V8 epigenetics during mamalian development

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V8 epigenetics during mamalian development
Key feature of multi-cellular organisms ability
to develop specialized cells with specific
functions. Seen the other way around Mammalian
development is a unidirectional process during
which there is a progressive loss of
developmental potential. It begins with the
formation of a unicellular zygote and ends with
the establishment of the 220 specialized cell
types of the mammalian body.

de la Serna et al. Nat. Rev. Gen. 7, 461 (2006)
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Development potential epigenetic states of cells
A modification of C. H. Waddington's epigenetic
landscape model, showing cell populations with
different developmental potentials and their
respective epigenetic states. Developmental
restrictions can be illustrated as marbles
rolling down a landscape into one of several
valleys (cell fates). Colored marbles
correspond to different differentiation states
(purple, totipotent blue, pluripotent red,
multipotent green, unipotent). Examples of
reprogramming processes are shown by dashed
arrows.

Hochedlinger, Development 136, 509 (2009)
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Glossary I
Totipotency Ability of a cell to give rise to all
cells of an organism, including embryonic and
extraembryonic tissues. Zygotes are
totipotent. Pluripotency Ability of a cell to
give rise to all cells of the embryo. Cells of
the inner cell mass (ICM see below) and its
derivative, embryonic stem (ES) cells, are
pluripotent. Multipotency Ability of a cell to
give rise to different cell types of a given cell
lineage. These cells include most adult stem
cells, such as gut stem cells, skin stem cells,
hematopoietic stem cells and neural stem
cells. Unipotency Capacity of a cell to sustain
only one cell type or cell lineage. Examples are
terminally differentiated cells, certain adult
stem cells (testis stem cells) and committed
progenitors (erythroblasts).

Hochedlinger, Development 136, 509 (2009)
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Chromatin-remodelling enzymes
Inner cell mass (ICM) Cells of the blastocyst
embryo that appear transiently during development
and give rise to the three germ layers of the
developing embryo. Embryonic stem (ES) cells
Pluripotent cell line derived from the ICM upon
explantation in culture, which can differentiate
in vitro into many different lineages and cell
types, and, upon injection into blastocysts, can
give rise to all tissues including the
germline. Primordial germ cells (PGCs) PGCs give
rise to oocytes and sperm in vivo and to
embryonic germ (EG) cells when explanted in
vitro. Embryonic germ (EG) cells Pluripotent
cell line derived from explanted PGCs. In
contrast to pluripotent ICM and ES cells, PGCs
are unipotent but become pluripotent upon
explantation in culture.

Hochedlinger, Development 136, 509 (2009)
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Chromatin-remodelling enzymes
Induced pluripotent stem (iPS) cells Cells
generated by the overexpression of specific
transcription factors in mouse or human somatic
cells, which are molecularly and functionally
highly similar to ES cell counterparts. Insertion
al mutagenesis Insertion of a viral genome near
endogenous genes, resulting in gene activation or
silencing. Retrovirus-mediated insertional
mutagenesis in hematopoietic cells can enhance
self-renewal in vitro and cause cancer in vivo.

Hochedlinger, Development 136, 509 (2009)
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epigenetics during mamalian development
Mammalian development depends on cellular
differentiation pathways. Initiation of such
pathways is determined by coordinated regulation
of - silent genes that in many cases have never
been expressed must be activated, - a number of
transcriptionally competent genes must be
repressed. Essential in understanding
differentiation identify tissue-specific genes
and regulatory proteins that directly control
their expression.

de la Serna et al. Nat. Rev. Gen. 7, 461 (2006)
Biological Sequence Analysis
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SS 2009 lecture 8
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specialisation of cells
However, tissue-specific transcriptional
regulatory proteins are not sufficient to
initiate differentiation. Also essential -
changes at the level of both higher-order
chromatin structure and - chromatin organization
at individual genes. Proteins are needed that
alter the structure of chromatin at
lineage-specific genes to facilitate the function
of tissue-specific regulators.

de la Serna et al. Nat. Rev. Gen. 7, 461 (2006)
Biological Sequence Analysis
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SS 2009 lecture 8
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Methylation reprogramming in the germ line

Primordial germ cells (PGCs) in the mouse become
demethylated early in development.
Remethylation begins in prospermatogonia on E16
in male germ cells, and after birth in growing
oocytes.
Reik, Dean, Walter. Science 293, 1089 (2001)
Biological Sequence Analysis
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SS 2009 lecture 8
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Methylation reprogramming in preimplantation
embryos
The paternal genome (blue) is demethylated by an
active mechanism immediately after fertilization.
The maternal genome (red) is demethylated by a
passive mechanism that depends on DNA
replication. Both are remethylated around the
time of implantation to different extents in
embryonic (EM) and extraembryonic (EX) lineages.

