REGULATORY INTERACTIONS BETWEEN THE HUMAN HOXB1, HOXB2, AND HOXB3 PROTEINS AND THE UPSTREAM SEQUENCE OF THE OTX2 GENE IN EMBRYONAL CARCINOMA-CELLS

Vertebrate Hox and Otx genes encode homeodomain-containing transcripti on factors thought to transduce positional information along the body axis in the segmental portion of the trunk and in the rostral brain, r espectively. Moreover, Hox and Otx2 genes show a complementary spatial regulation during embryogenesis. In this report, we show that a 1821- base pair (bp) upstream DNA fragment of the Otx2 gene is positively re gulated by co-transfection with expression vectors for the human HOXB1 , HOXB2, and HOXB3 proteins in an embryonal carcinoma cell line (NT2/D 1) and that a shorter fragment of only 534 bp is able to drive this re gulation. We also identified the HOXB1, HOXB2, and HOXB3 DNA-binding r egion on the 534-bp Otx2 genomic fragment using nuclear extracts from Hox-transfected COS cells and 12.5 days postcoitum mouse embryos or HO XB3 homeodomain containing bacterial extracts. HOXB1, HOXB3, and nucle ar extracts from 12.5 days postcoitum mouse embryos bind to a sequence containing two palindromic TAATTA sites, which bear four copies of th e ATTA core sequence, a common feature of most HOM-C/HOX binding sites , HOXB2 protected an adjacent site containing a direct repeat of an AC TT sequence, quite divergent from the ATTA consensus. The region bound by the three homeoproteins is strikingly conserved through evolution and necessary (at least for HOXB1 and HOXB3) to mediate the up-regulat ion of the Otx2 transcription. Taken together, our data support the hy pothesis that anteriorly expressed Rot genes might play a role in the refinement of the Otx2 early expression boundaries in vivo.