Transcript Computational Biology

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.
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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.
Insertional 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)
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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)
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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)
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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)
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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 histone–DNA contacts.
Both classes have significant roles in gene regulation,
including differentiation-specific gene expression.
ATP-dependent remodellers don‘t 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 chromatinremodelling 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-remodelling
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
(1) histone acetyl transferase (HAT) enzymes — which acetylate (Ac) both
promoter histones and MyoD — and
(2) 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.
www.wikipedia.org
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http://symatlas.gnf.org/SymAtlas/
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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.
www.wikipedia.org
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http://symatlas.gnf.org/SymAtlas/
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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)
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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)
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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)
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