Methylated imprinted genes and some repeat
sequences (dashed line) do not become
demethylated. Unmethylated imprinted genes
(dashed line) do not become methylated.
Reik, Dean, Walter. Science 293, 1089 (2001)
Biological Sequence Analysis
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SS 2009 lecture 8
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Chromatin-remodelling enzymes

• Two main classes
• - enzymes that covalently modify histone proteins
  (see V1), and
• - enzymes that use ATP hydrolysis to alter
  histoneDNA contacts.
• Both classes have significant roles in gene
  regulation,
• including differentiation-specific gene
  expression.
• ATP-dependent remodellers dont function
  similarly in all cell types.
• Instead, they have a range of specific and
  context-dependent roles in differentiation. E.g.
  they have functions in
• - recombination,
• - cell-cycle regulation and
• genome organization,
• indicating important links between chromatin
  remodelling and other cellular processes during
  differentiation.

de la Serna et al. Nat. Rev. Gen. 7, 461 (2006)
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ATP-dependent chromatin-remodelling enzymes
The three best-characterized classes of
ATP-dependent chromatin-remodelling enzyme are
the families of - SWI/SNF, - CHD (chromodomain
and helicase-like domain) and - ISWI (imitation
SWI). Each has a unique domain (bromo, chromo
and sant) that likely interact with specific
chromatin substrates. Each enzyme class forms
complexes with other proteins - SWI/SNF
proteins interact with brahma (BRM)- or
brahma-like 1 (BRG1)-containing enzymes. - CHD
proteins can form part of the NuRD (nucleosome
remodelling and histone deacetylase) complex,
which can include CHD3- or CHD4-containing
enzymes, or possibly both. - ISWI
SNF2H-containing enzymes are found in several
complexes (for example, ACF (ATPutilizing
chromatin assembly and remodelling factor) and
RSF (remodelling and spacing factor)), and SNF2L
enzymes form part of the NuRF (nucleosome-remodell
ing factor) and CERF (CECR2-containing
remodelling factor) complexes.

de la Serna et al. Nat. Rev. Gen. 7, 461 (2006)
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Example skeletal muscle differentiation

• The myogenin gene (Myog) is expressed
  specifically during skeletal muscle
  differentiation. The locus is constitutively
  bound by a heterodimer of 2 homeodomain proteins
  from the PBX/MEIS family in undifferentiated
  cells.
• E binding sites for the transcription factor
  MyoD
• P binding sites for the transcription factor
  PBX
• M binding sites for the transcription factor
  MEF2
• T the TATA box for Myog.
• In undifferentiated cells, several of these sites
  are inaccessible to the proteins that bind them
  due to the conformation of chromatin at this
  locus (indicated by crosses).
• Initial targeting of the skeletal muscle
  regulator, MyoD, to the myogenin promoter occurs
  in part through physical interactions with PBX.
• MyoD then sequentially targets
• histone acetyl transferase (HAT) enzymes which
  acetylate (Ac) both promoter histones and MyoD
  and
• a BRG1-based SWI/SNF enzyme, which is activated
  through the p38 kinase-mediated phosphorylation
  (yellow circle) of the BAF60 subunit.
• The SWI/SNF enzyme mediates ATP-dependent
  chromatin remodelling at the myogenin promoter,
  which results in changes in accessibility that
  permit the stable binding of heterodimers of MyoD
  and an E-box binding protein (EBP), and another
  factor, MEF2, to their cognate binding sites in
  the myogenin promoter.
• Then transcription of Myog can take place.

