Cdx Protein Interaction with Hoxa5 Regulatory Sequences Contributes to Hoxa5 Regional Expression along the Axial Skeleton

Abstract

Hox gene functions are intimately linked to correct developmental expression of the genes. The identification of cis-acting regulatory sequences and their associated trans-acting factors constitutes a key step in deciphering the mechanisms underlying the correct positioning of the functional domain of Hox genes along the anterior-posterior axis. We have identified DNA elements driving Hoxa5 regionalized expression in mice, using the 2.1-kb mesodermal enhancer (MES) localized in Hoxa5 3′ flanking sequences as a starting point. The MES sequence comprises regulatory elements targeting Hoxa5 expression in the limbs, the urogenital and gastrointestinal tracts, and the cervical-upper thoracic region of the prevertebral column. A 164-bp DNA fragment within the MES caudally restricts Hoxa5 expression at the level of prevertebra 10, corresponding to the posterior limit of its functional domain. Cdx proteins directly bind to this element in vitro via two conserved sites. Preventing Cdx binding by mutating the sites causes caudal expansion of the transgene expression domain. Of all three murine Cdx proteins that bind this element in vitro, Cdx4 has emerged as a potential regional posterior repressor of Hoxa5 expression. The restrictive control provided by Cdx interactions with Hoxa5 regulatory sequences may be one of the critical events in cervicothoracic axial specification.

ACKNOWLEDGMENTS

We thank J. Aubin, J. Charron, and C. Séguin for helpful comments on the manuscript and M. Lemieux for technical assistance. We are also grateful to J. Deschamps for providing Cdx plasmids, P. Gruss and D. Lohnes for the Cdx1 mutant mouse line, F. Beck and K. Chawengsaksophak for the Cdx2 mutant mouse line, and E. Lonergan, C. Wright, and D. Lohnes for the calsequestrin, Cdx4, and Cdx1 and Cdx2 antibodies, respectively.

This work was supported by a grant from the National Institutes of Health (RO1-HD38463 to L.J. and C.K.T.). L.J. holds a Chercheur National Award from the Fonds de la Recherche en Santé du Québec.