DNA甲基化修饰与内源性miRNA对珠蛋白基因表达调控的研究
|
摘要
珠蛋白基因簇在个体发育中表现为依次表达,并具有高度的组织特异性和发育阶段特异性。珠蛋白基因的转录受染色质结构、编码基因与其旁侧序列甲基化程度、顺式调节元件及细胞内反式作用因子的调节控制。Locus control regiongs (LCRs,基因座控制区)在功能上被定义为一类发育中的哺乳动物红细胞α珠蛋白和β珠蛋白基因表达所必需的上游调控序列。LCR可提高与其相衔接的基因的表达并具有组织特异型和拷贝数依赖的功能元件,是最重要调节珠蛋白基因表达的一类顺式作用元件。有关珠蛋白基因组织特异性表达的各种研究和理论模型中,都暗示了珠蛋白表观遗传修饰在调控珠蛋白基因的特异性表达中的重要功能。由于缺乏合适的实验模型,以往有关组蛋白修饰与珠蛋白基因表达关心的研究大多在细胞系中进行,因而无法重现珠蛋白基因的组织特异性表达。本论文利用先前构建的由LCR的HS3-2元件和β-珠蛋白基因启动子驱动表达EGFP报告基因的小鼠模型,对DNA的差异性甲基化修饰和内源性的miRNA在珠蛋白基因的特异性表达中的作用进行了研究。
首先我们运用定量RT-PCR的技术检测了报告基因在不同组织中的表达水平;在该模型小鼠呈现良好的组织特异性表达的基础上,应用DNA亚硫酸盐修饰的方法,测定了人LCR和β-珠蛋白启动子区域在转基因小鼠各组织中的甲基化水平差异,以揭示DNA的甲基化修饰在组织特异性表达中的作用。这部分工作获得的主要进展是:
1.以人珠蛋白基因的LCR和启动子驱动表达EGFP的转基因小鼠在红系组织中高效表达人珠蛋白基因,是在活体水平研究珠蛋白基因表达调控的良好模型;
2.运用亚硫酸盐测序的方法,对转基因小鼠不同组织中珠蛋白基因的LCR和启动子区的CpG的甲基化状态进行了检测和比较。结果证实,对应的CpG位点在6种红系组织和非红系组织中的甲基化程度和模式有显著差异。对于区域的甲基化程度,红系组织中较非红系组织中低,而这种较低的甲基化修饰与报告基因EGFP的活跃转录对应;
3.运用定量RT-PCR技术,对三种DNA甲基化酶在转基因小鼠各组织内的表达进行了比较分析。结果显示,“起始性”(de novo)的Dnmt3a和Dnmt3b在红系组织和非红系组织中的表达模式有显著差异,而“维持性”(maintenance)的甲基化酶Dnmt1的表达则没有明显的红系特异性。这不仅是DNA甲基化修饰影响珠蛋白基因的表达的直接证据,也说明珠蛋白基因的甲基化修饰是Dnmt3a和Dnmt3b作用的结果;
4珠蛋白基因的启动子甲基化存在偏爱性,有若干个甲基化化敏感位点。这些位点是一些转录因子的调节区域。表观遗传修饰可能是作用于转录因子和DNA的结合来调节珠蛋白基因的表达。
为了探讨转基因小鼠报告基因EGFP组织特异性表达是否与内源性miRNA的功能相关,本研究对靶向作用于人珠蛋白基因的内源性的miRNA进行了初步研究。应用生物信息学和实验验证的方法,找到了针对珠蛋白基因3’UTR区的25个小鼠内源性miRNA,并对其中5个miRNA进行了较深入的分析。结果显示,miR-34c和miR-379与组织中EGFP mRNA及Dnmt3a和Dnmt3b转录本的水平密切相关。由于转基因小鼠各组织中EGFP的表达水平与该组织中的甲基化状态相关,因此推论组织特异性表达的转基因可能被内源性的miRNA所诱发,进而发生转录本的抑制和甲基化修饰,导致其表达水平呈现红系特异性的特征。
Human globin gene cluster shows erythroid tissue-specific and developmental stages-specific expression. The transcription of globin genes is controlled by the chromatin structure, methylation status of the coding sequence and the flanking regions, cis-regulatory elements and trans-factors. Locus control region (LCR) is the most important cis-element in regulation ofβ-globin gene expression. Several models (including looping, tracking, linking, topologic alterations, and modification of proteins associated with chromatin) have been proposed to explain the mechanism of humanβ-globin tissue-specific expression. All the models, directly or indirectly, implicate the methylation pattern of DNA and dynamic chromatin configuration. We previously generated a transgenic mouse strain, in which the spatial and hematopoietic-specific expression of the transgene (enhanced green fluorescent protein, EGFP) is driven by the humanβ-globin promoter and under controlled of LCR elements (HS2-HS3). The present study extends the previous work to further investigate the mechanism of tissue-specific expression of the gene.
To explore the potential role of DNA methylation in expression of humanβ-globin, we analyzed the EGFP mRNA expression profiles among tissues of the transgenic mice using real-time RT-PCR. The results showed that the EGFP expression was highly specific in hematopoietic tissues. We then investigated the methylation status of the LCR and promoter regions by using sodium bisulfite genomic sequencing. These researches provided significant clues to elucidate the mechanism of the regulation on tissue-specific expression of genes. The main progresses are as follow:
1. Expression patterns of the plasmid HS3-2-βP-EGFP-βE are different among various tissues of the transgenic mice. This hematopoietic-specific transgenic mouse model can be used to investigate the mechanism of regulation of humanβ-globin gene expression in vivo.
