Download epigenetic control of cellular differentiation

Chapter twelve
12
EPIGENETIC CONTROL
OF CELLULAR
DIFFERENTIATION
All the information needed to construct an adult organism can be found
in the genomes of any number of its nucleated cells, but because the
body is composed of a wide variety of different cell types capable of performing very different functions, there must be mechanisms to control
the way in which information encoded in the genome can be selectively
controlled. Different types of cells arise early in embryonic development,
and epigenetic control of gene expression is used to establish specific
gene expression patterns that distinguish individual types of cells. The
process by which different cell types arise is known as differentiation.
12.1 FROM CELLULAR TOTIPOTENCY TO
PLURIPOTENCY
The early embryos of most multicellular eukaryotes begin as groups of
cells that are termed totipotent because each cell seems to have an
equal ability to become any of the cell types generated as the embryo
develops. Figure 12.1 shows some of the stages of pre-implantation
development that apply to mammals. The blue spheroids are referred to
as blastomeres and are totipotent. The first differentiation of these cells
takes place between the morula and early blastocyst stages, when those
blastomeres that happen to be on the outside of the morula become trophoblast cells, which are destined to become parts of the placenta. Only
the cells referred to as the inner cell mass (ICM) of the blastocyst will
give rise to the tissues of the developing embryo.
12.1 FROM CELLULAR
TOTIPOTENCY TO
PLURIPOTENCY
12.2 MAINTENANCE OF
PLURIPOTENCY IN
EMBRYONIC STEM CELLS
12.3 DIFFERENTIATION OF
EMBRYONIC STEM CELLS
12.4 BIVALENT CHROMATIN
DOMAINS IN NEURAL STEM
CELLS
12.5 CHROMATIN PROFILE
OF HEMATOPOIETIC
PROGENITORS
Once formed, the trophoblast cannot produce any of the cells that normally derive from the inner cell mass. Moreover, trophoblast cells can
never revert to totipotent blastomeres under normal circumstances. This
brings up an important point that must be borne in mind in any consideration of the process of cellular differentiation: it is almost always
unidirectional. Similarly, the ICM cannot produce trophoblast cells (in a
normal embryo); its cells are therefore referred to a pluripotent (that is,
able to give rise to many cell types but not all).
The ICM is of great interest to science because removing this structure
and culturing it under special growth conditions gives rise to embryonic
stem cells, which may be thought of as a “snapshot” of a very brief period
of embryonic development. It is brief indeed, because a few days after the
blastocyst has implanted in the wall of the uterus, the ICM differentiates
into three primordial layers that will produce the organs and structures
of the body (Figure 12.2) and pluripotent cells no longer exist in the body.
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Oocyte
EPIGENETIC CONTROL OF CELLULAR DIFFERENTIATION
1-cell
2-cell
4-cell
8–16-cell
16–32-cell
early blastocyst
first fate decision
late blastocyst
second fate decision
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Figure 12.1 The stages of pre-implantation
development. The totipotent fertilized
oocyte undergoes a series of divisions to
generate an 8–16-cell stage referred to as
the morula. After this stage, the outer cells
differentiate to become the trophectoderm
(the first fate decision) while the inner cells are
destined to become the inner cell mass, which
subsequently differentiates into the primitive
endoderm and epiblast by the late blastocyst
stage of development. [Adapted from ZernickaGoetz M, Morris SA & Bruce AW (2009) Nat. Rev.
Genet. 10, 467. With permission from Macmillan
Publishers Ltd.]
There are some possible exceptions to this rule, because recent studies suggest that the bone marrow and reproductive organs may contain
small populations of apparently pluripotent cells. However, the evidence
for this is inconclusive so far.
With the exception of erythrocytes and post-meiotic gametes, most of the
cells of the developing organism contain an identical copy of the original genome that was generated by the combination of the two parental
genomes shortly after fertilization. The basis of cell differentiation is that
specific patterns of gene expression apply to different types of cells, but
the fact that they all probably possess the same genome implies that epigenetic regulation must control the cell-type-specific expression pattern.
It is important to understand how this works, but the subject is worthy of
an entire volume in its own right and we can only give a few select examples. A useful first step is to examine how the pluripotent state is maintained, because this will help us to understand how the cells of the ICM
may be able to make fate decisions that allow them to produce multiple
cell types. We will follow this with some examples of the pathways leading to the differentiation of specific cell types, although it must be said
skin cells
of epidermis
neuron
of brain
pigment
cell
gastrula
ectoderm
Figure 12.2 Three primordial germ layers
arise during embryonic development.
