**3. Epigenetic reprogramming by cell fusion**

mammals [22–24]. Since the SCNT process does not increase the frequency of genetic alterations, it is suggested that the variable phenotypes observed in cloned embryos are associated with the reprogrammed epigenetic status of the donor nuclei [25]. This is supported by the findings that the developmental defects in cloned animals were not transmittable to the next offspring generation, indicating the presence of aberrant epigenetic reprogramming [26]. Aberrant DNA methylation patterns were indeed observed in NT embryos [27]. It was also demonstrated that the bovine NT blastocysts lack asymmetric patterns of both H3K9 methylation and acetylation between the inner cell mass and trophectoderm [28], which may account

Although the rate of successful SCNT is very low, the reprogramming ability of factors in the egg/oocyte is highly efficient as the transplanted nuclei take less than 1 day to initiate cell division and trigger the "normal" developmental program. The donor cell epigenetic status has to be reprogrammed in order to support the embryonic program of development. In fact, genomewide demethylation was observed in the cloned blastocysts [29]. It has been shown that the *Oct4* promoter of somatic cells undergoes DNA demethylation after nuclear transplantation into the germinal vesicle of *Xenopus* oocytes [30]. This demethylation of the *Oct4* promoter was found to be mediated by Tet3 in a mouse SCNT study [31], which is essential for the reactivation of *Oct4* expression for successful SCNT. In addition, chromatin remodeling factors, such as ISWI and BRG1, are documented in facilitating the reprogramming of cell fate. It has been shown that ISWI, which is a chromatin remodeling ATPase, is able to dissociate TATA binding protein in somatic nuclei after incubation in the *Xenopus* egg extract, suggesting that ISWI-containing complexes are facilitating epigenetic reprogramming in an egg environment [32]. Besides, *Xenopus* egg extract depleted of BRG1 protein showed an abolishment of the reprogramming ability and hence failed to induce *Oct4* expression in the somatic nuclei [33]. Therefore, chromatin structure remodeling is believed to be one of the reprogramming mechanisms. In addition, the maternalderived histone H3 variant H3.3 in the enucleated egg was found to replace the canonical histone H3 in the donor nuclei after SCNT, leading to the reactivation of key pluripotent genes that are originally associated with repressive histone marks [34]. Histone H2A variant, macroH2A, also plays an important role in the reactivation of female donor cell's inactive X chromosome during reprogramming. It was shown that knockdown of macroH2A facilitates X-reactivation and the expression of pluripotent genes in cloned *Xenopus* embryos [35]. More recently, it has been demonstrated that the H3K9 tri-methylation (H3K9me3) of the donor cell genome is a major epigenetic barrier to SCNT. Ectopic expression of H3K9 demethylases *Kdm4b* or *Kdm4d* in the mouse donor cells de-repressed the genomic regions that are resistant to reprogramming and thus significantly improving SCNT efficiency [36, 37]. Similarly, removal of H3K9me3 by ectopic expression of other H3K9 demethylases also demonstrated improved reprogramming

Interestingly, it has been shown that the epigenetic state of a differentiated cell is directly correlated with its reprogrammability [40]. SCNT with ES cell nuclei demonstrated a much higher efficiency of generation of NT blastocysts than using other somatic cell types [41]. This could be associated with a more relaxed chromatin configuration in ES cells that may render their epigenome more susceptible for reprogramming [42]. Alternatively, it has been demonstrated that the cloning efficiency can be significantly improved by pre-treating the more

for abnormal cloned embryo development.

168 Chromatin and Epigenetics

efficiency in human and bovine SCNT experiments [38, 39].

Cell fusion is a natural event that is crucial for fertilization and in various organs such as placenta, skeletal muscles and bones [47]. It has been proposed that the fusion of bone marrowderived stem cells and tissue cells, e.g. hepatocytes, is one of the mechanisms of tissue repair [48, 49]. Cell fusion experiments using pluripotent cells, e.g. an ES or embryonic germ (EG) cell, were shown to be able to reprogram a differentiated cell type [3–5]. Both ES and EG cells possess reprogramming ability and are able to reactivate pluripotent genes and silence differentiation genes in the somatic cell nucleus within a tetraploid hybrid cell after cell fusion. It is indeed the case that the new transcription profile of a hybrid cell is partly contributed by the reprogrammed somatic nucleus to a pluripotent-like state. Moreover, injection of hybrid cells into normal diploid blastocysts demonstrated their contribution to all three germ layers in the chimeras [4, 50], indicating the pluripotent nature of hybrid cells. Similar to the SCNT, different somatic cell types show different kinetics of reprogramming by the cell fusion approach, which could be associated with the somatic chromatin accessibility status [51, 52].

Cell fusion with a pluripotent cell can trigger extensive epigenetic reprogramming in the differentiated cell nucleus. It has been shown that reactivation of *Oct4* from the somatic nucleus occurs before DNA replication after cell fusion [53], suggesting the involvement of active demethylation process [54]. This was further supported by the functional roles of Tet1 and Tet2 in the demethylation of *Oct4* and imprinted control regions by fusing somatic cells with EG cells [55]. Besides, in a cell fusion experiment using thymocytes and ES cells from two different mouse strains, it was observed that the epigenetic profile of the somatic cell nucleus was reprogrammed to a similar pattern to that of the ES cell. Global histone H3 and H4 acetylation and H3K4 di- and tri-methylation were increased in the hybrid cell to a level comparable to ES cells, whereas these modifications are weak in the parental somatic thymocytes. Examination of gene specific loci showed that the *Oct4* promoter was enriched with H3 acetylation and the promoter of the thymocyte marker *Thy-1* was enriched with H3K27 tri-methylation in both ES cell and hybrid cell chromatin, whereas these epigenetic modifications are missing in the thymocyte [56]. Hence, the somatic genome has undergone epigenetic reprogramming triggered by fusion with the ES cell, suggesting that the process of cell fusion mediates a transcription activation-permissive chromatin state in the hybrid genome. In addition, silencing the somatic differentiation genes was shown to be associated with polycomb repressive complexes in the cell fusion experiment using ES cells [57].

