Regulation of Target Genes by PC

<div><p>In situ hybridizations in WT and in <i>Pc^XL5</i> mutant embryos for the <i>gt</i> and <i>peb</i> genes. The developmental stage of the embryos is indicated on the left. Arrowheads indicate regions of increased or ectopic labeling in <i>Pc</i> mutants compared to WT. </p> <p>(A) <i>gt</i> expression at embryonic stages 9, 10, 11, and 14. </p> <p>(B) <i>peb</i> expression at embryonic stages 9, 10, 11, and 14. </p></div

Developmental Comparison of the Distribution Profiles of PC, PH, and GAF

<div><p>PC is shown in light blue, PH in blue, and GAF in red. All signals that are not significantly enriched are set to one in these graphs. Thus, only the significant targets detected by RDAM at FDR 10% are shown. The correlation coefficient for each comparison is indicated above the graph.</p> <p>(A) A comparison between PH and PC at the embryonic stage shows the extensive overlap between the two proteins.</p> <p>(B) A comparison between GAF and PH in embryos shows the fundamentally different distribution profile for the two proteins.</p> <p>(C) Comparison between the distributions of PC in embryos over PC in pupae.</p> <p>(D) Comparison between the distributions of GAF in embryos over GAF in pupae.</p> <p>(E) Comparison between the distribution of PH males versus PH in females.</p> <p>(F) Comparison between the distributions of GAF in males versus GAF in females.</p></div

High Resolution Distribution Profiles

<div><p>GAF profiles are in red, PH in blue, and PC in light blue. Only significantly enriched signals detected by RDAM at a FDR of 10% are represented. Above each graph, the annotated genes of each genome region are shown.</p> <p>(A) Distribution profiles of PC, PH, and GAF in the <i>en</i>/ <i>inv</i> locus at the embryonic stage. </p> <p>(B) Distribution of PC, PH, and GAF in the <i>en</i>/ <i>inv</i> locus at the adult stage (females). The <i>en</i> PRE used as positive control is indicated by an asterisk. </p> <p>(C) Distribution of PC, PH, and GAF in the <i>gt</i>/ <i>z</i> locus at the embryonic stage. </p> <p>(D) Distribution of PC, PH, and GAF in the <i>gt</i>/ <i>z</i> locus in adult females. Note the disappearance of a strong PcG binding site, while GAF remains stable. </p> <p>(E) Distribution of PC, PH, and GAF in the <i>futsch</i> locus in embryos. </p> <p>(F) PC, PH, and GAF in the <i>futsch</i> locus in adult females. Note that a new binding site for PcG that was absent at the embryonic stage appears at the adult stage, while GAF remains stable. </p></div

Immuno-FISH Mapping of PcG Protein Binding at Four Different Target Loci

<p>DAPI labeling of DNA is shown in light blue. The immunostainings of PC and PH (as indicated to the right of each row) are shown in red. DNA FISH staining is shown in green, and the merge of the red and the green channels is shown in right panels. The name and cytological position for each probe is indicated on the right. The numbers identifying the probes used correspond to those indicated in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040170#pbio-0040170-g002" target="_blank">Figure 2</a>. 1 corresponds to the <i>bifid</i> gene locus, 2 to the CG4136 locus, 3 to the <i>mab-2</i> locus, and 4 to the <i>cut</i> locus. The arrows point to the bands that co-localize with the FISH signal. </p

ChIP on Chip Mapping of the PH Protein Using MY Microarrays

<div><p>(A) Schematic representation of the four <i>Drosophila</i> chromosomes, with the Montpellier tiling path assembly shown in red, and the Yale tiling path regions shown in light blue. </p> <p>(B) The distribution of the PH protein along the tiling path of the X chromosome in <i>Drosophila</i> embryos. Numbers 1 to 4 indicate the regions for which FISH probes used in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040170#pbio-0040170-g003" target="_blank">Figure 3</a> were designed. The <i>ph</i> locus is a known PcG target that served as a positive control. The other arrows point to the major binding sites. Below the graphs, a scheme of the corresponding chromosomal region is shown; with the cytological location of known PH bands in polytene chromosomes indicated as red ovals. </p> <p>(C) The distribution of the PH protein along the Adh region of Chromosome 2L in <i>Drosophila</i> embryos. Symbols are as in (B). </p></div

