Transcript Computational Biology

V2 epigenetics during development
Review of lecture V1 ...
What is:
- bisulfite treatment of DNA
- methylation-specific PCR
- Restriction landmark genomic scanning (RLGS)
- chromatin immunoprecipitation using the ChIP-on-chip approach?
Biological Sequence Analysis
SS 2008
lecture 2
1
epigenetics during specialisation of cells
Key feature of multi-cellular organisms:
ability to develop specialized cells with specific functions.
Process 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
SS 2008
lecture 2
2
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
SS 2008
lecture 2
3
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)
Biological Sequence Analysis
SS 2008
lecture 2
4
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)
Biological Sequence Analysis
SS 2008
lecture 2
5
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)
Biological Sequence Analysis
SS 2008
lecture 2
6
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.
Biological Sequence Analysis
SS 2008
lecture 2
7
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
Biological Sequence Analysis
SS 2008
lecture 2
8
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
Biological Sequence Analysis
SS 2008
lecture 2
9
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)
Biological Sequence Analysis
SS 2008
lecture 2
10
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)
Biological Sequence Analysis
SS 2008
lecture 2
11
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)
Biological Sequence Analysis
SS 2008
lecture 2
12
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/
SS 2008
lecture 2
Biological Sequence Analysis
13
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
SS 2008
lecture 2
Biological Sequence Analysis
http://symatlas.gnf.org/SymAtlas/
14
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
SS 2008
lecture 2
Biological Sequence Analysis
http://symatlas.gnf.org/SymAtlas/
15
PGC development in mouse
Scheme summarising chromatin
changes occurring in PGCs during
the reprogramming process. The
chromatin changes in PGCs occur in
2 steps.
The first step is characterized by
loss of H3K2me2 and gain of
H3K27me3, H3K9ac and H4/H2A
R3me2s at E8.5.
The second step 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)
Biological Sequence Analysis
SS 2008
lecture 2
16
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)
Biological Sequence Analysis
SS 2008
lecture 2
17
methylation of CpG islands
What does the differential methylation mean?
Hajkova et al. Nature 452, 877 (2008)
Biological Sequence Analysis
SS 2008
lecture 2
18
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)
Biological Sequence Analysis
SS 2008
lecture 2
19