To date, more than 20 species of mammals have been successfully cloned by somatic cell nuclear transfer [2], which plays an important role in many fields [3]. However, the birth rate of such animals is low [16], and the individuals that are born exhibit more health defects than naturally bred individuals, which seriously hinders their further development [17–19]. It is generally believed that this phenomenon is caused by aberrant epigenetic modification in the process of somatic reprogramming, and the abnormal epigenetic modification of reprogrammed cells also causes abnormalities in the forward programming process, which is the root cause of low birth rates and developmental defects in cloned animals.
Previous studies have focused on the effects of only a single type of epigenetic modification, but there are many kinds of epigenetic modifications, and they often coordinate with each other to make chromatin denser or looser by changing the structure of chromatin. Dense chromatin is correlated with gene silencing, while open chromatin promotes gene expression. Thus, compared with studying a single modification, the study of whole-genome chromatin openness may provide a more comprehensive understanding of the effects of epigenetic modification differences on cloned cattle. Therefore, in this experiment, ATAC-seq technology, which is currently a relatively advanced and efficient method for detecting chromatin openness [23], was used in a joint analysis with RNA-seq to provide a more comprehensive analysis of the differences in the epigenetic modifications of cloned cattle at the chromatin level. We found that cloned bovine samples from the same source showed a clustering tendency in both ATAC-seq and RNA-seq analyses, whereas the data of the cloned cattle from different sources were not clustered together. Even after the processes of reprogramming and differentiation, epigenetic imprinting was observed to affect each individual (i.e., the epigenetic memory phenomenon), similar to the findings of previous studies [24, 25].
We further analyzed the related genes encoding the enzymes responsible for epigenetic modifications. In this study, we analyzed 16 related genes, and our RNA-seq results showed that the expression levels of all genes were higher in the FFB group than in the FOV group. It was also found that the expression of enzymes related to inhibiting gene expression was higher than that of modifying enzymes related to promoting gene expression; e.g., the expression of TET1, 2, 3, KDM5A, KDM6A and other genes related to the promotion of gene expression was lower, while DNMT1, EZH2, and KDM4A exhibited higher expression. Since the expression level of inhibitory epigenetic modification-related enzymes in the FFB group was higher than that in the FOV group, we inferred that during the forward programming differentiation of somatic cloned cattle, with the continuous action of inhibitory epigenetic modification-related enzymes, some of the open chromatin in the genome would gradually become restricted. However, the expression of enzymes mediating inhibitory epigenetic modifications was higher in the FFB group than in the FOV group, so the chromatin was more open in cloned bovine ear tissue samples derived from the FOV group. In addition, our ATAC-seq results showed that although FOV chromatin was more open, the difference in chromatin openness between the cloned cattle from two different sources occurred in intergenic regions rather than in regions of gene expression. These data also showed that the large number of differences in intergenic regions did not directly affect the life or death of cloned cattle.
Studies have reported that regions with a high content of 5hmC modification are often accompanied by chromatin structure opening. Transcription factors and other cofactors can bind open DNA more easily, so genes near such regions tend to be highly expressed. We started with the analysis of the 5hmc modification of genomic DNA, which is an important demethylation modification that less studied than other modification in cloned animals, and we hoped to discover the role of 5hmc in somatic reprogramming and differentiation. By comparing the 5mC and 5hmC contents of the whole genomes of live and dead cloned cattle, we found that the 5hmC content of donor FOV cells was higher than that of FFB cells. The analysis of the individuals that survived after overprogramming and differentiation also showed that the 5hmC content of the FOV group was higher than that of the FFB group.
Our cloned bovine samples came from Dai Yunping's research group at the State Key Laboratory of Agrobiotechnology, China Agricultural University. As early as 2004, Professor Dai Yunping was studying the effects of different donor cell types on the production efficiency of somatic cloned cattle. By referring to their research [10] in combination with the results of our study, we found that FOV cells presented a significant advantage in terms of the blastocyst development rate, but the final individual birth rates were not as high as those obtained from FFB cells. Combined with the transcriptome sequencing results of this study, it could be inferred that aberrant modification during reprogramming influenced forward programming differentiation. We know that normal zygotic development proceeds through two stages: reprogramming and forward programming differentiation [3]. The early stage of SCNT requires reprogramming, and all previous epigenetic markers need to be removed from the cells need before they can develop normally to the blastocyst stage. At this stage, the FOV group exhibited a more open chromatin state (its 5hmC content was higher than that of the FFB group, suggesting that the chromatin was more open), which may have promoted this process, and showed a higher blastocyst formation rate. However, in the subsequent stage of differentiation and development, this relatively open chromatin may impede the process of differentiation, and the expression levels of enzymes mediating epigenetic modifications related to gene expression inhibition were also lower in the FOV group. Therefore, some genes that need to be silenced may not be shut down in time or may maintain high expression levels, ultimately leading to lower individual survival rates and developmental abnormalities, which may be similar to the results of previous H3K4me3 studies [5].
In conclusion, we hypothesized that differences in epigenetic modifications existed in cloned cattle because of their different donor cell types, and even after reprogramming and differentiation, some of these differences were retained in the cloned animals. In the reprogramming process of somatic cloned cattle, a more open chromatin state was beneficial for reprogramming. As a result, we further hypothesized that the high expression of epigenetic modification-related enzymes that promote gene expression or low expression of enzymes mediating inhibition-related modifications would facilitate the preimplantation development of somatic cloned cattle. In the process of differentiation and development, some open chromatin needs to be closed via a specific process, which occurs via the gradual addition of inhibitory epigenetic modifications that antagonize chromatin opening. When a chromatin region that needs to be closed cannot be closed in time, it may cause the abnormal development or death of cloned animals. Thus, in the process of somatic reprogramming, greater chromatin openness can promote the formation of blastocysts. However, in the development and differentiation period, this relatively open chromatin impedes the normal development of cloned animals.