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Hoxb1 Enhancer and Control of Rhombomere 4 Expression: Complex Interplay between PREP1-PBX1-HOXB1 Binding Sites

Elisabetta Ferretti,^1, Francisco Cambronero,² Stefan Tümpel,² Elena Longobardi,¹ Leanne M. Wiedemann,^2,3 Francesco Blasi,^1,5* and Robb Krumlauf^2,4

Molecular Genetics Unit, Department of Molecular Biology and Functional Genomics, Istituto Scientifico H. San Raffaele, Università Vita Salute San Raffaele, Milano, Italy,¹ Stowers Institute for Medical Research, Kansas City, Missouri 64110,² Department of Pathology and Laboratory Medicine, Kansas University Medical School, Kansas City, Kansas,³ Department of Anatomy and Cell Biology, Kansas University Medical School, Kansas City, Kansas,⁴ IFOM (FIRC Institute of Molecular Oncology), via Adamello 16, Milano, Italy⁵

Received 25 February 2005/ Returned for modification 10 April 2005/ Accepted 14 July 2005

The Hoxb1 autoregulatory enhancer directs segmental expression in vertebrate hindbrain. Three conserved repeats (R1, R2, and R3) in the enhancer have been described as Pbx-Hoxb1 (PH) binding sites, and one Pbx-Meinox (PM) binding site has also been characterized. We have investigated the importance and relative roles of PH and PM binding sites with respect to protein interactions and in vivo regulatory activity. We have identified a new PM site (PM2) and found that it cooperates with the R3 PH site to form ternary Prep1-Pbx1-Hoxb1 complexes. In vivo, the combination of the R3 and PM2 sites is sufficient to mediate transgenic reporter activity in the developing chick hindbrain. In both chicken and mouse transgenic embryos, mutations of the PM1 and PM2 sites reveal that they cooperate to modulate in vivo regulatory activity of the Hoxb1 enhancer. Furthermore, we have shown that the R2 motif functions as a strong PM site, with a high binding affinity for Prep1-Pbx1 dimers, and renamed this site R2/PM3. In vitro R2/PM3, when combined with the PM1 and R3 motifs, inhibits ternary complex formation mediated by these elements and in vivo reduces and restricts reporter expression in transgenic embryos. These inhibitory effects appear to be a consequence of the high PM binding activity of the R2/PM3 site. Taken together, our results demonstrate that the activity of the Hoxb1 autoregulatory enhancer depends upon multiple Prep1-Pbx1 (PM1, PM2, and PM3) and Pbx1-Hoxb1 (R1 and R3) binding sites that cooperate to modulate and spatially restrict the expression of Hoxb1 in r4 rhombomere.

Present address: Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021.

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