2). The methylation status of HS3-2 elements in LCR as well asβ-globin promoter with upstream region was analyzed. The results showed that all CpGs of the LCR and promoter region in each hematopoietic tissue revealed a tendency toward a hypomethylation pattern, while there is a hypermethylation pattern in non-hematopoietic tissues. The results also demonstrated that different levels of the expression of EGFP transgene in hematopoietic and non-hematopoietic tissues were tightly correlated with different methylated patterns of theβ-globin LCR and promoter.
3. Differential expression patterns of Dnmt3a and Dnmt3b, but not Dnmt1, in hematopietic and non-henatopoietic tissues, that corresponding to the EGFP expression. It provided the evidence that the differ expression of Dnmt3a and Dnmt3b may constitute one of the mechanisms of the methylations status at CpG sites inβ-globin LCR and promoter regions of various tissues.
4) Methylation frequencies at some CpG sites ofβ-globin LCR and promoter region were more predominant, and the methylation status was biased along the CpG positions. The nearer the transcription start site of the transgene plasmid, the higher the methylation frequencies were. The transcription initiation complex plays a critical role in methylation and demethylation, possibly be interfering with the methylation modification after DNA replication.
We also investigate whether the transgene tissue-specific expression may be related with the functions of the endogenous microRNA. The 3’-UTR region of humanβ-globin gene was searched for complimentarily with the existing human miRNAs from miRBase using RNAhybrid software, and 25 miRNAs were identified to be complementary to 3′-UTR region of humanβ-globin. The analysis of the expression patterns of 5 miRNAs among tissues showed that miR-34c and miR-379 expression profiles were closely correlated with the target EGFP mRNA as well as Dnmt3a and Dnmt3b transcripts in LCR elements andβ-globin promoter. Since the expression of the transgene (EGFP) was tightly linked with the methylation status of the individual tissues, we therefore deduced that the differential expression of the transgene in each tissue may be induced by the endogenous miRNAs, causing the differention of the transcription and methylation modification and then impacting on the gene expression.
首先我们运用定量RT-PCR的技术检测了报告基因在不同组织中的表达水平;在该模型小鼠呈现良好的组织特异性表达的基础上,应用DNA亚硫酸盐修饰的方法,测定了人LCR和β-珠蛋白启动子区域在转基因小鼠各组织中的甲基化水平差异,以揭示DNA的甲基化修饰在组织特异性表达中的作用。这部分工作获得的主要进展是:
1.以人珠蛋白基因的LCR和启动子驱动表达EGFP的转基因小鼠在红系组织中高效表达人珠蛋白基因,是在活体水平研究珠蛋白基因表达调控的良好模型;
2.运用亚硫酸盐测序的方法,对转基因小鼠不同组织中珠蛋白基因的LCR和启动子区的CpG的甲基化状态进行了检测和比较。结果证实,对应的CpG位点在6种红系组织和非红系组织中的甲基化程度和模式有显著差异。对于区域的甲基化程度,红系组织中较非红系组织中低,而这种较低的甲基化修饰与报告基因EGFP的活跃转录对应;
3.运用定量RT-PCR技术,对三种DNA甲基化酶在转基因小鼠各组织内的表达进行了比较分析。结果显示,“起始性”(de novo)的Dnmt3a和Dnmt3b在红系组织和非红系组织中的表达模式有显著差异,而“维持性”(maintenance)的甲基化酶Dnmt1的表达则没有明显的红系特异性。这不仅是DNA甲基化修饰影响珠蛋白基因的表达的直接证据,也说明珠蛋白基因的甲基化修饰是Dnmt3a和Dnmt3b作用的结果;
4珠蛋白基因的启动子甲基化存在偏爱性,有若干个甲基化化敏感位点。这些位点是一些转录因子的调节区域。表观遗传修饰可能是作用于转录因子和DNA的结合来调节珠蛋白基因的表达。
为了探讨转基因小鼠报告基因EGFP组织特异性表达是否与内源性miRNA的功能相关,本研究对靶向作用于人珠蛋白基因的内源性的miRNA进行了初步研究。应用生物信息学和实验验证的方法,找到了针对珠蛋白基因3’UTR区的25个小鼠内源性miRNA,并对其中5个miRNA进行了较深入的分析。结果显示,miR-34c和miR-379与组织中EGFP mRNA及Dnmt3a和Dnmt3b转录本的水平密切相关。由于转基因小鼠各组织中EGFP的表达水平与该组织中的甲基化状态相关,因此推论组织特异性表达的转基因可能被内源性的miRNA所诱发,进而发生转录本的抑制和甲基化修饰,导致其表达水平呈现红系特异性的特征。
Human globin gene cluster shows erythroid tissue-specific and developmental stages-specific expression. The transcription of globin genes is controlled by the chromatin structure, methylation status of the coding sequence and the flanking regions, cis-regulatory elements and trans-factors. Locus control region (LCR) is the most important cis-element in regulation ofβ-globin gene expression. Several models (including looping, tracking, linking, topologic alterations, and modification of proteins associated with chromatin) have been proposed to explain the mechanism of humanβ-globin tissue-specific expression. All the models, directly or indirectly, implicate the methylation pattern of DNA and dynamic chromatin configuration. We previously generated a transgenic mouse strain, in which the spatial and hematopoietic-specific expression of the transgene (enhanced green fluorescent protein, EGFP) is driven by the humanβ-globin promoter and under controlled of LCR elements (HS2-HS3). The present study extends the previous work to further investigate the mechanism of tissue-specific expression of the gene.
