332
Xenopus
23.4.4. DNA METHYLATION AS A BARRIER
TO NUCLEAR REPROGRAMMING
DNA methylation, specifcally 5-methylcytosine (5mC) methylation, is a fairly stable epigenetic mark that is widely associated with the repressive state of gene expression (Greenberg
and Bourc’his, 2019). Nuclear reprogramming depends on the
ability to activate expression of several stably repressed genes
in the donor nucleus in order to attain totipotency. It has been
shown using multiple reprogramming systems that the repressive nature of DNA methylation poses a roadblock to eff cient
transcriptional reprogramming and that DNA demethylation is necessary for reprogramming somatic cell nuclei in
Xenopus and in mammals (Simonsson and Gurdon, 2004;
Mikkelsen et al., 2008). Aberrant erasure of DNA methylation marks in the donor nucleus causes ineffcient cloning and
may also lead to anomalies in development, as demonstrated
in mammalian systems, including mouse and bovine embryos
(Dean et al., 2001; Kang et al., 2001). Similar incomplete
DNA methylation reversal has been reported in human iPSCs
(Lister et al., 2011), showing that DNA methylation is a def -
nite cause of resistance to reprogramming, regardless of the
approach. However, the dependence of DNA demethylation
during successful reprogramming in the Xenopus system
remains to be fully elucidated.
23.4.5. HISTONE MODIFICATIONS AND RESISTANCE
TO NUCLEAR REPROGRAMMING
Chromatin organization and the epigenetic signature of the
donor nucleus contribute to the resistance against reprogramming. Histone modifcations affect both, contribute to maintenance of cellular identity, and can infuence the ability of
genes to be successfully reprogrammed (Kouzarides, 2007).
Several studies have been conducted on the role of repressive histone marks as reprogramming barriers in Xenopus. In
mouse SCNT-embryos, these barriers are thought to prevent
early developmental genes from being reprogrammed into
active expression. The histone marks H3K27me3, H3K9me3,
and H2AK119 monoubiquitylation and the histone variant macro H2A, for example, are often associated with
gene repression. In Xenopus and in mammals, H3K9me3 is
enriched in reprogramming-resistant regions, and removal
of this mark using either ectopic expression of its specif c
demethylase Kdm4d or knock-down of the H3K9 methyltransferase Suv39h in Xenopus and mouse NT experiments
has been shown to be promising for reprogramming (Jullien
et al., 2017; Matoba et al., 2014; Liu et al., 2016). H3K27me3
is enriched at genes important for embryonic development and also acts as an epigenetic barrier to reprogramming ( Zhang et al., 2009; Jullien et al., 2017) in Xenopus
and in mouse. Overexpression of the H3K27 demethylase
KDM6A improved overall transcriptional reprogramming
in Xenopus and mouse, as well as the developmental potential of mouse NT embryos. (Jullien et al., 2017; Zhou et al.,
2019). H2AK119 monoubiquitylation is a repressive mark
that confers reprogramming resistance (Jullien et al., 2017),
as its removal via USP21 overexpression improved transcriptional reprogramming effciencies in Xenopus oocyte NT.
The histone variant macroH2A is involved in the stability
of repressed states during Xenopus oocyte-NT, as seen by its
role in resisting the reversal of X chromosome inactivation in
mouse donor cells (Pasque et al., 2011). A role of macroH2A
as reprogramming barrier was confrmed in the iPSC reprogramming system (Pasque et al., 2012).
However, aberrant gene expression patterns in the NT
embryo cannot be exclusively attributed to repressive modif -
cations resisting reprogramming—they may be also due to the
transmission of active gene states. Intriguingly, the low eff -
ciency of cell-fate reprogramming Xenopus NT embryos has
been linked to the retention of donor cell type-specif c memory of an active chromatin state stabilized by histone 3 lysine
4 (H3K4) methylation. The number of genes with persisting
active and inactive gene states in Xenopus embryos is surprisingly similar (Hörmanseder et al., 2017). The genes resistant to reprogramming that are thus inappropriately active
are implicated in functions related to the donor cell type,
such as cell type-specifc transcription factors, and showed
increased H3K4me3 intensities and domain breadth when
compared to genes that were properly reprogrammed, that
is, downregulated (Hörmanseder et al., 2017). When nuclei
with experimentally reduced H3K4 methylation were used
to generate NT-embryos, a reduction of ON-memory and an
improvement of cell-fate conversion was observed. This phenomenon was found to be conserved in mouse NT-embryos
(Hörmanseder et al., 2017; Liu et al., 2016). These studies
suggest that H3K4me3 stabilizes donor cell-fate memory in
Xenopus NT-embryos and that active histone marks could
contribute, in combination with repressive histone marks, to
the epigenetic mechanisms of cellular memory.
The previously described chromatin modif cations are
largely observed in resistant genes regardless of the means
of reprogramming—oocyte-NT, egg-NT, iPS, and cell
fusion alike, suggesting similar mechanisms of epigenetic
resistance (Jullien et al., 2017). Although it is well accepted
that silent chromatin states characterized by modif cations
such as H3K9me3 contribute to cellular memory, it is still
under debate whether active histone marks like H3K4me3
also have the potential for this (Stewart-Morgan et al., 2020)
and whether other active histone marks impose barriers to
successful nuclear reprogramming. Furthermore, it is not
well understood how the different chromatin marks interact
with each other, potentially in combination with transcription factors, to safeguard cellular identities and to prevent
effcient nuclear reprogramming.
