IN-VITRO AND TRANSGENIC ANALYSIS OF A HUMAN HOXD4 RETINOID-RESPONSIVEENHANCER

Expression of vertebrate Hox genes is regulated by retinoids in cell c ulture and in early embryonic development. We have identified a 185-bp retinoid-responsive transcriptional enhancer 5' of the human HOXD4 ge ne, which regulates inducibility of the gene in embryonal carcinoma ce lls through a pattern of DNA-protein interaction on at least two disti nct elements. One of these elements contains a direct repeat mediating ligand-dependent interaction with retinoic acid receptors, and is nec essary though not sufficient for the enhancer function. The HOXD4 enha ncer directs expression of a lacZ reporter gene in the neural tube of transgenic mouse embryos in a time-regulated and regionally restricted fashion, reproducing part of the anterior neuroectodermal expression pattern of the endogenous Hoxd-4 gene. Administration of retinoic acid to developing embryos causes alterations in the spatial restriction o f the transgene expression domain, indicating that the HOXD4 enhancer is also a retinoid-responsive element in vivo. The timing of the retin oic acid response differs from that seen with more 3' Hox genes, in th at it occurs much later. This shows that the temporal window of compet ence in the ability to respond to retinoic acid differs between Hox ge nes and can be linked to specific enhancers. Mutations in the direct r epeat or in a second element in the enhancer affect both retinoid resp onse in culture and developmental regulation in embryos, suggesting th at co-operative interactions between different factors mediate the enh ancer activity. These data provide further support for a role of endog enous retinoids in regulation and spatial restriction of Hox gene expr ession in the central nervous system.