To explore the potential role of DNA methylation in expression of humanβ-globin, we analyzed the EGFP mRNA expression profiles among tissues of the transgenic mice using real-time RT-PCR. The results showed that the EGFP expression was highly specific in hematopoietic tissues. We then investigated the methylation status of the LCR and promoter regions by using sodium bisulfite genomic sequencing. These researches provided significant clues to elucidate the mechanism of the regulation on tissue-specific expression of genes. The main progresses are as follow:
1. Expression patterns of the plasmid HS3-2-βP-EGFP-βE are different among various tissues of the transgenic mice. This hematopoietic-specific transgenic mouse model can be used to investigate the mechanism of regulation of humanβ-globin gene expression in vivo.
2). The methylation status of HS3-2 elements in LCR as well asβ-globin promoter with upstream region was analyzed. The results showed that all CpGs of the LCR and promoter region in each hematopoietic tissue revealed a tendency toward a hypomethylation pattern, while there is a hypermethylation pattern in non-hematopoietic tissues. The results also demonstrated that different levels of the expression of EGFP transgene in hematopoietic and non-hematopoietic tissues were tightly correlated with different methylated patterns of theβ-globin LCR and promoter.
3. Differential expression patterns of Dnmt3a and Dnmt3b, but not Dnmt1, in hematopietic and non-henatopoietic tissues, that corresponding to the EGFP expression. It provided the evidence that the differ expression of Dnmt3a and Dnmt3b may constitute one of the mechanisms of the methylations status at CpG sites inβ-globin LCR and promoter regions of various tissues.
4) Methylation frequencies at some CpG sites ofβ-globin LCR and promoter region were more predominant, and the methylation status was biased along the CpG positions. The nearer the transcription start site of the transgene plasmid, the higher the methylation frequencies were. The transcription initiation complex plays a critical role in methylation and demethylation, possibly be interfering with the methylation modification after DNA replication.
We also investigate whether the transgene tissue-specific expression may be related with the functions of the endogenous microRNA. The 3’-UTR region of humanβ-globin gene was searched for complimentarily with the existing human miRNAs from miRBase using RNAhybrid software, and 25 miRNAs were identified to be complementary to 3′-UTR region of humanβ-globin. The analysis of the expression patterns of 5 miRNAs among tissues showed that miR-34c and miR-379 expression profiles were closely correlated with the target EGFP mRNA as well as Dnmt3a and Dnmt3b transcripts in LCR elements andβ-globin promoter. Since the expression of the transgene (EGFP) was tightly linked with the methylation status of the individual tissues, we therefore deduced that the differential expression of the transgene in each tissue may be induced by the endogenous miRNAs, causing the differention of the transcription and methylation modification and then impacting on the gene expression.
引文
1. Van Driel R, Fransz PF, Verschure PJ. The eukaryotic genome:a system regulated at different hierarchical level. J Cell Sci, 2003, 116:4067-4075
2. Jones PA , L aird PW. Cancer epigenetics comes of age. Nat Genet, 1999, 21 (2) : 163- 167.
3. Sugimura T , U shijima T. Genetic and epigenetic alterations in carcinogenesis. Mutation Res, 2000, 462 (2) : 235- 246.
4. Klose R J,Bird A P. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci, 2006, 31 (2): 89297.
5. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development, Science,2001, 293 :1089–1093.
6. M. Ehrlich, Expression of various genes is controlled by DNA methylation during mammalian development, J Cell Biochem,2003, 88 :. 899–910
7. Masaki Okano, Daphne W. Bell, Daniel A. Haber, En Li. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 1999, 99:247-252
8. Aviva Levine Fridman, Rita Rosati, Qunfang Li,Michael A Tainsky. Epigentic and functional analysis of IGFBP# and IGFBPrP1 in cellular immortalization. BBRC.2007;357:785-791
9. O.I. Kulaeva, S. Draghici L. Tang, J.M. Kraniak, S.J. Land, M.A. Tainsky. Epigenetic silencing of multiple interferon pathway genes after celluar immortalizatation. Oncogene. 2003;22:4118-4127
10.曾溢滔.人类血红蛋白,北京,科学出版社,2002,1-16
11. Li Q, Harju S, Peterson KR. Locus control regions: conning of age at a decade plus. Trans Genet. 1999; 15:403-408
12. Engel JD, Tanimoto K. Looping, linking, and chromatin activity: new insights into beta-globin locus regulation. Cell. 2000;100:499-502
13. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell.2004;116:281-297
14. Sood P, Krek A, Zavolan M, Msvino G, Rajuwsky N. Cell-type-specific signaturesof microRNAs on target mRNA expression. PNAS. 2006;103:2746-2751
15. Thermann R., Hentze MW. Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation Nature.2007;447:875-878
16. Wakiyama M, Takinoto K, Ohara O, Yokoyama S. Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system. Genes Dev. 2007;21:1587-1862
