Abstract
The rat Hst70 gene and its mouse counterpart Hsp70.2 are expressed specifically in pachytene primary spermatocytes and spermatids. Here we demonstrate that a 165 bp fragment of the Hst70 gene promoter, containing the T1 transcription start site region, entire exon 1 and 42 bp 5' region of the intron, is sufficient to drive testis-specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice with the same developmentally regulated pattern as the endogenous Hsp70.2 gene. We show further that high-level tissue-specific gene expression requires additional sequences localized upstream of the T2 transcription start site. Electrophoretic mobility-shift assay analysis revealed that only testes of juvenile rats, when Hst70 gene expression is repressed, contain proteins that specifically bind to the Oct (octamer) sequence localized directly downstream of the T1 site.
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- Alastalo T. P., Lönnström M., Leppä S., Kaarniranta K., Pelto-Huikko M., Sistonen L., Parvinen M. Stage-specific expression and cellular localization of the heat shock factor 2 isoforms in the rat seminiferous epithelium. Exp Cell Res. 1998 Apr 10;240(1):16–27. doi: 10.1006/excr.1997.3926. [DOI] [PubMed] [Google Scholar]
- Bonnycastle L. L., Yu C. E., Hunt C. R., Trask B. J., Clancy K. P., Weber J. L., Patterson D., Schellenberg G. D. Cloning, sequencing, and mapping of the human chromosome 14 heat shock protein gene (HSPA2). Genomics. 1994 Sep 1;23(1):85–93. doi: 10.1006/geno.1994.1462. [DOI] [PubMed] [Google Scholar]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Dix D. J., Allen J. W., Collins B. W., Mori C., Nakamura N., Poorman-Allen P., Goulding E. H., Eddy E. M. Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3264–3268. doi: 10.1073/pnas.93.8.3264. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dix D. J., Rosario-Herrle M., Gotoh H., Mori C., Goulding E. H., Barrett C. V., Eddy E. M. Developmentally regulated expression of Hsp70-2 and a Hsp70-2/lacZ transgene during spermatogenesis. Dev Biol. 1996 Mar 15;174(2):310–321. doi: 10.1006/dbio.1996.0076. [DOI] [PubMed] [Google Scholar]
- Ezashi T., Ghosh D., Roberts R. M. Repression of Ets-2-induced transactivation of the tau interferon promoter by Oct-4. Mol Cell Biol. 2001 Dec;21(23):7883–7891. doi: 10.1128/MCB.21.23.7883-7891.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fiorenza M. T., Farkas T., Dissing M., Kolding D., Zimarino V. Complex expression of murine heat shock transcription factors. Nucleic Acids Res. 1995 Feb 11;23(3):467–474. doi: 10.1093/nar/23.3.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Frazar Tiffany F., Weisbein Jessica L., Anderson Stacie M., Cline Amanda P., Garrett Lisa J., Felsenfeld Gary, Gallagher Patrick G., Bodine David M. Variegated expression from the murine band 3 (AE1) promoter in transgenic mice is associated with mRNA transcript initiation at upstream start sites and can be suppressed by the addition of the chicken beta-globin 5' HS4 insulator element. Mol Cell Biol. 2003 Jul;23(14):4753–4763. doi: 10.1128/MCB.23.14.4753-4763.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grimes S. R., Wolfe S. A., Koppel D. A. Tissue-specific binding of testis nuclear proteins to a sequence element within the promoter of the testis-specific histone H1t gene. Arch Biochem Biophys. 1992 Aug 1;296(2):402–409. doi: 10.1016/0003-9861(92)90590-s. [DOI] [PubMed] [Google Scholar]
