CHO细胞重组蛋白表达失稳的DNA甲基化-H3K9me3协同作用及机制

利用中国仓鼠卵巢（CHO）细胞生产重组药物蛋白，保持细胞传代培养过程中重组蛋白表达稳定性、避免表达量降低是目前亟待解决的问题。申请者前期建立的DNA甲基转移酶Dnmt3a、3b缺陷型CHO细胞系能够显著改善重组蛋白表达失稳性，但仍存在启动子差异引起的稳定异质性，其原因及机制尚未阐明。组蛋白H3K9三甲基化（H3K9me3）也是导致基因转录沉默发生的关键因素，与DNA甲基化存在着密切联系。我们提出重组蛋白表达失稳的“DNA甲基化-H3K9me3协同作用”假说。本项目拟在前期研究的基础上，分析重组蛋白表达时DNA甲基化、H3K9me3表观遗传修饰的协同作用；ChIP-seq高通量测序筛选协同作用关键因子；采用基因过表达或基因敲除研究关键因子对CHO细胞重组蛋白表达不稳定性的影响。本项目对于阐明影响重组蛋白表达失稳的分子机制和建立高效稳定表达的CHO细胞系统具有重要的理论意义和应用价值。

It is an urgent issue to maintain the expression stability and to avoid the decrease of recombinant protein produced by Chinese hamster ovary (CHO) cells. In our previous study, the DNA methyltransferase Dnmt3a- or 3b-deficient CHO cell lines established by gene knockout can significantly improve the expression instability of the recombinant protein. However, there are still stable heterogeneity caused by different promoters, and the reason and the underlying mechanism have not been clarified. Trimethylation of histone H3K9 (h3k9me3) is also a key factor of gene transcription silencing, which is closely related to DNA methylation. Therefore, we propose the hypothesis of "synergistic effect of DNA methylation-H3K9 trimethylation" on the expression instability of recombinant protein. Based on previous studies, we plan to investigate the synergistic effect of DNA methylation and H3K9me3 via epigenetic modification on the recombinant protein expression; to screen and identify the key factors involved in the process of DNA methylation and H3K9me3 by high-throughput ChIP-seq; the influence of key factors on the expression instability of recombinant protein in CHO cells will be studied through gene overexpression or gene knockout. Our study will provide a novel strategy for theoretical basis and application basis for elucidating the epigenetic molecular mechanism related to the expression instability of recombinant protein and establishing a new high-efficient and stable CHO cell expression system.