哈尔滨和湛江雄性褐家鼠Gsk3β差异甲基化区域DNA序列多态性分析与调控功能鉴定
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摘要
糖原合成酶激酶-3β(glycogen synthase kinase-3β,Gsk3β)基因能够参与多条细胞周期信号传导通路,从而通过调控细胞分裂过程,影响组织与器官的发育。本实验室前期研究发现,与湛江褐家鼠种群相比,哈尔滨褐家鼠种群在秋冬季存在睾丸发育受抑制的现象,并通过基因组与甲基化组联合分析,在Gsk3β基因内含子区域筛选到1个差异甲基化区域(differentially methylated region, DMR)。为分析该DMR的DNA序列多态性及其在Gsk3β基因表达调控中的可能作用,本研究选取了哈尔滨(n=52)和湛江(n=39)共91个性成熟褐家鼠睾丸样品。采用PCR测序方法在群体水平上分析了该DMR的DNA序列多态性,在2个褐家鼠亚种的分化特征,并选取2个群体中具有代表性的单倍型,通过荧光素酶基因报告系统在293T细胞中检测该DMR不同单倍型的调控活性。结果表明,该DMR存在2个SNP位点,共组成3个单倍型、6个基因型。卡方检验表明,其频率在2个群体间发生显著分化(单倍型,P=1.13E-29;基因型,P=1.15E-14)。Gsk3β基因的-2 218/+238 bp区具有明显启动子活性(P<0.05);哈尔滨和湛江2个单倍型都显示显著的沉默子活性(P<0.05),同时表现显著的调控活性差异(P<0.05),表明这2个单倍型可以在293T细胞中作为沉默子抑制Gsk3β基因的启动子活性。2个单倍型调控活性差异,可能与SNP导致的转录因子结合位点的获得/丢失,以及2个单倍型在不同种群中甲基化状态差异有关。本研究结果表明,该DMR可能通过调控褐家鼠Gsk3β基因表达,在睾丸发育过程中发挥作用。2个种群主要单倍型的调控活性差异,及其甲基化状态差异可能是2个种群睾丸发育表型差异的重要调控因素之一。
By participating in multiple signaling pathways related to cell cycle control, glycogen synthase kinase-3β(Gsk3β) gene plays an important role in tissue and organ development. We previously found that inhibition of testis development during Autumn and Winter in the rat subspecies, Rattus norvegicus caraco, at city Harbin rather than the rat subspecies, Rattus norvegicus norvegicus, at city Zhanjiang, and detected a differentially methylated region(DMR) located on intron 1 of Gsk3β gene by genome and methylome sequencing. To investigate the DNA polymorphism in this DMR and its possible role in regulation of Gsk3β expression, we selected a total of 91 testis samples of sexual mature individuals from Harbin(n=52) and Zhanjiang(n=39), obtained two subspecies-dominant haplotypes by PCR-sequencing, and analyzed their regulatory activities by luciferase reporter assay in 293 T cells. By two SNP loci, we identified three haplotypes and six genotypes in this DMR, which significantly diverged between two subspecies(Pearson's Chi-squared test: haplotype, P =1.13 E-29; genotype, P=1.15 E-14). Luciferase reporter assay showed that the-2 218/+238 bp region of Gsk3β gene displayed the significant promoter activity(P<0.05), both subspecies-dominant haplotypes displayed the significant silencer activities(P<0.05), and these two haplotypes displayed the significantly different activities(P<0.05). These results demonstrated that these two haplotypes could act as silencers suppressing the Gsk3β expression in 293 T cells. The significantly different regulatory activities between two haplotypes might be caused by two SNPs related gain/loss of transcription factor binding sites(TFBS) and methylation level difference of two haplotypes. This study demonstrated that this DMR may play an important role in testis development by regulating Gsk3β expression. The DNA polymorphism related difference of regulatory activities and methylation states may be the important factors regulating the phenotypic difference of testis development between two subspecies.