- Gross D. S., Garrard W. T. Nuclease hypersensitive sites in chromatin. Annu Rev Biochem. 1988;57:159–197. doi: 10.1146/annurev.bi.57.070188.001111. [DOI] [PubMed] [Google Scholar]
- Kleene Kenneth C., Cataldo Leah, Mastrangelo Mary-Ann, Tagne Jean-Bosco. Alternative patterns of transcription and translation of the ribosomal protein L32 mRNA in somatic and spermatogenic cells in mice. Exp Cell Res. 2003 Nov 15;291(1):101–110. doi: 10.1016/s0014-4827(03)00339-2. [DOI] [PubMed] [Google Scholar]
- Krawczyk Z., Mali P., Parvinen M. Expression of a testis-specific hsp70 gene-related RNA in defined stages of rat seminiferous epithelium. J Cell Biol. 1988 Oct;107(4):1317–1323. doi: 10.1083/jcb.107.4.1317. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Krawczyk Z., Schmid W., Härkönen P., Wolniczek P. The ERE-like sequence from the promoter region of the testis specific hsp70-related gene is not estrogen responsive. Cell Biol Int. 1993 Mar;17(3):245–253. doi: 10.1006/cbir.1993.1061. [DOI] [PubMed] [Google Scholar]
- Krawczyk Z., Wiśniewski J., Biesiada E. A hsp70-related gene is constitutively highly expressed in testis of rat and mouse. Mol Biol Rep. 1987;12(1):27–34. doi: 10.1007/BF00580647. [DOI] [PubMed] [Google Scholar]
- Liu Tiffany Yi-Chen, Chen Huang-Hui, Lee Kun-Hsiung, Choo Kong-Bung. Display of different modes of transcription by the promoters of an early embryonic gene, Zfp352, in preimplantation embryos and in somatic cells. Mol Reprod Dev. 2003 Jan;64(1):52–60. doi: 10.1002/mrd.10218. [DOI] [PubMed] [Google Scholar]
- Murphy S. P., Gorzowski J. J., Sarge K. D., Phillips B. Characterization of constitutive HSF2 DNA-binding activity in mouse embryonal carcinoma cells. Mol Cell Biol. 1994 Aug;14(8):5309–5317. doi: 10.1128/mcb.14.8.5309. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pesce M., Gross M. K., Schöler H. R. In line with our ancestors: Oct-4 and the mammalian germ. Bioessays. 1998 Sep;20(9):722–732. doi: 10.1002/(SICI)1521-1878(199809)20:9<722::AID-BIES5>3.0.CO;2-I. [DOI] [PubMed] [Google Scholar]
- Pothier F., Ouellet M., Julien J. P., Guérin S. L. An improved CAT assay for promoter analysis in either transgenic mice or tissue culture cells. DNA Cell Biol. 1992 Jan-Feb;11(1):83–90. doi: 10.1089/dna.1992.11.83. [DOI] [PubMed] [Google Scholar]
- Raab L. S., Polakoski K. L., Hancock L. W., Hamilton D. W. Characterization of the heat shock protein P70 in rat spermatogenic cells. Mol Reprod Dev. 1995 Feb;40(2):186–195. doi: 10.1002/mrd.1080400207. [DOI] [PubMed] [Google Scholar]
- Rosario M. O., Perkins S. L., O'Brien D. A., Allen R. L., Eddy E. M. Identification of the gene for the developmentally expressed 70 kDa heat-shock protein (P70) of mouse spermatogenic cells. Dev Biol. 1992 Mar;150(1):1–11. doi: 10.1016/0012-1606(92)90002-x. [DOI] [PubMed] [Google Scholar]
- Sarge K. D., Park-Sarge O. K., Kirby J. D., Mayo K. E., Morimoto R. I. Expression of heat shock factor 2 in mouse testis: potential role as a regulator of heat-shock protein gene expression during spermatogenesis. Biol Reprod. 1994 Jun;50(6):1334–1343. doi: 10.1095/biolreprod50.6.1334. [DOI] [PubMed] [Google Scholar]
