Integrated RNA-seq and ATAC-seq analysis of miR-101 in bovine somatic cell reprogramming

Somatic cell nuclear transfer (SCNT) technology represents a cutting-edge advancement in animal biotechnology and reproductive science, carrying significant implications for livestock breeding and human medicine. Despite over two decades of development, SCNT still encounters challenges such as low cloning efficiency, compromised embryo development, and elevated malformation rates. The suboptimal efficiency of nuclear transfer is believed to be linked to incomplete reprogramming post-transfer, with epigenetic modifications playing a critical role in this process. miR-101, a non-coding RNA, is known to regulate late embryonic development. In donor bovine fetal fibroblasts, we demonstrated that overexpression of miR-101 enhances nuclear transfer embryo developmental potential. In this study, we constructed a miR-101 overexpression vector and screened for monoclonal positive cell lines. We successfully generated the miR-101 overexpression vector, miR-101-CD-513B1, and established stable monoclonal cell lines exhibiting increased levels of miR-101. RNA-seq analysis was performed to identify genes associated with reprogramming, while ATAC-seq analysis revealed that miR-101 overexpression resulted in an increased number of open chromatin regions. Based on the donor cell findings, we inferred candidate genes that may contribute to early nuclear reprogramming in SCNT embryos. We predicted the target genes of miR-101 and conducted Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Ultimately, we identified genes that are closely associated with early nuclear reprogramming in cloned embryos. This study provides a foundational understanding of the role of miR-101 in enhancing nuclear transfer efficiency and contributes to further elucidating the regulatory mechanisms involved.