REGULATION OF THE HUMAN HOXD4 GENE BY RETINOIDS

Hox genes are developmentally regulated in mammalian embryogenesis, ac cording to temporally and spatially restricted patterns which are affe cted by retinoids, vitamin A derivatives which have a function as, or at least mimic the action of, axis-specifying morphogens. In the human embryonal carcinoma cell line NT2/D1, HOX gene clusters are activated by at least two retinoids, all-trans- and 9-cis-retinoic acid (RA), i n a 3' to 5' sequential cascade which reproduces the activation patter n observed in early embryogenesis. We have studied the regulation of t he early activated HOXD4 gene, which is expressed in human embryogenes is in multiple transcripts generated by the developmentally controlled use of alternative transcription start sites and polyadeny!ation sign als. Transfection of a 2.9 kb HOXD4 upstream genomic region linked to a reporter gene in NT2/D1 cells, allowed the identification of two dif ferent promoters and a distal enhancer element necessary for RA-depend ent gene activation. This element confers to a heterologous promoter t he ability to be induced by RA in NT2/D1 cells, and transactivated by alpha, beta and gamma retinoic acid receptors (RARs), but not retinoid X receptor (RXR), in COS-7 cells. DNase I footprinting analysis allow ed the identification of four sequences which bind nuclear factors fro m both RA-induced NT2/D1 cells and embryonic tissues with similar patt erns. The use of specific antibodies allowed the identification of at least RAR beta in some of the DNA-protein complexes, although the four sequences bind single RARs transfected in COS cells much less efficie ntly, or not at all, when compared to a canonical RAR responsive eleme nt. Induction of the HOXD4 promoter-enhancer in the presence of a sele ctive RAR alpha antagonist indicated that the RAR alpha-dependent RAR beta activation is nevertheless a necessary step in HOX gene activatio n. Our results indicate that the ligand-dependent activity of RARs upo n specific, cis-acting regulatory elements may have a key role in the induction of early activated HOX genes in response to retinoids. Howev er, RARs represent only a fraction of the transcription factors intera cting with the RA-responsive HOXD4 enhancer, which appears to be a com plex element requiring specific combinations of nuclear factors for it s proper function.