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. 2002 Apr 16;99(8):5442-7.
doi: 10.1073/pnas.082096999.

Suppressor of Fused represses Gli-mediated transcription by recruiting the SAP18-mSin3 corepressor complex

Steven Yan Cheng  1 , J Michael Bishop
Affiliations

Suppressor of Fused represses Gli-mediated transcription by recruiting the SAP18-mSin3 corepressor complex

Steven Yan Cheng et al. Proc Natl Acad Sci U S A. .

Abstract

The Suppressor of Fused [Su(fu)] protein plays a conserved role in the regulation of Gli transcription factors of the hedgehog (Hh) signaling pathway that controls cell fate and tissue patterning during development. In both Drosophila and mammals, Su(fu) represses Gli-mediated transcription, but the mode of its action is not completely understood. Recent evidence suggests that Su(fu) physically interacts with the Gli proteins and, when overexpressed, sequesters Gli in the cytoplasm. However, Su(fu) also traverses into the nucleus under the influence of a serine-threonine kinase, Fused (Fu), and has the ability to form a DNA-binding complex with Gli, suggesting that it has a nuclear function. Here we report that the mouse homolog of Su(fu) [mSu(fu)] specifically interacts with SAP18, a component of the mSin3 and histone deacetylase complex. In addition, we demonstrate that mSu(fu) functionally cooperates with SAP18 to repress transcription by recruiting the SAP18-mSin3 complex to promoters containing the Gli-binding element. These results provide biochemical evidence that Su(fu) directly participates in modulating the transcriptional activity of Gli.

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Figures

Figure 1
Figure 1
Physical interaction between mSu(fu) and SAP18. (A) Association of mSu(fu) with SAP18 in yeast two-hybrid assays. The cDNAs of full-length mSu(fu) and the cytoplasmic domain of transforming growth factor-β (TGFβ) type I receptor were inserted downstream of the coding sequences for the LexA DNA-binding domain to generate the “bait” constructs. These plasmids were transformed into yeast strain EGY48/pSH18–4 along with SAP18, or FKBP12 cDNA that were fused in frame to the coding sequences of the activation domain B42. Yeast transformants were tested on galactose-containing X-Gal plates, and protein–protein interactions were determined by scoring for β-galactosidase activity. (B) Association of mSu(fu) with SAP18 in vitro. The translated, [35S]methionine-labeled mSin3A, mSu(fu), or luciferase was incubated with partially purified recombinant GST-SAP18 or GST alone immobilized on glutathione-Sepharose beads. The radioactively labeled proteins bound on beads were separated by SDS/PAGE and visualized by autoradiography. (C) Association of mSu(fu) with SAP18 in mammalian cells. HEK293T cells were transfected with Myc-tagged mSu(fu), FLAG-tagged SAP18 expression constructs individually or together. Total cell lysates and pellets from anti-Myc immunoprecipitation were separated by SDS/PAGE and followed by protein blot analysis by using anti-Myc 9E10 or anti-FLAG M2 antibodies.
Figure 2
Figure 2
Requirement for histone deacetylase activity in the repression of Gli1-mediated transcription by mSu(fu). To measure transcriptional activation from the Gli-dependent promoter, 8xGli-BS-Luc was used as the reporter. Plasmid pTK-RL (Promega), which expresses Renilla luciferase under the control of TK promoter, was included in all samples to normalize transfection efficiency. The total plasmid concentration was kept constant, and whenever needed, vector DNA was added. (A) Repression of Gli1-mediated transcription by mSu(fu). HEK293T cells were transfected with 8xGli-BS Luc reporter together with the indicated amount of plasmids encoding Gli1 or mSu(fu). (B) TSA, an inhibitor of histone deacetylase reversed repression by mSu(fu) on Gli1-mediated transcription. HEK293T cells were transfected with 8x Gli-BS Luc and Gli1 together with either a control-vector plasmid or a mSu(fu)-expression vector. Twenty-four hours before analysis, the indicated concentrations of TSA were added. The luciferase activity in the presence of cotransfected mSu(fu) relative to that obtained with the control plasmid was calculated for each concentration of TSA.
Figure 3
Figure 3
Functional cooperation of mSu(fu) and SAP18 in the repression of Gli-mediated transcription. Transcriptional assays were performed as in Fig. 2. (A) SAP18 cooperates with mSu(fu) to repress Gli1-mediated transcription. HEK293T cells were transfected with 8xGli-BS Luc reporter together with the indicated plasmids encoding Gli1, mSu(fu), or SAP18. (B) mSu(fu) potentiates repression of Shh-induced transcription by Gli3. In HEK293T cells, the transcription from Gli-dependent promoter, 8xGli-BS Luc, was induced by expression of Shh. Gli1 enhanced Shh-induced transcription, whereas Gli3 inhibited Shh-induced transcription. Expression of mSu(fu) inhibited transcription activated by Shh and Gli1, and potentiated repression of Shh-induced transcription by Gli3. (C) Coexpression of mSu(fu) with SAP18 and/or mSin3 enhanced Gli3-mediated repression of Shh-induced transcription.
Figure 4
Figure 4
Recruitment of mSin3 to the consensus Gli DNA-binding sequence through interaction of mSu(fu) and SAP18. Nuclear extracts from HEK293T cells transfected with indicated plasmids were incubated with biotinylated DNA oligonucleotides containing either wild-type (lanes 5–8) or mutant (lanes 9–12) Gli-binding site (Gli-BS). DNA-bound proteins were selected by using streptavidin-coated magnetic beads (Promega) in the presence of 2 μg/ml poly(dI-dC) and subjected to Western blotting with corresponding antibodies. The expression levels of the transfected proteins or endogenous mSin3 were assessed by direct Western blot analysis of total nuclear extracts (lanes 1–4).
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References

    1. Kalderon D. Cell. 2000;103:371–374. - PubMed
    1. Ruiz i Altaba A. Nat Cell Biol. 1999;1:323–391. - PubMed
    1. Ingham PW. EMBO J. 1998;17:3505–3511. - PMC - PubMed
    1. Kinzler K W, Vogelstein B. Mol Cell Biol. 1990;10:634–642. - PMC - PubMed
    1. Dominguez M, Brunner M, Hafen E, Basler K. Science. 1996;272:1621–1625. - PubMed

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