A Comparative Analysis of Single-Cell Transcriptome Identifies Reprogramming Driver Factors for Efficiency Improvement

Abstract

Terminally differentiated somatic cells can be reprogrammed into a totipotent state through somatic cell nuclear transfer (SCNT). The incomplete reprogramming is the major reason for developmental arrest of SCNT embryos at early stages. In our studies, we found that pathways for autophagy, endocytosis, and apoptosis were incompletely activated in nuclear transfer (NT) 2-cell arrest embryos, whereas extensively inhibited pathways for stem cell pluripotency maintenance, DNA repair, cell cycle, and autophagy may result in NT 4cell embryos arrest. As for NT normal embryos, a significant shift in expression of developmental transcription factors (TFs) Id1, Pou6f1, Cited1, and Zscan4c was observed. Compared with pluripotent gene Ascl2 being activated only in NT 2-cell, Nanog, Dppa2, and Sall4 had major expression waves in normal development of both NT 2-cell and 4-cell embryos. Additionally, Kdm4b/4d and Kdm5b had been confirmed as key markers in NT 2-cell and 4-cell embryos, respectively. Histone acetylases Kat8, Elp6, and Eid1 were co-activated in NT 2-cell and 4-cell embryos to facilitate normal development. Gadd45a as a key driver functions with Tet1 and Tet2 to improve the efficiency of NT reprogramming. Taken together, our findings provided an important theoretical basis for elucidating the potential molecular mechanisms and identified reprogramming driver factor to improve the efficiency of SCNT reprogramming.

Characterization of DNA Methylation Patterns and Mining of Epigenetic Markers During Genomic Reprogramming in SCNT Embryos

“…Among them, Rrn3 is a specific RNA polymerase transcription initiation factor and necessary for mammalian RNA polymerase formation ( Herdman et al, 2017 ). Obox6 is a key transcription factor that enhances reprogramming efficiency ( Li et al, 2020 ). Taken together, our WGCNA analysis identified a number of developmental key genes whose expression was impaired in SCNT embryos probably due to hyper DNA methylation, further suggesting that these genes may act as epigenetic barrier during genomic reprogramming in SCNT embryos ( Figure 4C ).…”

Transcriptome Analyses Reveal Differential Transcriptional Profiles in Early- and Late-Dividing Porcine Somatic Cell Nuclear Transfer Embryos

“…Therefore, it is of great significance to analyze the molecular mechanisms leading to differences in the time of the first cleavage of pig cloned embryos. Considering that the use of single-cell RNA-seq is already well established for the analysis of gene expression in large livestock embryos [ 24 , 27 , 28 , 29 ], early-dividing and late-dividing pig SCNT embryos were collected and sequenced in this study.…”

Going up the hill: chromatin‐based barriers to epigenetic reprogramming

“…TETmediated DNA demethylation is required for mesenchymal-to-epithelial transition (MET) during early reprogramming, as MEFs deficient for TET1-3 fail to undergo this crucial step of the process [14]. Interestingly, TET1/2 are significantly upregulated in 2-cell embryos and interact with NANOG to facilitate maintenance of pluripotency in ES cells by binding to and enhancing the expression of key pluripotency genes [15,16]. TET3-deficient SCNT embryos showed higher methylation levels of the Oct4 promoter, which was associated with lower activation of the gene, thus pointing to the importance of the TET3 activity in remodeling the epigenetic landscape [17].…”

“…On the other hand, siRNA-mediated knockdown of Kdm4b decreases cloning efficiency and injection of oocytes with Kdm4b mRNA before SCNT greatly enhances it by reducing the reprogramming-resistant H3K9me3 regions in the SCNT embryo [40]. Kdm4b/ 4d have also been validated as factors that promote nuclear reprogramming in recent studies [15]. Taken together, these studies establish H3K9 methylation as a major mechanism to block pluripotency-specific gene expression and show that experimental perturbation of this chromatin mark can significantly increase reprogramming.…”