These originate from the ICM and are known as
ectoderm, mesoderm, and endoderm. These
produce different parts of the body, as shown.
For example, the cells of the ectoderm give
rise to the skin, peripheral nerves, and CNS,
whereas the mesoderm produces bones, blood,
kidneys, muscles, and heart. The endoderm
produces organs such as the lungs, liver, and
digestive tract.
mesoderm
cardiac
muscle
endoderm
skeletal
muscle
cells
tubule
cell of
the kidney
red
blood
cells
lung cell
(alveolar
cell)
thyroid
cell
pancreatic
cell
smooth
muscle
(in gut)
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MAINTENANCE OF PLURIPOTENCY IN EMBRYONIC STEM CELLS
173
that the information provided is incomplete because the science simply
has not progressed to the level where step-by-step descriptions of all the
required epigenetic controls are possible at this time.
12.2 MAINTENANCE OF PLURIPOTENCY IN
EMBRYONIC STEM CELLS
Embryonic stem cells (ESCs) are derived from the ICM of day 5–8 blastocyst-stage or morula-stage embryos (see Figure 12.1). ESCs are pluripotent and are capable of karyotypically stable, prolonged self-renewal.
They are characterized by their potential to differentiate into cells of the
three germ layers both in vitro and in vivo. In contrast to the specific gene
expression programs observed in differentiated cells, ESCs are defined
by their potential to activate all of the gene expression programs that are
found in embryonic and adult cell lineages.
Various studies have endeavored to understand the molecular mechanisms that give ESCs their properties. These attempts to identify a molecular “signature” of ESCs have borne some fruit, including the identification
of the transcription factors OCT4 and NANOG as markers for ESCs.
However, these experiments also show that there is very little other overlapping of gene expression among different ESC lines and also among
the same ESC lines studied in different laboratories. This lack of overlap
raises the question of whether a common molecular identity for ESCs
can be uncovered. However, recent studies have indicated that epigenetic mechanisms may regulate self-renewal, pluripotency, and lineagespecific differentiation, giving ESCs their unique characteristics. Recall
that epigenetic mechanisms include modification of histone proteins,
DNA methylation, ATP-dependent remodeling, incorporation of variant
histones, changes in local and higher-order conformation of DNA, and
RNAi. Through the combined efforts of these epigenetic mechanisms,
gene expression patterns can be tightly and dynamically regulated.
As an example of this regulation, the chromatin structure of mouse
embryonic stem cells (mESCs) has now been demonstrated to be hyperdynamic, in that major architectural proteins, for example H1, H2B, H3,
and HP1, are loosely bound to chromatin with very short residency times.
Differentiation has been found to lead to a decrease in the dynamic nature
of these proteins, as demonstrated by an increase in their residency time.
These findings suggest that this dynamic nature of chromatin is specific
to pluripotent cells and that differentiation leads to a restructuring of the
genome. Additionally, it was demonstrated that heterochromatic markers change from a dispersed localization in mESCs to more concentrated,
distinct foci in differentiated cells, with increased global levels of trimethylated lysine 9 H3 (H3K9me3)—a heterochromatic histone modification
linked to gene repression (see Table 6.2)—and decreased levels of acetylated histones H3 and H4 (H3ac and H4ac). Recall that acetylation of histones is linked to euchromatin and permissivity of gene expression, and
so the noted decrease in this modification is in keeping with the dampening of gene expression. Analyses of global histone modification patterns
in ESCs have previously suggested that the ESC genome is subject to
generalized histone acetylation and lysine 4 H3 methylation (H3K4me).
As these are both transcription-activating modifications, these changes
in global genomic architecture and global histone modifications suggest
that the chromatin environment in ESCs is highly euchromatic, and the
genome is therefore highly permissive for gene expression. This would
account for the pluripotent nature of ESCs, with the genome becoming
more structured, condensed, and heterochromatic during differentiation,
leading to loss of pluripotency.
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Another study of mESCs sought to understand regulatory mechanisms
involved in development by examining highly conserved noncoding elements (HCNEs). HCNEs were studied because they are present in regions
where genes encoding developmentally important transcription factors
are concentrated. Large-scale chromatin immunoprecipitation (ChIP)
assays found large areas of a repressive histone modification, dimethylation and trimethylation of lysine 27 histone H3 (H3K27me2/me3), alongside smaller regions of a permissive modification, H3K4me, at HCNEs.
