Project name: Drosophila melanogaster

Description: 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. Here we report the use of a YFP-tagged Drosophila protein-trap line together with chromatin immunoprecipitation and microarray analysis to identify genome-wide binding sites for the Hox protein Ultrabithorax (Ubx). We find 1,147 genes bound by Ubx 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 showing differential expression between wing and haltere imaginal discs. The Ubx-bound genes are highly enriched in genes involved in developmental processes. They contain both high-level regulatory genes as well as genes involved in more basic cell functions supporting the idea that Hox genes regulate many levels of developmental pathways. Several signalling pathways are highly enriched and within these pathways Ubx target genes occur at multiple level from ligands to transcriptional effectors. We also performed genome-wide analysis of the binding sites for the Hox cofactor Homothorax (Hth), revealing a striking similarity in the binding profiles of Hth and Ubx. We suggest that these binding profiles may be strongly influenced by chromatin accessibility and we provide evidence of a clear link between Ubx/Hth binding and the chromatin state at genes regulated by Polycomb silencing. The role of chromatin accessibility has important implications for Hox gene function and for genomic target selection by transcription factors in general.Overall design: In this study, we used Ubx-YFP (CPTI-000601) and Hth-YFP (CPTI-000378) protein trap lines from the Cambridge protein trap project, FlyProt (Ryder et al. 2009, Bioinformatics 25(4):p548-549). For both Ubx-YFP and Hth-YFP, genome-wide binding was assayed using chromatin samples from 0-16hr embryos and 3rd larval instar haltere imaginal discs; for Hth-YPF we also assayed binding in 3rd larval instar wing imaginal disc chromatin. For the ChIP-array analysis, we assayed ChIP enrichment comparing the specific signal derived from immunoprecipitation of chromatin from the YFP-protein trap line with anti-GFP/YFP antibody versus the control signal from chromatin from the isogenic wild-type progenitor immunoprecipitated with the same anti-GFP/YFP antibody. We performed three biological replicates for each sample. Purified DNAs were then fragmented, TdT labeled and hybridized to the Affymetrix Drosophila genome Tiling Array 2.0, as previously described (Manak et al. 2006, Nat. Gen. 38: p1151).

Data type: Epigenomics

Sample scope: Multiisolate

Relevance: ModelOrganism

Release date: 2011-04-14

Last updated: 2010-08-30