基于供体细胞染色质开放性分析的Brg1对猪体细胞克隆胚胎早期发育的作用研究

Brg1是SWI/SNF染色质重塑复合物的中心组成单位，通过改变染色质的结构参与基因的转录调控，在胚胎早期发育、干细胞多能性维持、iPS重编程过程中均发挥重要作用，然而Brg1在体细胞克隆重编程过程中的作用尚未见报道。本课题在前期研究基础上，首先构建正负表达Brg1的载体，筛选获得稳定表达Brg1的猪体细胞系。通过ATAC-seq和RNA-seq方法分析Brg1对供体成纤维细胞染色质开放度和基因转录活性的影响，联合分析获得染色质开放区域的基因数目及其表达量变化、显著富集的转录因子及其表达量等信息。采用核移植方法探讨调控Brg1表达对克隆猪胚胎早期发育潜能及相关基因表达的影响，通过分析Brg1表达是否影响H3K9me3对猪胚胎ZGA标志性基因启动子区富集量等初步阐明其对克隆胚胎发育的作用机理。研究结果可为了解染色质重塑重要基因对SCNT重编程的调控作用、并提高核移植生产效率打下基础。

Brg1 is a central component of SWI/SNF chromatin remodeling complex. It is involved in transcriptional regulation by changing the structure of chromatin and plays an important role in early embryonic development, stem cell pluripotency maintenance and iPS reprogramming. However, the role of Brg1 in somatic cloning reprogramming has not been reported. On the basis of previous studies, this project firstly constructs the positive and negative Brg1 expression vectors, screens and obtains cell lines of stable expressing pig Brg1. ATAC-seq and RNA-seq methods are used to analyze the effects of Brg1 on the chromatin openness and gene transcriptional activity of donor fibroblasts, and the number of genes and their expression changes in the open chromatin region, as well as the significantly enriched transcription factors and their expression levels are obtained by combined analysis. Nuclear transfer method is used to investigate the effects of Brg1 expression regulation on the early developmental potential of cloned pig embryos and the expression of related genes, and the mechanism of its effect on the development of cloned pig embryos is preliminarily clarified by analyzing whether the expression of Brg1 affects the enrichment of H3K9me3 on the promoter region of ZGA marker genes in pig embryos. The results can lay a foundation for understanding the regulation role of important genes of chromatin remodeling on SCNT reprogramming and improving the production efficiency of nuclear transfer.