Role of de novo DNA methyltransferases in initiation of genomic imprinting and X-chromosome inactivation
Role of de novo DNA methyltransferases in initiation of genomic imprinting and X-chromosome inactivation
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DOI：
10.1101/sqb.2004.69.125
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发表时间：
2004-01-01
期刊：
影响因子：
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通讯作者：
Sasaki, H
中科院分区：
文献类型：
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作者：
Kaneda, M;Sado, T;Sasaki, H
#现地址：马萨诸塞州剑桥市诺华生物医学研究所，02139。提出Dnmt3L可能通过从头甲基转移酶调控母体印迹。与这一模型一致，将[DNMT3A-/-，DNMT3B+/-]卵巢移植到野生型雌性体内后，未能建立卵母细胞特有的(母体)甲基化印记(Hata等人。2002年)。我们希望通过破坏雄性和雌性生殖细胞中的基因，更详细地研究DNMT3A和/或DNMT3B在印记起始步骤中的作用。一个很大的问题是，传统的DNMT3B基因敲除小鼠是胚胎致死的，而传统的DNMT3A基因敲除小鼠在达到生殖阶段之前大约3-4周死亡(Okano等人。1999年)。因此，我们无法检查这些小鼠的配子或它们衍生的后代。为了克服这个问题，我们利用了Cre-loxP条件敲除技术，以生殖细胞特有的方式破坏了Dnmt3基因，使大多数体细胞中的基因保持不变(图1)。我们使用了组织非特异性碱性磷酸酶(TNAP)-Cre敲门小鼠，它在E9的生殖细胞中表达Cre重组酶。5至妊娠晚期(Lomeli等人)2000)。虽然TNAP-Cre的表达不是严格意义上的生殖细胞特异性的，但我们可以获得能够存活到成年的条件性基因敲除小鼠([Dnmt3a2lox/1lox，TNAP-Cre]和[Dnmt3b2lox/1lox，TNAP-Cre]，其中2lox代表功能等位基因，1lox代表非功能等位基因)(图1)(Kaneda等人)。2004年)。当[Dnmt3a2lox/1lox，TNAP-Cre]雌性与野生型雄性杂交时，没有获得活的幼崽。随后的研究显示，所有胚胎都在胚胎10岁左右死亡。5有各种发育缺陷，如神经管开放，缺乏鳃弓，血液循环障碍(Kaneda等人)。2004年)。我们检测了E10中印迹基因的差异甲基化区域(DMRS)的甲基化状态。5个胚胎。通常在母体等位基因上甲基化的DMRS，如Snrpn、Igf2r和Peg1，被发现是未甲基化的(Kaneda等人。2004年)。相比之下，父系甲基化的H19和Rasgrf1DMRS的甲基化状态没有受到影响。然后，我们检查了母系印记基因在相同胚胎中的表达，发现p57Kip2(CDKN1c)和Igf2r是沉默的，这与正常活跃的母系等位基因的表达缺失一致(Kaneda等人)。2004年)。我们还发现，Peg1、Snrpn和Peg3的表达增加，而通常沉默的母体等位基因的表达降低(Kaneda等人)。2004年)。因此，DNMT3A条件突变雌性不能在母体印记的位置上建立卵母细胞特有的印记。[Dnmt3a2lox/1lox，TNAP-Cre]雄性显示精子发生受损(Kaneda等人)。2004年)。组织学检查显示，在出生后第11天，突变雄鼠的睾丸在生精小管中的精原细胞数量略有减少(P11)(Kaneda等人)。2004年)。然而，在11周龄时，它们只含有很少的精原细胞，没有精母细胞、精子细胞或精子(Kaneda等人。2004年)。由于无精子症，我们无法获得后代进行检查。因此，我们使用激光显微解剖技术从P11睾丸的组织切片中收集精原细胞，并通过亚硫酸氢盐测序分析了三个父系甲基化的精原细胞。我们发现DNMT3A条件突变雄性精原细胞在正常甲基化的H19DMR和Dlk1-Gtl2基因间隔区DMR缺乏甲基化(Kaneda等人)。2004年)。The…
# Present address: Novartis Institute for Biomedical Research, Cambridge, Massachusetts 02139. proposed that Dnmt3L may regulate maternal imprinting via the de novo methyltransferases. Consistent with this model, a [Dnmt3a–/–, Dnmt3b+/–] ovary transplanted into a wild-type female failed to establish the oocyte-specific (maternal) methylation imprints (Hata et al. 2002). We wanted to investigate the role of Dnmt3a and/or Dnmt3b in the initiation step of imprinting in more detail by disrupting the genes in male and female germ cells. A big problem was that the conventional Dnmt3b knockout mice are embryonic lethal and the conventional Dnmt3a knockout mice die around 3–4 weeks of age, before reaching the reproductive stage (Okano et al. 1999). We therefore could not examine the gametes of these mice or the offspring derived from them. To overcome this problem, we took advantage of the Cre-loxP conditional knockout technology and disrupted the Dnmt3 genes in a germ-cell-specific way, leaving the genes intact in most somatic cells (Fig. 1). We used the tissue nonspecific alkaline phosphatase (TNAP)-Cre knockin mice, which express the Cre recombinase in germ cells from E9. 5 to late gestation (Lomeli et al. 2000). Although expression of TNAP-Cre was not strictly germ-cell-specific, we could derive conditional knockout mice ([Dnmt3a2lox/1lox, TNAP-Cre] and [Dnmt3b2lox/1lox, TNAP-Cre], where 2lox represents the functional allele and 1lox represents the nonfunctional allele) that can survive to adulthood (Fig. 1)(Kaneda et al. 2004). When the [Dnmt3a2lox/1lox, TNAP-Cre] females were crossed with wild-type males, no live pups were obtained. Subsequent studies revealed that all embryos died around E10. 5 with various developmental defects, such as open neural tube, lack of branchial arches, and impediment of blood circulation (Kaneda et al. 2004). We examined the methylation status of the differentially methylated regions (DMRs) of the imprinted genes in the E10. 5 embryos. The DMRs normally methylated on the maternal allele, such as those of Snrpn, Igf2r, and Peg1, were found to be unmethylated (Kaneda et al. 2004). By contrast, the methylation status of the paternally methylated H19 and Rasgrf1 DMRs was unaffected. We then examined the expression of the maternally imprinted genes in the same embryos and found that p57kip2 (Cdkn1c) and Igf2r are silenced, consistent with a loss of expression from the normally active maternal alleles (Kaneda et al. 2004). We also found that expression of Peg1, Snrpn, and Peg3 is increased, with a derepression of the normally silent maternal alleles (Kaneda et al. 2004). Thus, the Dnmt3a conditional mutant females fail to establish the oocyte-specific imprints at the maternally imprinted loci. The [Dnmt3a2lox/1lox, TNAP-Cre] males showed impaired spermatogenesis (Kaneda et al. 2004). Histological examinations showed that the testes from the mutant males contain a slightly reduced number of spermatogonia in the seminiferous tubules at postnatal day 11 (P11)(Kaneda et al. 2004). However, at 11 weeks of age, they contained only few spermatogonia and no spermatocytes, spermatids, or spermatozoa (Kaneda et al. 2004). Because of the azoospermia, we could not obtain offspring to examine. We therefore used the laser-microdissection technology to collect spermatogonia from histological sections of the P11 testes and analyzed three paternally methylated DMRs by bisulfite sequencing. We found that the spermatogonia from the Dnmt3a conditional mutant males lacked methylation at the normally methylated H19 DMR and Dlk1-Gtl2 intergenic DMR (Kaneda et al. 2004). The …