腺苷酸环化酶3缺失对小鼠主要嗅觉表皮组织内DNA甲基化的影响
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摘要
腺苷酸环化酶3 (adenylate cyclaseⅢ,AC3)是嗅觉系统中的重要成分,AC3缺失后小鼠的主要嗅觉表皮组织(main olfactory epidermal,MOE)随年龄增长逐渐变薄,MOE内基因表达谱发生改变. DNA甲基化在动物发育、基因表达调控中具有重要作用.为了探讨AC3缺失后小鼠MOE内基因启动子甲基化水平的改变以及对基因表达的影响,本文采用DNA甲基化免疫共沉淀芯片(methylated DNA immunoprecipitation chip,MeDIP-chip)筛选AC3缺失小鼠MOE内启动子区甲基化差异表达基因,利用甲基化特异PCR (methylation-specific PCR,MSP)、实时荧光定量PCR (qRT-PCR)进一步检测部分甲基化差异基因的DNA甲基化水平改变和表达差异.结果表明,AC3缺失小鼠中有1 978个基因启动子的甲基化水平发生了改变,占总探针数的9%,其中727个基因启动子甲基化水平升高,1 251个甲基化水平降低.功能分析表明,这些启动子甲基化发生改变的基因主要涉及的功能分别与嗅觉受体、神经发育、cAMP信号通路、ATP结合、钙离子调控、乙酰化修饰、转录因子等相关. MSP检测表明,嗅觉受体基因Olfr1153、Olfr231、Olfr378、Olfr651、Olfr691启动子区的甲基化水平升高,Cngb1、Pde4a和Olfr1394基因启动子区的甲基化水平降低. qRT-PCR结果显示,基因Cngb1、Hcn4、Olfm1、Olfr1394、Olfr1153、Olfr231、Olfr378、Olfr691的表达水平显著下降,而Pde4a和Olfr651基因的表达水平显著升高.总之,AC3缺失后MOE内嗅觉受体基因、神经发育相关基因、cAMP信号通路等相关基因启动子甲基化水平发生显著改变,影响核苷酸切除修复、DNA复制、错配修复等信号通路的传导,从而综合调控小鼠MOE内的基因表达数量和水平.
Adenylate cyclase Ⅲ(AC3) is an important component of odorant perception signaling in the olfactory system. The thinness of main olfactory epithelium(MOE) become thin with ageing, and the gene expression profile alters after AC3 deletion. DNA methylation plays a key role in animal development and regulation of gene expression. In the present study, whether the DNA methylation level of gene promoter, as well as their associations with the expression of the genes in MOE will be altered after AC3 deletion, was investigated by using methylated DNA immunoprecipitation chip(MeDIP-chip), methylation-specific PCR(MSP) and realtime fluorescence quantification PCR. The data showed that the DNA methylation levels of promoters of 1 978 genes were altered in AC3-deficient mice, accounting for 9% of the total number of genes. Of which 727 genes with their promoter's DNA methylation levels were elevated, 1 251 genes with their promoter's methylation levels were lowered. The functions of these genes are mainly involved with olfactory receptor, neurodevelopmental,cAMP signaling pathway, ATP-binding, calcium regulation, acetylation modification, and transcription factors. It was further confirmed by MSP that methylation levels of promoter of the olfactory receptor genes Olfr1153,Olfr231, Olfr378, Olfr651 and Olfr691 were increased, whereas methylation level of the promoters of Cngb1,Pde4 a and Olfr1394 were decreased. In line with MSP results, qRT-PCR data showed that the expression levels of Cngb1, Hcn4, Olfm1, Olfr1394, Olfr1153, Olfr231, Olfr378 and Olfr691 were significantly decreased, whereas the expression levels of Pde4 a and Olfr651 were significantly increased. In conclusion, the methylation levels of promoters of olfactory receptor genes, neurodevelopmental related genes and cAMP signaling pathways in MOE are modified significantly after AC3 deletion, which affected the transduction of signal pathways such as nucleotide excision and repair, DNA replication and mismatch repair, thus comprehensively regulating the number and level of gene expression in MOE of mice.
