Epigenetic regulation during early embryogenesis

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Epigenetic regulation during early embryogenesis
reprogramming and remodelling

• Helena Fulkova
• Istitute of Animal Science, www.vuzv.cz
• helena.fulkova_at_vuzv.cz

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Basic facts

• No loss/gain of genomic DNA during development
  and differentiation
• Somatic cells possess full developmental
  potential demonstrated by SCNT

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• 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 expressionand nuclear
organisation
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DNA proteins chromatin
This cannot simply be achived by different TFs!
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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

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Histones

• H2A, H2B, H3 and H4 (canonical core histones -
  octamer)
• 146bp DNA nucleosome

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Histones II

• Basic proteins
• Conserved among Eukaryotes
• C- and N- terminal tails globular domain
• Subjected to post-translational covalent
  modifications

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H1 (linker histone) other non-histone
proteins? chromatin
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Histone modifications

• Acetylation - HATs/HDACs
• methylation - HMTs/demethylases
• Phosphotylation kinases/phosphatases
• Ubiquitination

Activating vs. repressive modifications
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(No Transcript)
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• H3/K9, H3/K27, H4/K20 repression of
  transcription and silencing
• H3/K4 activation

• Methylation
• Acetylation
• Phoshorylation
• Ubiquitination

• 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

• Ubiquitin H2A X chromosome silencing

The histone code hypothesis
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Histone variants

• H3 ? H3.3 ? CENP A

Canonical core histone Transcriptional
activation Kinetochore assembly
Canonical core histone DNA repair and
recombination Gene expression X chromosome
inactivation Transcriptional activation?

• H2A ? H2AX ? H2AZ ?
  macroH2A ? H2ABBD

• H2B

Canonical core histone Canonical core histone

• H4

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Maintaining histone modifications over DNA
replication phase
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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

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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

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DNA methylation DNA methyltransferases

• Dnmt1 (maintenance function)
• Dnmt3 family(de novo methylation)
• Dnmt2

• Dnmt1o, p, s isoform
• Dnmt 3a
• Dnmt 3b
• Dnmt 3L

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N-terminal regulatory domain
C-terminal catalytic domain
Substrate S-Adenosyl-Methionin Base flipping
(like repair enzymes)
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De novo methylation vs. maintenance methylation
Active vs. passive demethylation
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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)

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Adaptor proteins

• e.g. Methyl CpG binding proteins
• MBD 1-4
• MeCP2

MBD1 transcriptional repression (not in MeCP1
complex) MBD2 associated with HDAC1? repression
(found in MeCP1, Sin3, Mi2/NURD complexes) MBD3
species specific binding of methylated/nonmethyl
ated DNA (found in Mi2) MBD4 repair?, can
induce nicks, has glycosylase activity, binds
TpG (deamination of 5-MeC)
Rett syndrome?X linked, predomimantly ?
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HP1
Targeting methylated H3/K9 (triMe)

• HP1 alpha
• HP1 beta
• HP1 gamma

• Centromeric heterochromatin
• Pericentric heterochromatin, but also euchromatin
  ?
• Euchromatin, active genes?

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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)
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Composition of remodelling complexes
Swi/Snf family
CHD family
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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)

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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)
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Epigenetics and development
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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)

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Epigenetic life cycle
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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

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Post-fertilization epigenetic remodelling
? ?
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DNA methylation active demethylation
All species tested excluding Sheep!
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Histone modifications

• Asymmetric distribution
• Symmetric distribution
• Associated with specific DNA sequences (chromatin
  structures)

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(No Transcript)
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What happens next passive demethylation
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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)

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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
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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

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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

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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

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• Human syndromes (Prader-Willi/Angelman,
  Silver-Russel) mostly mental retardation
• Higher frequency in ART conceived children!

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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

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Chromatin organisation Fibroblast vs. Embryo
SCNT
vs.
Anti 5-MeC
Santos and Dean, Reproduction 2004
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Consequence

• Low SCNT efficiency
• LOS (Igf2?)
• Placental hyperplasia
• Death soon after birth
• TSA, 5-AzaC better efficiency
• ESCs better donors for SCNT

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Methods

• DNA methylation
• Immunofluorescence
• Methylation sensitive restriction
• Bisulfite sequencing
• COBRA
• Histone modifications
• Immunofluorescence
• Chromatin IP

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DNA methylation

• Convertion of DNA by sodium bisulfite5-MeC
  unreactive, C ? U
• PCR 5-MeC(C)/G and U(T)/A,Sequence and compare
  differences
• COBRA sequence differences based restriction

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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! ?

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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

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Main problems

• Usually HIGH amount of starting material
  required!!! ?
• Good antibody necessary
• Modification Carrier ChIP (starting material
  still high, radioactively labelled primers )

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Recommended literature

• Epigenetics, Ed. Allis, Jenuwein, Reinberg, Cold
  Spring Harbor Press, 2006
• Gene Expression at the Beginning of Animal
  Development, Ed. DePamphilis, Elsevier, 2002

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Thank you for your attention!