The homeoprotein Alx3 contains discrete functional domains and exhibits cell-specific and selective monomeric binding and transactivation

Abstract

AlxS is a paired class aristaless-like homeoprotein expressed during embryonic development. Transcriptional transactivation by aristaless-like proteins has been associated with cooperative dimerization upon binding to artificially generated DNA consensus sequences known as P3 sites, but natural target sites in genes regulated by AlxS are unknown. We report the cloning of a cDNA encoding the rat homolog of AlxS, and we characterize the protein domains that are important for transactivation, dimerization, and binding to DNA. Two proline-rich domains located amino-terminal to the homeodomain (Pro1 and Pro2) are necessary for Alx3-dependent transactivation, whereas another one (Pro3) located in the carboxyl terminus is dispensable but contributes to enhance the magnitude of the response. We confirmed that transcriptional activity of Alx3 from a P3 site correlates with cooperative dimerization upon binding to DNA. However, AlxS was found to bind selectively to non-P3-related TAAT-containing sites present in the promoter of the somatostatin gene in a specific manner that depends on the nuclear protein environment. Cell-specific transactivation elicited by AlxS from these sites could not be predicted from in vitro DNA-binding experiments by using recombinant AlxS. In addition, transactivation did not depend on cooperative dimerization upon binding to cognate somatostatin DNA sites. Our data indicate that the paradigm according to which AlxS must act homodimerically via cooperative binding to PS-like sites is insufficient to explain the mechanism of action of this homeoprotein to regulate transcription of natural target genes. Instead, AlxS undergoes restrictive or permissive interactions with nuclear proteins that determine its binding to and transactivation from TAAT target sites selected in a cell-specific manner.