Cite this paper:
WANG Qian, CUI Zhongkai, GUO Hua, ZHANG Nianwei, XU Wenteng, YANG Yingming, CHEN Songlin. Functional analysis of the promoter of the dmrt1 gene in Chinese tongue sole, Cynoglossus semilaevis[J]. HaiyangYuHuZhao, 2019, 37(4): 1333-1341

Functional analysis of the promoter of the dmrt1 gene in Chinese tongue sole, Cynoglossus semilaevis

WANG Qian1,2, CUI Zhongkai1,2, GUO Hua1,2, ZHANG Nianwei1,2, XU Wenteng1,2, YANG Yingming1,2, CHEN Songlin1,2
1 Key Lab of Sustainable Development of Marine Fisheries, Ministry of Agriculture; Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;
2 Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266273, China
The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is considered to play an essential role in testis differentiation and development among metazoan species. As an economically important marine fish in China, Chinese tongue sole (Cynoglossus semilaevis) possess a ZW/ZZ sex-determining system, and the females usually grow two to four times bigger than males. Previous studies have confirmed that Z-linked dmrt1 gene is the male determining gene in C. semilaevis and TALENs-mediated knocking-out individuals showed increased growth phenotype. In order to investigate the function of dmrt1 regulatory regions and its potential application, we have cloned the promoter of C. semilaevis dmrt1. The acquired sequence consisted of a 5'-upstream sequence and a partial exon1; functional analysis showed that there were two positive regulatory regions in the promoter, as well as one negative region and one neutral region. These positive regions were then combined to obtain several newly constructed promoters. Both in vitro and in vivo analysis confirmed one of them (dmrt1-Δ3+Δ4) showed elevated promoting activity compared to the full-length promoter and could drive exogenous gene expression in tongue sole gonads. Our results will provide useful information for understanding the regulatory mechanism of dmrt1 gene during C. semilaevis sex determination, and the endogenous promoter will facilitate the transgenic research for analyzing sex- and growth-related genes.
Key words:    Cynoglossus semilaevis|dmrt1|promoter|transcription regulation|luciferase assay|gonad   
Received: 2018-07-01   Revised: 2018-08-14
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Åkerfelt M, Morimoto R I, Sistonen L. 2010. Heat shock factors:integrators of cell stress, development and lifespan. Nat. Rev. Mol. Cell. Biol., 11(8):545-555.
Chakraborty T, Zhou L Y, Chaudhari A, Iguchi T, Nagahama Y. 2016. Dmy initiates masculinity by altering Gsdf/Sox9a2/Rspo1 expression in medaka (Oryzias latipes). Sci. Rep., 6:19 480.
Chen S L, Tian Y S, Yang J F, Shao C W, Ji X S, Zhai J M, Liao X L, Zhuang Z M, Su P Z, Xu J Y, Sha Z X, Wu P F, Wang N. 2009. Artificial gynogenesis and sex determination in half-smooth tongue sole (Cynoglossus semilaevis). Mar.Biotechnol., 11(2):243-251.
Chen S L, Zhang G J, Shao C W, Huang Q F, Liu G, Zhang P, Song W T, An N, Chalopin D, Volff J N, Hong Y H, Li Q Y, Sha Z X, Zhou H L, Xie M S, Yu Q L, Liu Y, Xiang H, Wang N, Wu K, Yang C G, Zhou Q, Liao X L, Yang L F, Hu Q M, Zhang J L, Meng L, Jin L J, Tian Y S, Lian J M, Yang J F, Miao G D, Liu S S, Liang Z, Yan F, Li Y Z, Sun B, Zhang H, Zhang J, Zhu Y, Du M, Zhao Y W, Schartl M, Tang Q S, Wang J. 2014. Whole-genome sequence of a flatfish provides insights into ZW sex chromosome evolution and adaptation to a benthic lifestyle. Nat.Genet., 46(3):253-260.
Cook T, Gebelein B, Urrutia R. 1999. Sp1 and its likes:biochemical and functional predictions for a growing family of zinc finger transcription factors. Ann. N. Y.Acad. Sci., 880(1):94-102.
Cui Z K, Liu Y, Wang W W, Wang Q, Zhang N, Lin F, Wang N, Shao C W, Dong Z D, Li Y Z, Yang Y M, Hu M Z, Li H L, Gao F T, Wei Z F, Meng L, Liu Y, Wei M, Zhu Y, Guo H, Cheng C H K, Schartl M, Chen S L. 2017. Genome editing reveals dmrt1 as an essential male sex-determining gene in Chinese tongue sole (Cynoglossus semilaevis). Sci.Rep., 7:42 213.
Friedenreich H, Schartl M. 1990. Transient expression directed by homologus and heterologous prometer and enhancer sequences in fish cells. Nucleic Acids Res., 18(11):3 299-3 305.