By participating in multiple signaling pathways related to cell cycle control, glycogen synthase kinase-3β(Gsk3β) gene plays an important role in tissue and organ development. We previously found that inhibition of testis development during Autumn and Winter in the rat subspecies, Rattus norvegicus caraco, at city Harbin rather than the rat subspecies, Rattus norvegicus norvegicus, at city Zhanjiang, and detected a differentially methylated region(DMR) located on intron 1 of Gsk3β gene by genome and methylome sequencing. To investigate the DNA polymorphism in this DMR and its possible role in regulation of Gsk3β expression, we selected a total of 91 testis samples of sexual mature individuals from Harbin(n=52) and Zhanjiang(n=39), obtained two subspecies-dominant haplotypes by PCR-sequencing, and analyzed their regulatory activities by luciferase reporter assay in 293 T cells. By two SNP loci, we identified three haplotypes and six genotypes in this DMR, which significantly diverged between two subspecies(Pearson's Chi-squared test: haplotype, P =1.13 E-29; genotype, P=1.15 E-14). Luciferase reporter assay showed that the-2 218/+238 bp region of Gsk3β gene displayed the significant promoter activity(P<0.05), both subspecies-dominant haplotypes displayed the significant silencer activities(P<0.05), and these two haplotypes displayed the significantly different activities(P<0.05). These results demonstrated that these two haplotypes could act as silencers suppressing the Gsk3β expression in 293 T cells. The significantly different regulatory activities between two haplotypes might be caused by two SNPs related gain/loss of transcription factor binding sites(TFBS) and methylation level difference of two haplotypes. This study demonstrated that this DMR may play an important role in testis development by regulating Gsk3β expression. The DNA polymorphism related difference of regulatory activities and methylation states may be the important factors regulating the phenotypic difference of testis development between two subspecies.
引文
[1] Wang DW, Cong L, Yue LF, et al. Seasonal variation in population characteristics and management implications for brown rats (Rattus norvegicus) within their native range in Harbin, China[J]. J Pest Sci, 2011, 84(4): 409-418
[2] Shen W, Taylor B, Jin Q, et al. Inhibition of DYRK1A and GSK3B induces human β-cell proliferation[J]. Nat Commun, 2015, 6: 8372
[3] Fisher DL, Morin N, Doree M. A novel role for glycogen synthase kinase-3 in Xenopus development: maintenance of oocyte cell cycle arrest by a beta-catenin-independent mechanism[J]. Development, 1999, 126(3): 567-576
[4] Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase[J]. Eur J Biochem, 1980, 107(2): 519-527
[5] Woodgett JR, Cohen P. Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II(glycogen synthase kinase-5)[J]. Biochim Biophys Acta, 1984, 788(3): 339-347
[6] Woodgett JR. Molecular cloning and expression of glycogen synthase kinase-3/factor A[J]. EMBO J, 1990, 9(8): 2431-2438
[7] G?tschel F, Kern C, Lang S, et al. Inhibition of GSK3 differentially modulates NF-kappaB, CREB, AP-1 and beta-catenin signaling in hepatocytes, but fails to promote TNF-alpha-induced apoptosis[J]. Exp Cell Res, 2008, 314(6): 1351-1366
[8] Shin SH, Lee EJ, Chun J, et al. The nuclear localization of glycogen synthase kinase 3β is required its putative PY-nuclear localization sequences[J]. Mol Cells, 2012, 34(4): 375-382
[9] Guo TB, Chan KC, Hakovirta H, et al. Evidence for a role of glycogen synthase kinase-3 beta in rodent spermatogenesis[J]. J Androl, 2003, 24(3): 332-342
[10] Rossi P, Lolicato F, Grimaldi P, et al. Transcriptome analysis of differentiating spermatogonia stimulated with kit ligand[J]. Gene Expr Patterns, 2008, 8(2): 58-70
[11] Uzbekova S, Salhab M, Perreau C, et al. Glycogen synthase kinase 3B in bovine oocytes and granulosa cells: possible involvement in meiosis during in vitro maturation[J]. Reproduction, 2009, 138(2): 235-246
