Transforming growth factor beta -inducible independent binding of SMAD to the Smad7 promoter - PubMed (original) (raw)

Transforming growth factor beta -inducible independent binding of SMAD to the Smad7 promoter

N G Denissova et al. Proc Natl Acad Sci U S A. 2000.

Abstract

SMAD proteins can mediate transforming growth factor beta (TGF-beta)-inducible transcriptional responses. Whereas SMAD can recognize specific DNA sequences, it is usually recruited to a promoter through interaction with a DNA-binding partner. In an effort to search for TGF-beta-inducible genes, we used a subtractive screening method and identified human Smad7, which can antagonize TGF-beta signaling and is rapidly up-regulated by TGF-beta. In this report, we show that TGF-beta can stabilize Smad7 mRNA and activate Smad7 transcription. The Smad7 promoter is the first TGF-beta responsive promoter identified in vertebrates that contains the 8-bp palindromic SMAD-binding element (SBE), an optimal binding site previously identified by a PCR-based selection from random oligonucleotides by using recombinant Smad3 and Smad4. We demonstrate that on TGF-beta treatment, endogenous SMAD complex can bind to a Smad7 promoter DNA as short as 14 or 16 bp that contains the 8-bp SBE in gel mobility shift and supershift assays. Our studies provide strong evidence that SMAD proteins can bind to a natural TGF-beta responsive promoter independent of other sequencespecific transcription factors. We further show that, whereas recombinant Smad3 binds to the SBE, endogenous or even transfected Smad3 cannot bind to the SBE in the absence of Smad4. These findings have important implications in the identification of target genes of the TGF-beta/SMAD signaling pathways.

PubMed Disclaimer

Figures

Figure 1

Figure 1

(A) TGF-β rapidly induces Smad7 mRNA levels. Poly(A)+ RNA (4 μg) from HaCaT cells that were untreated or treated with TGF-β for different lengths of time was analyzed by Northern blot assay by using the N-terminal domain of Smad7 as a probe. (B) TGF-β can increase the stability of Smad7 RNA. Poly(A)+ RNA (4 μg) from HaCaT cells that were treated with actinomycin D (1 μg/ml) in the absence or presence of TGF-β (500 pM) for various times was analyzed by Northern blot analysis with the Smad7 probe. In both A and B, each blot was subsequently hybridized with a glyceraldehyde-3-phosphate dehydrogenase probe to normalize the amount of RNA loaded.

Figure 2

Figure 2

Determination of transcription start site(s) of human Smad7 promoter. Poly(A)+ RNA (0.4 μg) from HaCaT cells that were untreated or treated with TGF-β (500 pM) for 1 h was used with a 22-bp oligonucleotide in the primer extension analysis. The products were analyzed on a denaturing gel (see A) with a sequencing reaction (not shown). The two transcription start sites (P1 and P2), the SBE, and the position of the 22-bp primer are marked.

Figure 3

Figure 3

(A) The SBE is essential for TGF-β inducibility of the Smad7 promoter. Luciferase constructs containing different lengths of the Smad7 promoter that bear wild-type or mutated SBE were transfected into HaCaT cells and treated with TGF-β as indicated. Luciferase activity was then analyzed. (B) The Smad3–Smad4 complex activates transcription from the Smad7 promoter optimally. Smad2, Smad3, and Smad4 were cotransfected with the −339 to +641 Smad7-luciferase construct into SW480.7 cells and treated with TGF-β as indicated. Luciferase activity was then determined.

Figure 4

Figure 4

Endogenous SMAD can bind to the SBE in response to TGF-β. Whole-cell extracts from HaCaT cells that were untreated or treated with TGF-β for 1 h were incubated with the 16-bp probe in a gel mobility shift assay shown in A and B. (A) Antibodies against Smad2/Smad3 or Smad4 can cause supershift of the TGF-β-inducible DNA–protein complex. (B) Specific competitor refers to an oligo with mutated flanking sequence but containing intact 8-bp SBE. Nonspecific competitor refers to an oligo with a 2-bp mutation in the GTCT sequence of the SBE. (C) HaCaT cells were untreated or treated with TGF-β for various times and then analyzed for SBE-binding activity in a gel mobility shift assay.

Figure 5

Figure 5

(A and B) Endogenous or transfected Smad3 cannot bind to the SBE in the absence of Smad4, and the Smad3–Smad4 complex binds optimally to the SBE. (A) SW480.7 cells were cotransfected with Flag-Smad2, Flag-Smad3, and Smad4-HA, and treated with TGF-β as indicated. Whole-cell extracts were used in a gel mobility shift assay with the 16-bp probe. (B) Antibodies against the Flag and HA epitopes were included for supershift assay. (C) Recombinant Smad3 and Smad4 can bind to the 16-bp probe. GST-SMAD proteins (0.8 μg) was used in a gel mobility shift assay.

References

    1. Heldin C-H, Miyazono K, ten Dijke P. Nature (London) 1997;390:465–471. - PubMed
    1. Massagué J. Annu Rev Biochem. 1998;67:753–791. - PubMed
    1. Whitman M. Genes Dev. 1998;12:2445–2462. - PubMed
    1. Derynck R, Zhang Y, Feng X. Cell. 1998;95:737–740. - PubMed
    1. Roberts A B. Microb Infect. 1999;1:1265–1273. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources