Transcript Epigentic regulation during early embryogenesis

Epigenetic regulation during early
embryogenesis – reprogramming
and remodelling
Helena Fulkova
Istitute of Animal Science, www.vuzv.cz
[email protected]
Basic facts
• No loss/gain of genomic DNA during
development and differentiation
• Somatic cells possess full developmental
potential – demonstrated by SCNT
•Organisation of DNA in nucleus is nonrandom and
heritable
•Gene expression is not reset every cell division
→ A mechanism that is flexible
but heritably regulates gene
expression
and nuclear organisation
…
DNA + proteins =
chromatin
This cannot simply be achived by different TFs!
What is epigenetic inheritance?
A heritable change in gene expression that is not
caused by changes in the DNA seguence
→ DNA methylation
→ Histone post-translational modifications
→ Histone variants
→ non-histone protein composition of chromatin
→ chromatin remodelling complexes
→ (RNA – antisense transcripts…)
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Role in gene expression
Differentiation
Development
Genome integrity, nuclear organisation
Recombination
DNA repair …
Histones
• H2A, H2B, H3 and H4 (canonical core
histones - octamer)
+146bp DNA = nucleosome
Histones II
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Basic proteins
Conserved among Eukaryotes
C- and N- terminal tails + globular domain
Subjected to post-translational covalent
modifications
+ H1 (linker histone) + other non-histone proteins→ chromatin
Histone modifications
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Acetylation - HATs/HDACs
methylation - HMTs/demethylases
Phosphotylation – kinases/phosphatases
Ubiquitination…
Activating vs. repressive modifications
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• Methylation
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H3/K9, H3/K27, H4/K20 …
repression of transcription and
silencing
H3/K4 … activation
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• Acetylation
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• Phoshorylation
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• Ubiquitination
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H3/K9, H4/K16, H4/K12, H4/K8
and H4/K5 … activation
H4 acetylation … DS break repair
H3/S10 … active transcription,
chromosome condensation
H2AX/S139 … DS breaks repair
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Ubiquitin H2A … X chromosome
silencing
The histone code hypothesis
Histone variants
• H3
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H2A
Canonical core histone
 H3.3
 CENP A
 H2AX
 H2AZ
 macroH2A
 H2ABBD
• H2B
• H4
Transcriptional activation
Kinetochore assembly
Canonical core histone
DNA repair and recombination
Gene expression
X chromosome inactivation
Transcriptional activation?
Canonical core histone
Canonical core histone
Maintaining histone modifications
over DNA replication phase
Chromatin
• Euchromatin – active histone
modifications, low DNA methylation …
• Heterochromatin
– Constitutive – repressive histone
modifications, high DNA methylation, specific
histone variants
– Facultative – repressive histone modifications,
high DNA methylation
DNA methylation
• Cytosine  5-methylcytosine
• In CpG dinucleotides
(exception: most CpG islands)
• Highly mutagenic: mCT
• Repression of transcription,
mobile element silencing,
host defence,
genomic imprinting,
genome stability …
• Genome instability and global hypomethylation
is linked to diseases such as cancer
DNA methylation – DNA
methyltransferases
• Dnmt1
• Dnmt1o, p, s isoform
(maintenance function)
• Dnmt3 family
(de novo methylation)
• Dnmt2
• Dnmt 3a
• Dnmt 3b
• Dnmt 3L
N-terminal regulatory domain
C-terminal catalytic domain
Substrate: S-AdenosylMethionin
Base flipping (like repair
enzymes)
De novo methylation vs.
maintenance methylation
Active vs. passive demethylation
How do these systems
intergate?
• Adaptor proteins –
chromodomain/bromodomain (TFs)…
• Heterochromatin/Euchromatin (chromatin
remodelling)
• X chromosome inactivation
• Change of gene expression (imprinting,
development associated genes – Oct4,
Nanog…)
Adaptor proteins
e.g. Methyl CpG binding proteins:
MBD1 …transcriptional repression (not
• MBD 1-4
in MeCP1 complex)
MBD2 …associated with HDAC1
repression (found in MeCP1,
Sin3, Mi2/NURD complexes)
MBD3 …species specific binding of
methylated/nonmethylated DNA
(found in Mi2)
MBD4 …repair?, can induce nicks, has
glycosylase activity, binds TpG
(deamination of 5-MeC)
• MeCP2
Rett syndromeX linked, predomimantly ♀
HP1
Targeting: methylated H3/K9 (triMe)
• HP1 alpha
• Centromeric
heterochromatin
• HP1 beta
• Pericentric
heterochromatin, but
also euchromatin ?
• HP1 gamma
• Euchromatin, active
genes?
