178
Xenopus
genes such as sia1, nodal5, and nodal6 for expression in
the dorsal region of blastula-stage embryos (Blythe et al.
2010). In addition, β-Catenin works together with Foxh1 and
Nodal/Tgfβ signaling in the activation of Wnt target genes,
as shown by knockdown and pharmacological inhibition
experiments (Afouda et al. 2020). The endoderm-specif c
transcription factor Sox17 and β-Catenin also co-occupy
many regulatory elements, which modifes the response to
Wnt/β-Catenin signaling in endoderm ( Mukherjee et al.
2020 ).
These data illustrate how some transcription factors gain
early access to generally inaccessible chromatin to orchestrate embryonic gene regulation. Chromatin accessibility at
promoter and enhancer regions generally increases during
blastula and gastrula stages (Bright et al. 2021). At the early
gastrula stage, extensive chromatin accessibility is observed
in different regions of the embryo, supporting the extensive
lineage potential of the cells in these regions. Within this
relatively open chromatin, transcription factors operate in a
combinatorial fashion (Bright et al. 2021), allowing the cells
to respond to inductive signaling. This involves so-called
feed-forward circuitry in which (maternal) factors activate genes both directly and indirectly (Gazdag et al. 2016;
Charney et al. 2017b; Afouda et al. 2020). Collectively, these
studies document how establishing chromatin accessibility
is a key step in establishing developmental competence and
the ability to respond to inductive signals.
11.4. HISTONE MODIFICATIONS:
ACQUISITION AND DYNAMICS
DURING EARLY DEVELOPMENT
11.4.1. OOCYTE AND EGG STORAGE
HISTONES AND REPLICATION-COUPLED
CHROMATIN ASSEMBLY DYNAMICS
The histone proteins are extensively modifed in cell-type
and locus-specifc ways. The modifcations provide a scaffold for binding proteins that are recruited to sequences
wrapped in nucleosomes with these modif cations. These
“reader” proteins can be effector molecules, involved in activation or repression of transcription (reviewed in Smith and
Shilatifard 2010; Soshnev et al. 2016). As discussed above
(3.1), oocytes and early embryos contain storage forms of
histone protein that are not associated with genomic DNA.
Early experiments indicated that pre-deposition (i.e. maternally stored) histones H3 and H4 are acetylated (Woodland
1979). These marks are linked to gene activity when present
in chromatin, but H4, and to some extent H3, is deacetylated upon deposition. This corresponds to removal of some
of the storage modifcations. Mass spectrometry and western blotting experiments showed that these are not the only
modifcations on storage histones, some of which are not
removed during deposition. H2A and H3 arginine methyl
marks, for example, are no different before and after deposition in chromatin. Conversely, some modifcations are not
present in storage histones. Examples of these are H3K4
methylation (permissive mark for transcription), H3K27
trimethylation (facultative heterochromatin), and H3K9
trimethylation (constitutive heterochromatin), which are
not found on pre-deposition histones (Nicklay et al. 2009;
Shechter et al. 2009).
DNA replication-coupled chromatin assembly exerts
a major effect on histone modifcations in chromatin, as
it determines to what extent the existing marks are maintained. During DNA replication in Drosophila embryos,
chromatin of nascent DNA does not contain H3K4me3 and
H3K27me3 until one hour after DNA replication, when they
are re-established (Petruk et al. 2012). In mammalian cells,
some di- and tri-methylation modifcations in “old” nucleosomes may be recycled, as they are reduced two-fold upon
DNA replication, to be gradually restored during the ensuing cell cycle (Alabert et al. 2015). In both cases, this means
that there is a major delay in restoring histone modif cations
after DNA replication. Contributing to such a delay, in both
Drosophila embryos and differentiating mouse ES cells,
is the Utx histone H3K27demethylase, which prevents reestablishing of H3K27me3 on newly replicated DNA (Petruk
et al. 2013; Petruk et al. 2017). These mechanisms have not
been studied in Xenopus, but such delays are likely to have
a dramatic impact on histone modifcation dynamics during
the cleavage stages, when the cell cycles are extremely short.
11.4.2. ACQUISITION OF THE ANTAGONISTIC H3K4ME3
AND H3K27ME3 MARKS IN THE EMBRYO
H3K4me3 and H3K27me3 are the marks associated with
respectively the Trithorax and Polycomb group genes,
originally identifed in Drosophila. Using mass spectrometry, the permissive H3K4me3 promoter mark appears
to be relatively abundant in late blastula embryos. In later
development, its share in embryonic chromatin decreases
(Schneider et al. 2011). H3K27me3, the Polycomb repressive
mark, on the other hand, is quite low in blastula embryos
but increases during subsequent development. This suggests
that chromatin becomes less permissive and more repressive as development proceeds. As a general notion, this
is in line with chromatin accessibility data that link open
chromatin to multi-lineage potential in blastula and gastrula
embryos (Bright et al. 2021). Chromatin immunoprecipitation (ChIP) experiments have provided independent results
on these dynamics. H3K4 methylation accumulates rapidly
in early blastula embryos, whereas H3K27me3 follows a
bit later: it accumulates between mid-blastula and late gastrula stages (Akkers et al. 2009; van Heeringen et al. 2014;
Hontelez et al. 2015). We have referred to this as a hierarchy of activation and repression (Figure 11.2), as opposed to
co-occurring H3K4me3 and H3K27me3, referred to as bivalency. Bivalent chromatin does occur in Xenopus embryos,
as determined by ChIP-re-ChIP experiments, but it is not a
predominant feature, and it tends to be quickly resolved spatially within the embryo (Akkers et al. 2009). These observations initially caused some debate because bivalency had
attracted some attention as a special type of chromatin state.
