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J Biol Chem, Vol. 273, Issue 18, 11092-11099, May 1, 1998

Regulatory Interactions between the Human HOXB1, HOXB2, and HOXB3 Proteins and the Upstream Sequence of the Otx2 Gene in Embryonal Carcinoma Cells

Stefania Guazzi, Maria Luisa Pintonello, Alessandra Viganò, and Edoardo Boncinelli^¶

From the   Department of Biology and Biotechnology, H. San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milano, Italy and ^¶ Centro Infrastrutture Cellulari, Consiglio Nazionale delle Ricerche, Via Vanvitelli 32, 20129 Milano, Italy

Vertebrate Hox and Otx genes encode homeodomain-containing transcription factors thought to transduce positional information along the body axis in the segmental portion of the trunk and in the rostral brain, respectively. 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 regulated by co-transfection with expression vectors for the human HOXB1, HOXB2, and HOXB3 proteins in an embryonal carcinoma cell line (NT2/D1) and that a shorter fragment of only 534 bp is able to drive this regulation. We also identified the HOXB1, HOXB2, and HOXB3 DNA-binding region on the 534-bp Otx2 genomic fragment using nuclear extracts from Hox-transfected COS cells and 12.5 days postcoitum mouse embryos or HOXB3 homeodomain-containing bacterial extracts. HOXB1, HOXB3, and nuclear extracts from 12.5 days postcoitum mouse embryos bind to a sequence containing two palindromic TAATTA sites, which bear four copies of the ATTA core sequence, a common feature of most HOM-C/HOX binding sites. HOXB2 protected an adjacent site containing a direct repeat of an ACTT 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-regulation of the Otx2 transcription. Taken together, our data support the hypothesis that anteriorly expressed Hox genes might play a role in the refinement of the Otx2 early expression boundaries in vivo.

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