Gao S, Zhang T, Zhou X, Zhao Y, Li Q, Guo Y Q, Cheng H H. 2005. Molecular cloning, expression of Sox5 and its down-regulation of Dmrt1 transcription in zebrafish. J.Exp. Zool. B Mol. Dev. Evol., 304B(5):476-483.
Guan G J, Kobayashi T, Nagahama Y. 2000. Sexually dimorphic expression of two types of DM (Doublesex/Mab-3)-domain genes in a teleost fish, the Tilapia (Oreochromis niloticus). Biochem. Biophys. Res. Commun., 272(3):662-666.
Gubbay J, Collignon J, Koopman P, Capel B, Economou A, Münsterberg A, Vivian N, Goodfellow P, Lovell-Badge R. 1990. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature, 346(6281):245-250.
Ji X S, Liu H W, Chen S L, Jiang Y L, Tian Y S. 2011. Growth differences and dimorphic expression of growth hormone(GH) in female and male Cynoglossus semilaevis after male sexual maturation. Mar. Genomics, 4(1):9-16.
Jørgensen A, Morthorst J E, Andersen O, Rasmussen L J, Bjerregaard P. 2008. Expression profiles for six zebrafish genes during gonadal sex differentiation. Reprod. Biol.Endocrinol., 6(1):25.
Kfoury N, Kapatos G. 2009. Identification of neuronal target genes for CCAAT/enhancer binding proteins. Mol. Cell.Neurosci., 40(3):313-327.
Kobayashi T, Matsuda M, Kajiura-Kobayashi H, Suzuki A, Saito N, Nakamoto M, Shibata N, Nagahama Y. 2004.Two DM domain genes, DMY and DMRT1, involved in testicular differentiation and development in the medaka, Oryzias latipes. Dev. Dyn., 231(3):518-526.
Koopman P. 2009. Sex determination:the power of DMRT1.Trends Genet., 25(11):479-481.
Lambeth L S, Raymond C S, Roeszler K N, Kuroiwa A, Nakata T, Zarkower D, Smith C A. 2014. Over-expression of DMRT1 induces the male pathway in embryonic chicken gonads. Dev. Biol., 389(2):160-172.
Lei N, Heckert L L. 2002. Sp1 and Egr1 regulate transcription of the Dmrt1 gene in Sertoli cells. Biol. Reprod., 66(3):675-684.
Lei N, Karpova T, Hornbaker K I, Rice D A, Heckert L L. 2009. Distinct transcriptional mechanisms direct expression of the rat Dmrt1 promoter in sertoli cells and germ cells of transgenic mice. Biol. Reprod., 81(1):118-125.
Luo D J, Liu Y, Chen J, Xia X Q, Cao M X, Cheng B, Wang X J, Gong W M, Qiu C, Zhang Y S, Ki Cheng, C H, Zhu Z Y, Hu W. 2015. Direct production of XYDMY- sex reversal female medaka (Oryzias latipes) by embryo microinjection of TALENs. Sci. Rep., 5:14 057.
Marchand O, Govoroun M, D'Cotta H, McMeel O, Lareyre J J, Bernot A, Laudet V, Guiguen Y. 2000. DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss. Biochim. Biophys. Acta, 1493(1-2):180-187.
Marin M, Karis A, Visser P, Grosveld F, Philipsen S. 1997.Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation. Cell, 89(4):619-628.
Matsuda M, Nagahama Y, Shinomiya A, Sato T, Matsuda C, Kobayashi T, Morrey C E, Shibata N, Asakawa S, Shimizu N, Hori H, Hamaguchi S, Sakaizumi M. 2002. DMY is a Y-specific DM-domain gene required for male development in the medaka fish. Nature, 417(6888):559-563.
Matsuda M, Shinomiya A, Kinoshita M, Suzuki A, Kobayashi T, Paul-Prasanth B, Lau E L, Hamaguchi S, Sakaizumi M, Nagahama Y. 2007. DMY gene induces male development in genetically female (XX) medaka fish. Proc. Natl. Acad.Sci. USA, 104(10):3 865-3 870.
Mawaribuchi S, Musashijima M, Wada M, Izutsu Y, Kurakata E, Park M K, Takamatsu N, Ito M. 2017. Molecular evolution of two distinct dmrt1 promoters for germ and somatic cells in vertebrate gonads. Mol. Biol. Evol., 34(3):724-733.
Nanda I, Kondo M, Hornung U, Asakawa S, Winkler C, Shimizu A, Shan Z H, Haaf T, Shimizu N, Shima A, Schmid M, Schartl M. 2002. A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes. Proc. Natl. Acad. Sci. USA, 99(18):11 778-11 783.
