Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China; Guangzhou Key Laboratory of Insect Development Regulation and Applied Research, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou, 510631, China.
Division of Plant Sciences, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, MO, 65211, USA.
Insect Biochem Mol Biol. 2021 Jul;134:103583. doi: 10.1016/j.ibmb.2021.103583. Epub 2021 May 16.
DNA methylation is an important epigenetic modification. DNA methyltransferases (Dnmts), which catalyze the formation of 5-methylcytosine, play a role in ovarian and embryonic development in some insects. However, the underlying mechanism of Dnmt in mediating ovarian and embryonic development remains unclear. In this study, the regulation and function of Bombyx mori Dnmt1 were investigated. By progressively deleting the sequence upstream of Dnmt1, a region located between -580 and -560 region from the transcription initiation site was found to have the most transcriptional activity. Electrophoretic mobility shift assay and chromatin immunoprecipitation demonstrated that transcription factor Y box binding protein (YBP), a homolog of human Y box binding protein 1 (YBX1), bound to the -580 to -560 region. YBP knockdown and overexpression in a Bombyx cell line indicated that YBP activates Dnmt1 expression. Furthermore, GST-pulldown and co-immunoprecipitation demonstrated that YBP and ovarian CCAAT/enhancer binding protein (C/EBPg) could bind each other. Simultaneous knockdown of C/EBPg and YBP was more effective than single-gene RNAi in inhibiting Dnmt1 expression and reducing the hatching rate. These results demonstrated that the interaction of C/EBPg and YBP activated Dnmt1 expression. Correlated with the expression profiles of the studies genes, our results suggest that high-level expression and interaction of C/EBPg and YBP in ovaries and embryos enhance the expression of Dnmt1, thus ensuring high reproduction rate in B. mori.
DNA 甲基化是一种重要的表观遗传修饰。DNA 甲基转移酶（Dnmts），它催化 5-甲基胞嘧啶的形成，在一些昆虫的卵巢和胚胎发育中起作用。然而，Dnmt 介导卵巢和胚胎发育的潜在机制尚不清楚。在这项研究中，研究了家蚕 Dnmt1 的调节和功能。通过逐步删除 Dnmt1 上游的序列，发现转录起始位点上游的-580 到-560 区域具有最强的转录活性。电泳迁移率变动分析和染色质免疫沉淀表明，转录因子 Y 盒结合蛋白（YBP），一种与人 Y 盒结合蛋白 1（YBX1）同源的蛋白，与-580 到-560 区域结合。在一种家蚕细胞系中敲低和过表达 YBP 表明，YBP 激活 Dnmt1 的表达。此外，GST 下拉和共免疫沉淀表明，YBP 和卵巢 CCAAT/增强子结合蛋白（C/EBPg）可以相互结合。同时敲低 C/EBPg 和 YBP 比单独的基因 RNAi 更有效地抑制 Dnmt1 的表达和降低孵化率。这些结果表明，C/EBPg 和 YBP 的相互作用激活了 Dnmt1 的表达。与研究基因的表达谱相关，我们的结果表明，在家蚕的卵巢和胚胎中，C/EBPg 和 YBP 的高水平表达和相互作用增强了 Dnmt1 的表达，从而确保了家蚕的高繁殖率。
