Mechanism Of Chromatin Accessibility Memory Of Donor Cells Disrupts Bovine Somatic Cell Nuclear Transfer Blastocysts Development

Somatic cell nuclear transfer(SCNT)technology has immense potential in preserving valuable genetic resources and improving breeding through genome-edited animals.However,its widespread application and adoption are severely hindered by extremely low efficiency.Among all successfully cloned species,bovine exhibit relatively high efficiency compared to other animals,with their cloned embryos showing better development rates in early stage.Nevertheless,a considerable portion of well-formed cloned embryos still degenerate or disappear rapidly after being transferred into the uterus of the recipient cow,indicating abnormal gene regulation still exists during embryonic development to the blastocyst stage.Chromatin accessibility,serving as a hallmark of gene transcription regulatory elements and a predictor of gene transcription activity,has been rarely reported in SCNT embryos to date.In this study,we utilized single-cell ATAC-seq(Assay for Transposase-Accessible Chromatin with high throughput sequencing)technology to obtain the chromatin accessibility profiles for bovine in vivo and SCNT blastocysts,providing a comprehensive analysis of their differences.By correlating with the chromatin accessibility and histone modifications of donor cells,chromatin accessibility memory was identified from the donor cells to the SCNT blastocysts.Finally,the abnormal chromatin accessibility was corrected by manipulating the histone modification levels,and functionally validated the identified genes related to chromatin accessibility memory.The main experimental content and results are as follows:1.Utilizing single-cell ATAC-seq,chromatin accessibility profiles were generated for both in vivo and SCNT blastocysts in bovine.The results showed that both in vivo and SCNT blastocysts consisted of two cell populations,the inner cell mass(ICM)and the trophectoderm(TE).However,it was noteworthy that the proportion of ICM cells in SCNT blastocysts has decreased.2.The ICM and TE of in vivo and SCNT blastocysts were paired and compared separately to analyze their respective differences in chromatin accessibility(differentially accessible regions,DARs).The results revealed that there were numerous significantly differentially accessible regions(DARs,P<0.05)between the ICMs of the two types of blastocysts.These DARs were adjacent to many transcription factors(TFs)involved in regulating ICM development.In contrast,there were relatively fewer DARs between the TEs of the two types of blastocysts.When analyzed from the perspective of motifs accessibility related to TF binding sites,it became evident that some TF involved in lineage development exhibited more pronounced differences in accessibility between the two types of ICMs.3.By conducting pseudo-time analysis,lineage separation cell trajectories were constructed for in vivo and SCNT blastocysts.The results revealed significantly greater differences in the ICM trajectories between the two types of blastocysts.For both in vivo and SCNT blastocysts,positive TFs involved in lineage separation were identified.Interestingly,14 positive TFs,including 12 ICM TFs and 2 TE TFs,exhibited dysregulated gene activity in the trajectories of SCNT blastocysts.After analyzing the co-accessibility patterns of the putative cis-regulatory elements for these positive TFs,the majority of ICM-related TFs were found to exhibit less sophisticated co-accessibility patterns in SCNT blastocysts compared to those in in vivo ones.4.By comparing the chromatin accessibility of donor cells with the DARs between in vivo and SCNT blastocysts,a series of chromatin accessibility memories retained from the donor cells to SCNT blastocysts were identified.The regions that were highly accessible in donor cells and exhibited abnormally increased accessibility in SCNT embryos were defined as active memory regions,while the regions that were low in accessibility in donor cells and displayed decreased accessibility in SCNT blastocysts were defined as silent memory regions.Through ChIP-seq,virous histone modification profiles were generated for donor cells,and it was found that H3K4me3 and H3K27ac were enriched in the active memory regions,while H3K9me3 and H3K27me3 were enriched in the silent memory regions.Typically,H3K4me3 was significantly enriched in the active memory regions that specific to the ICM of SCNT blastocysts(P<0.01).5.Aiming at H3K4me3-enriched regions in active memory regions,inject H3K4me3specific demethylase KDM5B mRNA into SCNT embryos to detect the effects of overexpression of KDM5B on active memory regions.The results showed that overexpression of KDM5B significantly reduced the H3K4me3 level in SCNT blastocysts(P<0.01),significantly improved their developmental rate(P<0.05),and preferentially increased the number of ICM cells(P<0.05).The results of ATAC-seq and RNA-seq showed that KDM5B overexpression could effectively correct the accessibility levels of the active memory regions of donor cells that were retained in the ICM and the expression of related genes(P<0.05),such as the active memory region located in the BCL11B promoter region and the expression level of BCL11B.However,the effect of KDM5B on the abnormal chromatin accessibility and expression pattern of TE is relatively weak.6.Through UliNChIP-qPCR,it was detected that overexpression of KDM5B indeed decreased the H3K4me3 levels in the BCL11B promoter in ICM of SCNT blastocysts(P<0.05).Gene function validation experiments showed that disrupting BCL11B in SCNT embryos significantly promoted ICM proliferation(P<0.05),while overexpressing BCL11B in IVF embryos significantly inhibited embryo development(P<0.05).Furthermore,BCL11B was positively correlated with the level of cell cycle inhibitor p21.In summary,this study analyzed the differences between SCNT and in vivo blastocysts from the perspective of chromatin accessibility,and identified more abnormal accessibility related to ICM development in SCNT blastocysts.These abnormalities were associated with highly enriched H3K4me3 open sites in donor cells.Decreasing H3K4me3 levels with KDM5B corrected the abnormal accessibility and preferentially improved ICM development in SCNT embryos.These findings provide insight into the chromatin accessibility defects during SCNT reprogramming,and may facilitate the identification of new targets to correct accessibility abnormalities and improve the efficiency of SCNT production.