Chromatin remodelling complexes
• ATP dependent
Three classes:
SWI/SNF-like
ISWI-like
CHD-like
All three possess in vitro nucleosome remodelling activity in
the absence of associated subunits (2-12 in the complex)
Composition of remodelling
complexes
Swi/Snf family
CHD family
Possible mechanism
• Trans-transfer of nucleosomes (SWI/SNF)
• Cis-transfer = nucleosome sliding (ISWI)
• generating superhelical torsion (SWI/SNF,
ISWI, Mi2)
• Generating loops (RSF – a member of
ISWI family; SWI/SNF binds DNA in two
positions)
Snf5p subunit of SWI/SNF can interact
with H2B; SWR1 complex (Swr1p)
directs specifity towards H2A.Z
Other complexes contain histone
chaperones (ACF and CHRAC –
NAP1)
Epigenetics and development
Critical developmental time points:
• Fertilization: gametes vs. zygote
• Differentiation: ICM vs. TE (~ 3,5 dpc),
stem cells vs. specific cell types
• PGC establishment and migration
(~ 10 dpc)
Epigenetic „life cycle“
PGCs
TE
Embryo
ICM
+ placenta
Fertilization
Gametes – terminally differentiated and
highly specialized
Genomes - (oocyte, sperm) transcriptionally
inactive after fertilization
•One cell stage embryo is totipotent
•Fertilization represents RADICAL
REPROGRAMMING OF BOTH GENOMES
Post-fertilization epigenetic
remodelling
♂
♀
•Protamines are exchanged for histones
•DNA is demethylated
•High histone acetylation, methylated histones are rare
Histones are highly methylated
DNA is highly methylated
Acetylation of histones rises more slowly
DNA methylation –
active demethylation
5-MeC
FPN
MPN
FPN
MPN
PB
Mouse
Human
Pig
All species tested excluding Sheep!
Histone modifications
• Asymmetric distribution
• Symmetric distribution
• Associated with specific DNA sequences
(chromatin structures)
What happens next –
passive demethylation
Differentiation
• First differentiation – Blastocyst (ICM vs.
TE)
• Pluripotent stem cells → specific cell types
• X chromosome inactivation
• Associated with the change of gene
expression (silencing of Oct4, Nanog,
Sox2 by DNA methylation…+ activation of
tissue specific genes)
X chromosome inactivation
• In female mammals
• Imprinted vs. random (TE vs. ICM)
• Xist non-coding RNA (~15kb - mouse)– in cis
(„way-stations“?)
• Xist regulated by antisense transcript Tsix
• Inactive X form „Barr Body“ – H3K27-3Me, macroH2A,
Ub-H2A, promoter hypermethylation, general
transcriptional silencing …
46 XX
47 XXX
Clemson et al,
1996, J Cell Biol
Bivalent domains
• Regions that contain both activating and
repressive histone marks (prior to
differentiation)
• H3/K4-3Me + H3/K27-3Me
• Typical for tissue-specific genes with CpG
island promoters (PRC targets)
• Upon differentiation the modifications
stabilize to either active or repressive state
Primordial Germ Cells (PGCs)
• Epiblast (embryonic ectoderm) – 7.25 dpc
• Expression of Stella and Fragilis
• Oct4 and other pluripotency-associated
genes (AP…)
• Migration to genital rigdes
• Erasure of imprinting and establishment
according to the sex of the embryo
(8.5 -10.5-11.5 dpc),
X chromosome reactivation
Imprinting
• Gene expression according to parental
origin
• Localized in clusters
• +/- 80 genes (predicted 150)
• Expression based on methylation of
parental alleles (DMRs/DMDs)
• Growth of embryonic/extraembryonic
tissues
•Human syndromes (Prader-Willi/Angelman, Silver-Russel)
– mostly mental retardation
•Higher frequency in ART conceived children!
Epigenetics and SCNT
• Chromatin organisation maintained
(according to donor cell)
• Initial global demethylation, abnormal
remethylation (aberrant Dnmt1
localization)
• Imprinted genes dysregulated
• X chromosome initially reactivated, later
on mosaicism
Chromatin organisation:
Fibroblast vs. Embryo
SCNT
vs.
Anti 5-MeC
Santos and Dean,
Reproduction 2004
Consequence
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Low SCNT efficiency
LOS (Igf2?)
Placental hyperplasia
Death soon after birth
…TSA, 5-AzaC – better efficiency
…ESCs better donors for SCNT
Methods
DNA methylation:
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Immunofluorescence
Methylation sensitive restriction
Bisulfite sequencing
COBRA
Histone modifications:
– Immunofluorescence
– Chromatin IP
DNA methylation
• Convertion of DNA by sodium bisulfite:
5-MeC unreactive, C → U
• PCR: 5-MeC(C)/G and U(T)/A,
Sequence and compare differences
• COBRA – sequence differences based
restriction
Main problems
• PCR is often biased towards unmethylated
teplate!!! 
• Primer design is not easy 
• High quality DNA required …OK
• DNA degraded during treatment, most
template unconverted 
…but if working – lots of information! 
Chromatin IP - ChIP
• Crosslink of DNA and associated proteins
• Antibody against desired protein –
pulldown of all fragments crosslinked
• PCR with primers for certain region
• Or microarray experiment
Main problems
• Usually HIGH amount of starting material
required!!! 
• Good antibody necessary
• Modification – „Carrier ChIP“
(starting material still high, radioactively
labelled primers …)
Recommended literature
• Epigenetics, Ed. Allis, Jenuwein, Reinberg,
Cold Spring Harbor Press, 2006
• Gene Expression at the Beginning of
Animal Development, Ed. DePamphilis,
Elsevier, 2002
Thank you for your attention!