17. Sood P, Krek A, Zavolan M, Msvino G, Rajuwsky N. Cell-type-specific signatures of microRNAs on target mRNA expression. PNAS. 2006;103:2746-2751
18. Babak T, Zhang W, Mossis Q, Blencowe BJ, Hugahes TR, Probing microRNAs with microarrays: tissue specificity and functional inference. RNA. 2004;10:1813-1819
19. Jia CP, Huang SZ, Yan JB, Xiao YP, Ren ZR, Zeng YT. Effects of human locus control region elements HS2 and HS3 on human beta-globin expression in transgenic mouse. BLOOD CELL MOL DIS. 2003;31:360-369
1. Jia CP, Huang SZ, Yan JB, Xiao YP, Ren ZR, Zeng YT. Effects of human locus control region elements HS2 and HS3 on human beta-globin expression in transgenic mouse. Blood Cell, Molecular Disease. 2003;31:360-369
2.贾春平,颜景斌,曾溢滔等.红系特异表达载体在转基因小鼠中表达的研究,自然科学进展,2003,703-708
3. Qiliang Li, Kenneth R. Peterson, Xiangdong Fang. Locus control regions. 2002, Blood, 2002, 100( 9) :3077-3086
4. Qiliang Li, Susanna Harju, Kenneth R Peterson. locus control regions: coming of age at a decade plus. Trends Genet. 1999,15(10): 403-408
5.M. A. Bender, Michael Bulger, Jennie Close.β-globin Gene Switching and DNase I sensitivity of the Endogenousβ-globin locus in mice do not require the locus control region. Molecular Cell. 2000,5 (2):387-393
6. Reik A, Telling A, Zitnik G., et al. Theβ-globin LCR is not necessary for an open chromatin structure or developmentally Regulated Transcription of the native mouseβ-Globin Locus. Molecular and Cellular Biology. 1998,2 ( 4) 447-455
7. V.K. Rakyan , S. Beck. Epigenetic variation and inheritance in mammals, Curr Opin Genet Dev .2006,16:573–577
1. V.K. Rakyan , S. Beck. Epigenetic variation and inheritance in mammals. Curr Opin Genet Dev .2006,16:573-577
2. Klose R.J , Bird A.P. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci .2006,31 :89-97
3. Bernstein B.E. The mammalian epigenome. Cell. 2007, 128 : 669-681.
4. Timothy H. B. The DNA methyltransferases of mammals. Human Molecular Genetics. 2000, 16: 2395-2402
5. M.Okano, D.W.Bell, D.A.Haber,E.Li. DNA methyltransgferase Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 1999,99: 247-257
6. B.H. Ramsahoye . Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. PNAS. 2000, 97:5237-5242
7.Rhee I, Bachman KE, Park BH, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. 2002, 416(6880):552-556.
8. Frommer M, McDonald LE, Millar DS, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl Acad. Sci. USA.1992, 89:1827–1831
9.Herman J G, Graff J R, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl Acad. Sci. USA. 1996, 93: 9821–9826.
10. Snow K, Doud L K, Hagerman R, et al. Analysis of a CGG sequence at the FMR-1 locus in fragile X families and in the general population. American Journal of Human Genetics. 1993, 53 (6):1217-1228.
11. Cottrell S E, Laird P W. Sensitive detection of DNA methylation. Ann. NY Acad, Sci. 2003, 983:120-130.
12. Christina D ,Per G. DNA methylation analysis techniques. Biogerontology. 2003, 4 (4):233-250
13. Gonzalgo M L , Jones P A . Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Research. 1997,12: 2529-2531
14. Xiong Z., Laird P W. COBRA: A sensitive and quantitative DNA methylation assay. Nucleic Acids Research.1997,25 (12):2532-2534
15. Eads C A, Danenberg K D, Kawakami K, et al. Methylight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res, 2000,28(8)E32
16. Rand K, Qu W, Ho T, et al. Conversion-specific detection of DNA methylation using real-time polymerase chain reaction(ConLight-MSP) to avoid false posives. Methods, 2002,27(2):114-120
17. Nicola Harte, Ville Silventoinen, Emmanuel Quevillon,et al. Public web-based services from the European Bioinformatics Institute. Nucleic Acids Research. 2004, 32:W3-W9
18. Long-Cheng Li , Rajvir Dahiya. MethPrimer: Designing primers for methylation PCRs. Bioinformatics,2002, 18 (11):1427-1431
19. Eads C A, Danenberg K D, Kawakamik, et al. Methylation: a high-throughput assay to measure DNA methylation. Nucleic Acids Res.2000,28:E32
20. Tamura G, Yin J,Wang S,et al. E-Cadherin gene promoter hypermethylation in primary human gastric carcinomas. J Natl Cancer Inst, 2000,92:569-573
1. Lee R C, Feinbaum R L, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 1993, 75(5):843-854.
2. Bartel D P. MiRNA: genomics, biogenesis,mechanism and function. Cell, 2004,116(2):281–297.
3. Ambros V, Bartel B, Bartel D P, et al. A uniforn system for microRNA annotation. RNA,2003, 9(3): 277-279.
4. Berezikov E, Cuppen E, Plasterk R H. Approaches to microRNA discovery. Nature Genet,2006,38 suppl:S2-S7.
5. Ambros V. The functions of animal miRNA. Nature,2004,431:.350-355.
6. Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human miRNA . Nature Genet,2005, 37(7): 766-770.
7. Kim V N. Small RNAs: classification, biogenesis, and function. Mol Cells,2005, 19(1):1-15.
8. Kim V N, Nam J W. Genomics of microRNA. Trends in Genetics,2006, 22(3):165-173.
9. Gregory R I, Yan K P, Amuthan G, et al. The microprocessor complex mediates the genesis of miRNA. Nature,2004, 432,235–240.
10.Lund E, Guttinger S, Calado A, et al. Nuclear export of microRNA precursors. Science,2004, 303, 95–98.
