Direct Binding of Smad1 and Smad4 to Two Distinct Motifs Mediates Bone Morphogenetic Protein-specific Transcriptional Activation ofId1 Gene

Abstract

Bone morphogenetic proteins (BMPs) are potent inhibitors of myoblast differentiation and inducers of bone formation both in vivo and in vitro. Expression of Id1, a negative regulator of basic helix-loop-helix transcription factors, is up-regulated by BMPs and contributes to the antimyogenic effects of this family of cytokines. In this report, we have identified a specific BMP-2 immediate early response enhancer in the human Id1 gene. Transcriptional activation of the enhancer was increased by overexpression of BMP-responsive Smads, and Smad4 and was completely abrogated in Smad4-deficient cells. Deletion analysis demonstrates that the responsive region is composed of two separate DNA binding elements, a set of overlapping GC boxes, which bind BMP-regulated Smads upon BMP stimulation, and three repeats of CAGAC boxes. Gel shift and oligonucleotide pull-down assays demonstrated that these two types of motifs were capable of binding their corresponding Smads. However, deletion or mutation of either DNA binding element was nonadditive, since disruption of either GC or CAGAC boxes resulted in complete or severe loss of BMP-2 responsiveness. These data suggest the simultaneous requirement of two independent DNA binding elements to allow functional cooperativity of BMP-regulated Smads and Smad4 in BMP-activated gene promoters.

Finding partners: How BMPs select their targets

“…GC-rich boxes also differentially favor BMP activation in cell culture transfections and reporter gene induction is inefficient with SBE sites alone (Korchynskyi and ten Dijke, 2002;Katagiri et al, 2002;López-Rovira et al, 2002). Perhaps the similarity in in vitro affinities of isolated MH1 domains to DNA motifs does not accurately reflect the preferences of the full-length proteins complexed in R-Smad/Smad4 heterotrimers.…”

“…This may explain why relatively long concatemers of Smadbinding sites are often needed to obtain transcriptional activation in cell culture (Jonk et al, 1998;. The promoters of some BMP inducible genes (e.g., bambi, and the id and vent family genes) have numerous Smad-binding sites (López-Rovira et al, 2002;Korchynskyi and ten Dijke, 2002;Karaulanov et al, 2004), presumably to more efficiently recruit the Smads to enhance transcriptional regulation. While isolated MH1 domains of Smad3 do not show cooperative binding to palindromic SBEs (Shi et al, 1998), it is reasonable to believe that full-length Smads would behave differently.…”

Smad3 mediates immediate early induction of Id1 by TGF-β

“…We found that Smad2-null MEFs responded well to TGF-β1 to induce Smad3 phosphorylation and Id1 expression ( Figure 3C, lane 4). In addition, we included BMP2 as a control because it is a well-known inducer of Id1 expression [29,30]. As expected, BMP2 normally induces Smad1 phosphorylation and does not require Smad2 for Id1 induction ( Figure 3C, lane 2).…”

“…The BMP-responsive element of the Id1 promoter has been localized to a small fragment containing a cluster of SBE and binding sites of other transcription factors [29,30]. To determine the TGF-β1 responsive element in the Id1 promoter, we analyzed the ~2-kb sequence upstream of the transcriptional initiation site of the Id1 gene.…”

Regulation of Hex Gene Expression by a Smads-dependent Signaling Pathway

“…This motif can also bind phosphorylated Smad1 in the presence of Smad4, and heterologous reporter genes containing concatamers of this sequence respond to BMP but not TGF␤/ activin signaling (10). A similar motif is responsible for BMP responsiveness of the mouse Smad6 and Id1 gene promoters (11)(12)(13). Other Smad binding elements include the sequence CAGAC, multimers of which are found in TGF␤-responsive promoter elements and are required for TGF␤ responsiveness (14 -16).…”