小鼠生殖细胞特异性转录因子SOHLH1调控Sox30基因表达的机制
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摘要
目的 SOHLH1 (Spermatogenesis-and oogenesis-specific bHLH transcription factor-1)和SOX30(SRY box)是与精子发生相关的转录因子,Sohlh1与Sox30的基因敲除雄鼠均丧失生育能力。出生后7天Sohlh1基因敲除雄鼠DNA芯片显示Sox30表达显著下调,本文探究了早期发育相关转录因子SOHLH1对精子发生后期的关键基因Sox30基因的转录调控作用。方法构建Sox30启动子荧光素酶表达质粒,瞬时共同转染Sox30荧光素酶表达质粒和Sohlh1真核表达质粒并利用荧光素酶活性测定实验检测荧光值。通过染色质免疫共沉淀实验检测SOHLH1与Sox30启动子DNA序列特异序列的结合情况筛选主要激活位点。结果共转染Sohlh1真核表达质粒和Sox30启动子区域荧光素酶质粒的实验组荧光素酶活性高于对照组。SOHLH1与Sox30启动子上的特异性位点结合,在-489 bp位点的结合作用最强。结论精子发生早期重要转录因子SOHLH1可以直接激活顶体形成相关基因Sox30转录,-489 bp (CAGGTG)为主要特异性结合位点,丰富了精子发生中延时翻译表达调控的现象并进行了调控机制的初步探索。
Objective The spermatogenesis-and oogenesis-specific bHLH transcription factor-1(SOHLH1) and SRY box(SOX30) are transcription factors related to spermatogenesis. Sohlh1 and Sox30 knockout male mice lose fertility. At 7 days after birth, the Sohlh1 knockout male mouse DNA chip exhibits significant down-regulation of Sox30 expression. This study explored the role of the early development-related transcription factor SOHLH1 in the transcriptional regulation of the key gene Sox30 during late spermatogenesis. Methods The Sox30 promoter luciferase expression plasmid was constructed, and then together with the Sohlh1 eukaryotic expression plasmid was transiently co-transfected. The fluorescence value was then detected by a luciferase activity assay. The primary activation site was screened using a chromatin immunoprecipitation assay to detect the binding of SOHLH1 to the Sox30 promoter DNA sequence. Results The luciferase activity was higher in the experimental group co-transfected with the Sohlh1 eukaryotic expression plasmid and the Sox30 promoter region luciferase plasmid than in the control group. SOHLH1 binding to specific sites was strongest on the Sox30 promoter binding sites at-489 bp. Conclusions The early important transcription factor SOHLH1 can directly activate acrosome-forming gene Sox30 transcription, with-489 bp(CAGGTG) as the main specific binding site. This action enriches the regulation of delayed translation expression during spermatogenesis. The study thus provides a preliminary exploration of the regulatory mechanism.
Objective The spermatogenesis-and oogenesis-specific bHLH transcription factor-1(SOHLH1) and SRY box(SOX30) are transcription factors related to spermatogenesis. Sohlh1 and Sox30 knockout male mice lose fertility. At 7 days after birth, the Sohlh1 knockout male mouse DNA chip exhibits significant down-regulation of Sox30 expression. This study explored the role of the early development-related transcription factor SOHLH1 in the transcriptional regulation of the key gene Sox30 during late spermatogenesis. Methods The Sox30 promoter luciferase expression plasmid was constructed, and then together with the Sohlh1 eukaryotic expression plasmid was transiently co-transfected. The fluorescence value was then detected by a luciferase activity assay. The primary activation site was screened using a chromatin immunoprecipitation assay to detect the binding of SOHLH1 to the Sox30 promoter DNA sequence. Results The luciferase activity was higher in the experimental group co-transfected with the Sohlh1 eukaryotic expression plasmid and the Sox30 promoter region luciferase plasmid than in the control group. SOHLH1 binding to specific sites was strongest on the Sox30 promoter binding sites at-489 bp. Conclusions The early important transcription factor SOHLH1 can directly activate acrosome-forming gene Sox30 transcription, with-489 bp(CAGGTG) as the main specific binding site. This action enriches the regulation of delayed translation expression during spermatogenesis. The study thus provides a preliminary exploration of the regulatory mechanism.
