SPECIFICITIES OF HOMEODOMAIN PROTEINS IN DEVELOPMENT

DESCRIPTION (provided by applicant): Homeodomain containing proteins are a pervasive family of DNA binding transcription factors, regulating the expression of developmental and tissue specific genes in a wide range of eukaryotic organisms. We propose structural and functional studies on Hox, Extradenticle (Exd), Homothorax (Hth) and Pit-1 proteins, with the long term goal of understanding how they achieve their functional specificities in vivo. Exd is highly homologous to the oncoprotein Pbx1 in humans, and like Pbx1 it can modulate the specificities of Hox proteins. Pit-1 is expressed exclusively in the anterior pituitary gland. Many of the mutations causing combined pituitary hormone deficiency in humans have been mapped to Pit-1. Together, these studies represent a continuation of our earlier work on homeodomain containing proteins and the crucial roles they play in development and organogenesis. The specific aims of this proposal are as follows: 1) Determine the basis of Hox/Exd selectivity. In particular, we will determine the structures of Hox/Exd pairs bound to a bone fide in vivo element and quantitate DNA binding in solution. 2) Determine the basis of Exd-Hth partnership, as a mechanism for understanding how the heterodimer enters the nucleus and establishes of a trimeric Hox complex. 3) Characterize the role of phosphorylation in Pit-1 cell specificity. In particular, we will test a novel hypothesis that Pit-1 is phosphorylated in a cell specific manner as a basis for its dual activation and repression functions. These aims will be accomplished by a combination of crystallographic and biophysical methods, complemented by in vivo studies in the laboratories of collaborators.