**5. "Epigenetic memory" in reprogrammed cells**

"Epigenetic memory" refers to the persistent expression of parental genes in the daughter cells through the inheritance of distinctive epigenetic marks. Consequently, the epigenetic profile of a parent cell is faithfully passed on to its daughter cells such that the gene expression pattern is memorized and maintained throughout cell generations. In the situation of reprogramming cell fate, the persistent somatic cell epigenetic signature and the expression of lineage genes in the reprogrammed cells is thus regarded as an example of epigenetic memory.

Even though it has been shown that prolonged *in vitro* culture of mammalian embryos can lead to aberrant expression of imprinted and non-imprinted genes owing to associated epigenetic 'errors' [104, 105]. It has been shown that many cloned embryos demonstrate different degrees of resemblance with donor cell gene expression patterns. The aberrant epigenetic pattern in cloned embryos is thought to be the result of persistence of the epigenetic memory of the donor cells. Indeed, the resemblance of DNA and histone modification patterns of NT embryos to those of the donor cell types supports this conclusion [45–47]. For example, both global and gene-specific patterns of DNA methylation in cloned bovine and mouse embryos were shown to be similar to those of their respective donor somatic cell types [28, 106, 107]. The phenomenon of epigenetic memory is also highlighted by the X inactivation pattern in cloned embryos. In normal fertilized embryos, the paternal X chromosome is preferentially silenced in the trophectoderm and extraembryonic endoderm lineages, whereas random X inactivation occurs in the inner cell mass. However, in NT embryos generated from female donor nuclei, the inactive X chromosome of the donor cell is preferentially chosen for inactivation in the trophectoderm, which is in contrast to the random X inactivation in the embryo proper, demonstrating a certain extent of memory of the inactive X chromosome status [48]. Random X inactivation in the placenta was found in deceased cloned bovine embryos, which suggests that the persistence of this inactive X chromosome memory in the placenta may be crucial for fetal survival [49].

Dnmt3a/b were found to be dispensable [92], DNA demethylation of key pluripotency loci

Previous studies demonstrated that the partially reprogrammed iPS cells contained significantly fewer genes marked by the bivalent chromatin signature (co-existence of both H3K4 and H3K27 methylation) and an enrichment of DNA hyper-methylated loci when compared to the wild-type ES cells and the fully reprogrammed iPS cells [70]. Therefore, it is proposed that completion of the epigenetic reprogramming process is pre-requisite for the acquisition of pluripotency. This is supported by the observation that treatment of partially reprogrammed iPS cells with the DNA methyltransferase inhibitor 5-aza-cytidine was able to promote their transition into the fully reprogrammed pluripotent state [70]. Besides, inhibition of H3K27 methyltransferase Ezh2 by small molecule, GSK-126, reduced reprogramming efficiency [84], whereas inhibition of DOT1L by small molecule, EPZ004777, showed enhancement of reprogramming [83]. Various histone deacetylase inhibitors (HDACi) were also shown to improve reprogramming [96–100]. In combination with HDACi valproic acid, human iPS cells can be generated only with Oct4 and Sox2 with a comparable reprogramming efficiency by the four Yamanaka factors [101]. Interestingly, it was found that vitamin C can increase reprogramming efficiency by promoting the transition of pre-iPS cells to fully reprogrammed cells [102], potentially through acting as a cofactor of Kdm2b to induce H3K36me2/3 demethylation [78], activation of H3K9 demethylases (Kdm3a, 3b, 4c and 4d) to remove H3K9me3 [89], and promoting Tet-mediated DNA demethylation [103]. With the aid of small chemical molecules, the

mediated by Tet proteins is required for efficient reprogramming [93–95].

172 Chromatin and Epigenetics

iPS cell reprogramming efficiency and duration could be further improved.

"Epigenetic memory" refers to the persistent expression of parental genes in the daughter cells through the inheritance of distinctive epigenetic marks. Consequently, the epigenetic profile of a parent cell is faithfully passed on to its daughter cells such that the gene expression pattern is memorized and maintained throughout cell generations. In the situation of reprogramming cell fate, the persistent somatic cell epigenetic signature and the expression of lineage genes in the reprogrammed cells is thus regarded as an example of epigenetic memory. Even though it has been shown that prolonged *in vitro* culture of mammalian embryos can lead to aberrant expression of imprinted and non-imprinted genes owing to associated epigenetic 'errors' [104, 105]. It has been shown that many cloned embryos demonstrate different degrees of resemblance with donor cell gene expression patterns. The aberrant epigenetic pattern in cloned embryos is thought to be the result of persistence of the epigenetic memory of the donor cells. Indeed, the resemblance of DNA and histone modification patterns of NT embryos to those of the donor cell types supports this conclusion [45–47]. For example, both global and gene-specific patterns of DNA methylation in cloned bovine and mouse embryos were shown to be similar to those of their respective donor somatic cell types [28, 106, 107]. The phenomenon of epigenetic memory is also highlighted by the X inactivation pattern in cloned embryos. In normal fertilized embryos, the paternal X chromosome is preferentially silenced in the trophectoderm and extraembryonic endoderm lineages, whereas random X inactivation occurs in the inner cell mass. However, in NT embryos generated from female donor nuclei, the

