Identification Of New Molecular Markers For Somatic Reprogramming By Using Transcriptomes Of Single-cell Cloned Embryos

Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer(SCNT).However,the majority of SCNT embryos will developmental arrest in the early stages due to many defects in reprogramming process.For the problem about low rate of nuclear transfer embryos,the focus of research is basically on the molecular mechanism in early embryonic development stage of SCNT.To date,only the reduction of H3K9me3 by ectopic expression of histone demethylases has been shown to greatly promote the development of SCNT embryos,and its potential molecular regulation mechanism has not yet been precisely resolved.In this paper,we first comprehensively analyzed the transcriptome expression profiles of normal fertilized and somatic cell nuclear transfer embryos in mice.With systematic analysis of molecular differences inwhole genome expression of diverse developing fate cloned embryos,we found that abnormal activation of important functional pathways such as apoptosis,autophagy,endocytosis,and DNA repair is a key factor for2-cell arrest of nuclear transfer embryos;and abnormal activation of stem cell population maintence,DNA repair,cell cycle,and autophagy is a hallmark event for 4-cell arrest of cloned embryos.By comparing the transcriptomes between SCNT embryos with different fate,some nuclear reprogramming related gene clusters of 2-cell and 4-cell embryos were screened out from the angles of transcription factors,pluripotency maintenance genes,histone modification factors,DNA/RNA demethylase genes.For example,Ascl2?Bmp7?Kdm4d?Kat2b?Srcap and Zfp217 belong to the key regulatory factors of cloned 2-cell embryo;Sall4 ?Vegfa?Hinfp?Kdm3b?Kdm5a?Kdm5b?Ctnnb1?Hdac4?Brca2?Yeats4?Atxu7 and Arid4 b play an important regulatory role in cloned 4-cell embryo;Zscan4c?Id1?Pou6f1?Cited1?Nanog?Dppa2?Fgf4?Med17?Cnot1?Kdm4b?Kdm3a?Kdm7a?Kat8?Elp6?Eid1?Gadd45a?Tet1and Tet2 belong to the molecular markers which are not normally activated in cloned embryos,due to the differences between normal and cloned embryos of 2-cell and 4-cell,and a small number of these genes have been reported.These molecular differences may explain the phenomenon of developmental arrest in SCNT embryos.In addition,we compared and analyzed the potential molecularmechanism of histone demethylases Kdm4b/4d and Kdm5 b in overcoming the embryonic developmental arrest of nuclear transfer and promoting cloned embryonic developmental potential.and found that activation of key pathways and core gene clusters played an important role.Kdm4b/4d can promote the expression of the key regulatory factors Zscan4 c and Zscan4 d that maintain telomeres in 2-cells.In addition,we also found that Kdm4 d can specifically activate Kdm5b?Tet1 and Id1 in the 2-cell stage,which indicates that the molecular markers of Kdm4 d significantly improves the blastocyst rate of SCNT embryo cells are identificated.Moreover,we discovered that kdm5 b could activate the pathways of stem cell maintenance and DNA repair and expression of key genes of Ubtfl1?Nr5a2?Prdm14?Thoc5?Dppa3 and Klf5 in 4-cell,the activation of these key genes may be one of the important factors that histone demethylase can overcome the blockage of SCNT embryos.Our findings provide important theoretical basis for exploring the potential molecular mechanisms and key factors in SCNT-mediated somatic cell reprogramming,and provide some help for further elucidating the molecular regulation mechanisms of cell reprogramming.