CRL4调控的DNA去甲基化与去甲基化DNA结合蛋白CXXC1在小鼠受精卵基因组重编程中的作用

Epigenetic regulation is particularly important in germ cell development. Therefore, the genome reprogramming processes in fertilized eggs are ideal model systems to study the mechanisms of epigenetic regulation. Recently we reported that CRL4 ubiquitin E3 ligase activated TET family DNA demethylase, and regulated demethylation of male pronuclear DNAs in mouse zygotes. DNA binding protein CXXC1 is a key subunit of SET1 histone methyl-transferase, which recognizes the unmethylated CpG islands in genomes, and mediates the trymethylation of histone H3 at lysine-4 (H3K4me3) at specific chromatin regions. Our recent results showed that oocyte-specific knockout of Cxxc1 caused arrest of early embryo development at 1-cell or 2-cell stages. In this study, we aim to investigate the relationship between CRL4-regulated DNA demethylation and CXXC1 functions, as well as the functions and mechanisms of CRL4/CXXC1 in regulating genome reprogramming of mouse zygotes. We also aim to identify the target genes downstream of CRL4 and CXXC1, and give insights into the epigenetic regulation network during fertilization. This study will provide new knowledge to the molecular mechanisms of DNA methylation and demethylation.

表观遗传学调控在生殖细胞中尤为重要，受精卵基因组重编程过程是研究表观遗传调控机制的理想模型。我们发现 CRL4 泛素连接酶通过激活TET家族DNA去甲基化酶，调节受精卵雄原核去甲基化和体细胞克隆过程中基因组重编程，维持雌性生殖力。DNA结合蛋白CXXC1是SET1组蛋白甲基化酶的重要亚基，识别基因组中处于非甲基化状态的CpG岛，介导特定染色体区域组蛋白H3的第4位赖氨酸发生3甲基化（H3K4me3）。前期工作发现在卵母细胞中敲除Cxxc1造成胚胎发育一细胞到二细胞的阻滞。本课题将研究CRL4调控的DNA去甲基化与CXXC1之间的关系，进一步研究CRL4调控的DNA去甲基化与去甲基化DNA结合蛋白CXXC1在小鼠受精卵基因组重编程中功能和作用机制，并找到其下游调节的靶基因，以阐明受精过程中表观遗传调控的分子网络。本研究与重大研究计划中“DNA甲基化和去甲基化的分子机制”密切相关。

表观遗传学调控在生殖细胞中尤为重要，受精卵基因组重编程过程是研究表观遗传调控机制的理想模型。组蛋白H3的第4位赖氨酸发生3甲基化（H3K4me3）是一种常见的组蛋白修饰，通常位于真核生物的基因启动子区域，促进相应基因的转录激活。DNA结合蛋白CXXC1是SET1组蛋白甲基化酶的重要亚基，识别基因组中处于非甲基化状态的CpG岛，介导特定染色体区域组蛋白发生H3K4me3。为了验证在没有DNA复制情况下，H3K4me3调节组蛋白构象和基因转录激活的体内功能，我们在生长的卵母细胞中特异性敲除CXXC结合蛋白（CFP1）。卵母细胞中特异性敲除Cxxc1造成胚胎发育一细胞到二细胞阻滞。在卵母细胞成熟和受精卵基因激活过程中，CFP1对于H3K4me3的积累以及染色体构象的维持是必需的。CFP1作用的母源性基因参与减数分裂，母体-胚胎转换，和母源基因印记的建立。因此，卵母细胞中缺少CFP1不能完成卵母细胞成熟和受精后早期胚胎继续发育。我们的研究结果证明了体内条件下在没有DNA复制体系中，组蛋白H3K4me3修饰对于维持转录调控，染色体构象和正常的胚胎发育进程的重要作用。