DNA binding specificities and transcriptional activity of mirror, an Iroquois transcription factor, in Drosophila melanogaster
Iroquois family genes regulate diverse developmental processes in higher eukaryotes. The Drosophila Iroquois complex consists of three closely related genes: mirror (mirr), araucan (ara) and caupolican (caup). Iroquois genes have been shown to be essential in various systems for processes such as neural development, patterning of the vertebrate heart, establishment of planar cell polarity and axon pathfinding. However, despite extensive work demonstrating the importance of Iroquois transcription factors in diverse aspects of development, it is still unclear how they act to control transcription or, which are their downstream targets. Iroquois proteins are atypical Homeodomain (HD) transcription factors of the TALE class. To determine the DNA-binding preference of the Iroquois family members I conducted a DNA binding site selection experiment using Drosophila Mirror and defined a novel consensus sequence ACAnnTGT, which is different from the classic HOX motif. I provide evidence that this motif is the minimum requirement for Mirror binding to DNA. Other members of the Iroquois family can recognise this site in vitro suggesting that it may be a universal Iroquois binding site (IBS). Mirror binds the IBS as a homodimer and can also form heterodimers with other Iroquois proteins. To test if the site is functional I made in vivo reporter constructs and showed that the site can mediate transcriptional repression in transgenic flies. Genome-wide searches for occurrences of transcription factor binding sites can help identifying their downstream targets. In combination with a microarray screen, recently carried out in our lab to identify Mirror downstream targets, we have looked for Mirror binding sites in genomic regions of candidate genes. The case of a candidate for direct Mirror target will be presented.