Xenopus
genes such as sia1, nodal5, and nodal6 for expression in
the dorsal region of blastula-stage embryos (Blythe et al.
2010). In addition, β-Catenin works together with Foxh1 and
Nodal/Tgfβ signaling in the activation of Wnt target genes,
as shown by knockdown and pharmacological inhibition
experiments (Afouda et al. 2020). The endoderm-specif c
transcription factor Sox17 and β-Catenin also co-occupy
many regulatory elements, which modifes the response to
Wnt/β-Catenin signaling in endoderm ( Mukherjee et al.
2020 ).
These data illustrate how some transcription factors gain
early access to generally inaccessible chromatin to orchestrate embryonic gene regulation. Chromatin accessibility at
promoter and enhancer regions generally increases during
blastula and gastrula stages (Bright et al. 2021). At the early
gastrula stage, extensive chromatin accessibility is observed
in different regions of the embryo, supporting the extensive
lineage potential of the cells in these regions. Within this
relatively open chromatin, transcription factors operate in a
combinatorial fashion (Bright et al. 2021), allowing the cells
to respond to inductive signaling. This involves so-called
feed-forward circuitry in which (maternal) factors activate genes both directly and indirectly (Gazdag et al. 2016;
Charney et al. 2017b; Afouda et al. 2020). Collectively, these
studies document how establishing chromatin accessibility
is a key step in establishing developmental competence and
the ability to respond to inductive signals.
11.4. HISTONE MODIFICATIONS:
ACQUISITION AND DYNAMICS
DURING EARLY DEVELOPMENT
11.4.1. OOCYTE AND EGG STORAGE
HISTONES AND REPLICATION-COUPLED
CHROMATIN ASSEMBLY DYNAMICS
The histone proteins are extensively modifed in cell-type
and locus-specifc ways. The modifcations provide a scaffold for binding proteins that are recruited to sequences
wrapped in nucleosomes with these modif cations. These
“reader” proteins can be effector molecules, involved in activation or repression of transcription (reviewed in Smith and
Shilatifard 2010; Soshnev et al. 2016). As discussed above
(3.1), oocytes and early embryos contain storage forms of
histone protein that are not associated with genomic DNA.
Early experiments indicated that pre-deposition (i.e. maternally stored) histones H3 and H4 are acetylated (Woodland
1979). These marks are linked to gene activity when present
in chromatin, but H4, and to some extent H3, is deacetylated upon deposition. This corresponds to removal of some
of the storage modifcations. Mass spectrometry and western blotting experiments showed that these are not the only
modifcations on storage histones, some of which are not
removed during deposition. H2A and H3 arginine methyl
marks, for example, are no different before and after deposition in chromatin. Conversely, some modifcations are not
present in storage histones. Examples of these are H3K4
methylation (permissive mark for transcription), H3K27
trimethylation (facultative heterochromatin), and H3K9
trimethylation (constitutive heterochromatin), which are
not found on pre-deposition histones (Nicklay et al. 2009;
Shechter et al. 2009).
DNA replication-coupled chromatin assembly exerts
a major effect on histone modifcations in chromatin, as
it determines to what extent the existing marks are maintained. During DNA replication in Drosophila embryos,
chromatin of nascent DNA does not contain H3K4me3 and
H3K27me3 until one hour after DNA replication, when they
are re-established (Petruk et al. 2012). In mammalian cells,
some di- and tri-methylation modifcations in “old” nucleosomes may be recycled, as they are reduced two-fold upon
DNA replication, to be gradually restored during the ensuing cell cycle (Alabert et al. 2015). In both cases, this means
that there is a major delay in restoring histone modif cations
after DNA replication. Contributing to such a delay, in both
Drosophila embryos and differentiating mouse ES cells,
is the Utx histone H3K27demethylase, which prevents reestablishing of H3K27me3 on newly replicated DNA (Petruk
et al. 2013; Petruk et al. 2017). These mechanisms have not
been studied in Xenopus, but such delays are likely to have
a dramatic impact on histone modifcation dynamics during
the cleavage stages, when the cell cycles are extremely short.
11.4.2. ACQUISITION OF THE ANTAGONISTIC H3K4ME3
AND H3K27ME3 MARKS IN THE EMBRYO
H3K4me3 and H3K27me3 are the marks associated with
respectively the Trithorax and Polycomb group genes,
originally identifed in Drosophila. Using mass spectrometry, the permissive H3K4me3 promoter mark appears
to be relatively abundant in late blastula embryos. In later
development, its share in embryonic chromatin decreases
(Schneider et al. 2011). H3K27me3, the Polycomb repressive
mark, on the other hand, is quite low in blastula embryos
but increases during subsequent development. This suggests
that chromatin becomes less permissive and more repressive as development proceeds. As a general notion, this
is in line with chromatin accessibility data that link open
chromatin to multi-lineage potential in blastula and gastrula
embryos (Bright et al. 2021). Chromatin immunoprecipitation (ChIP) experiments have provided independent results
on these dynamics. H3K4 methylation accumulates rapidly
in early blastula embryos, whereas H3K27me3 follows a
bit later: it accumulates between mid-blastula and late gastrula stages (Akkers et al. 2009; van Heeringen et al. 2014;
Hontelez et al. 2015). We have referred to this as a hierarchy of activation and repression (Figure 11.2), as opposed to
co-occurring H3K4me3 and H3K27me3, referred to as bivalency. Bivalent chromatin does occur in Xenopus embryos,
as determined by ChIP-re-ChIP experiments, but it is not a
predominant feature, and it tends to be quickly resolved spatially within the embryo (Akkers et al. 2009). These observations initially caused some debate because bivalency had
attracted some attention as a special type of chromatin state.