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An upstream regulatory element confers orientation-independent enhancement of the TSG101 promoter activity in transformed cells

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Abstract

Tumor susceptibility gene 101 (TSG101), a mammalian homologue of yeast vps23, is involved in protein sorting, vesicular trafficking and maintenance of genomic integrity. Upregulation of the TSG101 gene was found in human thyroid papillary and breast tumors. Here, we define the proximal promoter of human TSG101 at −1 to −436 by reporter assay. Intact Sp1 and MAZ binding sequences within this region are essential, and mutation of both sites eliminates proximal promoter activity implying cooperation between these two cis-elements. Chromatin immunoprecipitation and DNA affinity precipitation assay confirmed in vivo Sp1 binding on the GGGGCGGGTT sequence. MAZ protein was essential for TSG101 promoter activity because its knockdown using siRNA decreased reporter activity. An upstream regulatory element (URE) at the −1280 to −1757 region was identified to confer the orientation-independent enhancement of the promoter activity in transformed COS-1, ARO and WRO cell lines but not in a normal thyroid FRTL cell line. The sequence of this URE region contains putative binding sites for thyroid transcription factor 2 (TTF-2) and thyroid hormone receptor (T3R), which might be relevant to differential regulation of TSG101 promoter activity in transformed and primary cells.

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Acknowledgments

We would like to thank Professor Margaret Dah-Tsyr Chang for kindly providing us with MAZ antiserum and Drs. J.D. Lin and G.J.F. Juillard for the thyroid carcinoma cell lines ARO and WRO. This work was supported by grants from National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, National Science Council of Republic of China, NSC 95-2320-B-110-009 to J.-T. Cheng, and grant VGHNSU92-05 to Y.-G., Goan and J.-T. Cheng.

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Authors and Affiliations

  1. Department of Biological Sciences, National Sun Yat-Sen University, 70 Lien Hai Road, Kaohsiung, 80424, Taiwan, ROC

    Shih-Fang Hsu, Hsien-Yu Tsai, Yi-Ru Lin & Jiin-Tsuey Cheng

  2. Department of Nursing, Tzu Hui Institute of Technology, Ping Dong Hsuan, Taiwan, ROC

    Shih-Fang Hsu

  3. Division of Thoracic Surgery, Veterans General Hospital, Kaohsiung, Taiwan, ROC

    Yih-Gang Goan

  4. Division of Metabolism, Chang Gung Memorial Hospital, Kaohsiung, Taiwan, ROC

    Rue-Tsuan Liu

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  1. Shih-Fang Hsu
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  2. Yih-Gang Goan
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  3. Hsien-Yu Tsai
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  4. Yi-Ru Lin
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  5. Rue-Tsuan Liu
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  6. Jiin-Tsuey Cheng
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Correspondence to Jiin-Tsuey Cheng.

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Hsu, SF., Goan, YG., Tsai, HY. et al. An upstream regulatory element confers orientation-independent enhancement of the TSG101 promoter activity in transformed cells. Mol Biol Rep 39, 517–525 (2012). https://doi.org/10.1007/s11033-011-0766-6

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  • DOI: https://doi.org/10.1007/s11033-011-0766-6

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