This is unusual inasmuch as these modifications are usually mutually
exclusive, and so these regions were termed “bivalent” domains (Figure
12.3). Interestingly, the bivalent domains coincided with differentiationassociated transcription factor genes expressed at very low levels in the
ESCs. It was therefore proposed that the bivalent domains act to silence
such genes so as to maintain pluripotency while also allowing the genes
to remain poised for transcription, so they can be rapidly activated on
differentiation. This echoes the presence of activating histone modification such as H3K4me2 and H3K4me3 in the HOXA cluster, as described
in Chapter 9. On differentiation, these bivalent domains “resolve,” so that
silent genes become concentrated for H3K27me2/me3 only, and active
genes become enriched for euchromatic modifications.
12.3 DIFFERENTIATION OF EMBRYONIC
STEM CELLS
The earliest observable differentiation events for ESCs in vitro are the
decrease in pluripotent character and the appearance of characteristics
of the three primordial germ layers. It is probable that several changes in
gene expression precede these events, but either they are too transient
to be detected with our current techniques or they make no observable
changes to the morphology or behavior of the cells. Another problem is
that differentiation of ESCs seems to be partly random in the laboratory.
One would not expect this to be so in the developing embryo, in which
ES cells
Me
Me
Me
Ac
Me
key developmental regulators
‘bivalent’ domains
early replication
Figure 12.3 Bivalent chromatin domains.
In pluripotent stem cells, a large number of
genes encoding key developmental regulators
are marked by a combination of permissive
(H3K9me2 and H3K9ac, shown in blue and
green, respectively) and repressive (H3K27me3,
shown in red) epigenetic marks. Such domains
are usually resolved during cell differentiation
when specific genes must either be expressed
or permanently repressed. (Courtesy of
Véronique Azuara, Imperial College London.)
differentiation
active early
replication
repressed
late replication
lineage-committed cells
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individual cells should respond to precisely regulated developmental cues
telling them what to do and where to go; however, in a culture dish in the
laboratory, such information is scrambled or nonexistent, and the resulting mixture of differentiated cells contains small percentages of nearly
all the cell types found in the adult body. This makes the process leading
to differentiation very difficult to study because we have to separate out
individual cells for analysis, a task more easily done for some types of
cells than for others. For example, early hematopoietic cells can be isolated in abundance because some of the proteins they express on their
surfaces can be used to separate them from the cellular “soup” by using
flow cytometry, but we do not have this luxury for many other cell types.
Because epigenetic control systems are highly dependent on cell identity,
we must restrict our description of such systems to those that can be
studied in well-defined and easily accessible cells. That said, it is nevertheless worth describing some of the more global changes that take place
during ESC differentiation. These changes tell us little about the mechanisms that control individual genes, but they are useful for understanding
the differences in genome organization between pluripotent and differentiated cells.
We saw earlier that ESCs have characteristic blocks of euchromatin
throughout much of their genome with relatively little heterochromatin. Heterochromatin is detected by the presence of H3K9 dimethylation
and trimethylation, and although these histone modifications do exist
in the ESC genome, they do not accumulate into large blocks of H3K9
dimethylated chromatin as seems to be the case for differentiated cells.
These regions known as large organized chromatin K9 modifications
(LOCKs) can be up to 4.9 Mb in length and are highly conserved between
human and mouse. Although they cover only 4% of the genome in mouse
ESCs, they cover 31% of the genome in differentiated ESCs, 46% in
mouse liver cells and 10% in brain. The positions of these LOCKs vary
between cell types; some of these are shown in Figure 12.4, along with
the relative positions of the genes in these regions.
The level of acetylation at H3K9 is also decreased. This modification is
normally associated with euchromatin, and its progressive loss is needed
to make way for the H3K9 dimethylation and trimethylation associated
with heterochromatinization.
Chr. 8:
44000000
45000000
46000000
47000000
48000000
49000000
Figure 12.4 Summary of data from a 10 Mb
region of mouse chromosome 8. Locations
of LOCKs in undifferentiated mouse ES cells
(green bars), differentiated ES cells (red bars),
liver cells (orange bars), and brain cells (blue
bars) are shown. [From Wen B, Wu H, Shinkai
Y et al. (2009) Nat. Genet. 41, 246. Macmillan
Publishers Ltd.]