Adenylate cyclase Ⅲ(AC3) is an important component of odorant perception signaling in the olfactory system. The thinness of main olfactory epithelium(MOE) become thin with ageing, and the gene expression profile alters after AC3 deletion. DNA methylation plays a key role in animal development and regulation of gene expression. In the present study, whether the DNA methylation level of gene promoter, as well as their associations with the expression of the genes in MOE will be altered after AC3 deletion, was investigated by using methylated DNA immunoprecipitation chip(MeDIP-chip), methylation-specific PCR(MSP) and realtime fluorescence quantification PCR. The data showed that the DNA methylation levels of promoters of 1 978 genes were altered in AC3-deficient mice, accounting for 9% of the total number of genes. Of which 727 genes with their promoter's DNA methylation levels were elevated, 1 251 genes with their promoter's methylation levels were lowered. The functions of these genes are mainly involved with olfactory receptor, neurodevelopmental,cAMP signaling pathway, ATP-binding, calcium regulation, acetylation modification, and transcription factors. It was further confirmed by MSP that methylation levels of promoter of the olfactory receptor genes Olfr1153,Olfr231, Olfr378, Olfr651 and Olfr691 were increased, whereas methylation level of the promoters of Cngb1,Pde4 a and Olfr1394 were decreased. In line with MSP results, qRT-PCR data showed that the expression levels of Cngb1, Hcn4, Olfm1, Olfr1394, Olfr1153, Olfr231, Olfr378 and Olfr691 were significantly decreased, whereas the expression levels of Pde4 a and Olfr651 were significantly increased. In conclusion, the methylation levels of promoters of olfactory receptor genes, neurodevelopmental related genes and cAMP signaling pathways in MOE are modified significantly after AC3 deletion, which affected the transduction of signal pathways such as nucleotide excision and repair, DNA replication and mismatch repair, thus comprehensively regulating the number and level of gene expression in MOE of mice.
引文
[1]Brunet L J,Gold G H,Ngai J.General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide-gated cation channel.Neuron,1996,17(4):681-693
[2]Johnson J L,Leroux M R.c AMP and c GMP signaling:sensory systems with prokaryotic roots adopted by eukaryotic cilia.Trends Cell Biol,2010,20(8):435-444
[3]Defer N,Bestbelpomme M,Hanoune J.Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase.American Journal of Physiology Renal Physiology,2000,279(3):F400
[4]Wang Z,Zhou Y,Luo Y,et al.Gene Expression profiles of main olfactory epithelium in adenylyl cyclase 3 knockout mice.Int JMol Sci,2015,16(12):28320-28333
[5]Bird A.Perceptions of epigenetics.Nature,2007,447(7143):396-398
[6]Li E,Zhang Y.DNA methylation in mammals.Cold Spring Harb Perspect Biol,2014,6(5):a019133
[7]Suzuki M M,Bird A.DNA methylation landscapes:provocative insights from epigenomics.Nat Rev Genet,2008,9(6):465-476
[8]Zhang Z,Yang D,Zhang M,et al.Deletion of type 3 adenylyl cyclase perturbs the postnatal maturation of olfactory sensory neurons and olfactory cilium ultrastructure in mice.Front Cell Neurosci,2017,11:1
[9]周艳芬,韩绍芳,舒俐,等.腺苷酸环化酶3缺失对小鼠主要嗅觉表皮组织内相关因子及信号通路的影响.中国生物化学与分子生物学学报,2016,32(7):816-822Zhou Y F,Han S F,Shu L,et al.Chinese Journal of Biochemistry and Molecular Biology,2016,32(7):816-822
[10]周艳芬,王伟娜,袁焕娜等.腺苷酸环化酶3缺失下调小鼠嗅觉受体基因表达.中国生物化学与分子生物学报,2017,33(11):1143-1151Zhou Y F,Wang W N,Yuan H N,et al.Chinese Journal of Biochemistry and Molecular Biology,2017,33(11):1143-1151
[11]Wang Z,Storm D R.Extraction of DNA from mouse tails.Biotechniques,2006,41(4):410-412
[12]Weber M,Davies J J,Wittig D,et al.Chromosome-wide and promoter-specific analyses identify sites of differential DNAmethylation in normal and transformed human cells.Nat Genet,2005,37(8):853-862
[13]Meng H,Cao Y,Qin J,et al.DNA methylation,its mediators and genome integrity.International Journal of Biological Sciences,2015,11(5):604-617
[14]Schultz M D,He Y,Whitaker J W,et al.Corrigendum:human body epigenome maps reveal noncanonical DNA methylation variation.Nature,2016,530(7589):242
