Abstract:
The Hox genes are a family of transcription factors which regulate animal development along the anterior-posterior axis. While Hox genes are highly conserved both at genetic and biochemical levels across diverse animal species, they are thought to mediate evolution of body plan among closely related species. As all Hox genes bind to a commonly occurring motif containing a TAAT core sequence, mechanisms by which they select targets and diversify appendage structures still remains an intriguing problem. In all insects families studied so far, the Hox gene Ultrabithorax (Ubx) is shown to be responsible for differences between the second and third thoracic segments, including differences, if any, between the two pairs of flight organs. In Hymenopterans, such as Apis mellifera, both forewing and hindwing are identical in structure and function, except that hindwing is marginally smaller. However, in Dipterans such as Drosophila, Ubx is responsible for the repression of wing fate and specification of the haltere fate in the third thoracic segment. To understand the molecular mechanisms leading to the evolution of function of Ubx in these two families, we compared genome wide binding of Ubx in the hindwing primordia of Apis and haltere discs of Drosophila. Denovo motif analyses indicate that a motif with TAAAT core is significantly enriched in Ubx-pulled-down sequences in Drosophila, while the motif with TAAT core is not enriched in either Apis or in Drosophila. In vitro DNA binding assays, luciferase reporter gene assays and transgenic studies indicate that the motif with TAAAT core facilitates stronger and functionally relevant binding of Ubx compared to the motif with TAAT core sequence. Replacing TAAT core sequence with TAAAT core alone is sufficient to bring under the influence of Ubx, an otherwise unresponsive enhancer of vestigial gene of Apis in transgenic Drosophila. Comparison of Ubx-binding motifs in the enhancers of genes that are upregulated vs downregulated genes during haltere development did not reveal any signature sequence that could be serve as basis of target selection for Ubx. Nevertheless, motifs for cofactors such as mes2 are enriched in enhancers of genes that are upregulated during haltere development. Overall, our studies provide novel insights into the mechanism of evolution of Ubx function between distant species, while shed light on the role of a strong binding site in gene regulation by Ubx in Drosophila halteres.
