[Show abstract][Hide abstract] ABSTRACT: Ids function as negative regulators of basic helix-loop-helix transcription factors and their expression is rapidly induced by serum stimulation in various cell types. In this study, we investigated the molecular basis of serum-induced expression of the mouse Id2 gene in NIH3T3 cells. A small-molecule inhibitor of bone morphogenetic protein (BMP) type I receptor kinases blocked the serum induction of Id2 mRNA. The chemical compound and several inhibitory proteins specific for BMP signaling suppressed the serum-induced activation of the luciferase construct with the mouse Id2 4.6-kb promoter region. Importantly, serum stimulation evoked rapid phosphorylation of Smad1/5/8 and significant activation of the reporter plasmid containing the recently identified BMP-responsive element (BRE) of the mouse Id2. Mutation analysis demonstrated that the binding sites for Smad proteins in the Id2 BRE were critical for serum response of the 4.6-kb whole construct. Gel shift and chromatin immunoprecipitation (ChIP) assays confirmed the serum-inducible binding of Smad1/5/8 and Smad4 to the Id2 BRE in vitro and in vivo. Finally, a knockdown experiment revealed the functional importance of Smad1 in the serum induction of Id2 expression. Thus, we concluded that BMP signaling is primarily responsible for the serum-induced Id2 expression. Our results also suggest that some of the cellular effects caused by serum are mediated through BMP signaling.
No preview · Article · Mar 2012 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: Inhibitor of DNA binding/differentiation (Id) genes are the targets of bone morphogenetic protein (BMP) signals in various types of cells. We investigated the molecular basis of BMP6-induced gene expression of mouse Id2 in C2C12 myoblasts. BMP6-dependent Id2 expression occurred immediately without de novo protein synthesis and was blocked by an inhibitor of the BMP type I receptors. A reporter assay identified a BMP6-responsive region 3.0kb upstream of the transcription initiation site. The region showed sequence similarity to the mouse Id1 promoter and shared potential Smad binding sites with it, two GGCGCC palindromes and one GTCT element. Mutation analysis demonstrated the involvement of these elements in the BMP response. Gel shift and chromatin immunoprecipitation (ChIP) assays confirmed the physical binding of Smad proteins to these elements. The 3'-positioned GGCGCC palindrome and the GTCT element were separated by 5-bp and conformed to the canonical BMP-responsive sequence. In addition, the 5'-positioned GGCGCC was accompanied by a previously uncharacterized CGCC element, which were separated by a 5-bp space, and this configuration coincided with that of a similar but distinct sequence to which a Drosophila homolog of the Smad complex can bind. Reporter and gel shift assays revealed the importance of this bipartite sequence. Therefore, we have identified the BMP-responsive elements in mouse Id2 and also shown that the CGCC sequence contributes to target recognition by Smad proteins.
No preview · Article · Aug 2010 · Biochemical and Biophysical Research Communications