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山羊PHLDA2启动子克隆和甲基化分析
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Abstract:
为了研究山羊血小板白细胞C激酶底物同源性样结构域蛋白家族A成员2 (pleckstrin homology-like domain family A member 2, PHLDA2)基因表达水平与其启动子甲基化状态相关性，试验采用RT-PCR方法检测PHLDA2在各组织间的表达水平(心脏、肝脏、脾脏、肺脏、肾脏、肌肉、脂肪、舌、大脑、胎盘)，以及扩增了山羊PHLDA2基因启动子区域序列，并利用Methylation specific PCR技术检测CpG岛在山羊各组织间的甲基化水平。结果表明：克隆得到的山羊PHLDA2 1842 bp的启动子序列与绵羊、牛PHLDA2启动子同源性分别达93.2%和87.9%；启动区CpG岛内第三位点CpG的甲基化频率和胎盘第一外显子区域甲基化水平显著低于其他组织(P < 0.05)。说明PHLDA2的mRNA相对表达量水平与启动子区CpG岛(？302~？88 bp)内第三个CpG位点甲基化频率和第一外显子CpG岛(49~333 bp)甲基化水平呈负相关。
In order to investigate the correlation between gene expression level of pleckstrin homology-like domain family A member 2 (PHLDA2) and its promoter methylation status of goat, RT-PCR was applied to detect the expression level of PHLDA2 in various tissues (heart, liver, spleen, lung, kidney, muscle, fat, tongue, brain and placenta) and clone the sequence of PHLDA2 gene promoter region of goat. Methylation specific PCR was used to detect the methylation level of CpG island in goat tissues. The results showed that the cloned goat PHLDA2 1842 bp promoter sequence had 93.2% homology with sheep and 87.9% homology with cattle PHLDA2 promoter, respectively, and the methylation frequency of CpG at the third site of CpG in the promoter region and the methylation level of the first exon of placenta were significantly lower than those of other tissues (P < 0.05). These results suggest that the mRNA relative expression level of PHLDA2 was negatively correlated with the methylation frequency of the third CpG site and the methylation level of the first exon CpG island (49~333 bp) in the promoter region CpG island (？302~？88 bp).
| [1] | Moore, G.E., Ishida, M., Demetriou, C., et al. (2015) The Role and Interaction of Imprinted Genes in Human Fetal Growth. Philosophical Transactions of the Royal Society B: Biological Sciences, 370, Article ID: 20140074.
https://doi.org/10.1098/rstb.2014.0074 |
| [2] | Vallet, J.L., Miles, J.R. and Freking, B.A. (2009) Development of the Pig Placenta. Society of Reproduction and Fertility Supplement, 66, 265-279. |
| [3] | Millership, S.J., Van de Pette, M. and Withers, D.J. (2019) Genomic Imprinting and Its Effects on Postnatal Growth and Adult Metabolism. Cellular and Mo-lecular Life Sciences, 76, 4009-4021.
https://doi.org/10.1007/s00018-019-03197-z |
| [4] | Tunster, S.J., Jensen, A.B. and John, R.M. (2013) Imprinted Genes in Mouse Placental Development and the Regulation of Fetal Energy Stores. Reproduction, 145, R117-R137. https://doi.org/10.1530/REP-12-0511 |
| [5] | Qian, N., Frank, D., O’keefe, D., et al. (1997) The IPL Gene on Chro-mosome 11p15.5 Is Imprinted in Humans and Mice and Is Similar to TDAG51, Implicated in Fas Expression and Apoptosis. Human Molecular Genetics, 6, 2021-2029. https://doi.org/10.1093/hmg/6.12.2021 |
| [6] | Tunster, S.J., Tycko, B. and John, R.M. (2010) The Imprinted Phlda2 Gene Regulates Extraembryonic Energy Stores. Molecular and Cellular Biology, 30, 295-306. https://doi.org/10.1128/MCB.00662-09 |
| [7] | Janssen, A.B., Tunster, S.J., Heazell, A.E., et al. (2016) Placental PHLDA2 Expression Is Increased in Cases of Fetal Growth Restriction Following Reduced Fetal Movements. BMC Medical Genetics, 17, 17-21.
