Dysregulation of genome-wide gene expression and DNA methylation in abnormal cloned piglets

BMC Genomics

Changchun Li³  Randall S Prather¹  Shuhong Zhao³  Melissa S Samuel¹  Mei Yu³  Xinyun Li³  Jianguo Zhao²  Qitao Jia²  Guanglei Li³ 

[1] Animal Science Research Center (ASRC), Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA;Group of Genetic modifications and establishment of biomedical models in large animals, The State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, People's Republic of China;Key Lab of Agriculture Animal Genetics, Breeding, and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China

关键词: Gene expression;    DNA methylation;    Piglets;    SCNT;   

Others  :  1140503
DOI  :  10.1186/1471-2164-15-811

 received in 2013-09-24, accepted in 2014-09-19,  发布年份 2014

【 摘 要 】

Background

Epigenetic modifications (especially altered DNA methylation) resulting in altered gene expression may be one reason for development failure or abnormalities in cloned animals, but the underlying mechanism of the abnormal phenotype in cloned piglets remains unknown. Some cloned piglets in our study showed abnormal phenotypes such as large tongue (longer and thicker), weak muscles, and exomphalos. Here we conducted DNA methylation (DNAm) immunoprecipitation and high throughput sequencing (MeDIP-seq) and RNA sequencing (RNA-seq) of muscle tissues of cloned piglets to investigate the relationship of abnormal DNAm with gene dysregulation and the unusual phenotypes in cloned piglets.

Results

Analysis of the methylomes revealed that abnormal cloned piglets suffered more hypomethylation than hypermethylation compared to the normal cloned piglets, although the DNAm level in the CpG Island was higher in the abnormal cloned piglets. Some repetitive elements, such as SINE/tRNA-Glu Satellite/centr also showed differences. We detected 1,711 differentially expressed genes (DEGs) between the two groups, of which 243 genes also changed methylation level in the abnormal cloned piglets. The altered DNA methylation mainly affected the low and silently expressed genes. There were differences in both pathways and genes, such as the MAPK signalling pathway, the hypertrophic cardiomyopathy pathway, and the imprinted gene PLAGL1; all of which may play important roles in development of the abnormal phenotype.

Conclusions

The abnormal cloned piglets showed substantial changes both in the DNAm and the gene expression. Our data may provide new insights into understanding the molecular mechanisms of the reprogramming of genetic information in cloned animals.