[12] Wang X, Liu XT, Dunn R, et al. Glycogen synthase kinase-3 regulates mouse oocyte homologue segregation[J]. Mol Reprod Dev, 2003, 64(1): 96-105
[13] 段泽林. GSK3β参与中华绒螯蟹(Eriocheir sinensis)精子发生过程的相关研究[D]. 华东师范大学(Duan ZL. GSK3β involved in the spermatogenesis of Eriocheir sinensis [D]. East China Normal Univ), 2016
[14] Bulger M, Groudine M. Functional and mechanistic diversity of distal transcription enhancers[J]. Cell, 2011, 144(3): 327-339
[15] Bulger M, Groudine M. Enhancers: the abundance and function of regulatory sequences beyond promoters[J]. Dev Biol, 2010, 339(2): 250-257
[16] Krivega I, Dean A. Enhancer and promoter interactions-long distance calls[J]. Curr Opin Genet Dev, 2012, 22(2): 79-85
[17] Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development[J]. Science, 2001, 293(5532): 1089-1093
[18] Kel AE, G??ling E, Reuter I, et al. MATCHTM: a tool for searching transcription factor binding sites in DNA sequences[J]. Nucleic Acids Res, 31(13): 3576-3579
[19] 崔跃, 余榕捷, 彭星荷, 等. 启动子荧光素酶报告系统检测低浓度过氧化氢激活神经肽PACAP特异受体PAC1-R启动子的作用[J].中国生物化学与分子生物学报(Cui Y, Yu RJ, Peng XH, et al. Analyzing activation of the promoter of neuropeptide PACAP preferring receptor PAC1-R under low concentration hydrogen peroxide exposure using promoter luciferase reporter system[J]. Chin J Biochem Mol Biol), 2016, 32(9): 1020-1026
[20] Kleinjan DA, van Heyningen V. Long-range control of gene expression: emerging mechanisms and disruption in disease[J]. Am J Hum Genet, 2005, 76(1): 8-32
[21] Kimura-Yoshida C, Kitajima K, Oda-Ishii I, et al. Characterization of the pufferfish Otx2 cis-regulators reveals evolutionarily conserved genetic mechanisms for vertebrate head specification[J]. Development, 2004, 131(1): 57-71
[22] Shibata Y, Sheffield NC, Fedrigo O, et al. Extensive evolutionary changes in regulatory element activity during human origins are associated with altered gene expression and positive selection[J]. PLoS Genet, 2012, 8(6): e1002789
[23] Tirona RG, Kim RB. Nuclear receptors and drug disposition gene regulation[J]. J Pharm Sci, 2005, 94(6): 1169-1186
[24] Gasiewicz TA, Henry EC, Collins LL. Expression and activity of aryl hydrocarbon receptors in development and cancer[J]. Crit Rev Eukaryot Gene Expr, 2008, 18(4): 279-321
[25] Thurman RE, Rynes E, Humbert R, et al. The accessible chromatin landscape of the human genome[J]. Nature, 2012, 489(7414): 75-82
[26] Zhu H, Wang G, Qian J. Transcription factors as readers and effectors of DNA methylation[J]. Nat Rev Genet, 2016, 17(9): 551-565
[27] Aran D, Hellman A. DNA methylation of transcriptional enhancers and cancer predisposition[J]. Cell, 2013, 154(1): 11-13
[28] Clermont PL, Parolia A, Liu H H, et al. DNA methylation at enhancer regions: Novel avenues for epigenetic biomarker development[J]. Front Biosci(Landmark Ed), 2016, 21: 430-446
[29] Luo Z, Lin C. Enhancer, epigenetics, and human disease[J]. Curr Opin Genet Dev, 2016, 36: 27-33
[30] Plank JL, Dean A. Enhancer function: mechanistic and genome-wide insights come together[J]. Mol Cell, 2014, 55(1): 5-14
[2] Shen W, Taylor B, Jin Q, et al. Inhibition of DYRK1A and GSK3B induces human β-cell proliferation[J]. Nat Commun, 2015, 6: 8372
[3] Fisher DL, Morin N, Doree M. A novel role for glycogen synthase kinase-3 in Xenopus development: maintenance of oocyte cell cycle arrest by a beta-catenin-independent mechanism[J]. Development, 1999, 126(3): 567-576
[4] Embi N, Rylatt DB, Cohen P. Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase[J]. Eur J Biochem, 1980, 107(2): 519-527
[5] Woodgett JR, Cohen P. Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II(glycogen synthase kinase-5)[J]. Biochim Biophys Acta, 1984, 788(3): 339-347
[6] Woodgett JR. Molecular cloning and expression of glycogen synthase kinase-3/factor A[J]. EMBO J, 1990, 9(8): 2431-2438
