Nucleic Acids Research Advance Access published online on April 16, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn148
Molecular Biology |
An ultraconserved Hox–Pbx responsive element resides in the coding sequence of Hoxa2 and is active in rhombomere 4
1Unit of Developmental Genetics, Université Catholique de Louvain, 1200 Brussels, Belgium, 2Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104 CNRS/INSERM/ULP, Collège de France, BP 10142-CU de Strasbourg, 67404 Illkirch Cedex, France, 3Unité de Recherche en Biologie Moléculaire, Facultés Universitaires Notre-Dame de la Paix, 61 rue de Bruxelles, 5000 Namur and 4Unit of Veterinary Sciences, Institut des Sciences de la Vie, Université Catholique de Louvain, 5 (box 10) place Croix du Sud, 1348 Louvain-la-Neuve, Belgium
*To whom correspondence should be addressed. Tel: +32 10 47 3701; Fax: +32 10 47 3717; Email: rene.rezsohazy{at}uclouvain.be Present address: Xavier Lampe, Laboratory of Molecular Virology, Faculty of Medicine, Free University of Brussels, 808 route de Lennik, 1070 Brussels, Belgium Filippo M. Rijli, Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058, Basel, Switzerland
Received February 15, 2008. Revised March 12, 2008. Accepted March 17, 2008.
The Hoxa2 gene has a fundamental role in vertebrate craniofacial and hindbrain patterning. Segmental control of Hoxa2 expression is crucial to its function and several studies have highlighted transcriptional regulatory elements governing its activity in distinct rhombomeres. Here, we identify a putative Hox–Pbx responsive cis-regulatory sequence, which resides in the coding sequence of Hoxa2 and is an important component of Hoxa2 regulation in rhombomere (r) 4. By using cell transfection and chromatin immunoprecipitation (ChIP) assays, we show that this regulatory sequence is responsive to paralogue group 1 and 2 Hox proteins and to their Pbx co-factors. Importantly, we also show that the Hox–Pbx element cooperates with a previously reported Hoxa2 r4 intronic enhancer and that its integrity is required to drive specific reporter gene expression in r4 upon electroporation in the chick embryo hindbrain. Thus, both intronic as well as exonic regulatory sequences are involved in Hoxa2 segmental regulation in the developing r4. Finally, we found that the Hox–Pbx exonic element is embedded in a larger 205-bp long ultraconserved genomic element (UCE) shared by all vertebrate genomes. In this respect, our data further support the idea that extreme conservation of UCE sequences may be the result of multiple superposed functional and evolutionary constraints.