【 授权许可】

   
2014 Li et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Briggs R, King TJ: Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs’ Eggs. Proc Natl Acad Sci U S A 1952, 38(5):455-463.
• [2]Walker SC, Shin T, Zaunbrecher GM, Romano JE, Johnson GA, Bazer FW, Piedrahita JA: A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes. Cloning Stem Cells 2002, 4(2):105-112.
• [3]Cho SK, Kim JH, Park JY, Choi YJ, Bang JI, Hwang KC, Cho EJ, Sohn SH, Uhm SJ, Koo DB, Lee KK, Kim T, Kim JH: Serial cloning of pigs by somatic cell nuclear transfer: restoration of phenotypic normality during serial cloning. Dev Dyn 2007, 236(12):3369-3382.
• [4]Campbell KH, Alberio R, Choi I, Fisher P, Kelly RD, Lee JH, Maalouf W: Cloning: eight years after Dolly. Reprod Domest Anim 2005, 40(4):256-268.
• [5]Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, Ball S, Dai Y, Boone J, Walker S, Ayares DL, Colman A, Campbell KH: Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 2000, 407(6800):86-90.
• [6]Groenen MAM, Archibald AL, Uenishi H, Tuggle CK, Takeuchi Y, Rothschild MF, Rogel-Gaillard C, Park C, Milan D, Megens HJ, Li ST, Larkin DM, Kim H, Frantz LAF, Caccamo M, Ahn H, Aken BL, Anselmo A, Anthon C, Auvil L, Badaoui B, Beattie CW, Bendixen C, Berman D, Blecha F, Blomberg J, Bolund L, Bosse M, Botti S, Zhan BJ, et al.: Analyses of pig genomes provide insight into porcine demography and evolution. Nature 2012, 491(7424):393-398.
• [7]Prather RS: Pig genomics for biomedicine. Nat Biotechnol 2013, 31(2):122-124.
• [8]Walters EM, Wolf E, Whyte JJ, Mao J, Renner S, Nagashima H, Kobayashi E, Zhao J, Wells KD, Critser JK, Riley LK, Prather RS: Completion of the swine genome will simplify the production of swine as a large animal biomedical model. BMC Med Genomics 2012, 5:55.
• [9]Park KW, Lai L, Cheong HT, Cabot R, Sun QY, Wu G, Rucker EB, Durtschi D, Bonk A, Samuel M, Rieke A, Day BN, Murphy CN, Carter DB, Prather RS: Mosaic gene expression in nuclear transfer-derived embryos and the production of cloned transgenic pigs from ear-derived fibroblasts. Biol Reprod 2002, 66(4):1001-1005.
• [10]Jiang L, Jobst P, Lai L, Samuel M, Ayares D, Prather RS, Tian XC: Expression levels of growth-regulating imprinted genes in cloned piglets. Cloning Stem Cells 2007, 9(1):97-106.
• [11]Carter DB, Lai L, Park KW, Samuel M, Lattimer JC, Jordan KR, Estes DM, Besch-Williford C, Prather RS: Phenotyping of transgenic cloned piglets. Cloning Stem Cells 2002, 4(2):131-145.
• [12]Hwang KC, Cho SK, Lee SH, Park JY, Kwon DN, Choi YJ, Park C, Kim JH, Park KK, Hwang S, Park SB, Kim JH: Depigmentation of skin and hair color in the somatic cell cloned pig. Dev Dyn 2009, 238(7):1701-1708.
• [13]Weksberg R, Shuman C, Smith AC: Beckwith-Wiedemann syndrome. Am J Med Genet C: Semin Med Genet 2005, 137C(1):12-23.
• [14]Choufani S, Shuman C, Weksberg R: Beckwith-Wiedemann syndrome. Am J Med Genet C: Semin Med Genet 2010, 154C(3):343-354.
• [15]Tian XC, Park J, Bruno R, French R, Jiang L, Prather RS: Altered gene expression in cloned piglets. Reprod Fertil Dev 2009, 21(1):60-66.
• [16]Jaenisch R, Bird A: Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals. Nat Genet 2003, 33:245-254.
• [17]Bernstein BE, Meissner A, Lander ES: The Mammalian Epigenome. Cell 2007, 128(4):669-681.
• [18]Lewis JD, Meehan RR, Henzel WJ, Maurer-Fogy I, Jeppesen P, Klein F, Bird A: Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA. Cell 1992, 69(6):905-914.
• [19]Lee JH, Skalnik DG: CpG-binding protein is a nuclear matrix- and euchromatin-associated protein localized to nuclear speckles containing human trithorax. Identification of nuclear matrix targeting signals. J Biol Chem 2002, 277(44):42259-42267.
• [20]Ishikawa K, Fukuda E, Kobayashi I: Conflicts targeting epigenetic systems and their resolution by cell death: novel concepts for methyl-specific and other restriction systems. DNA Res 2010, 17(6):325-342.
