Anomalies In Accessible Chromatin Of Somatic Cell Cloned Cattle And Epigenetic Research Thereof

Only several parts of the chromatin DNA of each cell show biological activity.This chromatin structure is loose,and the genomic DNA is also in a loose structure.The loose region can be bound with transcription factors and is the active part of gene expression,which called the open chromatin regions(OCRs)or chromatin accessibility.The remaining inactive parts are tightly packed into highly condensed chromatin and silenced,not allowing gene transcription.The active and inactive state of chromatin directly affects many basic cellular processes including gene expression,DNA replication,and DNA repair.Furthermore,the OCRs are also the effect of a variety of epigenetic modifications,especially the results of epigenetic modifications related to gene expression.Cloned animals have low survival rates and abnormal development,which are usually considered to be caused by abnormal epigenetic modifications.Recently,there is no relevant research on the variation analysis of the OCRs of cloned animals.And it is not clear how abnormal epigenetic modifications can regulate and affect the chromatin state of cloned animals.In order to understand the differences in the open chromatin region of somatic cloned cattle,we selected 6 cloned bovine ear tissue samples that appeared healthy and survived as the research object.We divided them into two groups,one group includes 3 cloned cattle(huiwa(HW),xingwa(XW),longwa(LW)),which the donor nucleus of cloned cattle is derived from fetal fibroblasts.The other group includes3 cloned cattle(S1231,S1232,S1253).And the donor nucleus is derived from oviduct epithelial cells.We then used the 6 donor nuclei for the ATAC-seq and RNA-seq analysis.In ATAC-seq,each cow received more than 19.5G clean bases.And subsequent analysis revealed a difference of 40029 peaks,of which18893 were raised and 21136 were lowered.Based on these data,the peak annotations were obtained and3,940 differential genes among 8,653 genes were obtained.These genes were annotated on chromosomes and analyzed for pathways.Then,we found that they were distributed on 317 pathways.Among them,KMT in H3K4 trimethyltransferase was enriched in arginine and proline metabolism pathways and lysine degradation pathways respectively.In the process of histone methylation and demethylation,we analyzed the chromatin accessibility of several genes such as KMT2,KMT5 B,SUV39H1,SETDB,EZH,EED,and other related genes.In the process of DNA methylation and demethylation,we analyzed the chromatin accessibility of several genes such as APOBEC3Z1,TDE,SUMG1,DNMT1,DNMT3 B,and DNMT3 A.In addition,we also analyzed the motifs and the difference motifs existing on the different peaks.Through the analysis,161 new motifs were predicted,and the positions of these motifs on the difference of 19563 peaks were analyzed.Among them,113 motifs were overexpressed.In RNA-seq,we counted gene expression levels from both gene and subtype.We found 1805 differentially expressed genes,and there were 7221 differences among different gene subtypes.In the GO enrichment analysis,we found that the differential genes are abnormal in the process of cells,cells,cell parts,and binding.We further analyzed 27 biological processes,17 cell components,and 11 molecular functions,the three major categories of differences.There are 941 gene differences in the cell progression pathway;941 gene differences participate in both the cell pathway,and the cell part pathway;784 genetic differences were found in the combination pathway.In the subtype analysis,there are a total of 3804 gene differences in the "cell progression" pathway,3921 gene differences in the "cell" pathway and the "cell part" pathway and 3328 genes in the combination pathway.In addition,we selected the 20 pathways with the most significant enrichment and plotted the scatterplot of KEGG enrichment of differentially expressed genes.We analyzed the pathways of KEGG enrichment,counted the differentially expressed genes,and annotated the up-and down-regulated genes to proteins.In this way,you can take out the differential genesof any pathway in the database,and you can also visually see the change of different genes.What's more,we also analyzed and predicted variable spliceosomes,SNP / Indel,lnc RNA,and so on.We selected 6 cloned bovine ear tissue samples with a healthy appearance.Under normal circumstances,although the ATAC and RNA of the same ear tissue are different,there should be no obvious tendency to cluster.However,our analysis found that the ear tissues derived from fetal fibroblasts and oviduct epithelial cells were clearly clustered,and the results of ATAC-seq and RNA-seq analysis were more consistent,that is,the ear tissues derived from the three fetal fibroblasts were clustered together,and the ear tissues derived from the three oviduct epithelial cells also gathered together.This result shows that although the analysis tissues are derived from the ear tissue samples of the same site,due to their different sources of donor nuclear cells,they show differences in chromatin openness and gene expression.This aspect shows that the reprogramming of the different sources of the somatic cell has differences in the epigenetic modification,even for cloned cattle that appear to be healthy and alive,not to mention cloned dead and dysplasia cloned cattle.The Twostep cluster also shows that the reprogrammed somatic cells can be influenced by the memory of donor nuclear cells.