de la Serna et al. Nat. Rev. Gen. 7, 461 (2006)
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Abstract A unique feature of the germ cell
lineage is the generation of totipotency. A
critical event in this context is DNA
demethylation and the erasure of parental
imprints in mouse primordial germ cells (PGCs) on
embryonic day 11.5 (E11.5) after they enter into
the developing gonads. Little is yet known about
the mechanism involved, except that it is
apparently an active process. We have examined
the associated changes in the chromatin to gain
further insights into this reprogramming event.
Here we show that the chromatin changes occur in
two steps. The first changes in nascent PGCs at
E8.5 establish a distinctive chromatin signature
that is reminiscent of pluripotency. Next, when
PGCs are residing in the gonads, major changes
occur in nuclear architecture accompanied by an
extensive erasure of several histone
modifications and exchange of histone variants.
Furthermore, the histone chaperones HIRA and
NAP-1 (NAP111), which are implicated in histone
exchange, accumulate in PGC nuclei undergoing
reprogramming. We therefore suggest that the
mechanism of histone replacement is critical for
these chromatin rearrangements to occur. The
marked chromatin changes are intimately linked
with genome-wide DNA demethylation. On the basis
of the timing of the observed events, we propose
that if DNA demethylation entails a DNA
repair-based mechanism, the evident histone
replacement would represent a repair-induced
response event rather than being a prerequisite.
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some terms from developmental biology
somatic cells cells forming the body of an
organism germ cells (dt. Keimzelle, Ovolum) are
part of the germline. germline (dt. Keimbahn)
line of germ cells that have genetic material
that may be passed to a child/embryo. Germline
cells are immortal. Gametocyte eukaryotic germ
cell includes spermatocytes (male) and oocytes
(female) primordial germ cells predecessors of
germ cells. They migrate to the gonadal ridge.
They may be detected from expression of
Stella gonad (dt. Keimdrüse) gonadal ridge
precursor to the gonads

www.wikipedia.org
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Germ line development
Germline cells are produced by embryonic
cleavage. Cleavage division of cells in the
early embryo. The zygotes of many species undergo
rapid cell cycles with no significant growth. The
different cells derived from cleavage are called
blastomeres. Cleavage in mammals is slow. Cell
division takes 12 24 hours and is
asynchronous. In mammals, specification of germ
cells seems to proceed by induction. BMP (Bone
morphogenetic protein) signals from the
extraembryonic ectoderm activate expression of
fragilis and bias the cells toward PGC. The cells
expressing fragilis collectively express stella
and Blimp1, a general repressor of transcription.

www.wikipedia.org
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Working hypothesis

• The specification of about 40 primordial germ
  cells (PGCs) from Blimp1-expressing PGCs
  precursors is accompanied by expression of stella
  on E7.25.
• After their migration into the developing gonads,
  PGCs show genome-wide DNA demethylation between
  E11.5 and E12.5, including erasure of genomic
  imprints, which is supposedly an active process.
• The mechanism of this DNA demethylation process
  is unknown, but we reasoned that it might be
  linked with changes in chromatin and histone
  modifications.
• Investigate chromatin in nascent PGCs at E8.5
  (100 PGCs per embryo)

Hajkova et al. Nature 452, 877 (2008)
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PGC development in mouse
The nascent (dt. im Entstehen begriffenen) PGCs
are first identified on E7.5 as a group of about
40 stella expressing cells. On E8.5 (Step1 of
the reprogramming process) when there are about
1000 PGCs per embryo, they start to migrate along
the developing hindgut (dt. Dickdarm) and reach
the developing gonads at about E10.5. Soon after
the entry into the gonads the PGCs undergo
epigenetic reprogramming (as a second step of the
reprogramming process), which includes
genome-wide DNA demethylation, erasure of genomic
imprints and re-activation of the inactive X
chromosome (Xi) in female embryos.

Hajkova et al. Nature 452, 877 (2008)
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chromatin changes
- loss of dimethylation of Lys9 of histone H3
(H3K9me2) - in early PGCs enhanced
trimethylation of H3K27me3 - enriched
methylation of H3K4me2 and H3K4me3 and of many
histone acetylation marks, especially H3K9ac, as
well as symmetrical methylation of Arg3 on
histones H4 and H2A (H4/H2AR3me2s). Notably,
this germ cell chromatin signature is established
specifically in PGCs (not detected in the
contemporary somatic cells) before their entry
into the gonads, and is associated with the
expression of pluripotency-specific genes Sox2,
Oct4 (Pou5f1), Nanog and stella. What are these
4 genes Sox2, Oct4, Nanog, stella?