[7] G?tschel F, Kern C, Lang S, et al. Inhibition of GSK3 differentially modulates NF-kappaB, CREB, AP-1 and beta-catenin signaling in hepatocytes, but fails to promote TNF-alpha-induced apoptosis[J]. Exp Cell Res, 2008, 314(6): 1351-1366
[8] Shin SH, Lee EJ, Chun J, et al. The nuclear localization of glycogen synthase kinase 3β is required its putative PY-nuclear localization sequences[J]. Mol Cells, 2012, 34(4): 375-382
[9] Guo TB, Chan KC, Hakovirta H, et al. Evidence for a role of glycogen synthase kinase-3 beta in rodent spermatogenesis[J]. J Androl, 2003, 24(3): 332-342
[10] Rossi P, Lolicato F, Grimaldi P, et al. Transcriptome analysis of differentiating spermatogonia stimulated with kit ligand[J]. Gene Expr Patterns, 2008, 8(2): 58-70
[11] Uzbekova S, Salhab M, Perreau C, et al. Glycogen synthase kinase 3B in bovine oocytes and granulosa cells: possible involvement in meiosis during in vitro maturation[J]. Reproduction, 2009, 138(2): 235-246
[12] Wang X, Liu XT, Dunn R, et al. Glycogen synthase kinase-3 regulates mouse oocyte homologue segregation[J]. Mol Reprod Dev, 2003, 64(1): 96-105
[13] 段泽林. GSK3β参与中华绒螯蟹(Eriocheir sinensis)精子发生过程的相关研究[D]. 华东师范大学(Duan ZL. GSK3β involved in the spermatogenesis of Eriocheir sinensis [D]. East China Normal Univ), 2016
[14] Bulger M, Groudine M. Functional and mechanistic diversity of distal transcription enhancers[J]. Cell, 2011, 144(3): 327-339
[15] Bulger M, Groudine M. Enhancers: the abundance and function of regulatory sequences beyond promoters[J]. Dev Biol, 2010, 339(2): 250-257
[16] Krivega I, Dean A. Enhancer and promoter interactions-long distance calls[J]. Curr Opin Genet Dev, 2012, 22(2): 79-85
[17] Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development[J]. Science, 2001, 293(5532): 1089-1093
[18] Kel AE, G??ling E, Reuter I, et al. MATCHTM: a tool for searching transcription factor binding sites in DNA sequences[J]. Nucleic Acids Res, 31(13): 3576-3579
[19] 崔跃, 余榕捷, 彭星荷, 等. 启动子荧光素酶报告系统检测低浓度过氧化氢激活神经肽PACAP特异受体PAC1-R启动子的作用[J].中国生物化学与分子生物学报(Cui Y, Yu RJ, Peng XH, et al. Analyzing activation of the promoter of neuropeptide PACAP preferring receptor PAC1-R under low concentration hydrogen peroxide exposure using promoter luciferase reporter system[J]. Chin J Biochem Mol Biol), 2016, 32(9): 1020-1026
[20] Kleinjan DA, van Heyningen V. Long-range control of gene expression: emerging mechanisms and disruption in disease[J]. Am J Hum Genet, 2005, 76(1): 8-32
[21] Kimura-Yoshida C, Kitajima K, Oda-Ishii I, et al. Characterization of the pufferfish Otx2 cis-regulators reveals evolutionarily conserved genetic mechanisms for vertebrate head specification[J]. Development, 2004, 131(1): 57-71
[22] Shibata Y, Sheffield NC, Fedrigo O, et al. Extensive evolutionary changes in regulatory element activity during human origins are associated with altered gene expression and positive selection[J]. PLoS Genet, 2012, 8(6): e1002789
[23] Tirona RG, Kim RB. Nuclear receptors and drug disposition gene regulation[J]. J Pharm Sci, 2005, 94(6): 1169-1186
[24] Gasiewicz TA, Henry EC, Collins LL. Expression and activity of aryl hydrocarbon receptors in development and cancer[J]. Crit Rev Eukaryot Gene Expr, 2008, 18(4): 279-321
[25] Thurman RE, Rynes E, Humbert R, et al. The accessible chromatin landscape of the human genome[J]. Nature, 2012, 489(7414): 75-82
[26] Zhu H, Wang G, Qian J. Transcription factors as readers and effectors of DNA methylation[J]. Nat Rev Genet, 2016, 17(9): 551-565
[27] Aran D, Hellman A. DNA methylation of transcriptional enhancers and cancer predisposition[J]. Cell, 2013, 154(1): 11-13
[28] Clermont PL, Parolia A, Liu H H, et al. DNA methylation at enhancer regions: Novel avenues for epigenetic biomarker development[J]. Front Biosci(Landmark Ed), 2016, 21: 430-446
[29] Luo Z, Lin C. Enhancer, epigenetics, and human disease[J]. Curr Opin Genet Dev, 2016, 36: 27-33
[30] Plank JL, Dean A. Enhancer function: mechanistic and genome-wide insights come together[J]. Mol Cell, 2014, 55(1): 5-14