引文
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[21] Zhou Z,Shirakawa T,Ohbo K,et al.RNA binding protein Nanos2 organizes post-transcriptional buffering system to retain primitive state of mouse spermatogonial stem cells[J].Dev Cell,2015,34(1):96-107.
[2] Kierszenbaum AL,Tres LL.Structural and transcriptional features of the mouse spermatid genome[J].J Cell Biol,1975,65(2):258-270.
[3] Tuck AR,Robker RL,Norman RJ,et al.Expression and localisation of c-kit and KITL in the adult human ovary[J].J Ovarian Res,2015,8(1):31.
[4] Ballow D,Meistrich ML,Matzuk M,et al.Sohlh1 is essential for spermatogonial differentiation[J].Dev Biol,2006,294(1):161-167.
[5] Barrios F,Filipponi D,Campolo F,et al.SOHLH1 and SOHLH2 control Kit expression during postnatal male germ cell development[J].J Cell Sci,2012,125(6):1455-1464.
[6] Suzuki H,Ahn HW,Chu T,et al.SOHLH1 and SOHLH2 coordinate spermatogonial differentiation[J].Dev Biol,2012,361(2):301-312.
[7] Anderson EL,Baltus AE,Roepersgajadien HL,et al.Stra8 and its inducer,retinoic acid,regulate meiotic initiation in both spermatogenesis and oogenesis in mice[J].Proc Natl Acad Sci U S A,2008,105(39):14976-14980.
[8] 米美玲,杨蓓,徐斯凡,等.Stra8:生殖细胞有丝分裂转变为减数分裂前特异表达的基因 [J].中华男科学杂志,2009,15(1):51-55.
[9] Liu X,Gao Q,Zhao N,et al.Sohlh1 suppresses glioblastoma cell proliferation,migration,and invasion by inhibition of Wnt/β-catenin signaling[J].Mol Carcinog,2018,57(4):494-502.
[10] Li Y,Qi W,Liu G,et al.Sohlh1 is required for synaptonemal complex formation by transcriptionally regulating meiotic genes during spermatogenesis in mice[J].Mol Reprod Dev,2019,86(3):252-264
[11] Han F,Wang Z,Wu F,et al.Characterization,phylogeny,alternative splicing and expression of Sox30 gene[J].BMC Mol Biol,2010,11:98.
[12] Zhang D,Xie D,Lin X,et al.The transcription factor SOX30 is a key regulator of mouse spermiogenesis[J].Development,2018,145(11):dev164723.
[13] Bai S,Fu K,Yin H,et al.Sox30 initiates transcription of haploid genes during late meiosis and spermiogenesis in mouse testes[J].Development,2018,145(13):dev164855.
[14] Feng CWA,Spiller C,Merriner DJ,et al.SOX30 is required for male fertility in mice[J].Sci Rep,2017,7(1):17619.
[15] Li S,Du B,Luo G,et al.Application of the dual-luciferase reporter assay to the analysis of mouse kit and Yy1 promoter activation regulation by Sohlh1[J].J Anim Vet Adv,2012,11(14):2523-2528.
[16] Soumillon M,Necsulea A,Weier M,et al.Cellular source and mechanisms of high transcriptome complexity in the mammalian testis[J].Cell Rep,2013,3(6):2179-2190.
[17] Kwon JT,Jin S,Choi H,et al.TEX13 is a novel male germ cell-specific nuclear protein potentially involved in transcriptional repression[J].FEBS Lett,2016,590(20):3526.
[18] Wang H,Zhao R,Guo C,et al.Knockout of BRD7 results in impaired spermatogenesis and male infertility[J].Sci Rep,2016,6:21776.
[19] Wasik KA,Tam OH,Knott SR,et al.RNF17 blocks promiscuous activity of PIWI proteins in mouse testes[J].Genes Dev,2015,29(13):1403-1415.
[20] Pan J,Goodheart M,Chuma S,et al.RNF17,a component of the mammalian germ cell nuage,is essential for spermiogenesis[J].Development,2005,132(18):4029-4039.
[21] Zhou Z,Shirakawa T,Ohbo K,et al.RNA binding protein Nanos2 organizes post-transcriptional buffering system to retain primitive state of mouse spermatogonial stem cells[J].Dev Cell,2015,34(1):96-107.