Identification and Functional Characterization of a Smad Binding Element (SBE) in the JunB Promoter That Acts as a Transforming Growth Factor-β, Activin, and Bone Morphogenetic Protein-inducible Enhancer

doi:10.1074/jbc.273.33.21145

Identification and Functional Characterization of a Smad Binding Element (SBE) in the JunB Promoter That Acts as a Transforming Growth Factor-β, Activin, and Bone Morphogenetic Protein-inducible Enhancer*

From the ^‡Department of Developmental Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, P. O. Box 14, 9750 AA Haren, The Netherlands and the ^¶Ludwig Institute for Cancer Research, Box 595, Biomedical Center, S-75, 24 Uppsala, Sweden

Abstract

Smad proteins have been identified as mediators of intracellular signal transduction by members of the transforming growth factor-β (TGF-β) superfamily, which affect cell proliferation, differentiation, as well as pattern formation during early vertebrate development. Following receptor activation, Smads are assembled into heteromeric complexes consisting of a pathway-restricted Smad and the common Smad4 that are subsequently translocated into the nucleus where they are thought to play an important role in gene transcription. Here we report the identification of Smad Binding Elements (SBEs) composed of the sequence CAGACA in the promoter of theJunB gene, an immediate early gene that is potently induced by TGF-β, activin, and bone morphogenetic protein (BMP) 2. TwoJunB SBEs are arranged as an inverted repeat that is transactivated in response to Smad3 and Smad4 co-overexpression and shows inducible binding of a Smad3- and Smad4-containing complex in nuclear extracts from TGF-β-treated cells. Bacterial-expressed Smad proteins bind directly to the SBE. Multimerization of the SBE creates a powerful TGF-β-inducible enhancer that is also responsive to activin and BMPs. The identification of the sequence CAGACA as a direct binding site for Smad proteins will facilitate the identification of regulatory elements in genes that are activated by members of the TGF-β superfamily.

Footnotes

↵* This work was sponsored by grants from the European Unity (BioMed Program BMH-CT95-0995) and the Dutch Cancer Society (KWF92–84) (to L. J., and W. K.).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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) AJ004891.
↵§ Both authors contributioned equally to this work.
↵‖ To whom correspondence should be addressed. Tel.: 31-50-363-2092; Fax: 31-50-363-2348; E-mail:w.kruijer{at}biol.rug.nl.
↵2 L. J. C. Jonk and W. Kruijer, unpublished observations.
↵3 Dennler, S., Itoh, S., Vivien, D., ten Dijke, P., Huet, S., and Gauthier, J. M. (1998) EMBO J. 17, 3091–3100.
Abbreviations:

AP-1

activating protein-1

ActR

activin receptor

ARE

activin response element

BMP

bone morphogenetic protein

BMPR

BMP receptor

Erk

extracellular signal-regulated kinase

GDF5

growth/differentiation factor-5

GST

glutathione S-transferase

Mad

mother against decapentaplegic

MH

Mad homology

OP-1

osteogenic protein-1

PAI-1

plasminogen activator inhibitor-1

SBE

Smad binding element

TGF-β

transforming growth factor-β

TβR

TGF-β receptor

TRE

TPA response element

ARF

activin response factor

PCR

polymerase chain reaction

WT

wild type

DMEM

Dulbecco’s modified Eagle’s medium

TBE

Tris borate/EDTA

nt

nucleotide(s)

bp

base pair(s).
- Received March 25, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.