**5. "Epigenetic memory" in reprogrammed cells**

The epigenetic memory in NT embryos that maintains the donor expression gene states can be explained by the inheritance of DNA methylation patterns. Heritability of DNA methylation is mediated by the activity of the maintenance DNA methyltransferase Dnmt1 which preferentially targets hemi-methylated DNA. Thus, Dnmt1 restores the parental methylation status on the newly synthesized daughter DNA strand, thereby maintaining a silent gene state after cell division [108]. Methylated donor genes, such as pluripotency genes, remain inactivated after SCNT, apparently owing to the persistent donor-specific methylation pattern in the cloned embryos, possibility mediated by the residual somatic form of Dnmt in the donor nucleus. DNA methylation therefore provides a plausible mechanism for the propagation of a silent memory state in SCNT [109]. On the other hand, an active gene memory of the donor differentiation state is also observed in NT embryos. For example, both the donor endoderm and neurectoderm markers, *Edd* and *Ncam*, were found to be aberrantly expressed in *Xenopus* NT embryos derived from the respective donor cell types [18]. This active gene memory was further demonstrated to be associated with the incorporation of a histone H3 variant H3.3 at the active gene loci in *Xenopus* NT embryos [110]. Histone variant H3.3 is enriched in the regulatory region of active genes and is preferentially marked by modifications associated with an active chromatin state, such as H3K4 methylation, H3K9 acetylation and H3K79 methylation [111, 112]. Experiments using a mutant form of H3.3 demonstrated that the K4 residue on H3.3 plays a key role in the inheritance of active epigenetic memory [110], proposing a model in which H3.3 K4 methylation creates an "active histone environment" for the recovery of active chromatin configuration in daughter cells after chromosomal replication [113].

Early studies of iPS cells demonstrated that the Nanog-iPS cells displayed not only a highly similar transcriptome to wild-type ES cells, but also an ES cell histone modification profile. Genome-wide comparison of histone modifications (H3K4 and H3K27 trimethylation) between ES cells, MEFs and MEF-derived iPS cells demonstrated that more than 94% of the ES- or MEF-signature genes in iPS cells have identical histone methylation marks as in ES cells. Only 0.7% of these signature genes retain the histone methylation status of the original MEFs [63]. However, other gene expression profile studies of iPS cells showed that a significant number of differentiation genes have a similar expression pattern to that in the somatic cell of origin, but not in ES cells [114–117]. This transcriptional memory in iPS cells was found to be correlated with biased differentiation towards the original cell lineage, and with less competence in differentiation to other unrelated somatic lineages [115, 118–121]. Importantly, the persistent expression of somatic genes in iPS cells was associated with the somatic DNA methylation pattern [117, 122–124], highlighting the crucial role of epigenetic regulation in the retention of memory. This is in fact similar to the observation that an incomplete removal of donor cell DNA methylation pattern was observed in some aberrantly developed NT embryos [125]. Strikingly, the addition of epigenetic modifying agents, such as DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC), can enhance the iPS cell reprogramming efficiency [70] and improve the differentiation competency to other unrelated somatic lineages [120]. Interestingly, it has been demonstrated that continuous passaging of iPS cells abrogates somatic DNA methylation patterns [115], which suggests a passive replication-dependent mechanism in loss of the parental memory in iPS cells. Nevertheless, a study showed that the epigenetic memory in some iPS cell lines cannot be removed even after extended passages [124]. Apart from DNA methylation, microRNA expression pattern was also shown to have a role in the retention of somatic memory in iPS cells derived from hematopoietic progenitors [126]. However, it should be emphasized that other profiling studies of iPS cells failed to find the gene expression and epigenetic differences when compared to ES cells [127, 128]. It thus proposes that the "somatic memory" in iPS cells could be an artifact of incomplete reprogramming resulting in variation between iPS cell lines [129]. It is also possible that there are individual iPS cell lines expressing gene signatures owing to culture conditions and laboratory practices [130], similar to the scenario that some ES cell lines exhibit preferential differentiation towards specific lineages [131–133]. In summary, the epigenetic memory in iPS cells remains a contentious issue.
