Analysis Of Chromatin Three-dimensional Structural Reconstruction During Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer(SCNT)allows reprogramming of a differentiated somatic cell into a totipotent embryo.However,the dynamic changes of the chromatin three-dimensional(3D)structures during the SCNT embryonic development and the degree of chromatin 3D structures reprogramming in nuclear transfer embryonic stem cells(ntESCs)remain poorly understood.In addition,induced pluripotent stem cell(iPSC)is another common reprogramming method.There is no comparison between these two pluripotent stem cells in 3D structures.Therefore,we collect somatic cells,SCNT embryos and ntESCs for Hi-C to analyse the dynamic changes during early embryonic development of SCNT,the reprogramming degree of ntESCs and the comparison of ntESCs and iPSC in the 3D chromatin structures.The main results are as follows:1.Using data analysis of Hi-C for somatic cells,reconstituted embryos at 1 h after nucleus injection,early 1-cell,late 1-cell,late 2-cell and 8-cell of SCNT embryos in mice,the results show that the chromatin 3D structures of the somatic cell nucleus are disappeared,the compartments and TAD structures have undergone extensive reprogramming during embryonic development,and the global 3D structures are largely re-established at the SCNT 8-cell stage.The gobal dynamic changes of the 3D structures of SCNT embryos are similar to fertilized embryos.2.A comparative analysis of the SCNT embryos and fertilized embryos in 3D sturctures shows that the TAD strength of SCNT 1-cell is significantly stronger than that fertilized‘s.Transient knockout of the strucutre protein ?Cohesin‘ of somatic cells can significantly improve the efficiency of SCNT.And RNA-seq analysis of Cohesin knockout SCNT embryos show that the Cohesin impedes the expression of the minor zygotic genome activation(Minor ZGA)genes.In addition,there are many abnormal compartment switches and TADs in the SCNT 8-cell.3.The analysis of the chromatin structures of ntESCs reveals that the chromatin accessibility and overall 3D structures(including comaprtment,TAD and chromatin loop levels)are highly similar to the fESCs and undego extensive reprogramming.In addition,the results show that there are many abnormally reprogrammed compartments,TADs and loops in ntESCs.And the genes located in these abnormal structure regions are mainly association with the development of tissues and organs as well as biological processes.4.Performing embryoid body(EB)induced differentiation on ntESCs and fESCs in vitro shows that ntESCs have more abnormally gene expressions of three germ layers then fESCs.Moreover,overexpressing the gene Msc in fESCs,which is located in the abnormal compartment,shows abnormities in EB induced differentiation.It implies that the abnormal structures impair the differentiation potential of ntESCs.In addition,by further combine analyzing of the compartment switches and H3K9me3 modifications,the results show that partial compartments which modified by H3K9me3 in CCs showed abnormal switches in ntESCs,especially resisting the switch of compartment B to A.5.A comparative analysis of the 3D structures of ntESCs and iPSC reveals that the overall 3D structures of ntESCs and iPSC are highly similar,but there are some different chromatin switches between them,which are associated with different somatic cell types and mechanisms.The above results indicate that the somatic cell nucleus undergoes a series of the 3D chromatin structures reprogramming during the SCNT.Different with the fertilized embryos,SCNT 1-cell show stronger TAD strength.In addition,the structure protein Cohesin of somatic cells affects the efficiency of SCNT by preventing minor ZGA.Despite the completion of the global 3D genome reprogramming,there are still some abnormal chromatin structures in ntESCs.And these abnormal strucutres impair the differentiation potential of ntESCs.H3K9me3 modifications of somatic cell may be involved in the formation of abnormal compartments in ntESCs.In the comparison of ntESC and iPSC structure reprogramming,a few differences are detected due to different somatic cell origins and mechanisms.This study systematically analyses the 3D genome organization in the process of SCNT in mice and compares the differences of chromatin3 D structures reprogramming between ntESCs and iPSC,which can further facilitate the understanding on the SCNT at 3D genome level and improve the knowledge of ntESCs and iPSC.