50000000
51000000
52000000
ES LOCKs
Diff. ES LOCKs
Liver LOCKs
Brain LOCKs
Mtus1 Zfp353
Fgl1
Pcm1
Asah1
Genes
Frg1
Triml1
Zfp42
Adam26b
Adam26a
EG384813
Adam34
EG384814
Fat1
Pdim3
Casp3
Rwdd4a
Odz3
Mtnr1a
Ufsp2
lrf2
lng2
F11
Snx25
Enpp6
Wwc2
Klkb1
Slc25a4
Stox2
Dctd
Cyp4v3
Helt
Stox2
Fam149a
Acsl1
Cdkn2alp
Tir3
Mlf1ip
Cldn22
Sorbs2
D030016E14Rik
Ccdc110
LOC100039801
1700029J07Rik
Ankrd37
Lrp2bp
4933411K20Rik
Ccdc111
EpiG12f04
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12.4 BIVALENT CHROMATIN DOMAINS IN NEURAL
STEM CELLS
Generating specific differentiated cell types from ESCs can help us to
describe and perhaps better understand the mechanism by which epigenetic modification controls the gene expression pattern of that cell. A
commonly occurring developmental pathway from ESCs and embryonal
carcinoma cells is neurogenesis; given the prevalence of defined surfacemarker proteins for the neural progenitor stage and the neural stem cell
stage, separation and analysis of these cells is relatively straightforward.
During human embryonic stem cell (hESC) differentiation into the neural lineage, two stages of specification can be purified by the use of flow
cytometry for the CD133 surface marker and β-III tubulin (Figure 12.5).
(CD133 is a marker of multipotent stem cells of many tissue types (including multipotent neural progenitor cells), and β-III tubulin is neuronspecific and indicative of more terminally differentiated cells.)
The CD133-expressing neural progenitor cells show substantial downregulation of pluripotency-associated genes compared with their ESC
expression levels, and up-regulation of neural marker genes such as
SOX1, SOX3, PAX7, and Nestin. Even though these latter genes are differentiation-specific, they are still expressed at low levels in ESCs; however, these levels are so much lower than in the neural progenitor cells
that the expression is probably of no functional significance. In line with
(a)
H9-derived CD133+ve population
(b)
β-III-tubulin immunostaining of EC
and H9-derived neuronal lineages
105
H9 monolayer-positive
CD133+ve cells
CD133
H9-derived monolayers
103
104
CD133
CD34
102
103
104
CD34
102
102 103 104 105
H9 monolayer-negative
105
102
103
104
β-III-tubulin
105
H9-derived β-III-tubulin+ve population
β-III-tubulin-monolayer
poly-D-lysine/laminin
Merged
(ab+DAPI)
50
70
10 30 50 70
β-III-tubulin-control pDi
BIII+
10
30
BIII+
102
103
104
105
102
103
104
105
Figure 12.5 Flow cytometric analysis and sorting of CD133expressing neural progenitor cells and terminally differentiated
EpiG12f05
neurons from differentiating hESCs. (a) Multipotent neural
progenitors express the surface antigen CD133, whereas terminally
differentiated neurons arising from the progenitors express β-III-tubulin.
This allows the use of flow cytometry to separate neural progenitors
from other cell types produced during hESC differentiation and to
isolate relatively pure populations of mature neurons. The diagrams
in (a) are the outputs from a flow cytometer in which each of the dots
on the plotted data represents a single cell expressing the CD133
Epigenetics_Chapter_12.indd 176
protein on its surface. This expression is detected by a fluorescent
antibody that binds specifically to CD133. CD133-expressing cells are
represented by blue dots. The flow cytometer can recognize the cells
and direct them into a different container to the other non-CD133expressing cells. The histogram shows how the cytometer can detect
β-III-tubulin-expressing neurons, which may be sorted in the same way
as the CD133-expressing cells. (b) Immunostaining of hESC-derived
neurons to confirm the presence of β-III-tubulin. [From Golebiewska A,
Atkinson SP, Lako M, Armstrong L (2009) Stem Cells 27, 1298. With
permission from John Wiley and Sons.]
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177
expectations, the neural progenitors did not display H3K4me3 at the
pluripotency-associated gene promoters, which correlates with the much
lower expression levels of genes associated with pluripotency in these
cells; however, significant levels of H3K4me2 were still detected at the
pluripotency-associated gene promoters in cells expressing CD133 and
β-III-tubulin, in keeping with the possible need for rapid up-regulation of
these genes.
Most of the genes specific to the neural lineage have bivalent chromatin
domains at their promoters in the undifferentiated ESCs, and these show
the expected resolution on differentiation into the neural progenitor cells.
