Hyldig Sara M W, Croxall Nicola, Contreras David A, Thomsen Preben D, Alberio Ramiro
Division of Animal Sciences, School of Biosciences, University of Nottingham, Loughborough, LE12 5RD, UK.
BMC Dev Biol. 2011 Feb 25;11:11. doi: 10.1186/1471-213X-11-11.
Epigenetic reprogramming is critical for genome regulation during germ line development. Genome-wide demethylation in mouse primordial germ cells (PGC) is a unique reprogramming event essential for erasing epigenetic memory and preventing the transmission of epimutations to the next generation. In addition to DNA demethylation, PGC are subject to a major reprogramming of histone marks, and many of these changes are concurrent with a cell cycle arrest in the G2 phase. There is limited information on how well conserved these events are in mammals. Here we report on the dynamic reprogramming of DNA methylation at CpGs of imprinted loci and DNA repeats, and the global changes in H3K27me3 and H3K9me2 in the developing germ line of the domestic pig.
Our results show loss of DNA methylation in PGC colonizing the genital ridges. Analysis of IGF2-H19 regulatory region showed a gradual demethylation between E22-E42. In contrast, DMR2 of IGF2R was already demethylated in male PGC by E22. In females, IGF2R demethylation was delayed until E29-31, and was de novo methylated by E42. DNA repeats were gradually demethylated from E25 to E29-31, and became de novo methylated by E42. Analysis of histone marks showed strong H3K27me3 staining in migratory PGC between E15 and E21. In contrast, H3K9me2 signal was low in PGC by E15 and completely erased by E21. Cell cycle analysis of gonadal PGC (E22-31) showed a typical pattern of cycling cells, however, migrating PGC (E17) showed an increased proportion of cells in G2.
Our study demonstrates that epigenetic reprogramming occurs in pig migratory and gonadal PGC, and establishes the window of time for the occurrence of these events. Reprogramming of histone H3K9me2 and H3K27me3 detected between E15-E21 precedes the dynamic DNA demethylation at imprinted loci and DNA repeats between E22-E42. Our findings demonstrate that major epigenetic reprogramming in the pig germ line follows the overall dynamics shown in mice, suggesting that epigenetic reprogramming of germ cells is conserved in mammals. A better understanding of the sequential reprogramming of PGC in the pig will facilitate the derivation of embryonic germ cells in this species.
表观遗传重编程对于生殖细胞发育过程中的基因组调控至关重要。小鼠原始生殖细胞（PGC）中的全基因组去甲基化是一种独特的重编程事件，对于消除表观遗传记忆和防止表观突变传递给下一代至关重要。除了DNA去甲基化，PGC还经历了组蛋白标记的主要重编程，其中许多变化与G2期的细胞周期停滞同时发生。关于这些事件在哺乳动物中的保守程度的信息有限。在这里，我们报告了家猪发育中的生殖细胞中印迹基因座和DNA重复序列的CpG位点处DNA甲基化的动态重编程，以及H3K27me3和H3K9me2的全局变化。
我们的结果显示，定殖于生殖嵴的PGC中DNA甲基化缺失。对IGF2-H19调控区域的分析显示，在E22-E42之间逐渐去甲基化。相比之下，IGF2R的DMR2在雄性PGC中到E22时已经去甲基化。在雌性中，IGF2R去甲基化延迟到E29-31，并在E42时重新甲基化。DNA重复序列从E25到E29-31逐渐去甲基化，并在E42时重新甲基化。组蛋白标记分析显示，在E15和E21之间的迁移PGC中有强烈的H3K27me3染色。相比之下，到E15时PGC中的H3K9me2信号较低，并在E21时完全消失。性腺PGC（E22-31）的细胞周期分析显示出典型的循环细胞模式，然而，迁移的PGC（E17）显示G2期细胞比例增加。
我们的研究表明，表观遗传重编程发生在家猪迁移和性腺PGC中，并确定了这些事件发生的时间窗口。在E15-E21之间检测到的组蛋白H3K9me2和H3K27me3的重编程先于E22-E42之间印迹基因座和DNA重复序列处的动态DNA去甲基化。我们的发现表明，猪生殖细胞中的主要表观遗传重编程遵循小鼠中显示的总体动态，这表明生殖细胞的表观遗传重编程在哺乳动物中是保守的。更好地了解猪中PGC的顺序重编程将有助于该物种胚胎生殖细胞的衍生。