23.5. CONCLUSION AND FUTURE PERSPECTIVES
The search for an answer to the fundamental question of
whether all cells in an organism have an identical set of genes
opened the door to the untapped feld of nuclear reprogramming. This feld holds endless possibilities and immense
Xenopus
23.4.4. DNA METHYLATION AS A BARRIER
TO NUCLEAR REPROGRAMMING
DNA methylation, specifcally 5-methylcytosine (5mC) methylation, is a fairly stable epigenetic mark that is widely associated with the repressive state of gene expression (Greenberg
and Bourc’his, 2019). Nuclear reprogramming depends on the
ability to activate expression of several stably repressed genes
in the donor nucleus in order to attain totipotency. It has been
shown using multiple reprogramming systems that the repressive nature of DNA methylation poses a roadblock to eff cient
transcriptional reprogramming and that DNA demethylation is necessary for reprogramming somatic cell nuclei in
Xenopus and in mammals (Simonsson and Gurdon, 2004;
Mikkelsen et al., 2008). Aberrant erasure of DNA methylation marks in the donor nucleus causes ineffcient cloning and
may also lead to anomalies in development, as demonstrated
in mammalian systems, including mouse and bovine embryos
(Dean et al., 2001; Kang et al., 2001). Similar incomplete
DNA methylation reversal has been reported in human iPSCs
(Lister et al., 2011), showing that DNA methylation is a def -
nite cause of resistance to reprogramming, regardless of the
approach. However, the dependence of DNA demethylation
during successful reprogramming in the Xenopus system
remains to be fully elucidated.
23.4.5. HISTONE MODIFICATIONS AND RESISTANCE
TO NUCLEAR REPROGRAMMING
Chromatin organization and the epigenetic signature of the
donor nucleus contribute to the resistance against reprogramming. Histone modifcations affect both, contribute to maintenance of cellular identity, and can infuence the ability of
genes to be successfully reprogrammed (Kouzarides, 2007).
Several studies have been conducted on the role of repressive histone marks as reprogramming barriers in Xenopus. In
mouse SCNT-embryos, these barriers are thought to prevent
early developmental genes from being reprogrammed into
active expression. The histone marks H3K27me3, H3K9me3,
and H2AK119 monoubiquitylation and the histone variant macro H2A, for example, are often associated with
gene repression. In Xenopus and in mammals, H3K9me3 is
enriched in reprogramming-resistant regions, and removal
of this mark using either ectopic expression of its specif c
demethylase Kdm4d or knock-down of the H3K9 methyltransferase Suv39h in Xenopus and mouse NT experiments
has been shown to be promising for reprogramming (Jullien
et al., 2017; Matoba et al., 2014; Liu et al., 2016). H3K27me3
is enriched at genes important for embryonic development and also acts as an epigenetic barrier to reprogramming ( Zhang et al., 2009; Jullien et al., 2017) in Xenopus
and in mouse. Overexpression of the H3K27 demethylase
KDM6A improved overall transcriptional reprogramming
in Xenopus and mouse, as well as the developmental potential of mouse NT embryos. (Jullien et al., 2017; Zhou et al.,
2019). H2AK119 monoubiquitylation is a repressive mark
that confers reprogramming resistance (Jullien et al., 2017),
as its removal via USP21 overexpression improved transcriptional reprogramming effciencies in Xenopus oocyte NT.
The histone variant macroH2A is involved in the stability
of repressed states during Xenopus oocyte-NT, as seen by its
role in resisting the reversal of X chromosome inactivation in
mouse donor cells (Pasque et al., 2011). A role of macroH2A
as reprogramming barrier was confrmed in the iPSC reprogramming system (Pasque et al., 2012).
However, aberrant gene expression patterns in the NT
embryo cannot be exclusively attributed to repressive modif -
cations resisting reprogramming—they may be also due to the
transmission of active gene states. Intriguingly, the low eff -
ciency of cell-fate reprogramming Xenopus NT embryos has
been linked to the retention of donor cell type-specif c memory of an active chromatin state stabilized by histone 3 lysine
4 (H3K4) methylation. The number of genes with persisting
active and inactive gene states in Xenopus embryos is surprisingly similar (Hörmanseder et al., 2017). The genes resistant to reprogramming that are thus inappropriately active
are implicated in functions related to the donor cell type,
such as cell type-specifc transcription factors, and showed
increased H3K4me3 intensities and domain breadth when
compared to genes that were properly reprogrammed, that
is, downregulated (Hörmanseder et al., 2017). When nuclei
with experimentally reduced H3K4 methylation were used
to generate NT-embryos, a reduction of ON-memory and an
improvement of cell-fate conversion was observed. This phenomenon was found to be conserved in mouse NT-embryos
(Hörmanseder et al., 2017; Liu et al., 2016). These studies
suggest that H3K4me3 stabilizes donor cell-fate memory in
Xenopus NT-embryos and that active histone marks could
contribute, in combination with repressive histone marks, to
the epigenetic mechanisms of cellular memory.
The previously described chromatin modif cations are
largely observed in resistant genes regardless of the means
of reprogramming—oocyte-NT, egg-NT, iPS, and cell
fusion alike, suggesting similar mechanisms of epigenetic
resistance (Jullien et al., 2017). Although it is well accepted
that silent chromatin states characterized by modif cations
such as H3K9me3 contribute to cellular memory, it is still
under debate whether active histone marks like H3K4me3
also have the potential for this (Stewart-Morgan et al., 2020)
and whether other active histone marks impose barriers to
successful nuclear reprogramming. Furthermore, it is not
well understood how the different chromatin marks interact
with each other, potentially in combination with transcription factors, to safeguard cellular identities and to prevent
effcient nuclear reprogramming.
23.5. CONCLUSION AND FUTURE PERSPECTIVES
The search for an answer to the fundamental question of
whether all cells in an organism have an identical set of genes
opened the door to the untapped feld of nuclear reprogramming. This feld holds endless possibilities and immense