330
Xenopus
on differentiated chromatin that further prevent complete
cell-fate reprogramming to pluripotency.
23.4.2. CHANGE OF GENE EXPRESSION PATTERNS
DURING REPROGRAMMING
Specifc genes required during early development are
typically repressed within the genome of a differentiated cell, whereas genes characteristic of the cell type are
highly expressed. For reprogramming to be successful,
it appears necessary that the gene expression pattern of
the differentiated donor cell be fully changed to that of
a pluripotent cell and further to that of a specialized cell
in the developing embryo. Thus, when reprogramming
fails, it was hypothesized that this could be the result of
ineffcient silencing of genes that were expressed in the
differentiated donor cell, together with unsuccessful activation of genes typically expressed in the reprogrammed
cell type.
Indeed, Xenopus cell lineages isolated from an NT-embryo
continue to express genes that are strictly characteristic of
the donor cell type and should not be active in these reprogrammed cell lineages (Hörmanseder et al., 2017). The f rst
evidence for a failure in inactivating genes during reprogramming was obtained when muscle nuclei were used as
donor cells. Neurectoderm and endoderm lineages derived
from roughly half of the resulting NT embryo continued
to inappropriately express certain muscle genes (Ng and
Gurdon, 2005). A serial NT study showed that the expression of muscle genes persisted even in approximately 50% of
the second-generation NT embryos, addressing the stability
and potential transmission of the donor-like expression state
(Ng and Gurdon, 2008). The transcriptional quiescence of
Xenopus embryos for the frst 12 divisions until ZGA suggests that the “memory” of the past active transcriptional
state is transmitted through subsequent embryonic cell divisions, independently of ongoing gene expression and the
signal that induced the state. However, the molecular basis
that maintains this memory of past active states remains
to be elucidated. Ineffcient reactivation of certain pluripotency genes, such as Oct4 (Pou5f1), is observed in mouse
NT-embryos (Matoba and Zhang, 2018). Interestingly, such
decreased expression of pluripotency genes was found to be
associated with a subsequent failure in embryonic development in mouse-derived SCNT-embryos (Boiani et al., 2002;
Bortvin et al., 2003) and was later confrmed in human
NT-embryos (Chung et al., 2015). Similar anomalous expression patterns are observed in iPSCs in which differentiation
bias exists towards the donor cell type used (Polo et al, 2010).
This bias can be attenuated upon persistent cell divisions,
suggesting only transient infuence levied by the original cell
type in iPSCs. It is, however, clear that persistence of active
and inactive past transcriptional states points to conserved
mechanisms that restrict reprogramming of these genes.
There exists a confict between persistence of the memory
state and imposition of the reprogramming process. It has
been observed that the factors present in the oocyte strongly
favor reprogramming, whereas the epigenetic state of the
donor cell type contributes to the resistance against it.
23.4.3. OOCYTE FACTORS PROMOTE
NUCLEAR REPROGRAMMING
NT provides a unique experimental advantage to study the
mechanisms of nuclear reprogramming with the hope to apply
the obtained knowledge to improve reprogramming eff ciencies in other reprogramming systems. A majority of the transcriptional reprogramming that a somatic nucleus undergoes
takes place within the f rst 48 hours of its transfer to an egg,
as compared to the two to eight weeks required to generate an
iPSC line. In particular, the transfer of multiple somatic nuclei
to the GV of an oocyte produces a rapid transcriptional reprogramming system of high quality as well as high eff ciency.
The introduction of somatic nuclei to GV of Xenopus
oocytes or to egg/oocyte extracts immediately triggers a
cascade of events within the system, enabling the process
of transcriptional reprogramming (Figure 23.2). When differentiated nuclei are injected into Xenopus egg extracts,
decondensation of the somatic chromatin is accompanied by
certain modifcations to histone marks typically associated
with an open chromatin structure (such as H3K14 acetylation) as well as the mobilization of the heterochromatin proteins HP1β and TIF1β (Trim28) from the nuclei (Tamada et
al., 2006). Rapid exchange of somatic linker histones with
oocyte linker histones occurs almost immediately posttransfer. The somatic linker histone H1 is replaced by the
Xenopus oocyte-specifc B4, which is bound on somatic
chromatin within 24 hours post-transfer (Jullien et al.,
2010). In mammals, the orthologous linker histone is H1foo,
which acts in a similar manner (Teranishi et al., 2004). This
exchange is closely followed by the recruitment of the Pol
II subunit—oocyte RPB1 in its hypophosphorylated form.
Within 48 hours post-transfer, the Pol IIA is phosphorylated on the C-terminal domain (CTD) at Ser5 (for initiation
of transcription) and Ser2 (for elongation of transcription)
(Jullien et al., 2014). A genome-wide shift in transcription
follows, in which genes responsible for transcription and
development are upregulated, while those involved in signaling pathways (probably enriched according to the properties
of the somatic cell) are downregulated. Here, the Xenopus
orthologs of highly expressed genes in MEF-transplanted
oocytes closely resemble the expression pattern of wild-type
Xenopus oocytes, much more so than the expression pattern
of mouse ESCs. Due to this, it is evident that NT to oocytes
induces a shift from the somatic to oocyte cell type rather
than the pluripotent stem cell pattern (Jullien et al., 2014).
Besides the exchange of linker histones between the
somatic nucleus and the oocyte GV, deposition of other
histone factors is also affected. The recruitment of Pol IIA
coincides with a drop in somatic RBP1 as well as an increase
in the deposition of oocyte histone H2B (Jullien et al., 2014).
Histone chaperone HIRA-dependent deposition of oocytespecifc H3.3 occurs on transplanted somatic nuclear chromatin within the frst 24 hours (Jullien et al., 2012). ASF1A,
Xenopus
on differentiated chromatin that further prevent complete
cell-fate reprogramming to pluripotency.
