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.
Highlights
Hox genes play a key role in development as they are responsible for specifying the differences between segments along the body axis [1]; reviewed in [2]
For the ChIP-array analysis, we compared the specific signal derived from immunoprecipitation of chromatin from a YFP-protein trap line with anti-GFP/YFP antibody versus the control signal from chromatin taken from the isogenic wild-type progenitor immunoprecipitated with the same anti-GFP/YFP antibody
We used Drosophila 2.0 Affymetrix genome tiling arrays and performed three biological replicates for each sample. For both Ubx-YFP and Hth-YFP, genome-wide binding was assayed using chromatin samples from 0–16 hr embryos and 3rd larval instar haltere imaginal discs; for Hth-YPF we assayed binding in 3rd larval instar wing imaginal disc chromatin
Summary
Hox genes play a key role in development as they are responsible for specifying the differences between segments along the body axis [1]; reviewed in [2]. Different Hox genes are expressed in overlapping patterns along the antero-posterior axis forming a Hox code that specifies particular target gene activities in each segment and generates specific segmental morphologies. At several target genes there is good evidence that cofactors contribute to binding specificity [18], at others the cofactors appear to modify Hox protein function [19,20] and for some targets cofactors may not be required [21]. To understand the interactions between Hox proteins and other regulatory inputs that enable, for example, Hox genes to regulate target genes appropriately in different tissues [22,23,24]. To understand the gene networks that connect the Hox genes to the developmental processes that build particular segmental morphologies [11,25,26,27]
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