SIP1, a Novel Zinc Finger/Homeodomain Repressor, Interacts with Smad Proteins and Binds to 5′-CACCT Sequences in Candidate Target Genes

doi:10.1074/jbc.274.29.20489

SIP1, a Novel Zinc Finger/Homeodomain Repressor, Interacts with Smad Proteins and Binds to 5′-CACCT Sequences in Candidate Target Genes*

From the ^tOaDepartment of Cell Growth, Differentiation and Development (VIB-07), Flanders Interuniversity Institute for Biotechnology (VIB), Herestraat49, B-3000 Leuven, Belgium, the ^tObLaboratory of Molecular Biology (CELGEN), University of Leuven, Herestraat 49, B-3000 Leuven, Belgium, the ^tOfDivision of Developmental Biology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, United Kingdom, and the ^tOhDepartment of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048

Abstract

Activation of transforming growth factor β receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes. We describe a novel Smad-interacting protein, SIP1, which was identified using the yeast two-hybrid system. Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast andin vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation. SIP1 is a new member of the δEF1/Zfh-1 family of two-handed zinc finger/homeodomain proteins. Like δEF1, SIP1 binds to 5′-CACCT sequences in different promoters, including the Xenopus brachyury promoter. Overexpression of either full-length SIP1 or its C-terminal zinc finger cluster, which bind to the Xbra2promoter in vitro, prevented expression of the endogenousXbra gene in early Xenopus embryos. Therefore, SIP1, like δEF1, is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways. The identification of this Smad-interacting protein opens new routes to investigate the mechanisms by which transforming growth factor β members exert their effects on expression of target genes in responsive cells and in the vertebrate embryo.

Footnotes

↵* This work was supported by the Flanders Interuniversity Institute for Biotechnology, the Fund of Scientific Research-Flanders (G.0296.98), and EU-Training and Mobility of Researchers (CT98-0216). The VIB-07 group kindly acknowledges the support from Innogenetics S. A. (Gent, Belgium) within the framework of collaboration agreements with the University of Leuven and the Flanders Interuniversity Institute for Biotechnology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵FNc Recipient of a Pre-doctoral Fellowship from the Flemish Institute for the Promotion of Industrial Research and Technology Transfer (IWT).
↵FNd Present address: Innogenetics S. A., Industriepark 7 box 4, B-9052 Zwijnaarde, Belgium.
↵FNe Recipient of a Post-doctoral Fellowship from the National Fund of Scientific Research (NFWO) and of the Research Council of the University of Leuven.
↵FNg Present address: Laboratory for Skeletal Development and Joint Disorders, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium.
↵i Supported by a Fellowship from the Organogenesis Center at the University of Michigan.
↵FNj Supported by grants from the National Institutes of Health and the American Heart Association.
↵FNk Supported by the UK Medical Research Council and was an International Fellow of the Howard Hughes Medical Institute.
↵FNl Supported by VIB. To whom correspondence should be addressed. Tel: +32 16 34 59 16; Fax: +32 16 34 59 33; E-mail: dhu@sgi.celgen. kuleuven.ac.be.
↵2 M.-T. Su, M. Fujioka, and R. Bodmer, unpublished results.
↵3 M.-T. Su, M. Liu, and R. Bodmer, unpublished results.
↵4 W. Lerchner, B. V. Latinkic, L. Fairclough, J. E. Remacle, D. Huylebroeck, and J. C. Smith, unpublished results.
Abbreviations:

TGF-β

transforming growth factor β

bHLH

basic helix-loop-helix

BMP

bone morphogenetic protein

bra

brachyury

CZF

C-terminal zinc finger cluster

DBD

DNA-binding domain

GST

glutathione S-transferase

LacZ

β-galactosidase product of the E. coli LacZ gene

NZF

N-terminal zinc finger cluster

SBD

Smad-binding domain

SIP

Smad-interacting protein

PCR

polymerase chain reaction

X

Xenopus

dpc

days post coitum
- Received March 5, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.