23.4.2. CHANGE OF GENE EXPRESSION PATTERNS
DURING REPROGRAMMING
Specifc genes required during early development are
typically repressed within the genome of a differentiated cell, whereas genes characteristic of the cell type are
highly expressed. For reprogramming to be successful,
it appears necessary that the gene expression pattern of
the differentiated donor cell be fully changed to that of
a pluripotent cell and further to that of a specialized cell
in the developing embryo. Thus, when reprogramming
fails, it was hypothesized that this could be the result of
ineffcient silencing of genes that were expressed in the
differentiated donor cell, together with unsuccessful activation of genes typically expressed in the reprogrammed
cell type.
Indeed, Xenopus cell lineages isolated from an NT-embryo
continue to express genes that are strictly characteristic of
the donor cell type and should not be active in these reprogrammed cell lineages (Hörmanseder et al., 2017). The f rst
evidence for a failure in inactivating genes during reprogramming was obtained when muscle nuclei were used as
donor cells. Neurectoderm and endoderm lineages derived
from roughly half of the resulting NT embryo continued
to inappropriately express certain muscle genes (Ng and
Gurdon, 2005). A serial NT study showed that the expression of muscle genes persisted even in approximately 50% of
the second-generation NT embryos, addressing the stability
and potential transmission of the donor-like expression state
(Ng and Gurdon, 2008). The transcriptional quiescence of
Xenopus embryos for the frst 12 divisions until ZGA suggests that the “memory” of the past active transcriptional
state is transmitted through subsequent embryonic cell divisions, independently of ongoing gene expression and the
signal that induced the state. However, the molecular basis
that maintains this memory of past active states remains
to be elucidated. Ineffcient reactivation of certain pluripotency genes, such as Oct4 (Pou5f1), is observed in mouse
NT-embryos (Matoba and Zhang, 2018). Interestingly, such
decreased expression of pluripotency genes was found to be
associated with a subsequent failure in embryonic development in mouse-derived SCNT-embryos (Boiani et al., 2002;
Bortvin et al., 2003) and was later confrmed in human
NT-embryos (Chung et al., 2015). Similar anomalous expression patterns are observed in iPSCs in which differentiation
bias exists towards the donor cell type used (Polo et al, 2010).
This bias can be attenuated upon persistent cell divisions,
suggesting only transient infuence levied by the original cell
type in iPSCs. It is, however, clear that persistence of active
and inactive past transcriptional states points to conserved
mechanisms that restrict reprogramming of these genes.
There exists a confict between persistence of the memory
state and imposition of the reprogramming process. It has
been observed that the factors present in the oocyte strongly
favor reprogramming, whereas the epigenetic state of the
donor cell type contributes to the resistance against it.
23.4.3. OOCYTE FACTORS PROMOTE
NUCLEAR REPROGRAMMING
NT provides a unique experimental advantage to study the
mechanisms of nuclear reprogramming with the hope to apply
the obtained knowledge to improve reprogramming eff ciencies in other reprogramming systems. A majority of the transcriptional reprogramming that a somatic nucleus undergoes
takes place within the f rst 48 hours of its transfer to an egg,
as compared to the two to eight weeks required to generate an
iPSC line. In particular, the transfer of multiple somatic nuclei
to the GV of an oocyte produces a rapid transcriptional reprogramming system of high quality as well as high eff ciency.
The introduction of somatic nuclei to GV of Xenopus
oocytes or to egg/oocyte extracts immediately triggers a
cascade of events within the system, enabling the process
of transcriptional reprogramming (Figure 23.2). When differentiated nuclei are injected into Xenopus egg extracts,
decondensation of the somatic chromatin is accompanied by
certain modifcations to histone marks typically associated
with an open chromatin structure (such as H3K14 acetylation) as well as the mobilization of the heterochromatin proteins HP1β and TIF1β (Trim28) from the nuclei (Tamada et
al., 2006). Rapid exchange of somatic linker histones with
oocyte linker histones occurs almost immediately posttransfer. The somatic linker histone H1 is replaced by the
Xenopus oocyte-specifc B4, which is bound on somatic
chromatin within 24 hours post-transfer (Jullien et al.,
2010). In mammals, the orthologous linker histone is H1foo,
which acts in a similar manner (Teranishi et al., 2004). This
exchange is closely followed by the recruitment of the Pol
II subunit—oocyte RPB1 in its hypophosphorylated form.
Within 48 hours post-transfer, the Pol IIA is phosphorylated on the C-terminal domain (CTD) at Ser5 (for initiation
of transcription) and Ser2 (for elongation of transcription)
(Jullien et al., 2014). A genome-wide shift in transcription
follows, in which genes responsible for transcription and
development are upregulated, while those involved in signaling pathways (probably enriched according to the properties
of the somatic cell) are downregulated. Here, the Xenopus
orthologs of highly expressed genes in MEF-transplanted
oocytes closely resemble the expression pattern of wild-type
Xenopus oocytes, much more so than the expression pattern
of mouse ESCs. Due to this, it is evident that NT to oocytes
induces a shift from the somatic to oocyte cell type rather
than the pluripotent stem cell pattern (Jullien et al., 2014).
Besides the exchange of linker histones between the
somatic nucleus and the oocyte GV, deposition of other
histone factors is also affected. The recruitment of Pol IIA
coincides with a drop in somatic RBP1 as well as an increase
in the deposition of oocyte histone H2B (Jullien et al., 2014).
Histone chaperone HIRA-dependent deposition of oocytespecifc H3.3 occurs on transplanted somatic nuclear chromatin within the frst 24 hours (Jullien et al., 2012). ASF1A,