Nuclear reprogramming: the zygotic transcription program is established through an“erase-and-rebuild” strategy

Oocytes display a maternal-specific gene expression profile, which is switched to a zygotic profile when a haploid set of chromatin is passed on to the fertilized egg that develops into an embryo. The mechanism underlying this transcription reprogramming is currently unknown. Here we demonstrate that by the time when transcription is shut down in germinal vesicle oocytes, a range of general transcription factors and transcriptional regulators are dissociated from the chromatin. The global dissociation of chromatin factors （CFs） disrupts physical contacts between the chromatin and CFs and leads to erasure of the maternal transcription program at the functional level. Critical transcription factors and regulators remain separated from chromatin for a prolonged period, and become re-associated with chromatin shortly after pronuclear formation. This is followed temporally by the re-establishment of nuclear functions such as DNA replication and transcription. We propose that the maternal transcription program is erased during oogenesis to generate a relatively naive chromatin and the zygotic transcription program is rebuilt de novo after fertilization. This process is termed as the ＂erase-and-rebuild＂ process, which is used to reset the transcription program, and most likely other nuclear processes as well, from a maternal one to that of the embryo. We further show in the accompanying paper （Gao T, et al., Cell Res 2007; 17：135-150.） that the same strategy is also employed to reprogram transcriptional profiles in somatic cell nuclear transfer and parthenogenesis, suggesting that this model is universally applicable to all forms of transcriptional reprogramming during early embryogenesis. Displacement of CFs from chromatin also offers an explanation for the phenomenon of transcription silence during the maternal to zygotic transition.

Nuclear reprogramming: the strategy used in normal development is also used in somatic cell nuclear transfer and parthenogenesis

Somatic cell nuclear transfer （SCNT） and parthenogenesis are alternative forms of reproduction and development, building new life cycles on differentiated somatic cell nuclei and duplicated maternal chromatin, respectively. In the preceding paper （Sun F, et al., Cell Res 2007; 17：117-134.）, we showed that an ＂erase-and-rebuild＂ strategy is used in normal development to transform the maternal gene expression profile to a zygotic one. Here, we investigate if the same strategy also applies to SCNT and parthenogenesis. The relationship between chromatin and chromatin factors （CFs） during SCNT and parthenogenesis was examined using immunochemical and GFP-fusion protein assays. Results from these studies indicated that soon after nuclear transfer, a majority of CFs dissociated from somatic nuclei and were redistributed to the cytoplasm of the egg. The erasure process in oogenesis is recaptured during the initial phase in SCNT. Most CFs entered pseudo-pronuclei shortly after their formation. In parthenogenesis, all parthenogenotes underwent normal oogenesis, and thus had removed most CFs from chromosomes before the initiation of development. The CFs were subsequently re-associated with female pronuclei in time and sequence similar to that in fertilized embryos. Based on these data, we conclude that the ＂erase-and-rebuild＂ process observed in normal development also occurs in SCNT and in parthenogenesis, albeit in altered fashions. The process is responsible for transcription reprogramming in these procedures. The ＂erase＂ process in SCNT is compressed and the efficiency is compromised, which likely contribute to the developmental defects often observed in nuclear transfer （nt） embryos. Furthermore, results from this study indicated that the cytoplasm of an egg contains most, if not all, essential components for assembling the zygotic program and can assemble them onto appropriate diploid chromatin of distinct origins.

Delineating nuclear reprogramming

Nuclear reprogramming is described as a molecular switch,triggered by the conversion of one cell type to another.Several key experiments in the past century have provided insight into the field of nuclear repro-gramming.Previously deemed impossible,this re-search area is now brimming with new findings and developments.In this review,we aim to give a historical perspective on how the notion of nuclear reprogram-ming was established,describing main experiments that were performed,including(1)somatic cell nuclear transfer,(2)exposure to cell extracts and cell fusion,and(3)transcription factor induced lineage switch.Ultimately,we focus on(4)transcription factor induced pluripotency,as initiated by a landmark discovery in 2006,where the process of converting somatic cells to a pluripotent state was narrowed down to four tran-scription factors.The conception that somatic cells possess the capacity to revert to an immature status brings about huge clinical implications including per-sonalized therapy,drug screening and disease model-ing.Although this technology has potential to revolu-tionize the medical field,it is still impeded by technical and biological obstacles.This review describes the effervescent changes in this field,addresses bottle-necks hindering its advancement and in conclusion,applies the latest findings to overcome these issues.

Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model

This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57×CBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 μmol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

Oocyte-associated transcription factors in reprogramming after somatic cell nuclear transfer:a review

Oocytes are unique cells with the inherent capability to reprogram nuclei.The reprogramming of the somatic nucleus from its original cellular state to a totipotent state is essential for term development after somatic cell nuclear transfer.The nuclear-associated factors contained within oocytes are critical for normal fertilization by sperm or for somatic cell nuclear reprogramming.The chromatin of somatic nuclei can be reprogrammed by factors in the egg cytoplasm whose natural function is to reprogram sperm chromatin.The oocyte first obtains its reprogramming capability in the early fetal follicle,and then its capacity is enriched in the late growth phase and reaches its highest capability for reprogramming as fully-grown germinal vesicle oocytes.The cytoplasmic milieu most likely contains all of the specific transcription and/or reprogramming factors necessary for cellular reprogramming.Certain transcription factors in the cytoplast may be critical as has been demonstrated for induced pluripotent stem cells.The maternal pronucleus exerts a predominant,transcriptiondependent effect on embryo cytofragmentation,with a lesser effect imposed by the ooplasm and the paternal pronucleus.With deep analysis of transcriptomics in oocytes and early developmental stage embryos more maternal transcription factors inducing cellular reprogramming will be identified.

Overexpression of Stella improves the efficiency of nuclear transfer reprogramming

In mammalians, the state of a somatic cell can be reversed from the terminal state to the totipotent state by means of somatic cell nuclear transfer （SCNT） （Gurdon, 1962） or induced pluripotent stem cells （iPSCs） （Takahashi and Yamanaka, 2006）. The DNA methylation and transcriptome profiles of embryonic stern cells （ESCs） derived from SCNT embryos （NT-ESCs） correspond closely to those of ESCs derived from in vitro fertilization embryos （IVF- ESCs）. In contrast, iPSCs differ from both NT-ESCs and IVF-ESCs in that they retain the residual DNA methylation patterns of their parental somatic cells. As SCNT can be used to faithfully reprogram human somatic cells to pluripotency, it is ideal for cell replacement therapies （Ma et al., 2014）. Following the successful production of the first human NT-ESCs （Tachibana et al., 2013） and the later gen- eration of human NT-ESCs based on cells from elderly adults or pa- tient cells （Chung et al., 2014; Yamada et al., 2014）, a version of the SCNT technique for human therapeutics comes closer to reality. However, no matter what animal species or donor cell types are used in the cloned process, the cloning efficiency remains undesir- able. Besides, there are many phenotypic abnormalities in cloned animals, containing frequent embryonic and perinatal death and placentomegaly, and the underlying mechanisms remain unclear （Yang et al, 2007）.