Phage Alignments and Neighboring Genes

<p>Conserved gene order between the WO phage in <i>Wolbachia</i> sp. <i>w</i>Kue and prophage regions of <i>w</i>Mel. Putative proteins in <i>w</i>Kue (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020069#pbio-0020069-Masui2" target="_blank">Masui et al. 2001</a>) were searched using TBLASTN against the <i>w</i>Mel genome. Matches with an <i>E</i>-value of less than 1e⁻¹⁵ are linked by connecting lines. CDSs are colored as follows: brown, phage structural or replication genes; light blue, conserved hypotheticals; red, hypotheticals; magenta, transposases or reverse transcriptases; blue, ankyrin repeat genes; light gray, <i>radC</i>; light green, paralogous genes; gold, others. The regions surrounding the phage are shown because they have some unusual features relative to the rest of the genome. For example, WO-A and WO-B are each flanked on one side by clusters of genes in two paralogous families that are distantly related to phage repressors. In each of these clusters, a homolog of the <i>radC</i> gene is found. A third <i>radC</i> homolog (WD1093) in the genome is also flanked by a member of one of these gene families (WD1095). While the connection between <i>radC</i> and the phage is unclear, the multiple copies of the <i>radC</i> gene and the members of these paralogous families may have contributed to the phage rearrangements described above.</p

Long Evolutionary Branches in <i>w</i>Mel

<p>Maximum-likelihood phylogenetic tree constructed on concatenated protein sequences of 285 orthologs shared among <i>w</i>Mel, R. prowazekii, R. conorii, <i>C. crescentus,</i> and E. coli. The location of the most recent common ancestor of the α-Proteobacteria (<i>Caulobacter</i>, <i>Rickettsia</i>, <i>Wolbachia</i>) is defined by the outgroup <i>E. coli.</i> The unit of branch length is the number of changes per amino acid. Overall, the amino acid substitution rate in the <i>w</i>Mel lineage is about 63% higher than that of <i>C. crescentus</i>, a free-living α-Proteobacteria. <i>w</i>Mel has evolved at a slightly higher rate than the <i>Rickettssia</i> spp., close relatives that are also obligate intracellular bacteria that have undergone accelerated evolution themselves. This higher rate is likely in part to be due to an increase in the rate of slightly deleterious mutations, although we have not ruled out the possibility of G+C content effects on the branch lengths.</p

Alignment of <i>w</i>Mel with a 60 kbp Region of the <i>Wolbachia</i> from <i>B. malayi</i>

<p>The figure shows BLASTN matches (green) and whole-proteome alignments (red) that were generated using the “promer” option of the MUMmer software (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020069#pbio-0020069-Delcher1" target="_blank">Delcher et al. 1999</a>). The B. malayi region is from a BAC clone (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020069#pbio-0020069-Ware1" target="_blank">Ware et al. 2002</a>). Note the regions of alignment broken up by many rearrangements and the presence of repetitive sequences at the regions of the breaks.</p

Genomic Organization and expression of Type IV Secretion Operons in <i>w</i>Mel

<p>(A) Organization of the nine <i>vir</i>-like CDSs (white arrows) and five adjacent CDSs that encode for either putative membrane-spanning proteins (black arrows) or non-<i>vir</i> CDSs (gray arrows) of wMel, R. conorii, and A. tumefaciens. Solid horizontal lines denote RT experiments that have confirmed that adjacent CDSs are expressed as part of a polycistronic transcript. Results of these RT-PCR experiments are presented in (B). Lane 1, <i>virB3</i>-<i>virB4</i>; lane 2, RT control; lane 3, <i>virB6</i>-WD0856; lane 4, RT control; lane 5, WD0856-WD0855; lane 6, RT control; lane 7, WD0854-WD0853; lane 8, RT control; lane 9, <i>virB8</i>-<i>virB9</i>; lane 10, RT control; lane 11, <i>virB9</i>-<i>virB11</i>; lane 12, RT control; lane 13, <i>virB11</i>-<i>virD4</i>; lane 14, RT control; lane 15, <i>virD4</i>-<i>wspB</i>; lane 16, RT control; lane 17, <i>virB4</i>-<i>virB6</i>; lane 18, RT control; lane 19, WD0855-WD0854; lane 20, RT control. Only PCRs that contain reverse transcriptase amplified the desired products. PCR primer sequences are listed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020069#st009" target="_blank">Table S9</a>.</p

Circular Map of the Genome and Genome Features

<p>Circles correspond to the following: (1) forward strand genes; (2) reverse strand genes, (3) in red, genes with likely orthologs in both R. conorii and R. prowazekii; in blue, genes with likely orthologs in R. prowazekii, but absent from R. conorii; in green, genes with likely orthologs in R. conorii but absent from R. prowazekii; in yellow, genes without orthologs in either <i>Rickettsia</i> (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020069#st003" target="_blank">Table S3</a>); (4) plot is of χ² analysis of nucleotide composition; phage regions are in pink; (5) plot of GC skew (G–C)/(G+C); (6) repeats over 200 bp in length, colored by category; (7) in green, transfer RNAs; (8) in blue, ribosomal RNAs; in red, structural RNA.</p