• [21]Kang YK, Koo DB, Park JS, Choi YH, Kim HN, Chang WK, Lee KK, Han YM: Typical demethylation events in cloned pig embryos. Clues on species-specific differences in epigenetic reprogramming of a cloned donor genome. J Biol Chem 2001, 276(43):39980-39984.
• [22]Peat JR, Reik W: Incomplete methylation reprogramming in SCNT embryos. Nat Genet 2012, 44(9):965-966.
• [23]Dean W: Conservation of methylation reprogramming in mammalian development: Aberrant reprogramming in cloned embryos. Proc Natl Acad Sci 2001, 98(24):13734-13738.
• [24]Wei Y, Zhu J, Huan Y, Liu Z, Yang C, Zhang X, Mu Y, Xia P: Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets. Cell Reprogram 2010, 12(2):213-222.
• [25]Mann MR, Chung YG, Nolen LD, Verona RI, Latham KE, Bartolomei MS: Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos. Biol Reprod 2003, 69(3):902-914.
• [26]Feber A, Wilson GA, Zhang L, Presneau N, Idowu B, Down TA, Rakyan VK, Noon LA, Lloyd AC, Stupka E, Schiza V, Teschendorff AE, Schroth GP, Flanagan A, Beck S: Comparative methylome analysis of benign and malignant peripheral nerve sheath tumors. Genome Res 2011, 21(4):515-524.
• [27]Gu YR, Li MZ, Zhang K, Chen L, Jiang AA, Wang JY, Li XW: Evaluation of endogenous control genes for gene expression studies across multiple tissues and in the specific sets of fat- and muscle-type samples of the pig. J Anim Breed Genet 2011, 128(4):319-325.
• [28]Jacinto FV, Ballestar E, Esteller M: Methyl-DNA immunoprecipitation (MeDIP): hunting down the DNA methylome. Biotechniques 2008, 44(1):35. 37, 39 passim
• [29]Marioni JC, Mason CE, Mane SM, Stephens M, Gilad Y: RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res 2008, 18(9):1509-1517.
• [30]Cullum R, Alder O, Hoodless PA: The next generation: using new sequencing technologies to analyse gene regulation. Respirology 2011, 16(2):210-222.
• [31]Gao F, Luo Y, Li S, Li J, Lin L, Nielsen AL, Sorensen CB, Vajta G, Wang J, Zhang X, Du Y, Yang H, Bolund L: Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls. PLoS One 2011, 6(10):e25901.
• [32]Park J, Marjani SL, Lai L, Samuel M, Wax D, Davis SR, Bruno RS, Prather RS, Yang X, Tian XC: Altered gene expression profiles in the brain, kidney, and lung of deceased neonatal cloned pigs. Cell Reprogram 2010, 12(5):589-597.
• [33]Li M, Wu H, Luo Z, Xia Y, Guan J, Wang T, Gu Y, Chen L, Zhang K, Ma J, Liu Y, Zhong Z, Nie J, Zhou S, Mu Z, Wang X, Qu J, Jing L, Wang H, Huang S, Yi N, Wang Z, Xi D, Wang J, Yin G, Wang L, Li N, Jiang Z, Lang Q, Xiao H, et al.: An atlas of DNA methylomes in porcine adipose and muscle tissues. Nat Commun 2012, 3:850.
• [34]Xiang H, Zhu J, Chen Q, Dai F, Li X, Li M, Zhang H, Zhang G, Li D, Dong Y, Zhao L, Lin Y, Cheng D, Yu J, Sun J, Zhou X, Ma K, He Y, Zhao Y, Guo S, Ye M, Guo G, Li Y, Li R, Zhang X, Ma L, Kristiansen K, Guo Q, Jiang J, Beck S, et al.: Single base-resolution methylome of the silkworm reveals a sparse epigenomic map. Nat Biotechnol 2010, 28(5):516-520.
• [35]Zhang X, Yazaki J, Sundaresan A, Cokus S, Chan SW, Chen H, Henderson IR, Shinn P, Pellegrini M, Jacobsen SE, Ecker JR: Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 2006, 126(6):1189-1201.
• [36]Bird A, Taggart M, Frommer M, Miller OJ, Macleod D: A fraction of the mouse genome that is derived from islands of nonmethylated. CpG-rich DNA. Cell 1985, 40(1):91-99.
• [37]Niemann H, Tian XC, King WA, Lee RS: Epigenetic reprogramming in embryonic and foetal development upon somatic cell nuclear transfer cloning. Reproduction 2008, 135(2):151-163.
• [38]Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B: Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 2008, 5(7):621-628.
• [39]Audic S, Claverie JM: The significance of digital gene expression profiles. Genome Res 1997, 7(10):986-995.
• [40]Laurent L, Wong E, Li G, Huynh T, Tsirigos A, Ong CT, Low HM, Kin Sung KW, Rigoutsos I, Loring J, Wei CL: Dynamic changes in the human methylome during differentiation. Genome Res 2010, 20(3):320-331.
• [41]Wilmut I, Beaujean N, de Sousa PA, Dinnyes A, King TJ, Paterson LA, Wells DN, Young LE: Somatic cell nuclear transfer. Nature 2002, 419(6907):583-586.
