Title

Identification of BMP-response elements in the chicken Msx2 gene

Date of Completion

January 2006

Keywords

Biology, Molecular|Biology, Genetics

Degree

Ph.D.

Abstract

Msx2 is a homeodomain-containing transcription factor expressed during development at sites of ectodermal-mesenchymal interactions, including craniofacial structures, limb buds and cardiac tissues. Previous studies have demonstrated that Msx2 is expressed in various regions that overlap with BMP expression domains and that BMPs can lead to the up-regulation of Msx2 expression in a variety of cells and tissues. The purpose of this study is to identify the nuclear factors and their DNA binding sites involved in BMP regulation of the chicken Msx2 gene and to characterize their interactions in regulating Msx2 gene transcription. ^ We have shown that BMP2 treatment delays the normal decrease in expression of endogenous chicken Msx2 expression in high-density micromass cultures of undifferentiated chick limb mesenchymal cells. BMP2 also increased endogenous Msx2 expression in immortalized mouse MLB13MYC cells. To identify DNA sequences responsible for BMP-induced up-regulation of Msx2, we examined the expression of luciferase reporter constructs driven by chicken Msx2 regulatory sequences in cultured transfected MLB13MYC cells. By sequential deletion and sequence analysis, we identified a 215-bp BMP-responsive region located 3.1-kb upstream of the chicken Msx2 transcription start site. In this enhancer element, one Smad4 binding site (BRE) and one Smad1/5/8 binding site (SBE) are closely spaced in a 69-bp region. Inactivation of either site reduced BMP-induced response significantly. ^ A Lef1 binding site and a GC-rich region are also required for the full BMP-response of the 215-bp enhancer element of chicken Msx2 gene. The Lef1 site, together with SBE and BRE, is located in a 69-bp region of the 215-bp element. A blast search of the 69-bp region showed that this region is highly conserved between species. The GC-rich region is located at the 5' end of the 215-bp enhancer element though the factors binding on this site need to be characterized. ^ The results suggest that BMP regulation of the chicken Msx2 gene involves multiple binding sites, simultaneous presence of multiple factors and possible cross-talk with the Wnt signaling pathway. ^