Developmental Effects of Ectopic Expression of the Glucocorticoid Receptor DNA Binding Domain Are Alleviated by an Amino Acid Substitution That Interferes with Homeodomain Binding

Abstract

Steroid hormone receptors are distinguished from other members of the nuclear hormone receptor family through their association with heat shock proteins and immunophilins in the absence of ligands. Heat shock protein association represses steroid receptor DNA binding and protein-protein interactions with other transcription factors and facilitates hormone binding. In this study, we investigated the hormone-dependent interaction between the DNA binding domain (DBD) of the glucocorticoid receptor (GR) and the POU domains of octamer transcription factors 1 and 2 (Oct-1 and Oct-2, respectively). Our results indicate that the GR DBD binds directly, not only to the homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several other homeodomain proteins. As these results suggest that the determinants for binding to the GR DBD are conserved within the homeodomain, we examined whether the ectopic expression of GR DBD peptides affected early embryonic development. The expression of GR DBD peptides in one-cell-stage zebra fish embryos severely affected their development, beginning with a delay in the epibolic movement during the blastula stage and followed by defects in convergence-extension movements during gastrulation, as revealed by the abnormal patterns of expression of several dorsal gene markers. In contrast, embryos injected with mRNA encoding a GR peptide with a point mutation that disrupted homeodomain binding or with mRNA encoding the DBD of the closely related mineralocorticoid receptor, which does not bind octamer factors, developed normally. Moreover, coinjection of mRNA encoding the homeodomain of Oct-2 completely rescued embryos from the effects of the GR DBD. These results highlight the potential of DNA-independent effects of GR in a whole-animal model and suggest that at least some of these effects may result from direct interactions with homeodomain proteins.

ACKNOWLEDGMENTS

We thank the many people who provided the plasmids used in this work including, in particular, Q. Long, T. Zerucha, and M. Ekker but also K. Yamamoto, W. Herr, C. Schild-Poulter, D. Grunwald, M. Halpern, V. Korzh, U. Strähle, M. Tada, D. Wilson, and E. Weinberg. We are grateful to the members of the Hachélaboratory and to Y. Lefebvre and M. Ekker for critical comments on the manuscript.

This work was supported by an operating grant from the Medical Research Council of Canada. J.M.W. has been funded by an L. Siminovitch postdoctoral fellowship from The Loeb Health Research Institute at the Ottawa Hospital and a fellowship from the Natural Sciences and Engineering Research Council of Canada. G.G.P. is the recipient of an MRC studentship. M.E.L. holds a junior postdoctoral fellowship from the National Cancer Institute of Canada. R.J.G.H. is a Medical Research Council of Canada scientist.