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J Biol Chem, Vol. 275, Issue 5, 3510-3521, February 4, 2000

OUT, a Novel Basic Helix-Loop-Helix Transcription Factor with an Id-like Inhibitory Activity^*

Osamu Narumi^§, Seiichi Mori^¶, Shuken Boku, Yoshihito Tsuji, Nobuo Hashimoto^§, Shin-Ichi Nishikawa, and Yoshifumi Yokota

From the  Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Shogoin Kawahara-cho 53, Sakyo-ku, 606-8507 Kyoto and the ^§ Department of Neurosurgery, Kyoto University, Shogoin Kawahara-cho 54, Sakyo-ku, 606-8507 Kyoto, Japan

Transcription factors belonging to the basic helix-loop-helix (bHLH) family are involved in various cell differentiation processes. We report the isolation and functional characterization of a novel bHLH factor, termed OUT. OUT, structurally related to capsulin/epicardin/Pod-1 and ABF-1/musculin/MyoR, is expressed mainly in the adult mouse reproductive organs, such as the ovary, uterus, and testis, and is barely detectable in tissues of developing embryos. Physical association of OUT with the E protein was predicted from the primary structure of OUT and confirmed by co-immunoprecipitation. However, unlike other bHLH factors, this novel protein failed to bind E-box or N-box DNA sequences and inhibited DNA binding of homo- and heterodimers consisting of E12 and MyoD in gel mobility shift assays. In luciferase assays, OUT inhibited the induction of E-box-dependent transactivation by MyoD-E12 heterodimers. Deletion studies identified the domain responsible for the inhibitory action of OUT in its bHLH and C-terminal regions. Moreover, terminal differentiation of C2C12 myoblasts was inhibited by exogenous introduction of OUT. These inhibitory functions of OUT closely resemble those of the helix-loop-helix inhibitor Id proteins. Based on these findings, we propose that this novel protein functions as a negative regulator of bHLH factors through the formation of a functionally inactive heterodimeric complex.

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^* This work was supported in part by Grants-in aid from the Ministry of Education, Science, Sports and Culture of Japan 07CE2005, 06277102, 10670119, and 06NP1101.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^TM/EMBL Data Bank with accession number(s) AF142405.

^¶ Supported by the Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists.

To whom correspondence should be addressed: Dept. of Molecular Genetics, Graduate School of Medicine, Kyoto University, Shogoin Kawahara-cho 53, Sakyo-ku, 606-8507 Kyoto, Japan. Tel.: +81-75-751-4162; Fax: +81-75-751-4169; E-mail: yyokota@virus.kyoto-u.ac.jp.

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

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