https://doi.org/10.1186/s12881-016-0279-1 |
| [8] | Xing, Y., Liu, H., Cui, Y., Wang, X., et al. (2019) Abundances of Placental Imprinted Genes CDKN1C, PHLDA2 and IGF-2 Are Related to Low Birth Weight and Early Catch-Up Growth in Full-Term Infants Born Small for Gestational Age. PLoS ONE, 14, e0218278. https://doi.org/10.1371/journal.pone.0218278 |
| [9] | Tunster, S.J., Van de Pette, M. and John, R.M. (2014) Isolating the Role of Elevated Phlda2 in Asymmetric Late Fetal Growth Restriction in Mice. Disease Models & Mechanisms, 7, 1185-1191. https://doi.org/10.1242/dmm.017079 |
| [10] | Tunster, S.J., Cretth, H.D.J. and John, R.M. (2016) The Im-printed Phlda2 Gene Modulates a Major Endocrine Compartment of the Placenta to Regulate Placental Demands for Ma-ternal Resources. Developmental Biology, 409, 251-260. https://doi.org/10.1016/j.ydbio.2015.10.015 |
| [11] | Sunamura, N., Ohira, T., Kataoka, M., et al. (2016) Regulation of Functional KCNQ1OT1 lncRNA by β-Catenin. Scientific Reports, 6, Article No. 20690. https://doi.org/10.1038/srep20690 |
| [12] | Sikore, K.M., Magee, D.A., Berkowicz, E.W., et al. (2012) PHLDA2 Is an Imprinted Gene in Cattle. Animal Genetics, 43, 587-590. https://doi.org/10.1111/j.1365-2052.2011.02292.x |
| [13] | Hou, S., Chen, Y., Liang, J., et al. (2010) Developmental Stage-Specific Imprinting of IPL in Domestic Pigs (Sus scrofa). Journal of Biomedicine and Biotechnology, 2010, Article ID: 527539. https://doi.org/10.1155/2010/527539 |
| [14] | 苏鲁方, 彭学强, 蒋曹德. 大足黑山羊PHLDA2基因的克隆、组织表达与印记状况分析[J]. 中国农业科学, 2015, 48(2): 343-351. |
| [15] | Guo, S.W. (2009) Epigenetics of Endometriosis. Molecular Human Reproduction, 15, 587-607.
https://doi.org/10.1093/molehr/gap064 |
| [16] | Nasu, K., Kawano, Y., Tsukamoto, Y., et al. (2011) Aberrant DNA Methylation Status of Endometriosis: Epigenetics as the Pathogenesis, Biomarker and Therapeutic Target. Journal of Ob-stetrics and Gynaecology Research, 37, 683-695.
https://doi.org/10.1111/j.1447-0756.2011.01663.x |
| [17] | Lister, R., Pelizzola, M., Dowen, R.H., et al. (2009) Hu-man DNA Methylomes at Base Resolution Show Widespread Epigenomic Differences. Nature, 462, 315-322. https://doi.org/10.1038/nature08514 |
| [18] | Bestor, T.H. (2000) The DNA Methyltransferases of Mammals. Human Molecular Genetics, 9, 2395-2402.
https://doi.org/10.1093/hmg/9.16.2395 |
| [19] | Ishida, M., Monk, D., Duncan, A.J., et al. (2012) Maternal Inheritance of a Promoter Variant in the Imprinted PHLDA2 Gene Significantly Increases Birth Weight. The American Journal of Human Genetics, 90, 715-719.
https://doi.org/10.1016/j.ajhg.2012.02.021 |
| [20] | Ng, H.H. and Bird, A. (1999) DNA Methylation and Chromatin Modification. Current Opinion in Genetics and Development, 9, 158-163. https://doi.org/10.1016/S0959-437X(99)80024-0 |
| [21] | Robertson, K.D. (2002) DNA Methylation and Chroma-tin-Unraveling the Tangled Web. Oncogene, 21, 5361-5379.
https://doi.org/10.1038/sj.onc.1205609 |
| [22] | Bamidele, O., Omitogun, O.G. and Mumorin, I.G. (2015) DNA Se-quence Characteristics and Phylogenetics of Putative Imprinted Genes on Bovine Chromosome 29. The Journal of Agri-cultural Science, 7, 131-142.
https://doi.org/10.5539/jas.v7n8p131 |
| [23] | Thiaville, M.M. and Kim, J. (2011) Oncogenic Potential of Yin Yang 1 Mediated through Control of Imprinted Genes. Critical Reviews in Oncogenesis, 16, 199-209. https://doi.org/10.1615/CritRevOncog.v16.i3-4.40 |
| [24] | Mele, M., Ferreira, P.G., Reverter, F., et al. (2015) Human Genomics. The Human Transcriptome across Tissues and Individuals. Science, 348, 660-665. https://doi.org/10.1126/science.aaa0355 |
| [25] | Weintraub, A., Li, C.H., Zamudio, A.V., et al. (2017) YY1 Is a Structural Regulator of Enhancer Promoter Loops. Cell, 171, 1573-1588. https://doi.org/10.1016/j.cell.2017.11.008 |
| [26] | Ohki, I., Shimotake, N., Fujita, N., et al. (2001) Solution Structure of the Methyl-CpG Binding Domain of Human MBD1 in Complex with Methylated DNA. Cell, 105, 487-497. https://doi.org/10.1016/S0092-8674(01)00324-5 |
| [27] | Anastasiadi, D., Esteve-Codina, A. and Piferrer, F. (2018) Consistent Inverse Correlation between DNA Methylation of the First Intron and Gene Expression across Tissues and Species. Epigenetics & Chromatin, 11, 37.
https://doi.org/10.1186/s13072-018-0205-1 |
