Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Wolynska Poznan, Poland.
Institute of Human Nutrition and Dietetics, Poznan University of Life Sciences, Wojska Polskiego, Poznan, Poland.
PLoS One. 2019 Apr 16;14(4):e0215471. doi: 10.1371/journal.pone.0215471. eCollection 2019.
The nutritional factors acting during early life can affect the development of the organism. It has been hypothesized that such programmed traits can be inherited by later generations. In this work, we present for the first time the effect of food deprivation in pregnant dams and its consequences for the transcription and DNA methylation profiles in the offspring of the next three generations. We used a 50% reduction in dietary intake during pregnancy in the rat and determined whether this altered the hepatic DNA methylation and transcription levels in female fetuses over three generations. Targeted next-generation sequencing (tNGS) was used in the first generation for 1748 genes associated with six selected biological processes. The selected cytosines were then studied by pyrosequencing in F1-F3. The transcript level of the selected genes was determined by the real-time PCR. The tNGS approach indicated 394 cytosines, in close proximity to 374 genes, with a statistically significant difference in methylation levels between the control and restricted groups. A gene clustering analysis revealed 23 molecular pathways to which the studied genes were assigned. Only seven cytosines were differently methylated to more than 10%, and so these sites were studied next using pyrosequencing. The observation from NGS was confirmed for only one cytosine located near the St6galnac5 gene, though this was with the opposite effect. A difference was also observed for the Usp30 gene, though in proximity to the cytosine selected from NGS. In F3, the differences were observed for the Oxct2b gene. We also found differences in methylation levels between generations for the Grb10 and St6galnac5 genes, but independently of the diet used. The transcript levels of selected genes (Usp30, Grb10, Pld1, St6galnac5, Oxct2b, Khk, and Acsl4) were not altered in F1, while changes were detected for Pld1 and Oxct2b in F2 and F3, respectively. Prenatal food deprivation did not induce broad changes in hepatic DNA methylation of the genes involved in lipid or carbohydrate metabolism, and did not result in alterations in their transcription. Thus, the hypothesis that transgenerational inheritance is induced by dietary restriction was not confirmed.
生命早期的营养因素会影响机体的发育。有人假设，这种编程特征可以遗传给后代。在这项工作中，我们首次展示了怀孕期间母体食物剥夺及其对下一代三个世代的后代转录和 DNA 甲基化谱的影响。我们使用了在怀孕大鼠中减少 50%的饮食摄入，并确定这是否改变了三代雌性胎儿肝脏的 DNA 甲基化和转录水平。在第一代中，使用靶向下一代测序（tNGS）对与六个选定生物过程相关的 1748 个基因进行了研究。然后在 F1-F3 中通过焦磷酸测序研究了选定的胞嘧啶。通过实时 PCR 测定所选基因的转录水平。tNGS 方法表明，在对照组和限制组之间，有 394 个接近 374 个基因的胞嘧啶在甲基化水平上有统计学意义的差异。基因聚类分析显示，研究的基因被分配到 23 个分子途径。只有 7 个胞嘧啶的甲基化程度超过 10%，因此接下来使用焦磷酸测序研究这些位点。只有一个位于 St6galnac5 基因附近的胞嘧啶与 NGS 的观察结果一致，但效果相反。Usp30 基因也观察到差异，尽管接近从 NGS 选择的胞嘧啶。在 F3 中，差异发生在 Oxct2b 基因。我们还发现 Grb10 和 St6galnac5 基因在不同世代之间的甲基化水平存在差异，但与所使用的饮食无关。在 F1 中，选定基因（Usp30、Grb10、Pld1、St6galnac5、Oxct2b、Khk 和 Acsl4）的转录水平没有改变，而在 F2 和 F3 中分别检测到 Pld1 和 Oxct2b 的变化。产前食物剥夺没有诱导参与脂质或碳水化合物代谢的基因的肝 DNA 甲基化发生广泛变化，也没有导致其转录发生变化。因此，饮食限制诱导跨代遗传的假设没有得到证实。