• [42]Yang X, Smith SL, Tian XC, Lewin HA, Renard JP, Wakayama T: Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning. Nat Genet 2007, 39(3):295-302.
• [43]Chung YG, Eum JH, Lee JE, Shim SH, Sepilian V, Hong SW, Lee Y, Treff NR, Choi YH, Kimbrel EA, Dittman RE, Lanza R, Lee DR: Human somatic cell nuclear transfer using adult cells. Cell Stem Cell 2014, 14(6):777-780.
• [44]Lee K, Kwon DN, Ezashi T, Choi YJ, Park C, Ericsson AC, Brown AN, Samuel MS, Park KW, Walters EM: Engraftment of human iPS cells and allogeneic porcine cells into pigs with inactivated RAG2 and accompanying severe combined immunodeficiency. Proc Natl Acad Sci U S A 2014, 111(20):7260-7265.
• [45]Yamanaka K, Kaneda M, Inaba Y, Saito K, Kubota K, Sakatani M, Sugimura S, Imai K, Watanabe S, Takahashi M: DNA methylation analysis on satellite I region in blastocysts obtained from somatic cell cloned cattle. Anim Sci J 2011, 82(4):523-530.
• [46]Mason K, Liu Z, Aguirre-Lavin T, Beaujean N: Chromatin and epigenetic modifications during early mammalian development. Anim Reprod Sci 2012, 134(1–2):45-55.
• [47]Oswald J, Engemann S, Lane N, Mayer W, Olek A, Fundele R, Dean W, Reik W, Walter J: Active demethylation of the paternal genome in the mouse zygote. Curr Biol 2000, 10(8):475-478.
• [48]Renard J, Chastant S, Chesne P, Richard C, Marchal J, Cordonnier N, Chavatte P, Vignon X: Lymphoid hypoplasia and somatic cloning. Lancet 1999, 353(9163):1489-1491.
• [49]Park J, Lai L, Samuel M, Wax D, Bruno RS, French R, Prather RS, Yang X, Tian XC: Altered gene expression profiles in the brain, kidney, and lung of one-month-old cloned pigs. Cell Reprogram 2011, 13(3):215-223.
• [50]Deshmukh RS, Ostrup O, Ostrup E, Vejlsted M, Niemann H, Lucas-Hahn A, Petersen B, Li J, Callesen H, Hyttel P: DNA methylation in porcine preimplantation embryos developed in vivo and produced by in vitro fertilization, parthenogenetic activation and somatic cell nuclear transfer. Epigenetics 2011, 6(2):177-187.
• [51]Bourc’his D, Le Bourhis D, Patin D, Niveleau A, Comizzoli P, Renard JP, Viegas-Pequignot E: Delayed and incomplete reprogramming of chromosome methylation patterns in bovine cloned embryos. Curr Biol 2001, 11(19):1542-1546.
• [52]Chan MM, Smith ZD, Egli D, Regev A, Meissner A: Mouse ooplasm confers context-specific reprogramming capacity. Nat Genet 2012, 44(9):978-980.
• [53]Cadieux B, Ching TT, VandenBerg SR, Costello JF: Genome-wide hypomethylation in human glioblastomas associated with specific copy number alteration, methylenetetrahydrofolate reductase allele status, and increased proliferation. Cancer Res 2006, 66(17):8469-8476.
• [54]Shapiro JA, von Sternberg R: Why repetitive DNA is essential to genome function. Biol Rev 2005, 80(2):227-250.
• [55]Rideout WM 3rd, Eggan K, Jaenisch R: Nuclear cloning and epigenetic reprogramming of the genome. Science 2001, 293(5532):1093-1098.
• [56]Hemberger M, Dean W, Reik W: Epigenetic dynamics of stem cells and cell lineage commitment: digging Waddington’s canal. Nat Rev Mol Cell Biol 2009, 10(8):526-537.
• [57]Pai AA, Bell JT, Marioni JC, Pritchard JK, Gilad Y: A genome-wide study of DNA methylation patterns and gene expression levels in multiple human and chimpanzee tissues. PLoS Genet 2011, 7(2):e1001316.
• [58]Bonk AJ, Li R, Lai L, Hao Y, Liu Z, Samuel M, Fergason EA, Whitworth KM, Murphy CN, Antoniou E, Prather RS: Aberrant DNA methylation in porcine in vitro-, parthenogenetic-, and somatic cell nuclear transfer-produced blastocysts. Mol Reprod Dev 2008, 75(2):250-264.
• [59]Park MR, Im GS, Kim SW, Hwang S, Park JH, Kim H, Do YJ, Park SB, Yang BS, Song YM, Cho JH, Ko YG: Aberrant gene expression patterns in extraembryonic tissue from cloned porcine embryos. Res Vet Sci 2013, 94(3):531-538.
• [60]Jaenisch R, Hochedlinger K, Blelloch R, Yamada Y, Baldwin K, Eggan K: Nuclear cloning, epigenetic reprogramming, and cellular differentiation. Cold Spring Harb Symp Quant Biol 2004, 69:19-27.