11. Valencia-Sanchez M A, Liu J, Hannon G J, et al. Control of teanslation and mRNA degradation by miRNA and siRNAs. Genes Dev,2006, 20(5):515-524.
12. Ying S Y, Lin S L. Current perspectives in intronic microRNAs(miRNA). Journal of Biomedical Science,2006, 13:5-15.
13. Lin H, Gregory J H. MiRNA: Small RNAs with a big role in gene regulation. Nature,2006,5:522-531.
14. Aurora E K , Frank J S. Oncomis-miRNA with a role in cancer. Nature,2006,6:259-269.
15. Pillai R S, Bhattacharyya S N, Artus C G, et al. Inhibition of translational initiation by let-7 microRNA in human cells. Science,2005, 309,1573–1576.
16. Li M, Jones-Rhoades M W, Lau N C, et al. Regulatory mutations of mir-48, a C. elegans let-7 family MicroRNA, cause developmental timing defects. Dev Cell,2005,9(3):415-422.
17. Yoo A S, Greenwald I. LIN-12/Notch activation leads to microRNA-mediated down-regulation of Vav in C. elegans. Science,2005, 310:1330-1333.
18. Richard W C. Gene regulation by miRNA. Current Opinion in Genetics & Delvelopment ,2006,16:203-208.
19. Brown B D, Venner M A, Zingale A, et al. Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nature Medicine,2006, 12(5):585-591.
20. Royo H, Bortolin M L, Seitz H, et al. Small non-conding RNAs and genomicimprinting. Cytogenet Genome Res,2006, 113(1-4):99-108.
21. Ligang W, Jihua F, Joel G B. MicroRNAs direct rapid deadenylation of mRNA. PNAS,2006,103:4034-4039.
22. Hidenori I, Yutaka F, Isaac S K. Lower expression of genes near microRNA in C. elegans germiline.BMC Bioinformatics,2006,7:1-8
23. Doench J G, Sharp P A. Specificity of microRNA target selection in translational repression. Genes & Dev,2004,18(5):504-511.
24. Kloosterrman W P, Wienholds E, Ketting R F, et al. Substrate requriments for let-7 function in the developing zebrafish embryo. Nucleic Acids Res,2004, 32(21):6284-6291.
25. Haley B, Zamore P D. Kinetic analysis of the RNAi enzyme complex. Nature.Struct & Mol Biol, 2004,11(7):599-606.
26. Jing Q, Huang S, Guth S, Zarubin T, et al. Involvementof microRNA in AU-rich element-mediated mRNA instability. Cell,2005,120: 623–634.
27. Fillipowicz W, Jaskiewicz L. Post-transcription gene silencing by siRNAs and miRNA. Current Opinion in Structural Biology,2005,15:331-341.
28. Berezikov E, Guryev V, Van de B J, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell,2005, 120(1):21–24.
29. Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human miRNA. Nature Genet,2005,37(7):766–770.
30. Krek A, Grun D, Poy M N, et al. Combinatorial microRNA target predictions. Nature Genet,2005,37(5), 495–500.
31.Rajewsky N. microRNA target prediction in animals. Nature Genet,2006,38:S8-S13.
32. Jan K, Matthew N P, Markus S. Strategies to determine the biological function of microRNAs. Nature Genet,2006,38:S14-19.
33. Rehmsmeier M, Steffen P, Hochsmann M, et al. Fast and effective prediction of microRNA/target duplexs. RNA, 2004,10:1570-1571
34. Enright A J, John B, Gaul U, et al. MicroRNA targets in Drosophila. Genome Biol, 2003, 5:R1
35. Kiriakidou M, Nelson P T, Kouranov A, et al. A combined computational -experimental approach predicts human microRNA targets, Genes Dev,2004,18:1165-1178
36. Lewis B P, Burge C B, Bartel D P. Conserved seed pairing, often flanked byadenosines, indicates that thousands of human genes are microRNA targets. Cell, 2005,120;15-20
37. Rusinov V, Baev V, Minkov I N, et al. MicroInpector: a wev tool for detection of miRNA binding sites in an RNA sequence. Nucleic Acids Res, 2005,33:W696-W700
38. Krek A, Grun D, Poy M N, et al. Combinatorial microRNA target predictions Nat Genet, 2005,37:495-500
39. Saetrom O, Ola Snove J, Saetrom P. Weighted sequence motifs as an improved seeding step in microRNA target prediction algorithms. RNA, 200511:995-1003
40. Saetrom P, Predicting the efficacy of short oligonucletides in antisense and RNAi exeriments with boosted genetic programming. Bioinformatics, 2004, 20:3055-3063
41. Kim S K, Nam J W, Rhee J K, et al. MiTarget: microRNA target gene predication using a support vector machine, BMC bioinform, 2006,7:411
42. Zuker M, Mfold web server for nucleic and folding and bybridization prediction prediction. Nucleic Acids Res,2003,31:3406-3415
43. Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs. Science, 2001, 294:853-858
44. Lee R C, Ambros V. An extensive class of small RNAs in Caenorhabditis elesgans. Science, 2001,294:862-864
45. Lau N C, Lim L P, Weinstein E G, et al. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elesgans. Science, 2001, 294;858-862
46. John B, Enright A J, Aravin J, Ford LP, Antisense inhibition of human miRNA and indications for an involvement of miRNA in cell growth and apotosis, Nucleic Acids Res. 2005,33(4):1290-1297.
