An architect gene at word : identification of potential target genes of Hoxa2

(eng) Hox genes encode evolutionarily conserved transcription factors which control important pathways during embryo development and adult lifetime. The Hoxa2 gene plays crucial conserved functions in craniofacial and hindbrain patterning and morphogenesis. While its biological roles are well documented, very little is known about the precise way of action of this transcription factor. The aim of this study was to identify potential primary target genes of Hoxa2 among a subset of thirty two candidate genes previously revealed by a screen in a cellular model. The first step of the strategy we adopted was to validate candidate target genes and to quantify the modulation of their activity upon expression of Hoxa2 and its Pbx cofactor. The second step was to establish whether these target genes could define direct targets of Hoxa2. This consisted in identifying Hoxa2/Pbx binding sites in the vicinity of the validated targets and in assaying their functionality. The last step was to correlate the expression profile of target genes with what is known for Hoxa2. As a result, we demonstrated that Adamts1, Frg1, Lmo1 and Plekha6 are potential primary target genes of Hoxa2. We mapped Hoxa2/Pbx binding sites (Hox Response Element or HRE) corresponding to the designed TGATDBAWKD consensus in the vicinity of each target gene. One HRE was identified upstream Frg1, three upstream Lmo1and two upstream Plekha6. We then confirmed that these elements display Hoxa2-dependent transcriptional activity by a reporter gene assay. Finally, we provided evidence that Adamts1 is up-regulated in the spine of mouse embryos ectopically expressing Hoxa2 in cells entering chondrogenesis, that Hoxa2 and Phox2b, a neuronal specification factor, probably act together to regulate Lmo1 in the developing hindbrain, and that Plekha6 is expressed in discrete territories of the developing hindbrain. These expression data therefore support that Hoxa2 may specify neuronal subtypes in the developing hindbrain through Lmo1 and Plekha6 regulation, and could control morphogenesis through Adamts1 and Frg1. In conclusion, our work provides new insights into the mode of action of the architect gene Hoxa2 by the identification of Adamts1, Frg1, Lmo1 and Plekha6 as potential primary target genes.