Maenohara Shoji, Unoki Motoko, Toh Hidehiro, Ohishi Hiroaki, Sharif Jafar, Koseki Haruhiko, Sasaki Hiroyuki
Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan.
PLoS Genet. 2017 Oct 4;13(10):e1007042. doi: 10.1371/journal.pgen.1007042. eCollection 2017 Oct.
The methylation of cytosine at CG sites in the mammalian genome is dynamically reprogrammed during gametogenesis and preimplantation development. It was previously shown that oocyte-derived DNMT1 (a maintenance methyltransferase) is essential for maintaining and propagating CG methylation at imprinting control regions in preimplantation embryos. In mammalian somatic cells, hemimethylated-CG-binding protein UHRF1 plays a critical role in maintaining CG methylation by recruiting DNMT1 to hemimethylated CG sites. However, the role of UHRF1 in oogenesis and preimplantation development is unknown. In the present study, we show that UHRF1 is mainly, but not exclusively, localized in the cytoplasm of oocytes and preimplantation embryos. However, smaller amounts of UHRF1 existed in the nucleus, consistent with the expected role in DNA methylation. We then generated oocyte-specific Uhrf1 knockout (KO) mice and found that, although oogenesis was itself unaffected, a large proportion of the embryos derived from the KO oocytes died before reaching the blastocyst stage (a maternal effect). Whole genome bisulfite sequencing revealed that blastocysts derived from KO oocytes have a greatly reduced level of CG methylation, suggesting that maternal UHRF1 is essential for maintaining CG methylation, particularly at the imprinting control regions, in preimplantation embryos. Surprisingly, UHRF1 was also found to contribute to de novo CG and non-CG methylation during oocyte growth: in Uhrf1 KO oocytes, transcriptionally-inactive regions gained less methylation, while actively transcribed regions, including the imprinting control regions, were unaffected or only slightly affected. We also found that de novo methylation was defective during the late stage of oocyte growth. To the best of our knowledge, this is the first study to demonstrate the role of UHRF1 in de novo DNA methylation in vivo. Our study reveals multiple functions of UHRF1 during the global epigenetic reprogramming of oocytes and early embryos.
在哺乳动物基因组中，CG位点处胞嘧啶的甲基化在配子发生和植入前发育过程中会动态重编程。先前的研究表明，卵母细胞来源的DNMT1（一种维持性甲基转移酶）对于在植入前胚胎的印记控制区域维持和传播CG甲基化至关重要。在哺乳动物体细胞中，半甲基化CG结合蛋白UHRF1通过将DNMT1募集到半甲基化的CG位点，在维持CG甲基化方面发挥关键作用。然而，UHRF1在卵子发生和植入前发育中的作用尚不清楚。在本研究中，我们发现UHRF1主要但并非仅局限于卵母细胞和植入前胚胎的细胞质中。然而，细胞核中存在少量的UHRF1，这与它在DNA甲基化中的预期作用一致。然后，我们构建了卵母细胞特异性Uhrf1基因敲除（KO）小鼠，发现尽管卵子发生本身未受影响，但来自KO卵母细胞的大部分胚胎在到达囊胚阶段之前死亡（一种母源效应）。全基因组亚硫酸氢盐测序显示，来自KO卵母细胞的囊胚CG甲基化水平大幅降低，这表明母源UHRF1对于在植入前胚胎中维持CG甲基化至关重要，尤其是在印记控制区域。令人惊讶的是，我们还发现UHRF1在卵母细胞生长过程中也有助于从头CG和非CG甲基化：在Uhrf1 KO卵母细胞中，转录不活跃区域的甲基化增加较少，而包括印记控制区域在内的活跃转录区域未受影响或仅受到轻微影响。我们还发现，在卵母细胞生长后期，从头甲基化存在缺陷。据我们所知，这是第一项证明UHRF1在体内从头DNA甲基化中作用的研究。我们的研究揭示了UHRF1在卵母细胞和早期胚胎的整体表观遗传重编程过程中的多种功能。