[15]Prokhortchouk E,Defossez P A.The cell biology of DNAmethylation in mammals.Biochim Biophys Acta,2008,1783(11):2167-2173
[16]Eckhardt F,Lewin J,Cortese R,et al.DNA methylation profiling of human chromosomes 6,20 and 22.Nat Genet,2006,38(12):1378-1385
[17]Illingworth R,Kerr A,Desousa D,et al.A novel CpG island set identifies tissue-specific methylation at developmental gene loci.PLoS Biol,2008,6(1):e22
[18]Han F,Dong Y,Liu W,et al.Epigenetic regulation of sox30 is associated with testis development in mice.PLoS One,2014,9(5):e97203
[19]Rollins R A,Haghighi F,Edwards J R,et al.Large-scale structure of genomic methylation patterns.Genome Res,2006,16(2):157-163
[20]Hackett J A,Reddington J P,Nestor C E,et al.Promoter DNAmethylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline.Development,2012,139(19):3623-3632
[21]Roh T Y,Cuddapah S,Zhao K.Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping.Genes Dev,2005,19(5):542-552
[22]Weber M,Hellmann I,Stadler M B,et al.Distribution,silencing potential and evolutionary impact of promoter DNA methylation in the human genome.Nat Genet,2007,39(4):457-466
[23]Lovkvist C,Sneppen K,Haerter J O.Exploring the link between nucleosome occupancy and DNA methylation.Front Genet,2017,8:232
[24]Zhao S,Tian H,Ma L,et al.Activity-dependent modulation of odorant receptor gene expression in the mouse olfactory epithelium.PLoS One,2013,8(7):e69862
[25]Buck L,Axel R.A novel multigene family may encode odorant receptors:a molecular basis for odor recognition.Cell,1991,65(1):175-187
[26]Krautwurst D,Yau K W,Reed R R.Identification of ligands for olfactory receptors by functional expression of a receptor library.Cell,1998,95(7):917-926
[27]Gilad Y,Przeworski M,Lancet D.Loss of olfactory receptor genes coincides with the acquisition of full trichromatic vision in primates.PLoS Biology,2004,2(1):E5
[28]Laska M,Genzel D,Wieser A.The number of functional olfactory receptor genes and the relative size of olfactory brain structures are poor predi ctors of olfactory discrimination performance with enantiomers.Chemical Senses,2005,30(2):171-175
[29]Niimura Y,Nei M.Evolutionary dynamics of olfactory and other chemosensory receptor genes in vertebrates.Journal of Human Genetics,2006,51(6):505-517
[30]Mombaerts P.Odorant receptor gene choice in olfactory sensory neurons:the one receptor-one neuron hypothesis revisited.Curr Opin Neurobiol,2004,14(1):31-36
[31]Bozza T,Feinstein P,Zheng C,et al.Odorant receptor expression defines functional units in the mouse olfactory system.Journal of Neuroscience,2002,22(8):3033-3043
[32]Rodriguez-Gil D J,Bartel D L,Jaspers A W,et al.Odorant receptors regulate the final glomerular coalescence of olfactory sensory neuron axons.Proc Natl Acad Sci USA,2015,112(18):5821-5826
[33]Secundo L,Snitz K,Sobel N.The perceptual logic of smell.Current Opinion in Neurobiology,2014,25(4):107-115
[34]Zou D J,Chesler A,Firestein S.How the olfactory bulb got its glomeruli:a just so story?.Nature Reviews Neuroscience,2009,10(8):611-618
[35]Wang Z,Balet Sindreu C,Li V,et al.Pheromone detection in male mice depends on signaling through the type 3 adenylyl cyclase in the main olfactory epithelium.J Neurosci,2006,26(28):7375-7379
[2]Johnson J L,Leroux M R.c AMP and c GMP signaling:sensory systems with prokaryotic roots adopted by eukaryotic cilia.Trends Cell Biol,2010,20(8):435-444
[3]Defer N,Bestbelpomme M,Hanoune J.Tissue specificity and physiological relevance of various isoforms of adenylyl cyclase.American Journal of Physiology Renal Physiology,2000,279(3):F400
[4]Wang Z,Zhou Y,Luo Y,et al.Gene Expression profiles of main olfactory epithelium in adenylyl cyclase 3 knockout mice.Int JMol Sci,2015,16(12):28320-28333
[5]Bird A.Perceptions of epigenetics.Nature,2007,447(7143):396-398
[6]Li E,Zhang Y.DNA methylation in mammals.Cold Spring Harb Perspect Biol,2014,6(5):a019133
[7]Suzuki M M,Bird A.DNA methylation landscapes:provocative insights from epigenomics.Nat Rev Genet,2008,9(6):465-476
[8]Zhang Z,Yang D,Zhang M,et al.Deletion of type 3 adenylyl cyclase perturbs the postnatal maturation of olfactory sensory neurons and olfactory cilium ultrastructure in mice.Front Cell Neurosci,2017,11:1
