1.2. SCNT embryo and transcriptome reprogramming
The normal development of SCNT embryo depends largely on the sufficient reprogramming of the donor cell transcriptome. These gene expression changes mainly affect the erasure and establishment of epigenetics, which in turn promotes the silencing of somatic cell-related genes and the successful occurrence of recombinant embryo ZGA. For example, mouse SCNT embryos are often unable to effectively suppress the expression of some somatic-related genes accompanying donor cells, that is, there is residual epigenetic memory; there are many genes that cannot be effectively activated in SCNT embryos in the two-cell period. These factors hinder the reprogramming of recombinant embryos. Subsequent studies found that these reprogramming resistance regions (genes) that cannot be effectively silenced or activated are closely related to abnormal epigenetic modifications.
1.3. SCNT embryos and DNA methylation
It is generally believed that DNA methyltransferases (DNMTs) mainly include two types: DNA replication-dependent and recruited by Uhrf1 protein, Dnmt1, which is essential for the maintenance of progeny DNA methylation during replication, and DNA-independent Dnmt3a and Dnmt3b that can initiate de novo methylation at specific sites can be replicated. Incomplete reprogramming of DNA methylation status of donor cells is also one of the main reasons limiting the success rate of SCNT embryos. DNA undergoes a large amount of demethylation through passive and active demethylation, and the DNA methylation level reaches the minimum during the blastocyst stage. Passive DNA demethylation refers to the lack of Dnmt1 expression, which results in the newly synthesized DNA being unable to be methylated and making the genomic DNA methylation level decrease with DNA replication and cell division; DNA active demethylation refers to DNA methylation oxidase Tet1, Tet2 or Tet3 combined with thymine DNA glycosylase Tdg and DNA base repair pathway work together to remove DNA methylation modification. Compared with normal embryos, DNA methylation in SCNT embryos is abnormal. DNA re-methylation is the key reason why zygote genes and partial retrotransposons in SCNT embryos cannot be fully activated. In terms of DNA methylation, the inhibition of DNA methyltransferase activity and the use of DNA passive and active demethylation pathways to reduce high DNA methylation levels in SCNT embryos are two ways to increase the success rate of SCNT embryos.
1.4. SCNT embryo and histone modification
The success of SCNT also depends on whether the histone modification pattern of the donor cell can be reprogrammed to the histone modification state of the fertilized egg. Through immunofluorescence staining, it was found that the histone acetylation and methylation of SCNT embryos were different from those of IVF embryos. H3K9me3 modification of the donor cell genome is a major obstacle to efficient reprogramming of SCNT. Expression of H3K9me3 demethylase Kdm4d in vitro can remove the reprogramming resistance regions (RRRs) enriched in H3K9me3 in mouse donor cells, significantly improving the efficiency of SCNT. In addition, the use of donor nuclei that inhibit H3K9 methyltransferase activity can also significantly increase the efficiency of SCNT.
1.5. SCNT embryos and histone variants
In addition to several common histones, some histone variants can also participate in the formation of nucleosomes, greatly increasing the diversity and complexity of nucleosomes and even chromosome structures. Compared with classical histones, these histone variants have one or several amino acid differences; their expression levels are relatively low, mainly by changing the conformation of nucleosomes to perform different biological functions. During germ cell generation and fertilization, non-classical histone variants can be observed to largely replace classic histones, which is closely related to cell reprogramming and cell fate changes.
After fertilization of a normal embryo, the sperm genome packaged with protamine undergoes chromatin remodeling, and some maternal histone variants participate in the repacking of the sperm genome, such as H3.3, H2AFX. In SCNT embryos, similar donor cell histones were quickly replaced by maternal histones. Donor cell-derived histone H3 variants and H2A and H2A.Z are rapidly eliminated in the chromatin of SCNT embryos; with this removal, histone H3 variants stored in oocytes and H2A.X are incorporated into the nucleus of the SCNT embryo. The histone variant macroH2A was rapidly removed from the donor cell nucleus in SCNT embryos, and the classically linked histone H1 in somatic cells was also completely replaced by the oocyte-specific histone variant H1FOO. Knockdown of histone variant H3.3 before SCNT can affect the activation of pluripotency genes and the subsequent development of SCNT embryos.
1.6. SCNT embryos and Xist activation
There is also a close connection between SCNT embryos and X chromosome inactivation, and many X chromosome-linked genes are specifically suppressed in SCNT embryos, regardless of gender. In normal embryos, X chromosome inactivation is a female-specific dose compensation mechanism, usually in the form of imprints on the parental X chromosome of pre-implantation embryos and extra-embryonic tissues, but it is randomly appeared in embryonic ectoderm cells. X chromosome inactivation is controlled by the X chromosome-linked paternal allele noncoding RNA Xist. Xist covers the entire X chromosome and mediates the establishment of an inhibitory histone modification H3K27me3, which in turn heterochromatinizes the entire X chromosome. A similar situation does not occur with maternal X chromosomes, resulting in similar expression doses of X-linked genes in female (XX) and male (XY) cells. Abnormal activation of Xist has been observed in some dysplastic SCNT embryos. Both the maternal and paternal X chromosomes are silenced, which may be an important reason for affecting the normal development of SCNT embryos.
2. iPSCs technology-mediated reprogramming
In 2006, Takahashi and Yamanaka screened 24 transcription factors related to stem cells or pluripotency and successfully induced somatic cell reprogramming to pluripotency. Among the 24 factors, Oct4, Sox2, Klf4 and c-Myc are the four most important factors. Using these four classic factors OSKM, mouse embryonic fibroblasts or adult mouse fibroblasts are induced into pluripotent iPS cells with stem cell properties, and human iPS cells can also be obtained. iPS cells are similar to embryonic stem cells (ESCs) in morphology, proliferative capacity, pluripotent gene expression, epigenetic modification status, embryoid body and teratoma generation capacity, and differentiation potential. The establishment of iPS cell technology has broken the dependence of embryonic-derived stem cells in the field of stem cell research, and provided important theoretical support and technical support for the induction of patient-specific stem cells and regenerative medicine research.
2.1. The system and mechanism classic iPS cell induction
The combination of Oct4 on the genome plays an important role in the orderly activation of the pluripotency network during the reprogramming process, but the combination of Oct4 is affected by the original epigenetic modification on the genome, which shows that transcription factors and epigenetic modifications are interrelated with each other to jointly control the process of reprogramming. OSK can be directly combined with the pluripotency-related region to reprogram chromatin accessibility, or it can be directly combined with the active somatic enhancer region, by recruiting histone deacetylase HDAC1 to inhibit somatic cell related transcription factors. The activity of SPS can also be used to indirectly shut down somatic cell-related gene regions by activating Sap30 expression to promote the reprogramming of iPS cells. Sap30 can recruit histone deacetylase to reduce the modification level of H3K27ac in key gene regions of somatic cells. The lack of Sap30 during reprogramming will lead to an abnormal increase in H3K27ac, resulting in the continuous expression of somatic cell-related genes and iPS cell reprogramming’s failure. Fibroblasts are mesenchymal cells, ES cells are epithelial-like cells, and mesenchymal-to-epithelial transition (MET) that occurs during the induction of iPS cells is an important reprogramming event. OSKM directly participated in the MET process.
To be continued in Part Three…