Co-localization of a novel transcriptional repressor simiRP58 with RP58 Akiyo Takahashi a,b,c , Shinobu Hirai a,b,c , Chiaki Ohtaka-Maruyama a , Akiko Miwa a , Yutaka Hata c , Shigeo Okabe b , Haruo Okado a, * a Tokyo Metropolitan Institute for Neuroscience, Molecular Physiology, 2-6 Musashidai, Fuchu, Tokyo 183-8526, Japan b Department of Cell Biology, Graduate School of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan c Department of Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan Received 21 January 2008 Available online 8 February 2008 Abstract We have cloned a novel transcriptional repressor protein, termed simiRP58, which has high homology to RP58. Both simiRP58 and RP58 belong to the POZ domain and Kruppel Zn finger (POK) family of proteins. Using the luciferase assay system, we found that simiRP58 also has transcriptional repressor activity like RP58. Northern blotting and quantitative RT-PCR showed that simiRP58 was expressed in testes at the highest level. In situ hybridization of testes showed that simiRP58 is expressed by spermatocytes in only a portion of the seminiferous tubules. In contrast, expression of RP58 by spermatocytes was ubiquitous in all seminiferous tubules. Using COS-7 cells, we observed that simiRP58 was localized in the cytoplasm, which is in contrast to RP58 that was localized in the nucleus. Interestingly, co-transfection with simiRP58 and RP58 induced changes in the localization patterns of both proteins. Ó 2008 Elsevier Inc. All rights reserved. Keywords: RP58; Transcriptional repressor; Testis; Co-localization; Spermatogenesis Members of the POK family of proteins are transcrip- tional repressors that contain an N-terminal BTB/POZ domain and C-terminal DNA binding Kruppel-type Zn finger domains. The BTB/POZ domain reportedly medi- ates protein–protein interactions [1]. Many of the POK family proteins, Hypermethylated in cancer 1, B cell lym- phoma 6, promyelocytic leukemia Zn finger (PLZF), and Kaiso link cancer and development [1,2]. PLZF is also essential for stem cell self-renewal in the murine testis [3,4]. The sequence-specific transcriptional repressor RP58 also belongs to the POK family [5]. We previously reported that RP58 is highly expressed in the mouse embryonic cere- bral cortex, suggesting that it plays a critical role during cerebral cortical formation [6]. Many POK family transcriptional repressors become active after homo- or heteromerization with POK family proteins via binding through their BTB/POZ [7] or Zn fin- ger domains [8]. It is possible that RP58 may likewise bind POK family proteins. The PLZF interacts with its homolog PLZP, and they also bind the same DNA target sequences [9]. To elucidate the molecular mechanisms underlying RP58 activity, it is necessary to isolate the proteins that interact with RP58. Because the novel functions of the pro- teins ZBTB4 and ZBTB38 were also first found using BLAST searches for proteins containing Kaiso-like Zn fin- gers [10], we used a similar approach in identifying the function of RP58-like proteins. We search Genbank and identified the gene similar to RP58 (simiRP58), which shows high sequence homology with RP58. We cloned simiRP58, and examined it for transcriptional repressor activity and expression pattern. Materials and methods Molecular cloning. The mouse simiRP58 cDNA sequence was obtained from GenBank (Accession No. BC051084). A full-length cDNA of simiRP58 was prepared by RT-PCR with ReverTra Ace (TOYOBO, Japan) from day 16 mouse embryonic brain. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.147 * Corresponding author. Fax: +81 42 321 8678. E-mail address: hokado@tmin.ac.jp (H. Okado). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 368 (2008) 637–642