The OAR/aristaless domain of the homeodomain protein Cart1 has an attenuating role in vivo

Abstract

Aristaless-related genes encode a structurally defined group of homeoproteins that share a C-terminal stretch of amino acids known as the OAR- or aristaless domain. Many aristaless-related genes have been linked to major developmental functions, but the function of the aristaless domain itself is poorly understood. Expression and functional studies have shown that a subgroup of these genes, including Prx1, Prx2, Alx3, Alx4 and Cart1, is essential for correct morphogenesis of the limbs and cranium. We now demonstrate the function of the aristaless domain in vivo by ectopically expressing normal and mutated forms of Cart1 and Alx3. Ectopic expression of Cart1 in transgenic mice does not disturb development, whereas expression of a Cart1 form from which the aristaless domain has been deleted results in severe cranial and vertebral malformations. The Alx3 protein contains a divergent aristaless domain that appears not to be functional, as ectopic expression of Alx3 results in an altered phenotype irrespective of the presence of this aristaless domain. Linking the Cart1 aristaless domain to Alx3 extinguishes teratogenicity. We show that, at the molecular level, the most important consequence of deleting the aristaless domain is increased DNA binding to its palindromic target sequence. This demonstrates that the aristaless domain functions as an intra-molecular switch to contain the activity of the transcription factor that it is part of.

The Homeoprotein Alx3 Contains Discrete Functional Domains and Exhibits Cell-specific and Selective Monomeric Binding and Transactivation

“…This does not seem to be the case for Alx3, because deletion of the region that contains the aristaless domain (Alx3-(1-279)) does not result in transcriptional enhancement in transfected cells, consistent with the notion that Alx3 contains a divergent aristaless domain that appears not to be functional (46).…”

“…Proteins of this group bind preferentially to P3 sites as dimers in a cooperative manner, whereas binding to P5 sites occurs in monomeric form (22,47), a situation that has been confirmed for Alx4 and Cart1 (13,17,29,31). Alx3 has not been studied so extensively, but recently Brouwer et al (46) showed that binding of Alx3 to P3 is inhibited by the aristaless domain of Cart1, suggesting that sequences located outside the homeodomain can affect binding of aristaless-related proteins to their target DNA sites. Consistent with previous work on paired Q50 homeoproteins (22,29), our study shows that a segment of Alx3 spanning the homeodomain (Alx3-(143-228)) contains sufficient structural information for binding to P3 and P5 sites, and that monomeric binding to P5 is not sufficient for functional activity.…”

Expression of a Prrxl1 alternative splice variant during the development of the mouse nociceptive system

“…5), we hypothesized that the precise control of the amount of Prrxl1 relative to Prrxl1-b is a key element in the regulation of the molecular mechanisms that govern the establishment of accurate sensory circuits. The importance of accurate regulation of the amount of this type of paired-like homeodomain transcription factors during development has been previously demonstrated by overexpression of an OAR truncated form of Cart1, which resulted in severe cranial and vertebral malformations due to an increase in DNA binding activity (Brouwer et al, 2003). We therefore suggest the existence of a delicate balance between Prrxl1 and its OARlacking variant, Prrxl1-b, which is critical during the nociceptive circuit development.…”

Opposing roles of two isoforms of the Prx1 homeobox gene in chondrogenesis

“…Second, the OAR domain of Prx1a is likely involved in controlling chondrogenesis at the level of condensation. Several other paired-related homeoproteins, which have OAR domains, have been shown to have a role in chondrogenesis (Zhao et al, 1996;Heanue et al, 1997;Rao et al, 1997;Belo et al, 1998;Qu et al, 1998;ten Berge et al, 1998a;Rivera-Perez et al, 1999;Clement-Jones et al, 2000;Mavrogiannis et al, 2001;Ross et al, 2001;Brouwer et al, 2003). Within this group of OARcontaining proteins, a subset of genes classified as group I Aristaless, which include Prx2, Alx3, Alx4, and Cart1, are the most similar to Prx1a in terms of patterns of gene expression and sequence homology (Meijlink et al, 1999;.…”

“…Within this group of OARcontaining proteins, a subset of genes classified as group I Aristaless, which include Prx2, Alx3, Alx4, and Cart1, are the most similar to Prx1a in terms of patterns of gene expression and sequence homology (Meijlink et al, 1999;. Removal of the OAR domain in Cart1 was shown to increase DNA binding and transactivation potential in vitro in addition to producing ectopic cartilage in the mandible in vivo (Brouwer et al, 2003). Previous work from our lab has also shown that removal of the OAR domain of Prx1 and Prx2 increases DNA binding and transactivation potential (Norris and Kern, 2001a,b).…”