47. Barad O,Meiri E, Avniel A et al. MicroRNA expression detected by oligonucleotide microarrays: systemestablishment and expression profiling in human tissues. Genome Res 2004;14912),2486-2494
48. Shi R,Chiang V L. Facile means for quantifying microRNA expression by reat-time PCR. Biotechiques,2005,39:519-525
49. Hanjiang Fu, Yi Tie, Chengwang Xu, Zhuoyuan Zhang, et al. Identification of human fetal liver miRNAs by a novel method. FEBS, 2005,579:3849-3854
50. Chen C, Riazon D Broomer A J, Real-time quantification of miRNA by stem-loopRT-PCR. Nucleic Acids Res, 2005,33,3179
51. Rasymond C K, Robert B S, Garrett-Engele P et al. Simple quantitative primer-extension PCR assay for direct monitoring of microRNA ad short interfering RNAs. RNA, 2005, 11:1737-1744
52. Shi R,Chiang V L. Facile means for quantifying microRNA expression by reat-time PCR. Biotechiques,2005,39:519-525
53. Lasse S, Tabea H, Philipp B, Dimos G, Fabio M, , et al .MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nature Structural & Molecular Biology, 2008, 15: 259 - 267
54. Anja M. Duursma, M K, Mariette S, et al. miR-148 targets human DNMT3b protein coding region. RNA, 2008,14,872-877.
2. Jones PA , L aird PW. Cancer epigenetics comes of age. Nat Genet, 1999, 21 (2) : 163- 167.
3. Sugimura T , U shijima T. Genetic and epigenetic alterations in carcinogenesis. Mutation Res, 2000, 462 (2) : 235- 246.
4. Klose R J,Bird A P. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci, 2006, 31 (2): 89297.
5. Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development, Science,2001, 293 :1089–1093.
6. M. Ehrlich, Expression of various genes is controlled by DNA methylation during mammalian development, J Cell Biochem,2003, 88 :. 899–910
7. Masaki Okano, Daphne W. Bell, Daniel A. Haber, En Li. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 1999, 99:247-252
8. Aviva Levine Fridman, Rita Rosati, Qunfang Li,Michael A Tainsky. Epigentic and functional analysis of IGFBP# and IGFBPrP1 in cellular immortalization. BBRC.2007;357:785-791
9. O.I. Kulaeva, S. Draghici L. Tang, J.M. Kraniak, S.J. Land, M.A. Tainsky. Epigenetic silencing of multiple interferon pathway genes after celluar immortalizatation. Oncogene. 2003;22:4118-4127
10.曾溢滔.人类血红蛋白,北京,科学出版社,2002,1-16
11. Li Q, Harju S, Peterson KR. Locus control regions: conning of age at a decade plus. Trans Genet. 1999; 15:403-408
12. Engel JD, Tanimoto K. Looping, linking, and chromatin activity: new insights into beta-globin locus regulation. Cell. 2000;100:499-502
13. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell.2004;116:281-297
14. Sood P, Krek A, Zavolan M, Msvino G, Rajuwsky N. Cell-type-specific signaturesof microRNAs on target mRNA expression. PNAS. 2006;103:2746-2751
15. Thermann R., Hentze MW. Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation Nature.2007;447:875-878
16. Wakiyama M, Takinoto K, Ohara O, Yokoyama S. Let-7 microRNA-mediated mRNA deadenylation and translational repression in a mammalian cell-free system. Genes Dev. 2007;21:1587-1862
17. Sood P, Krek A, Zavolan M, Msvino G, Rajuwsky N. Cell-type-specific signatures of microRNAs on target mRNA expression. PNAS. 2006;103:2746-2751
18. Babak T, Zhang W, Mossis Q, Blencowe BJ, Hugahes TR, Probing microRNAs with microarrays: tissue specificity and functional inference. RNA. 2004;10:1813-1819
19. Jia CP, Huang SZ, Yan JB, Xiao YP, Ren ZR, Zeng YT. Effects of human locus control region elements HS2 and HS3 on human beta-globin expression in transgenic mouse. BLOOD CELL MOL DIS. 2003;31:360-369
1. Jia CP, Huang SZ, Yan JB, Xiao YP, Ren ZR, Zeng YT. Effects of human locus control region elements HS2 and HS3 on human beta-globin expression in transgenic mouse. Blood Cell, Molecular Disease. 2003;31:360-369
2.贾春平,颜景斌,曾溢滔等.红系特异表达载体在转基因小鼠中表达的研究,自然科学进展,2003,703-708
3. Qiliang Li, Kenneth R. Peterson, Xiangdong Fang. Locus control regions. 2002, Blood, 2002, 100( 9) :3077-3086
4. Qiliang Li, Susanna Harju, Kenneth R Peterson. locus control regions: coming of age at a decade plus. Trends Genet. 1999,15(10): 403-408
5.M. A. Bender, Michael Bulger, Jennie Close.β-globin Gene Switching and DNase I sensitivity of the Endogenousβ-globin locus in mice do not require the locus control region. Molecular Cell. 2000,5 (2):387-393
6. Reik A, Telling A, Zitnik G., et al. Theβ-globin LCR is not necessary for an open chromatin structure or developmentally Regulated Transcription of the native mouseβ-Globin Locus. Molecular and Cellular Biology. 1998,2 ( 4) 447-455
7. V.K. Rakyan , S. Beck. Epigenetic variation and inheritance in mammals, Curr Opin Genet Dev .2006,16:573–577
1. V.K. Rakyan , S. Beck. Epigenetic variation and inheritance in mammals. Curr Opin Genet Dev .2006,16:573-577
2. Klose R.J , Bird A.P. Genomic DNA methylation: the mark and its mediators. Trends Biochem Sci .2006,31 :89-97
3. Bernstein B.E. The mammalian epigenome. Cell. 2007, 128 : 669-681.
4. Timothy H. B. The DNA methyltransferases of mammals. Human Molecular Genetics. 2000, 16: 2395-2402
5. M.Okano, D.W.Bell, D.A.Haber,E.Li. DNA methyltransgferase Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 1999,99: 247-257
6. B.H. Ramsahoye . Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. PNAS. 2000, 97:5237-5242
7.Rhee I, Bachman KE, Park BH, et al. DNMT1 and DNMT3b cooperate to silence genes in human cancer cells. 2002, 416(6880):552-556.
