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Nucleic Acids Research Advance Access originally published online on January 9, 2009
Nucleic Acids Research 2009 37(5):1423-1437; doi:10.1093/nar/gkn1068
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Nucleic Acids Research, 2009, Vol. 37, No. 5 1423-1437
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Genetic screening for modifiers of the DREF pathway in Drosophila melanogaster: identification and characterization of HP6 as a novel target of DREF

Hiroyuki Ida1,2, Noriyuki Suzusho1,2, Osamu Suyari1,2, Hideki Yoshida1,3, Katsuhito Ohno1, Fumiko Hirose4, Masanobu Itoh1,2 and Masamitsu Yamaguchi1,2,*

1Department of Applied Biology, 2Insect Biomedical Research Center, 3Venture Laboratory, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 and 4Department of Life Science, Graduate School of Life Science, University of Hyogo, Kamigori, Hyogo 678-1297, Japan

*To whom correspondence should be addressed. Tel: +81 75 724 7781; Fax: +81 75 724 7760; Email: myamaguc{at}kit.ac.jp

Received October 10, 2008. Revised December 19, 2008. Accepted December 19, 2008.

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. By genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express DREF in the eye discs, we identified 24 genes that suppressed and 12 genes that enhanced the rough eye phenotype when heterozygous for mutations. Five genes, HP6, pigeon, lace, X box binding protein 1 and guftagu were found to carry replication-related element (DRE) sequences in their 5'-flanking regions. Of these, the HP6 gene carries two sequences that match seven out of eight nucleotides of DRE and two additional sequences that match six out of eight nucleotides of DRE in the 5'-flanking region. Band mobility shift assays using Drosophila Kc cell nuclear extracts demonstrated DREF binding to two of these sites and chromatin immunoprecipitation using anti-DREF antibodies confirmed that this occurs in vivo. Knockdown of DREF in Drosophila S2 cells decreased the HP6 mRNA level. The results, taken together, indicate that DREF directly regulates expression of the HP6 gene. HP6 mRNA was detected throughout development by RT-PCR with highest levels in adult males. In addition, immunostaining analyses revealed colocalization of HP6 and DREF in nuclei at the apical tips in the testes.

Present addresses: Hiroyuki Ida, Environmental Research Laboratory of Public Health, Kankyo Eisei Yakuhin Co. Ltd., 3-6-2 Hikaridai, Seika-cho, Soraku-gun, Kyoto 619-0237, Japan

Hideki Yoshida, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College St., Toronto, Ontario, M5S 3E1, Canada

Katsuhito Ohno, Department of Molecular Biology and Biochemistry, Nelson Biology Laboratory, Rutgers University, 604 Allison Road, Piscataway, NJ 08854, USA


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