- Scieglinska D., Widłak W., Konopka W., Poutanen M., Rahman N., Huhtaniemi I., Krawczyk Z. Structure of the 5' region of the Hst70 gene transcription unit: presence of an intron and multiple transcription initiation sites. Biochem J. 2001 Oct 1;359(Pt 1):129–137. doi: 10.1042/0264-6021:3590129. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scieglińska D., Widłak W., Rusin M., Markkula M., Krawczyk Z. Expression of the testis-specific HSP70-related gene (hst70 gene) in somatic non-testicular rat tissues revealed by RT-PCR and transgenic mice analysis. Cell Biol Int. 1997 Dec;21(12):813–821. doi: 10.1006/cbir.1997.0195. [DOI] [PubMed] [Google Scholar]
- Singer-Sam J., Robinson M. O., Bellvé A. R., Simon M. I., Riggs A. D. Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis. Nucleic Acids Res. 1990 Mar 11;18(5):1255–1259. doi: 10.1093/nar/18.5.1255. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Upadhyaya A. B., Lee S. H., DeJong J. Identification of a general transcription factor TFIIAalpha/beta homolog selectively expressed in testis. J Biol Chem. 1999 Jun 18;274(25):18040–18048. doi: 10.1074/jbc.274.25.18040. [DOI] [PubMed] [Google Scholar]
- Wang P. Jeremy, Page David C. Functional substitution for TAF(II)250 by a retroposed homolog that is expressed in human spermatogenesis. Hum Mol Genet. 2002 Sep 15;11(19):2341–2346. doi: 10.1093/hmg/11.19.2341. [DOI] [PubMed] [Google Scholar]
- Widłak W., Markkula M., Krawczyk Z., Kananen K., Huhtaniemi I. A 252 bp upstream region of the rat spermatocyte-specific hst70 gene is sufficient to promote expression of the hst70-CAT hybrid gene in testis and brain of transgenic mice. Biochim Biophys Acta. 1995 Nov 7;1264(2):191–200. doi: 10.1016/0167-4781(95)00135-4. [DOI] [PubMed] [Google Scholar]
- Wiśniewski J., Kordula T., Krawczyk Z. Isolation and nucleotide sequence analysis of the rat testis-specific major heat-shock protein (HSP70)-related gene. Biochim Biophys Acta. 1990 Jan 30;1048(1):93–99. doi: 10.1016/0167-4781(90)90027-y. [DOI] [PubMed] [Google Scholar]
- Wu C. Heat shock transcription factors: structure and regulation. Annu Rev Cell Dev Biol. 1995;11:441–469. doi: 10.1146/annurev.cb.11.110195.002301. [DOI] [PubMed] [Google Scholar]
- Yamauchi Masamichi, Itoh Hiroshi, Naganuma Seiji, Koono Masashi, Hasui Yoshihiro, Osada Yukio, Kataoka Hiroaki. Expression of hepatocyte growth factor activator inhibitor type 2 (HAI-2) in human testis: identification of a distinct transcription start site for the HAI-2 gene in testis. Biol Chem. 2002 Dec;383(12):1953–1957. doi: 10.1515/BC.2002.220. [DOI] [PubMed] [Google Scholar]
- Yoshimizu T., Sugiyama N., De Felice M., Yeom Y. I., Ohbo K., Masuko K., Obinata M., Abe K., Schöler H. R., Matsui Y. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev Growth Differ. 1999 Dec;41(6):675–684. doi: 10.1046/j.1440-169x.1999.00474.x. [DOI] [PubMed] [Google Scholar]
- Zakeri Z. F., Wolgemuth D. J., Hunt C. R. Identification and sequence analysis of a new member of the mouse HSP70 gene family and characterization of its unique cellular and developmental pattern of expression in the male germ line. Mol Cell Biol. 1988 Jul;8(7):2925–2932. doi: 10.1128/mcb.8.7.2925. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zhang D., Penttila T. L., Morris P. L., Roeder R. G. Cell- and stage-specific high-level expression of TBP-related factor 2 (TRF2) during mouse spermatogenesis. Mech Dev. 2001 Aug;106(1-2):203–205. doi: 10.1016/s0925-4773(01)00439-7. [DOI] [PubMed] [Google Scholar]