8. Frommer M, McDonald LE, Millar DS, et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl Acad. Sci. USA.1992, 89:1827–1831
9.Herman J G, Graff J R, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl Acad. Sci. USA. 1996, 93: 9821–9826.
10. Snow K, Doud L K, Hagerman R, et al. Analysis of a CGG sequence at the FMR-1 locus in fragile X families and in the general population. American Journal of Human Genetics. 1993, 53 (6):1217-1228.
11. Cottrell S E, Laird P W. Sensitive detection of DNA methylation. Ann. NY Acad, Sci. 2003, 983:120-130.
12. Christina D ,Per G. DNA methylation analysis techniques. Biogerontology. 2003, 4 (4):233-250
13. Gonzalgo M L , Jones P A . Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Research. 1997,12: 2529-2531
14. Xiong Z., Laird P W. COBRA: A sensitive and quantitative DNA methylation assay. Nucleic Acids Research.1997,25 (12):2532-2534
15. Eads C A, Danenberg K D, Kawakami K, et al. Methylight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res, 2000,28(8)E32
16. Rand K, Qu W, Ho T, et al. Conversion-specific detection of DNA methylation using real-time polymerase chain reaction(ConLight-MSP) to avoid false posives. Methods, 2002,27(2):114-120
17. Nicola Harte, Ville Silventoinen, Emmanuel Quevillon,et al. Public web-based services from the European Bioinformatics Institute. Nucleic Acids Research. 2004, 32:W3-W9
18. Long-Cheng Li , Rajvir Dahiya. MethPrimer: Designing primers for methylation PCRs. Bioinformatics,2002, 18 (11):1427-1431
19. Eads C A, Danenberg K D, Kawakamik, et al. Methylation: a high-throughput assay to measure DNA methylation. Nucleic Acids Res.2000,28:E32
20. Tamura G, Yin J,Wang S,et al. E-Cadherin gene promoter hypermethylation in primary human gastric carcinomas. J Natl Cancer Inst, 2000,92:569-573
1. Lee R C, Feinbaum R L, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell, 1993, 75(5):843-854.
2. Bartel D P. MiRNA: genomics, biogenesis,mechanism and function. Cell, 2004,116(2):281–297.
3. Ambros V, Bartel B, Bartel D P, et al. A uniforn system for microRNA annotation. RNA,2003, 9(3): 277-279.
4. Berezikov E, Cuppen E, Plasterk R H. Approaches to microRNA discovery. Nature Genet,2006,38 suppl:S2-S7.
5. Ambros V. The functions of animal miRNA. Nature,2004,431:.350-355.
6. Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human miRNA . Nature Genet,2005, 37(7): 766-770.
7. Kim V N. Small RNAs: classification, biogenesis, and function. Mol Cells,2005, 19(1):1-15.
8. Kim V N, Nam J W. Genomics of microRNA. Trends in Genetics,2006, 22(3):165-173.
9. Gregory R I, Yan K P, Amuthan G, et al. The microprocessor complex mediates the genesis of miRNA. Nature,2004, 432,235–240.
10.Lund E, Guttinger S, Calado A, et al. Nuclear export of microRNA precursors. Science,2004, 303, 95–98.
11. Valencia-Sanchez M A, Liu J, Hannon G J, et al. Control of teanslation and mRNA degradation by miRNA and siRNAs. Genes Dev,2006, 20(5):515-524.
12. Ying S Y, Lin S L. Current perspectives in intronic microRNAs(miRNA). Journal of Biomedical Science,2006, 13:5-15.
13. Lin H, Gregory J H. MiRNA: Small RNAs with a big role in gene regulation. Nature,2006,5:522-531.
14. Aurora E K , Frank J S. Oncomis-miRNA with a role in cancer. Nature,2006,6:259-269.
15. Pillai R S, Bhattacharyya S N, Artus C G, et al. Inhibition of translational initiation by let-7 microRNA in human cells. Science,2005, 309,1573–1576.
16. Li M, Jones-Rhoades M W, Lau N C, et al. Regulatory mutations of mir-48, a C. elegans let-7 family MicroRNA, cause developmental timing defects. Dev Cell,2005,9(3):415-422.
17. Yoo A S, Greenwald I. LIN-12/Notch activation leads to microRNA-mediated down-regulation of Vav in C. elegans. Science,2005, 310:1330-1333.
18. Richard W C. Gene regulation by miRNA. Current Opinion in Genetics & Delvelopment ,2006,16:203-208.
19. Brown B D, Venner M A, Zingale A, et al. Endogenous microRNA regulation suppresses transgene expression in hematopoietic lineages and enables stable gene transfer. Nature Medicine,2006, 12(5):585-591.
20. Royo H, Bortolin M L, Seitz H, et al. Small non-conding RNAs and genomicimprinting. Cytogenet Genome Res,2006, 113(1-4):99-108.
