Genome-wide analysis of the binding of the Hox protein Ultrabithorax and the Hox cofactor Homothorax in Drosophila

Abstract

Hox genes encode a family of transcription factors that are key developmental regulators with a highly conserved role in specifying segmental diversity along the metazoan body axis. Although they have been shown to regulate a wide variety of downstream processes, direct transcriptional targets have been difficult to identify and this has been a major obstacle to our understanding of Hox gene function. We report the identification of genome-wide binding sites for the Hox protein Ultrabithorax (Ubx) using a YFP-tagged Drosophila protein-trap line together with chromatin immunoprecipitation and microarray analysis. We identify 1,147 genes bound by Ubx at high confidence in chromatin from the haltere imaginal disc, a prominent site of Ubx function where it specifies haltere versus wing development. The functional relevance of these genes is supported by their overlap with genes differentially expressed between wing and haltere imaginal discs. The Ubx-bound gene set is highly enriched in genes involved in developmental processes and contains both high-level regulators as well as genes involved in more basic cellular functions. Several signalling pathways are highly enriched in the Ubx target gene set and our analysis supports the view that Hox genes regulate many levels of developmental pathways and have targets distributed throughout the gene network. We also performed genome-wide analysis of the binding sites for the Hox cofactor Homothorax (Hth), revealing a striking similarity with the Ubx binding profile. We suggest that these binding profiles may be strongly influenced by chromatin accessibility and provide evidence of a link between Ubx/Hth binding and chromatin state at genes regulated by Polycomb silencing. Overall, we define a set of direct Ubx targets in the haltere imaginal disc and suggest that chromatin accessibility has important implications for Hox target selection and for transcription factor binding in general.

Figure 1. Ubx-bound genes are widely distributed across the Drosophila 20K gene network.

(A) Ubx-bound genes (blue) are mapped onto the network visualised in Cytoscape . (B) Ubx-bound genes (294 gene set as diamonds and remaining genes of the 1,147 set as circles) with selected subclusters coloured.

Figure 2. Features of Ubx-bound genes.

(A) Wnt/wingless pathway components from Panther are listed and coloured according to presence of corresponding genes in: 294 gene set (Ubx-bound and supported by regulation; red), remaining genes of 1,147 Ubx-bound gene set (pink) and genes not in the 1% FDR Ubx-bound list (blue). (B) Genes from the 1,147 Ubx-bound gene set that overlap with differentially expressed genes from the Mohit et al. , Hersh et al. and the larval genes from Pavlopoulos and Akam classified according to direction of regulation by Ubx.

Figure 3. Temporal targets.

(A–D) Overlaps between the 1,147 Ubx-bound gene set (purple) and the differentially expressed genes from Pavlopoulos and Akam at the larval (brown), prepupal (green), pupal (teal) or combined (yellow) timepoints. (E) Overlaps between the Ubx-bound genes at the three different timepoints in the Pavlopoulos and Akam data.

Figure 4. Comparison of Ubx and Hth binding profiles.

(A) Schematic of Ubx and Hth expression in the wing and haltere discs. The wing disc pouch region gives rise to the wing blade and the haltere pouch region gives rise to the haltere capitellum. We use the term haltere hinge to encompass the pedicel and scabellum. (B) Log₂ enrichment ratio profiles for Ubx and Hth on representative regions from chromosome 3R. The peaks at approx 12,500,000 (asterisk) present in the haltere profiles and absent in the wing are associated with the Ubx gene (see Figure 7).

Figure 5. Sequence motifs identified.

Enriched motifs derived from the Ubx and Hth ChIP-array data are compared to previously identified motifs from Mann et al. and Noyes et al. .

Figure 6. Ubx and Hth binding at salm.

The red vertical indicates the imaginal disc enhancer identified by Galant et al. .

Figure 7. Ubx and Hth binding at the Bithorax complex and Antp.

The embryo chromatin represents a heterogenous mixture of epigenetic ON and OFF states at the Bithorax genes and at Antp imparted by the Pc/Trx machinery. In the haltere disc chromatin Ubx and Antp are epigenetically ON, abd-A and Abd-B are OFF. In the wing disc Ubx, abd-A and Abd-B are OFF, whereas Antp is ON. The Bithorax Complex is on the left. The blue verticals represent the position of insulator component binding sites (CP190 and CTCF; [63]). The Antp locus is on the right.

Figure 8. Ubx and Hth binding at Pc target genes.

Examples of Pc target genes that are active or repressed in imaginal discs. (A) ptc is expressed in wing and haltere disc and is associated with Ubx and Hth binding. (B) Awh is likely to be predominantly silenced in imaginal discs and is bound by Ubx and Hth in the embryo but not in the imaginal discs. The Pc binding data from embryo and from T3 (haltere and leg 3) imaginal discs is from Kwong et al. .