Ma Xin, Zhang Sheng, Zhang Meiling, Zhu Yiran, Ma Panpan, Yang Shubao, Su Liyan, Li Ziyi, Lv Wenfa, Luan Weimin
1College of Animal Science and Technology,Jilin Agricultural University,Changchun,Jilin,China.
2State & Local Joint Engineering Laboratory for Animal Models of Human Diseases,Academy of Translational Medicine,First Hospital,Jilin University,Changchun,Jilin,China.
Zygote. 2018 Dec;26(6):449-456. doi: 10.1017/S0967199418000424. Epub 2019 Jan 23.
SummaryTRIM28/KAP1/TIF1β was identified as a universal transcriptional co-repressor and is critical for regulating post-fertilization methylation reprogramming in preimplantation embryos. In this study, three siRNAs (si647, si742, and si1153) were designed to target the TRIM28 mRNA sequence. After transfection of the mixture of the three siRNA (siMix) into bovine fibroblast cells, the most effective one for TRIM28 knockdown was selected. By injecting RNAi directed against TRIM28 mRNA, we found that TRIM28 knockdown in oocytes had the most effect on the H19 gene, in which differentially methylated region (DMR) methylation was almost completely absent at the 2-cell stage (1.4%), while control embryos showed 74% methylation. In addition, global H3K9me3 levels at the 2-cell stage were significantly higher in the in vitro fertilization (IVF) group than in the TRIM28 knockdown group (P<0.05). We further show that TRIM28 is highly expressed during oocyte maturation and reaches peak levels at the 2-cell stage. In contrast, at this stage, TRIM28 expression in somatic cell nuclear transfer (SCNT) embryos decreased significantly (P<0.05), suggesting that Trim28 transcripts are lost during SCNT. TRIM28 is required for the maintenance of methylation imprints in bovine preimplantation embryos, and the loss of TRIM28 during SCNT may contribute to the unfaithful maintenance of imprints in cloned embryos.
TRIM28/KAP1/TIF1β被鉴定为一种通用的转录共抑制因子，对调控植入前胚胎受精后的甲基化重编程至关重要。在本研究中，设计了三种针对TRIM28 mRNA序列的小干扰RNA（si647、si742和si1153）。将这三种小干扰RNA的混合物（siMix）转染到牛成纤维细胞中后，选择了对TRIM28敲低最有效的一种。通过注射针对TRIM28 mRNA的RNA干扰，我们发现卵母细胞中TRIM28的敲低对H19基因影响最大，在2细胞阶段，其差异甲基化区域（DMR）甲基化几乎完全缺失（1.4%），而对照胚胎显示74%的甲基化。此外，体外受精（IVF）组2细胞阶段的整体H3K9me3水平显著高于TRIM28敲低组（P<0.05）。我们进一步表明，TRIM28在卵母细胞成熟过程中高度表达，并在2细胞阶段达到峰值水平。相比之下，在此阶段，体细胞核移植（SCNT）胚胎中TRIM28的表达显著下降（P<0.05），这表明在SCNT过程中Trim28转录本丢失。TRIM28是牛植入前胚胎甲基化印记维持所必需的，SCNT过程中TRIM28的缺失可能导致克隆胚胎中印记维持不准确。
