Characterization of Differentially Methylated Regions in 3 Bovine Imprinted Genes: A Model for Studying Human Germ-Cell and Embryo Development
Characterization of Differentially Methylated Regions in 3 Bovine Imprinted Genes: A Model for Studying Human Germ-Cell and Embryo Development
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DOI：
10.1159/000322627
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发表时间：
2011-01-01
影响因子：
1.7
通讯作者：
Haaf, T.
中科院分区：
文献类型：
--
作者：
Hansmann, T.;Heinzmann, J.;Haaf, T.
正确的印记对哺乳动物胎儿和胎盘的正常发育至关重要。动物模型和人类流行病学研究的实验证据表明，辅助生殖技术（ARTs）可以干扰配子发生和早期胚胎发生中的印迹基因调控。金牛是一种重要的农业物种，通常使用抗逆转录病毒。由于该物种表现出与人类相似的着床前发育和妊娠期，因此越来越多地被用作人类生殖细胞和胚胎发育的模型。然而，与人类和小鼠相比，关于牛印迹基因的信息相对较少。在这里，我们表征了牛基因间IGF2-H19印迹控制区（ICR），该区域大约跨越3 kb。我们在H19启动子上游约6 kb处发现了一个300 bp的差异甲基化区（DMR），包含一个具有ctcf结合位点的CpG岛，与人类基因间ICR序列高度相似。其他差异甲基化的CpG岛位于启动子上游-6 kb至-3 kb，但这些岛不太保守。经典亚硫酸氢盐测序和焦磷酸亚硫酸氢盐测序均表明，IGF2-H19 ICR在精子中完全甲基化，在只有雌性基因组的孤雌胚胎中完全去甲基化，并且在胎盘和体细胞组织中存在差异甲基化。此外，我们建立了先前报道的牛SNRPN和PEG3 DMRs的焦磷酸测序方法。在所有分析的组织/细胞类型中，观察到的甲基化模式与基因组印迹一致。所鉴定的IGF2-H19 ICR和所建立的定量甲基化分析可能为进一步研究ARTs与牛模型印迹缺陷之间的关系提供帮助。版权所有：S. Karger AG，巴塞尔
Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie -6 kb to -3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model. Copyright (C) 2010 S. Karger AG, Basel