Mihaly et al. 1997. Black boxes indicate strong nuclease hypersensitive sites (HS). Ovals mark putative nucleosomal core particles. The deleted regions of the class I (Fab-71), class II (Fab-72), and class III (ΔPRE316) Fab-7-specific mutant fly lines are indicated. Fab-71 removes the whole element, Fab-72 removes the boundary region, and ΔPRE316 removes the PRE. (B) Abdominal cuticle patterns of adult male flies. Abdomens were cut along the dorsal midline and flattened on a slide. On the far right, a wild-type (+/+) and a barrL305 heterozygous cuticle are shown (barrL305 cuticles appear brighter, as these flies carry the yellow marker). Wild-type males show normal sized tergites 4, 5, and 6. Tergites 5 and 6 are pigmented. Structures in the bottom of each panel are part of the genitalia. Heterozygous Fab-71 and Fab-72 males have rudimentary or significantly reduced sixth tergites, respectively (arrows), whereas ΔPRE316 and barrL305 heterozygotes show a wild-type pattern. Cuticles of Fab-71/barrL305, Fab-72/barrL305, and ΔPRE316/barrL305 double heterozygotes are shown in the second and third rows. The bottom panels are from crosses of barrL305 females with male Fab-7 mutants (cuticles appear brighter, as male flies take the yellow marker from the barrL305 mothers), and the upper panels are from the reverse cross. Molecular Cell 2001 7, 127-136DOI: (10.1016/S1097-2765(01)00161-7)

Figure 7 Chromosome Segregation Defects in ph Mutant Embryos Early embryos (0–3 hr after egg laying) from the ph503 stock were collected, stained with DAPI, and screened for mitotic figures. In a subset of embryos at stages 8 and 9, mitotic defects were observed as abnormal anaphases. Multiple chromosomal bridges (brightly staining material between the separating chromosomes) are clearly visible (A and B). In some cases, a few chromosomal breaks were also found. Molecular Cell 2001 7, 127-136DOI: (10.1016/S1097-2765(01)00161-7)