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Molecular and Cellular Biology, July 2002, p. 5182-5193, Vol. 22, No. 14
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.14.5182-5193.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Drosophila Mi-2 Negatively Regulates dDREF by Inhibiting Its DNA-Binding Activity

Fumiko Hirose,1* Nobuko Ohshima,1,2 Eun-Jeong Kwon,1,3 Hideki Yoshida,1,4 and Masamitsu Yamaguchi1,{dagger}

Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-8681,1 Department of Pathology, Nagoya Graduate School of Medicine, Showa-ku, Nagoya 466-8550,2 Division of Biological Science, Nagoya Graduate School of Science, Chikusa-ku, Nagoya 464-8602,3 Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Noda, Chiba 278-8510, Japan4

Received 26 November 2001/ Returned for modification 29 January 2002/ Accepted 16 April 2002

Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5'-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.

* Corresponding author. Mailing address: Division of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-8681, Japan. Phone: 81-52-762-6111, ext. 7220. Fax: 81-52-763-5233. E-mail: fsegawa{at}aichi-cc.jp.

{dagger} Present address: Division of Biotechnology, Faculty of Textile Sciences, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan.

Molecular and Cellular Biology, July 2002, p. 5182-5193, Vol. 22, No. 14
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.14.5182-5193.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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