Figure 1.
Overview of ChIP-chip analysis.
A) Ubx is responsible for all of the differences between the second (T2) and third (T3) thoracic segments. Ubx loss-of-function mutations transform halteres (green box, center panel) into wings (left panel); Ubx gain-of-function in T2 transforms the wings into halteres (right panel). B) In this study, tissue-specific chromatin immunoprecipitation was performed with pure populations of haltere or T3 leg imaginal discs, both of which express Ubx in all cells (green) and express Hth (red) in the subset of cells that will give rise to proximal appendages and body wall.
Table 1.
Ubx and Hth binding overview.
Figure 2.
Ubx and Hth target gene classifications.
A) Venn diagram comparing genes called as Ubx targets in haltere or T3 leg imaginal discs to genes called as Hth targets in those tissues. B) Overrepresentation of Gene Ontology biological processes or molecular functions among various subsets Ubx and Hth target genes. For the indicated tissue, columns represent enriched GO categories for all genes targeted by Ubx (All Ubx), all Hth targets (All Hth), genes targeted by both Ubx and Hth (Ubx+Hth), genes targeted by Ubx but not targeted by Hth (Ubx not Hth), and genes targeted by Hth but not targeted by Ubx (Hth not Ubx).
Figure 3.
Ubx-regulated gene expression and Ubx occupancy.
A) Fraction of Ubx-responsive genes based on gene expression profiling that is identified as Ubx and/or Hth target genes in the haltere using ChIP-chip. Ubx-responsive genes are those called as up- or downregulated in haltere discs or wing discs ectopically expressing Ubx (Cbx1 mutant discs) as compared to wild type wing discs (expression data from [12]). B) Heatmap representing expression levels in the haltere or Cbx1 wing relative to wild type wing. Higher expression in haltere or Cbx1 is red; lower expression in haltere or Cbx1 is blue. Genes are hierarchically clustered. Two columns to the right of the heatmap indicate binding by Ubx (light green) or Hth (dark green) at a given gene locus. Five clusters (indicated on the far right) representing unique binding and expression profiles were found to found to be enriched for one or more GO categories. Cluster A consists of genes upregulated by ectopic Ubx expression in the wing that are bound by Ubx, whereas cluster B contains genes with the same expression pattern that are bound by Ubx and Hth. Cluster C consists of Ubx-bound genes that are expressed at lower levels in the haltere or upon ectopic expression of Ubx in the wing. Clusters D and E consist of genes that are expressed at lower levels in the haltere relative to the wing; only Ubx binds genes in cluster D, whereas Ubx and Hth bind those in cluster E. C) Enrichment of Gene Ontology biological process or molecular function categories among clusters A through E (described above).
Figure 4.
Examples of Ubx bound genes encoding transcription factors.
Ubx and Hth binding profiles in the T3 leg and haltere imaginal discs at the Antennapedia Complex (ANT-C) (A), the Bithorax Complex (BX-C) (B), vestigial (vg) (C), blistered/DSRF (bs) (D), E2F (E), and cut (ct) (F). Regions called as bound by Ubx but not Hth are highlighted in turquoise, and regions bound by both Ubx and Hth are highlighted in blue. Selected known enhancers are represented as boxes along the chromosomal map. For (A), the Dfd EAE element is represented with a green box, Antp P2 with a purple box, and Antp P1 with yellow box. For (B), the Ubx abx and bx1 enhancers are represented by yellow and purple boxes, respectively. For (C), the vg boundary and quadrant enhancers are represented by yellow and purple boxes, respectively. A track showing Ubx binding in 0–12 hr embryos is shown at the bottom of each panel for comparison. Tracks represent −10×log(p-value) as generated by TAS (methods).
Figure 5.
Examples of Ubx bound genes encoding signaling pathway components.
Ubx and Hth binding profiles at the Dpp pathway components thickveins (tkv) (A), dally (B), and notum (C); at the Wg pathway components frizzled 2 (fz2) (D), and wingless (wg) (E); at the Hippo pathway components bantam (ban) (F) and expanded (ex) (G); and at the Notch pathway components Notch (N) (H), mind bomb 1 (mib1) (I), and fringe (fng) (J). Color scheme and tracks are as described in Figure 4.
Figure 6.
Expression analysis of predicted target genes in the haltere and wing.
A) Dll expression along the DV axis of the wing is missing from the homologous region of the haltere. B) bs/DSRF is expressed in the pouch of the wing but is not expressed in the haltere. C) In comparison to the wing, ct expression along the DV axis is lower overall in the haltere, and is completely absent from the posterior compartment of the haltere. D) The stripe of notum-lacZ expression along the DV axis of the wing is almost entirely missing in the haltere, except for a low level of expression in the most medial region of this axis. E) vn-lacZ is expressed in the medial wing but repressed in the medial haltere. F) wg-lacZ expression along the DV axis is absent in the posterior compartment of the haltere. G) fj-lacZ expression is greatly reduced in the pouch of the haltere as compared to the pouch of the wing. H) ex-lacZ is expression is reduced in the pouch of the haltere. I) ban-Gal4 is repressed along the AP and DV axes of the wing, but is expressed along these axes in the haltere.
Figure 7.
Tissue specificity of Ubx binding.
A) Ubx and Hth binding profiles across approximately 300 kb of chromosome 3L (18,900,000–19,200,000). Sites matching the Ubx monomer consensus binding site TAAT[t/g]G are indicated as blue bars along the chromosomal map. B) Hierarchical clustering of the significance of overlap in binding between pairs of transcription factors. Ubx and Hth were compared to a set of embryo transcription factor binding profiles generated by MacArthur et al. Values represent −log10 transformed p-value for each pair-wise transcription factor comparison. C) Venn diagram comparing Ubx-bound regions in the embryo, haltere disc, or T3 leg disc. D) Multispecies conservation score for Ubx-bound regions in imaginal discs that overlap with Ubx-binding in the embryo (red, blue), and for regions that do not overlap with Ubx-binding in the embryo. Average score for a 1000 bp window (center of called peak plus/minus 500 bp) is represented.
Figure 8.
DNA motif analysis of Ubx, Hth, and Ubx+Hth bound fragments in the T3 leg and haltere.
Select motifs identified using SeqPos/CEAS (Methods; all motifs p<0.01). The haltere and leg schematics on the left are divided into the proximal (blue, expressing Ubx and Hth) and distal (yellow, expressing Ubx but not Hth) regions to illustrate Ubx and Ubx+Hth expression patterns. Motifs in the left box were identified in regions bound by Ubx (but not Hth) in the indicated tissue(s); this box is green because Ubx (not Hth) binding could occur in either the proximal or distal regions of these discs. Motifs in the center box were identified in regions bound by Ubx+Hth in the indicated tissues(s), and motifs in the right box were identified in sequences bound by Hth (but not Ubx) in the in the indicated tissue(s). The top panel (A) represents sequences identified in the haltere-specific binding regions for Ubx and/or Hth. The middle panel (B) represents sequences identified in the leg-specific binding regions for Ubx and/or Hth. The bottom panel represents sequences identified in binding regions for the indicated factor(s) that are not tissue-specific (i.e., shared between the haltere and leg). For each motif, the percentage of peaks containing a matching sequence within +/− 250bp is given in red, below the motif name.