This causes some loss of the repressive H3K27me3 modification, with
retention of the activating H3K4me3 mark. Markers of other lineages,
such as GATA4 (which is expressed by cells found in the mesoderm and
endoderm), lost H3K4me3 and displayed increased levels of H3K27me3
(which correlates with their lower expression during neurogenesis), but
surprisingly the resolution does not seem to be absolute because at least
some level of both types of histone modifications seemed to be retained
for some of the bivalent genes after differentiation. This was unexpected
for genes that are not likely to require expression in cells that differentiate from the neural progenitors, so it is currently unknown why bivalent domains are needed. It is possible that the functionality of a bivalent
domain depends on relative levels of the two histone modifications,
but this has not yet been determined with any certainty. However, it is
interesting that H3K9 methylation levels also increase at the apparently
retained bivalent promoters. Perhaps this is related to the suppression of
gene expression at these loci, which would make the nature of bivalent
domains rather different in pluripotent cells than in the multipotent progenitors (such as neural stem cells) that derive from them.
12.5 CHROMATIN PROFILE OF HEMATOPOIETIC
PROGENITORS
Neural stem cells are not unique in having lineage-specific genes that
are required for differentiating into multiple cell types further down the
differentiation pathway. Hematopoietic stem cells (HSCs) are multi­
potent cells that at the single-cell level have the potential to differentiate into all cells of the erythromyeloid and lymphoid lineages, as well
as to maintain their numbers by means of controlled self-renewal. In
short, they are responsible for the lifelong regeneration of the blood and
immune systems.
Progression from HSCs to their differentiated progeny involves the coordinated regulation of multiple gene expression programs that lead to the
activation or repression of lineage-specific genes. At the single-cell level
in HSCs, low-level transcription of lineage-affiliated genes has been
observed, a phenomenon known as lineage priming. It is possible that
a specific chromatin structure exists at lineage-affiliated genes in HSCs
that mediates low-level expression for the propagation of transcriptional
memory during the differentiation process. As in the neural progenitors,
it is apparent that H3K4 dimethylation marks the genes that need to be
kept in a “poised” state for rapid up-regulation when HSC differentiation
takes place. Because multiple fate decisions are possible, many different
genes must be maintained in this state. An excellent example of this is
the mouse γ5–VpreB1 locus.
Transcription of the VpreB1 and γ5 genes in mice is activated during
the pre-B-cell stage, before the heavy-chain rearrangement that is central to the immune function of the B cells that are eventually produced.
The γ5-VpreB1 domain is already marked by histone H3 acetylation and
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histone H3K4 methylation at a discrete site in ES cells; these modifications are not present in the rest of the locus. The marked region expands
in early B-cell progenitors and becomes a localized center for the recruitment of transcription factors and RNA polymerase II, but it disappears in
mature B cells and is not present in cells of other lineages such as liver.
This is a rather extreme example because other elements of this type have
not been discovered, but it demonstrates the general principle that lineage-specific genes may have regions of H3K4 methylation that prevent
them from being incorporated into heterochromatin while not actually
having sufficient activation capacity to increase expression probability
beyond a threshold level. In any case, gene up-regulation probably needs
the activity of a specific transcription factor, but at least the promoter is
more likely to be accessible when required.
There is also evidence that differential levels of H3K4 dimethylation
and trimethylation mark developmentally poised hematopoietic genes.
Multipotent hematopoietic cells have a subset of genes that are differentially methylated (H3K4me2+/me3–). These genes are transcriptionally
silent, lineage-specific hematopoietic genes that are uniquely susceptible
to differentiation-induced H3K4 demethylation in developmental pathways other than that of the hematopoietic system. Many of the genes are
not formally recognized as having bivalent chromatin domains in ESCs.
These examples suggest that formal recognition of the presence of bivalent chromatin domains within a gene locus does not mean that gene
is not able to undergo up-regulation of expression at some point during
differentiation of a progenitor cell. It merely suggests that bivalency is
one mechanism by which some genes can be held in a “poised” state that
permits rapid expression when such genes are needed. The alternative
mechanisms controlling the spatio-temporal expression of genes without
bivalent chromatin domains remain to be fully elucidated.
We have attempted to introduce the basic concepts behind the cellular
differentiation that permits development of the embryo, but this discussion is far from exhaustive. More detailed information can be obtained
from the references given in the Further Reading section below.
KEY CONCEPTS
• Embryonic development begins with a totipotent cell produced by the
fusion of sperm and oocyte during fertilization.
• The resulting “zygote” is called totipotent because it can differentiate
into any of the cell types found in the developing embryo.
• Changes in gene expression patterns both cause and result from the
differentiation of totipotent cells, and this is mostly controlled by epigenetic modifications.
• Embryonic stem cell differentiation can be used to model the processes of cellular differentiation occurring in the embryo.
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FURTHER READING
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FURTHER READING
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