Hajkova et al. Nature 452, 877 (2008)
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Sox2 SRY (sex determining region Y)-box 2
SRY or SOX2 is a transcription factor that is
essential to maintain self-renewal of
undifferentiated embryonic stem cells.

Intronless gene. The encoded protein may act as a
transcriptional activator after forming a
protein complex with other proteins.
www.wikipedia.org
http//symatlas.gnf.org/SymAtlas/
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Oct4 (ASH1L, POU5F1)
Oct4 is expressed in developing embryos
throughout the preimplantation period. Knockout
of Oct-4 promotes differentiation. One of its
main functions is to keep the embryo from
differentiating. Too much or too little
expression will cause differentiation of the
cells. Therefore, it is frequently used as a
marker for undifferentiated cells.

http//symatlas.gnf.org/SymAtlas/
www.wikipedia.org
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Nanog

Nanog is another gene expressed in embryonic stem
cells (ESC) and is thought to be a key factor in
maintaining pluripotency. Nanog works together
with other factors as POU5F1 and SOX2 to
establish ESC identity. Human nanog 305 amino
acid protein, conserved homeodomain that
facilitates DNA binding.
http//symatlas.gnf.org/SymAtlas/
www.wikipedia.org
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ES cell TF network implications for
reprogramming

• (A) The reprogramming factors Oct4, Sox2 and Klf4
  often co-bind promoter regions with other TFs,
  including
• Nanog, Nr0b1 (nuclear receptor subfamily 0),
• Esrrb (estrogen-related receptor,beta),
• Zfp281 (zinc finger protein 281) and
• Nac1 (nucleus accumbens associated 1,
• as well as with Stat3 and Smad1
• (TFs downstream of the Bmp4 and Lif signaling
  pathways that maintain ES cell self-renewal and
  pluripotency).

The recruitment of co-activators, such as the
histone acetyltransferase (HAT) p300 is often
observed (yellow). This binding pattern is found
in transcriptionally active genes in ES cells. ES
cell target groups and implications for
reprogramming are also indicated.
Hochedlinger, Development 136, 509 (2009)
Biological Sequence Analysis
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ES cell TF network implications for
reprogramming
(B) In ES cells, genes bound by either Oct4, Sox2
or Klf4 are often repressed, potentially through
the recruitment of Polycomb group (PcG) proteins
or histone deacetylases (HDACs), but become
activated upon differentiation. (C) cMyc is
proposed to bind and activate largely different
sets of genes to Oct4, Klf4 and Sox2, but in
collaboration with other transcription factors.
Hochedlinger, Development 136, 509 (2009)
Biological Sequence Analysis
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SS 2009 lecture 8
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Chromatin changes occurring in PGCs during the
reprogramming process
The chromatin changes in PGCs occur in 2 steps.
Step (1) is characterized by loss of H3K2me2
and gain of H3K27me3, H3K9ac and H4/H2A R3me2s at
E8.5. Step (2) occurs at E11.5 and is
characterized by changes in nuclear architecture
(loss of chromocenters) and by the loss of
numerous histone modifications.

Hajkova et al. Nature 452, 877 (2008)
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DAPI staining labelling of cell nucleus
DAPI 4,6-diamidino-2-phenylindole is a
fluorescent stain that binds strongly to DNA. It
can pass through intact cell membranes. When
bound to ds-DNA, DAPI absorbs maximally at 358 nm
and emits fluorescent light at 461 nm (blue/cyan).

Hajkova et al. Nature 452, 877 (2008)
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methylation of CpG islands
What does the differential methylation mean?

Hajkova et al. Nature 452, 877 (2008)
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connections between chromatin and DNA methylation
- What do the two models describe? - How did the
authors arrive at the two models? - How could
one distinguish between these two models?

Hajkova et al. Nature 452, 877 (2008)