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J Biol Chem, Vol. 275, Issue 2, 913-920, January 14, 2000

The kappa B and V(D)J Recombination Signal Sequence Binding Protein KRC Regulates Transcription of the Mouse Metastasis-associated Gene S100A4/mts1*

Iben HjelmsoeDagger , Carl E. Allen§, Martin A. CohnDagger , Eugene M. TulchinskyDagger , and Lai-Chu Wu§par

From the Dagger  Danish Cancer Society, Department of Molecular Cancer Biology, Strandboulevarden 49, DK-2100 Copenhagen, Denmark and the § Molecular Cellular and Developmental Biology Program,  Departments of Internal Medicine, Medical Biochemistry, and Molecular Virology, and Immunology and Medical Genetics, and the Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210

A kappa B-like sequence, Sb, is integral to the composite enhancer located in the first intron of the metastasis-associated gene, S100A4/mts1. Oligonucleotides containing this sequence form three specific complexes with nuclear proteins prepared from S100A4/mts1-expressing CSML100 adenocarcinoma cells. Protein studies show the Sb-interacting complexes include NF-kappa B/Rel proteins, p50·p50 and p50·p65 dimers. Additionally, the Sb sequence was bound by an unrelated ~200-kDa protein, p200. Site-directed mutagenesis in conjunction with transient transfections indicate that p200, but not the NF-kappa B/Rel proteins, transactivates S100A4/mts1. To identify candidate genes for p200, double-stranded DNA probes containing multiple copies of Sb were used to screen a randomly primed lambda gt11 cDNA expression library made from CSML100 poly(A)+ RNA. Two clones corresponding to the DNA-binding proteins KRC and Alf1 were identified. KRC encodes a large zinc finger protein that binds to the kappa B motif and to the signal sequences of V(D)J recombination. In vitro DNA binding assays using bacterially expressed KRC fusion proteins, demonstrate specific binding of KRC to the Sb sequence. In addition, introduction of KRC expression vectors into mammalian cells induces expression of S100A4/mts1 and reporter genes driven by S100A4/mts1 gene regulatory sequences. These data indicate that KRC positively regulates transcription of S100A4/mts1.

* This research was supported by National Institutes of Health (NIH) Grant GM48798 (to L.-C. W.), NIH T32 predoctoral training grant (NCI) (to C. E. A.), and in part by Comprehensive Cancer Center of The Ohio State University NIH Grant P30 CA16058 (NCI), a Danish Cancer Society research grant, and a Danish Natural Research Council grant.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.

par To whom correspondence should be addressed: Rm. S2077, Davis Medical Center, Dept. of Internal Medicine, The Ohio State University, 480 West Ninth Ave., Columbus, OH 43210. Tel.: 614-293-3042; Fax: 614-293-5631; E-mail: lcwu@magnus.acs.ohio-state.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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