21. Ligang W, Jihua F, Joel G B. MicroRNAs direct rapid deadenylation of mRNA. PNAS,2006,103:4034-4039.
22. Hidenori I, Yutaka F, Isaac S K. Lower expression of genes near microRNA in C. elegans germiline.BMC Bioinformatics,2006,7:1-8
23. Doench J G, Sharp P A. Specificity of microRNA target selection in translational repression. Genes & Dev,2004,18(5):504-511.
24. Kloosterrman W P, Wienholds E, Ketting R F, et al. Substrate requriments for let-7 function in the developing zebrafish embryo. Nucleic Acids Res,2004, 32(21):6284-6291.
25. Haley B, Zamore P D. Kinetic analysis of the RNAi enzyme complex. Nature.Struct & Mol Biol, 2004,11(7):599-606.
26. Jing Q, Huang S, Guth S, Zarubin T, et al. Involvementof microRNA in AU-rich element-mediated mRNA instability. Cell,2005,120: 623–634.
27. Fillipowicz W, Jaskiewicz L. Post-transcription gene silencing by siRNAs and miRNA. Current Opinion in Structural Biology,2005,15:331-341.
28. Berezikov E, Guryev V, Van de B J, et al. Phylogenetic shadowing and computational identification of human microRNA genes. Cell,2005, 120(1):21–24.
29. Bentwich I, Avniel A, Karov Y, et al. Identification of hundreds of conserved and nonconserved human miRNA. Nature Genet,2005,37(7):766–770.
30. Krek A, Grun D, Poy M N, et al. Combinatorial microRNA target predictions. Nature Genet,2005,37(5), 495–500.
31.Rajewsky N. microRNA target prediction in animals. Nature Genet,2006,38:S8-S13.
32. Jan K, Matthew N P, Markus S. Strategies to determine the biological function of microRNAs. Nature Genet,2006,38:S14-19.
33. Rehmsmeier M, Steffen P, Hochsmann M, et al. Fast and effective prediction of microRNA/target duplexs. RNA, 2004,10:1570-1571
34. Enright A J, John B, Gaul U, et al. MicroRNA targets in Drosophila. Genome Biol, 2003, 5:R1
35. Kiriakidou M, Nelson P T, Kouranov A, et al. A combined computational -experimental approach predicts human microRNA targets, Genes Dev,2004,18:1165-1178
36. Lewis B P, Burge C B, Bartel D P. Conserved seed pairing, often flanked byadenosines, indicates that thousands of human genes are microRNA targets. Cell, 2005,120;15-20
37. Rusinov V, Baev V, Minkov I N, et al. MicroInpector: a wev tool for detection of miRNA binding sites in an RNA sequence. Nucleic Acids Res, 2005,33:W696-W700
38. Krek A, Grun D, Poy M N, et al. Combinatorial microRNA target predictions Nat Genet, 2005,37:495-500
39. Saetrom O, Ola Snove J, Saetrom P. Weighted sequence motifs as an improved seeding step in microRNA target prediction algorithms. RNA, 200511:995-1003
40. Saetrom P, Predicting the efficacy of short oligonucletides in antisense and RNAi exeriments with boosted genetic programming. Bioinformatics, 2004, 20:3055-3063
41. Kim S K, Nam J W, Rhee J K, et al. MiTarget: microRNA target gene predication using a support vector machine, BMC bioinform, 2006,7:411
42. Zuker M, Mfold web server for nucleic and folding and bybridization prediction prediction. Nucleic Acids Res,2003,31:3406-3415
43. Lagos-Quintana M, Rauhut R, Lendeckel W, et al. Identification of novel genes coding for small expressed RNAs. Science, 2001, 294:853-858
44. Lee R C, Ambros V. An extensive class of small RNAs in Caenorhabditis elesgans. Science, 2001,294:862-864
45. Lau N C, Lim L P, Weinstein E G, et al. An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elesgans. Science, 2001, 294;858-862
46. John B, Enright A J, Aravin J, Ford LP, Antisense inhibition of human miRNA and indications for an involvement of miRNA in cell growth and apotosis, Nucleic Acids Res. 2005,33(4):1290-1297.
47. Barad O,Meiri E, Avniel A et al. MicroRNA expression detected by oligonucleotide microarrays: systemestablishment and expression profiling in human tissues. Genome Res 2004;14912),2486-2494
48. Shi R,Chiang V L. Facile means for quantifying microRNA expression by reat-time PCR. Biotechiques,2005,39:519-525
49. Hanjiang Fu, Yi Tie, Chengwang Xu, Zhuoyuan Zhang, et al. Identification of human fetal liver miRNAs by a novel method. FEBS, 2005,579:3849-3854
50. Chen C, Riazon D Broomer A J, Real-time quantification of miRNA by stem-loopRT-PCR. Nucleic Acids Res, 2005,33,3179
51. Rasymond C K, Robert B S, Garrett-Engele P et al. Simple quantitative primer-extension PCR assay for direct monitoring of microRNA ad short interfering RNAs. RNA, 2005, 11:1737-1744
52. Shi R,Chiang V L. Facile means for quantifying microRNA expression by reat-time PCR. Biotechiques,2005,39:519-525
53. Lasse S, Tabea H, Philipp B, Dimos G, Fabio M, , et al .MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells. Nature Structural & Molecular Biology, 2008, 15: 259 - 267
54. Anja M. Duursma, M K, Mariette S, et al. miR-148 targets human DNMT3b protein coding region. RNA, 2008,14,872-877.