Reprogramming to pluripotency by cell fusion approach requires lengthy selection of the successfully reprogrammed hybrid cells. The reprogramming efficiency of cell fusion is usually less than 0.001%, depending on the somatic cell types [50, 58]. The low reprogramming efficiency in hybrid cells can be largely enhanced by manipulation of key pluripotency-associated genes like *Nanog* [58, 59] and *Sall4* [60], or by activation of the Wnt signaling pathway [61], emphasizing the importance of these factors in cell fusion reprogramming. Overexpression of *Nanog* or *Sall4* in ES cells demonstrated a several hundred-fold increase in reprogramming efficiency after cell fusion. Similarly, treatment of ES cells with Wnt3a for 24–48 hours enhanced the reprogramming of somatic cells by 20-fold. However, owing to the low reprogramming efficiency and the tetraploid genome of the resulting hybrid cells, reprogramming by the cell fusion approach becomes less promising in regenerative medicine.

*Pax5* or forced expression of *C/EBPα* resulted in efficient reprogramming by the Yamanaka factors [69], suggesting that the epigenetic status of differentiated cells is crucial in successful reprogramming. The dynamics of transcription profile and epigenetic patterns during the reprogramming process from somatic to iPS cells were studied in details. The reprogramming of somatic cell fate is a sequential stochastic event which involves a gradual silencing of the somatic lineage genes and the viral transgenes, and a sequential expression of alkaline phosphatase and SSEA1 in partially reprogrammed cells, whereas endogenous *Oct4* and *Nanog* are only activated in fully reprogrammed iPS cells. Induction of the four Yamanaka factors results in an immediate cellular response of inactivation of thousands of somatic lineage distal enhancers and, to a smaller extent, the H3K4me3-enriched somatic gene promoters [70, 71], leading to down-regulation of somatic identity genes. This initial phase of reprogramming is also accompanied by a global reduction of H3K27me3 resulting in loss of heterochromatin [72]. Meanwhile, mesenchymal transcription factors, such as *Snail1/2*, *Zeb1/2*, are repressed [70, 73, 74], whereas epithelial transcription factors, such as *Cdh1*, *Epcam*, are activated [75, 76], resulting in mesenchymal-epithelial transition (MET). This is associated with an increase in H3K4me2 at epithelial genes, but a decrease in H3K4me2 and H3K79me2 at mesenchymal genes [71, 77]. Despite an increase in global H3K36me2/3 level, loss of H3K36me2/3 was observed at the *Ink4-Arf* locus, leading to enhanced cell proliferation during reprogramming [78]. The following phase of reprogramming is marked by upregulation of endogenous pluripotency genes to establish transcriptional program, which is independent of transgene expression [73, 79]. The final phase of reprogramming involves elongation of telomeres, X-reactivation in female iPS cells, and upregulation of DNA methylation genes [79]. This coincides with loss of DNA methylation and downregulation of *Xist* expression in

Resetting Cell Fate by Epigenetic Reprogramming http://dx.doi.org/10.5772/intechopen.81015 171

A number of epigenetic remodeling factors are involved in the reprogramming events. Both polycomb (PcG) and trithorax (TrxG) group proteins were found to be crucial in the derivation of iPS cell colonies. Upon knockdown of *Wdr5*, which is a core component of TrxG protein complex, cells failed to establish H3K4me3 at the pluripotent genes, like *Oct4* and *Nanog*, for their reactivation [82]; whereas inhibition of the core components of the polycomb repressive complex 1 and 2 reduced reprogramming efficiency [83], partly because of the dysregulation of genes involved in the MET process [84]. This is similar to the findings that inhibition of H3K79 methyltransferase DOT1L facilitates the loss of H3K79me2 mark at the mesenchymal genes to promote MET during reprogramming [83]. The H3K27 demethylase Kdm6a (also known as Utx) can directly interact with Oct4/Sox2/Klf4 to remove the repressive H3K27me3 mark from the early pluripotent genes in somatic cells for their reactivation [85]. This is in agreement with the findings that depletion of histone H2A variant, macroH2A, enhances reprogramming, owing to its co-occupancy with H3K27me3 to repress pluripotent genes [86, 87]. Besides, the H3K36 demethylase Kdm2b (also known as Jhdm1b) enhances the activation of early responsive genes (*Cdh1*, *Epcam*, *Dsg2*, *Dsp*, *Irf6*) during reprogramming through the removal of H3K36me2 at their promoters [88]. Interestingly, H3K9me3 was also found to be one of the major epigenetic barriers in the generation of iPS cells [89], similar to the findings in SCNT experiments. Depletion of the H3K9 methyltransferases SUV39H1/H2, Ehmt1/2 and Setdb1 or inhibition of Cbx3, a protein that recognizes H3K9 methylation, enhances reprogramming by de-repressing *Nanog* and abolishing the cellular responses to BMP signaling [89–91]. Although

the somatic inactive X chromosome [80, 81].