[9]周艳芬,韩绍芳,舒俐,等.腺苷酸环化酶3缺失对小鼠主要嗅觉表皮组织内相关因子及信号通路的影响.中国生物化学与分子生物学学报,2016,32(7):816-822Zhou Y F,Han S F,Shu L,et al.Chinese Journal of Biochemistry and Molecular Biology,2016,32(7):816-822
[10]周艳芬,王伟娜,袁焕娜等.腺苷酸环化酶3缺失下调小鼠嗅觉受体基因表达.中国生物化学与分子生物学报,2017,33(11):1143-1151Zhou Y F,Wang W N,Yuan H N,et al.Chinese Journal of Biochemistry and Molecular Biology,2017,33(11):1143-1151
[11]Wang Z,Storm D R.Extraction of DNA from mouse tails.Biotechniques,2006,41(4):410-412
[12]Weber M,Davies J J,Wittig D,et al.Chromosome-wide and promoter-specific analyses identify sites of differential DNAmethylation in normal and transformed human cells.Nat Genet,2005,37(8):853-862
[13]Meng H,Cao Y,Qin J,et al.DNA methylation,its mediators and genome integrity.International Journal of Biological Sciences,2015,11(5):604-617
[14]Schultz M D,He Y,Whitaker J W,et al.Corrigendum:human body epigenome maps reveal noncanonical DNA methylation variation.Nature,2016,530(7589):242
[15]Prokhortchouk E,Defossez P A.The cell biology of DNAmethylation in mammals.Biochim Biophys Acta,2008,1783(11):2167-2173
[16]Eckhardt F,Lewin J,Cortese R,et al.DNA methylation profiling of human chromosomes 6,20 and 22.Nat Genet,2006,38(12):1378-1385
[17]Illingworth R,Kerr A,Desousa D,et al.A novel CpG island set identifies tissue-specific methylation at developmental gene loci.PLoS Biol,2008,6(1):e22
[18]Han F,Dong Y,Liu W,et al.Epigenetic regulation of sox30 is associated with testis development in mice.PLoS One,2014,9(5):e97203
[19]Rollins R A,Haghighi F,Edwards J R,et al.Large-scale structure of genomic methylation patterns.Genome Res,2006,16(2):157-163
[20]Hackett J A,Reddington J P,Nestor C E,et al.Promoter DNAmethylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline.Development,2012,139(19):3623-3632
[21]Roh T Y,Cuddapah S,Zhao K.Active chromatin domains are defined by acetylation islands revealed by genome-wide mapping.Genes Dev,2005,19(5):542-552
[22]Weber M,Hellmann I,Stadler M B,et al.Distribution,silencing potential and evolutionary impact of promoter DNA methylation in the human genome.Nat Genet,2007,39(4):457-466
[23]Lovkvist C,Sneppen K,Haerter J O.Exploring the link between nucleosome occupancy and DNA methylation.Front Genet,2017,8:232
[24]Zhao S,Tian H,Ma L,et al.Activity-dependent modulation of odorant receptor gene expression in the mouse olfactory epithelium.PLoS One,2013,8(7):e69862
[25]Buck L,Axel R.A novel multigene family may encode odorant receptors:a molecular basis for odor recognition.Cell,1991,65(1):175-187
[26]Krautwurst D,Yau K W,Reed R R.Identification of ligands for olfactory receptors by functional expression of a receptor library.Cell,1998,95(7):917-926
[27]Gilad Y,Przeworski M,Lancet D.Loss of olfactory receptor genes coincides with the acquisition of full trichromatic vision in primates.PLoS Biology,2004,2(1):E5
[28]Laska M,Genzel D,Wieser A.The number of functional olfactory receptor genes and the relative size of olfactory brain structures are poor predi ctors of olfactory discrimination performance with enantiomers.Chemical Senses,2005,30(2):171-175
[29]Niimura Y,Nei M.Evolutionary dynamics of olfactory and other chemosensory receptor genes in vertebrates.Journal of Human Genetics,2006,51(6):505-517
[30]Mombaerts P.Odorant receptor gene choice in olfactory sensory neurons:the one receptor-one neuron hypothesis revisited.Curr Opin Neurobiol,2004,14(1):31-36
[31]Bozza T,Feinstein P,Zheng C,et al.Odorant receptor expression defines functional units in the mouse olfactory system.Journal of Neuroscience,2002,22(8):3033-3043
[32]Rodriguez-Gil D J,Bartel D L,Jaspers A W,et al.Odorant receptors regulate the final glomerular coalescence of olfactory sensory neuron axons.Proc Natl Acad Sci USA,2015,112(18):5821-5826
[33]Secundo L,Snitz K,Sobel N.The perceptual logic of smell.Current Opinion in Neurobiology,2014,25(4):107-115
[34]Zou D J,Chesler A,Firestein S.How the olfactory bulb got its glomeruli:a just so story?.Nature Reviews Neuroscience,2009,10(8):611-618
[35]Wang Z,Balet Sindreu C,Li V,et al.Pheromone detection in male mice depends on signaling through the type 3 adenylyl cyclase in the main olfactory epithelium.J Neurosci,2006,26(28):7375-7379