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 3.0 kb 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. (C) 2010 Elsevier Inc. All rights reserved.