Paul-Prasanth B, Matsuda M, Lau E L, Suzuki A, Sakai F, Kobayashi T, Nagahama Y. 2006. Knock-down of DMY initiates female pathway in the genetic male medaka, Oryzias latipes. Biochem. Biophys. Res. Commun., 351(4):815-819.
Quitschke W W, Lin Z Y, DePonti-Zilli L, Paterson B M. 1989.The beta actin promoter. High levels of transcription depend upon a CCAAT binding factor. J. Biol. Chem., 264(16):9 539-9 546.
Raymond C S, Murphy M W, O'Sullivan M G, Bardwell V J, Zarkower D. 2000. Dmrt1, a gene related to worm and fly sexual regulators, is required for mammalian testis differentiation. Genes Dev., 14(20):2 587-2 595.
Raymond C S, Parker E D, Kettlewell J R, Brown L G, Page D C, Kusz K, Jaruzelska J, Reinberg Y, Flejter W L, Bardwell V J, Hirsch B, Zarkower D. 1999. A region of human chromosome 9p required for testis development contains two genes related to known sexual regulators. Hum. Mol.Genet., 8(6):989-996.
Raymond C S, Shamu C E, Shen M M, Seifert K J, Hirsch B, Hodgkin J, Zarkower D. 1998. Evidence for evolutionary conservation of sex-determining genes. Nature, 391(6668):691-695.
Shao C W, Li Q Y, Chen S L, Zhang P, Lian J M, Hu Q M, Sun B, Jin L J, Liu S S, Wang Z J, Zhao H M, Jin Z H, Liang Z, Li Y Z, Zheng Q M, Zhang Y, Wang J, Zhang G J. 2014.Epigenetic modification and inheritance in sexual reversal of fish. Genome Res., 24(4):604-615.
Shetty S, Kirby P, Zarkower D, Graves J A M. 2002. DMRT1 in a ratite bird:evidence for a role in sex determination and discovery of a putative regulatory element. Cytogenet.Genome Res., 99(1-4):245-251.
Sinclair A H, Berta P, Palmer M S, Hawkins J R, Griffiths B L, Smith M J, Foster J W, Frischauf A M, Lovell-Badge R, Goodfellow P N. 1990. A gene from the human sexdetermining region encodes a protein with homology to a conserved DNA-binding motif. Nature, 346(6281):240-244.
Slomiany B A, D'Arigo K L, Kelly M M, Kurtz D T. 2000. C/EBPα inhibits cell growth via direct repression of E2FDP-mediated transcription. Mol. Cell. Biol., 20(16):5 986-5 997.
Smith C A, McClive P J, Western P S, Reed K J, Sinclair A H. 1999. Conservation of a sex-determining gene. Nature, 402(6762):601-602.
Smith C A, Roeszler K N, Ohnesorg T, Cummins D M, Farlie P G, Doran T J, Sinclair A H. 2009. The avian Z-linked gene DMRT1 is required for male sex determination in the chicken. Nature, 461(7261):267-271.
Sunilkumar G, Connell J P, Smith C W, Reddy A S, Rathore K S. 2002. Cotton α-globulin promoter:isolation and functional characterization in transgenic cotton, Arabidopsis, and tobacco. Transgenic Res., 11(4):347-359.
Topilko P, Schneider-Maunoury S, Levi G, Trembleau A, Gourdji D, Driancourt M A, Rao C V, Charnay P. 1998.Multiple pituitary and ovarian defects in Krox-24 (NGFIA, Egr-1)-targeted mice. Mol. Endocrinol., 12(1):107-122.
Tourtellotte W G, Nagarajan R, Auyeung A, Mueller C, Milbrandt J. 1999. Infertility associated with incomplete spermatogenic arrest and oligozoospermia in Egr4-deficient mice. Development, 126(22):5 061-5 071.
Tsunoda T, Takagi T. 1999. Estimating transcription factor bindability on DNA. Bioinformatics, 15(7-8):622-630.
Xia W, Zhou L, Yao B, Li C J, Gui J F. 2007. Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers.Mol. Cell. Endocrinol., 263(1-2):156-172.
Yoshimoto S, Ito M. 2011. A ZZ/ZW-type sex determination in Xenopus laevis. FEBS J., 278(7):1 020-1 026.
Zhao L, Svingen T, Ng E T, Koopman P. 2015. Female-to-male sex reversal in mice caused by transgenic overexpression of Dmrt1. Development, 142(6):1 083-1 088.
Zhou L Q, Yang A G, Liu X Z, Du W, Zhuang Z M. 2005. The karyotype of the tonguefish Cynoglossus semilaevis. J.Fish. Sci. China, 29(3):417-419. (in Chinese with English abstract)