Title

Identification and functional characterization of the cis-acting regulatory regions in the chicken {\it Msx-2\/} gene functioning during limb development

Date of Completion

January 1997

Keywords

Biology, Molecular|Biology, Genetics

Degree

Ph.D.

Abstract

The Msx-2 gene is a vertebrate homologue of the Drosophila msh gene. During limb development, Msx-2 is expressed in the anterior peripheral mesenchyme, the AER (apical ectodermal ridge), the interdigital mesenchyme, and a very small region of posterior mesenchyme (Coelho et al., 1991). Previously, our laboratory identified an AER enhancer region in the chicken Msx-2 gene (Sumoy et al., 1995). We hypothesize that this enhancer region contains multiple cis-acting elements, and that Msx-2 expression in the AER is regulated by the concerted action of the trans-acting factors binding to these cis-acting elements.^ Within this AER enhancer, there are four TAAT sites named as B-, C-, D-, and E-TAAT sites. Because many homeodomain proteins bind to sequences with TAAT cores and since the Msx-2 and Dlx-5 genes are expressed in the AER, Msx-2 and Dlx-5 might regulate the expression of Msx-2 in the AER through these TAAT sequences. In support of this hypothesis, both Msx-2 and Dlx-5 proteins bind to this enhancer fragment, and the TAAT sequences are essential for the binding. The functions of the four TAAT sequences in vivo were studied using transgenic mice. The B-TAAT sequence has been identified to be important for AER enhancer activity. An oligonucleotide containing the B-TAAT sequence showed high-affinity binding to Msx-2 and Dlx-5 proteins in vitro, supporting the hypothesis that Msx-2 and Dlx-5 might be involved in regulating Msx-2 expression in the AER.^ To identify candidate cis-acting regulatory regions involved in Msx-2 expression, DHS (DNase I hypersensitive site) studies were employed. The DHSs from $-$6.1kb to +8.4kb relative to the translation start site of chicken Msx-2 gene were identified in the anterior limb mesenchymal cells, posterior limb mesenchymal cells, chicken calvarial osteoblasts, and chicken embryonic fibroblasts. Except for the DHS in the basal promoter region, none of the other DHSs is present in all four types of cells. These DHSs need to be further characterized by functional studies. ^