Identification and Functional Characterization of Distinct Critically Important Bone Morphogenetic Protein-specific Response Elements in the Id1 Promoter

Abstract

Transforming growth factor-␤ (TGF-␤) family members, which include bone morphogenetic proteins (BMPs) and TGF-␤s, elicit their cellular effects by activating specific Smad proteins, which control the transcription of target genes. BMPs and TGF-␤s have overlapping as well as specific effects on mesenchymal cell differentiation for which the mechanisms are incompletely understood. Here we report that Id1, a dominant negative inhibitor of basic helix-loop-helix proteins, is a direct target gene for BMP. BMP, but not TGF-␤, strongly activates the Id1 promoter in an Smad-dependent manner. We identified two BMP-responsive regions in the mouse Id1 promoter, which contain three distinct sequence elements; one region contains two Smad binding elements (SBEs), and the other region contains a GGCGCC palindromic sequence flanked by two CAGC and two CGCC motifs. Whereas SBEs and GGCGCC sequence are critically important, the CAGC and CGCC motifs are needed for efficient BMP-induced Id1 promoter activation. Smads are part of nuclear transcription factor complexes that specifically bind to SBEs and GGCGCC sequence in response to BMP but not TGF-␤. Multimerization of the all three distinct sequence motifs is needed to generate a highly sensitive and BMP/Smaddependent specific enhancer. Our results provide important new insights into how the BMP/Smad pathway can specifically activate target genes.

Inhibitory effects and target genes of bone morphogenetic protein 6 in Jurkat TAg cells

“…We used a reporter assay to demonstrate this also in Jurkat TAg cells. The BRE-Luc reporter contains elements from the ID1 promoter and is selectively activated by BMP-specific R-Smad [32]. This reporter was induced in Jurkat TAg cells by BMP-6 concentrations lower than normally used in functional assays, indicating that phosphorylation of R-Smad is an efficient signalling pathway in this cell line.…”

“…Jurkat TAg cells were transfected with the BRE-Luc plasmid (specific BMP/Smad transcriptional reporter [32]), in combination with pRL-CMV (Renilla luciferase), which was used as an internal transfection control. After transfection, the cells were treated with various concentrations of BMP-6 overnight.…”

Homozygous missense and nonsense mutations in BMPR1B cause acromesomelic chondrodysplasia-type Grebe

“…NIH/3T3 cells were seeded in a 96-well plate and co-transfected using Lipofectamine 2000 (Invitrogen, Life Technologies) at about 70% confluency with pCS2 þ expression constructs of Bmpr1b wt , Bmpr1b C53R , Bmpr1b dLBD , GDF5, BRE luciferase reporter construct 29 and the Renilla luciferase vector pRL-TK (Promega, Madison, WI, USA), which served as an internal control for transfection efficiency. Cells were cultivated in DMEM 4.5 g/l glucose (Lonza) supplemented with 1% FBS superior (Biochrom AG), 2 mM L-glutamine (Lonza), penicillin/streptomycin (Lonza) for 42 h after transfection and subsequently collected and lyzed in potassium phosphate buffer (9 mM potassium dihydrogen phosphate, 91 mM dipotassium phosphate, 0.2% Triton-X-100).…”

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.…”