• [61]Lui JC, Finkielstain GP, Barnes KM, Baron J: An imprinted gene network that controls mammalian somatic growth is down-regulated during postnatal growth deceleration in multiple organs. Am J Physiol Regul Integr Comp Physiol 2008, 295(1):R189-R196.
• [62]Bliek J, Verde G, Callaway J, Maas SM, De Crescenzo A, Sparago A, Cerrato F, Russo S, Ferraiuolo S, Rinaldi MM, Fischetto R, Lalatta F, Giordano L, Ferrari P, Cubellis MV, Larizza L, Temple IK, Mannens MMAM, Mackay DJG, Riccio A: Hypomethylation at multiple maternally methylated imprinted regions including PLAGL1 and GNAS loci in Beckwith–Wiedemann syndrome. Eur J Hum Genet 2008, 17(5):611-619.
• [63]Niederhoffer KY, Penaherrera M, Pugash D, Rupps R, Arbour L, Tessier F, Choufani S, Zhao C, Manokhina I, Shuman C, Robinson WP, Weksberg R, Boerkoel CF: Beckwith-Wiedemann syndrome in sibs discordant for IC2 methylation. Am J Med Genet A 2012, 158A(7):1662-1669.
• [64]Gu T, Su X, Zhou Q, Li X, Yu M, Ding Y, Zhao S, Li C: Molecular characterization of the Neuronatin gene in the porcine placenta. PLoS One 2012, 7(8):e43325.
• [65]Tilley RE, McNeil CJ, Ashworth CJ, Page KR, McArdle HJ: Altered muscle development and expression of the insulin-like growth factor system in growth retarded fetal pigs. Domest Anim Endocrinol 2007, 32(3):167-177.
• [66]Vadivelu SK, Kurzbauer R, Dieplinger B, Zweyer M, Schafer R, Wernig A, Vietor I, Huber LA: Muscle regeneration and myogenic differentiation defects in mice lacking TIS7. Mol Cell Biol 2004, 24(8):3514-3525.
• [67]Micheli L, Leonardi L, Conti F, Buanne P, Canu N, Caruso M, Tirone F: PC4 coactivates MyoD by relieving the histone deacetylase 4-mediated inhibition of myocyte enhancer factor 2C. Mol Cell Biol 2005, 25(6):2242-2259.
• [68]Wang L, Lei M, Xiong Y: Molecular characterization and different expression patterns of the muscle ankyrin repeat protein (MARP) family during porcine skeletal muscle development in vitro and in vivo. Anim Biotechnol 2011, 22(2):87-99.
• [69]Chen YW, Nader GA, Baar KR, Fedele MJ, Hoffman EP, Esser KA: Response of rat muscle to acute resistance exercise defined by transcriptional and translational profiling. J Physiol 2002, 545(Pt 1):27-41.
• [70]Hawke TJ, Atkinson DJ, Kanatous SB, Van der Ven PF, Goetsch SC, Garry DJ: Xin, an actin binding protein, is expressed within muscle satellite cells and newly regenerated skeletal muscle fibers. Am J Physiol Cell Physiol 2007, 293(5):C1636-C1644.
• [71]Kong Y, Flick MJ, Kudla AJ, Konieczny SF: Muscle LIM protein promotes myogenesis by enhancing the activity of MyoD. Mol Cell Biol 1997, 17(8):4750-4760.
• [72]Barash IA, Mathew L, Lahey M, Greaser ML, Lieber RL: Muscle LIM protein plays both structural and functional roles in skeletal muscle. Am J Physiol Cell Physiol 2005, 289(5):C1312-C1320.
• [73]Lee JH, Campbell KH: Effects of enucleation and caffeine on maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activities in ovine oocytes used as recipient cytoplasts for nuclear transfer. Biol Reprod 2006, 74(4):691-698.
• [74]Zhang W, Liu HT: MAPK signal pathways in the regulation of cell proliferation in mammalian cells. Cell Res 2002, 12(1):9-18.
• [75]Li J, Deng J, Yu S, Zhang J, Cheng D, Wang H: The virtual element in proximal promoter of porcine myostatin is regulated by myocyte enhancer factor 2C. Biochem Biophys Res Commun 2012, 419(2):175-181.
• [76]Dalkilic I, Schienda J, Thompson TG, Kunkel LM: Loss of FilaminC (FLNc) results in severe defects in myogenesis and myotube structure. Mol Cell Biol 2006, 26(17):6522-6534.
• [77]Lai L, Prather RS: Production of cloned pigs by using somatic cells as donors. Cloning Stem Cells 2003, 5(4):233-241.
• [78]Benjamini Y, Yekutieli D: The control of the false discovery rate in multiple testing under dependency. Ann Stat 2001, 29:1165-1188.
• [79]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25(4):402-408.
• [80]Chen H, Li C, Fang M, Zhu M, Li X, Zhou R, Li K, Zhao S: Understanding Haemophilus parasuis infection in porcine spleen through a transcriptomics approach. BMC Genomics 2009, 10:64.