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« Epigenetic regulation and DNA
methylation»
&
« Genomic Imprinting and Disease »
TC1: ‘Genetic and epigenetic information - molecular bases’
Université de Montpellier
14th September, 2016.
Robert Feil
IGMM, CNRS, Montpellier
E-mail: [email protected]
* Epigenetics & DNA methylation
* Genomic Imprinting
* Imprinting Disorders
------------------------------------------------* Epigenetic mechanisms in imprinting
* Evolutionary insights
Epigenetics?
«The study of somatically and/or
meiotically heritable, but reversible,
changes in gene function
that cannot be explained by
changes in DNA sequence »
Russo, Martienssen, Riggs, Epigenetic Mechanisms of Gene Regulation,
Cold Spring Harbor Press (1996)
A heritable DNA methylation change at a
gene involved in fruit ripening
Manning et al, Nature Genet. (2006)
Heritable expression and heritable repression of
the White gene in Drosophila (flies)
PRE
trxG
+
PcG
-
PRE
Renato Paro
Agouti (Avy) coat colour gene in mice:
MAJOR phenotypic effects of differential DNA methylation
Drs. Emma Whitelaw, Randy Jirtle, others
Genetic
Epigenetic
Information-phenotype
Information-phenotype
heritable: meiosis, mitosis
heritable: mitosis/meiosis
reversible
DNA
Unaltered DNA sequence
Epigenetic modification?
‘A heritable, but reversible,
modification on DNA or
chromatin which affects
gene expression and phenotype’
Epigenetic modifications ?
* DNA methylation
* Stable histone modifications
Environmental Factors
Environmental Factors,
Intrinsic Factors
Intrinsic Factors
GENOME
EPIGENOME(s)
EPIGENOME(s)
Developmental
establishment of
epigenetic marks
Somatic
maintenance of
epigenetic marks
PHENOTYPE
PHENOTYPE
Feil R, Mutation Research 2006
DNA methylation
Symmetrical
CG
GC
The CH3 groups are
exposed in the major groove
(G. Filion)
DNA methylation
Absent in S. cerevisiae and only little present in Drosophila
Present at high levels in plants, where it is highly complex
In mammals, present mostly in one form: 5-methylcytosine
Methylation
position 5
Cytosine
5meCytosine
DNA methyltransferase proteins (mammals)
DNMT1
DNMT2
Maintenance
-
DNMT3a
de novo
DNMT3b
de novo
DNMT3L
-
DNA methylation: an epigenetic modification
DNMT1
Réplication
DNMT3A, 3B
CG
GC
CG
GC
Link between
replication and
maintenance
of methylation:
CG
GC
CG
GC
CG
GC
CG
GC
DNMT1
PCNA
Roles of DNA methylation in mammals
CH3
Cytosine
5meCytosine
* Chromosome stability
* Repression of DNA elements of foreign origin
* Tissue-specific repression of genes
* ‘X-chromosome inactivation’ (Prof. Heard’s lecture)
* Genomic Imprinting
Oocyte and sperm are both required
for mammalian development
oocyte
sperm
oocyte
sperm
Genomic Imprinting:
~120 protein-coding genes
-hundreds of regulatory non-coding RNAs
(lncRNAs, miRNAs, snoRNAs)
genomic imprinting
Aberrant development of mono-parental embryos
Embryo
Yolk sac
Normal:
1 Maternal genome
1 Paternal genome
Androgenetic:
2 Paternal genomes
Parthenogenetic:
2 Maternal genomes
Placenta
Research on chimaeras:
Opposite developmental roles of
maternal and paternal genomes
Androgenetic
Wild type
Parthenogenetic
2 x paternal
genome
1 maternal
1 paternal
genome
2x maternal
genome
Bruce M. Cattanach,
MRC Harwell,
Oxford, UK.
Taking the hurdle of genomic imprinting :
completely female-derived mice
Tomohiro Kono (Tokyo):
Kawahara et al. Nature Biotech. 2007
Who needs daddies ?
« Fatherless mice live longer »
Kawahara & Kono, Human Reprod. 2010
‘bi-maternal’ mouse (to the right)
oocyte
sperm
Genomic Imprinting:
~120 protein-coding genes
-hundreds of regulatory non-coding RNAs
(lncRNAs, miRNAs, snoRNAs)
genomic imprinting
Imprinted genes influence nutrient transfer
Placental development and function
Foetal growth control
Postnatal fitness
Postnatal behaviour
Essential roles of imprinted gene expression
Plasschaert and Bartolomei,
Development 2014;141:1805-1813
Jo Peters, Nature Rev Genet 2014
Two imprinted domains
involved in fœtal growth
CDKN1C
H19
IGF2
ICR
ICR
KCNQ1 domain
Maternal
ICR
ICR
Paternal
IGF2-H19 domain
ICR
= ‘Imprinting Control Region’
= DNA methylation
Imprinting Control Regions (ICRs)
CH3 CH3 CH3 CH3 CH3 CH3
Parental allele WITH
DNA methylation
Parental allele WITHOUT
DNA methylation
Zygote
SOMATIC MAINTENANCE: DNMT1
Adult
ICR1
ICR2
ICR1
ICR2
ICR1
ICR2
ICR1
ICR2
Germinal
lineage
Sperm
ICR1
ICR1
Oocyte
ERASURE
ICR2
ICR2
ICR1
ICR2
ICR1
ICR2
ESTABLISHMENT:
DNMT3A and DNMT3L
ICR1
ICR2
ICR1
ICR2
Katia Delaval & Feil 2004
Curr Opin Genet Dev
Differential timing of imprint acquisition
in male and female germ cells
« Paternal » ICRs
DNMT3A
DNMT3L
« Maternal » ICRs
DNMT3A
DNMT3L
Image from
Déborah Bourc’his
DNA methylation landscapes of oocytes and sperm
Data from Kobayashi et al.
(2012) PLoS Genet. 8:e1002440.
DNA methylation dynamics in the early embryo:
only ICRs are stably maintained
ICRs
Kelsey and Feil, Phil. Trans. R. Soc. B (2013).
Smallwood and Kelsey, Trends Genet (2012).
HP1g
H3K9me3
H4K20me3
H4R3me2s ZFP57H3K64me3otherKAP1
proteins
DNMT1
CH3 CH3 CH3 CH3
H3/H4 acetylation
H3K4me2/3
DNA-methylation
protection against
DNA methylation
DNMT3A
DNMT3L
Collaboration with Rob Schneider on H3K64me3:
Vincenzo Di Cerbo et al., Elife 2014.
Maintenance of methylation imprints involves
specialized proteins and histone methylation
HP1g
ZFP57
KAP1/TRIM28
ESET/SETDB1
PRSET7
SUV4-20H1/H2
PRMT5
ATRX
Histone H3.3
H3K9me3
H3K64me3
H4K20me3
H4R3me2s
CH3 CH3 CH3 CH3
H3K4me2/3
H3/H4 acetylation
Parental allele WITH
DNA methylation
Parental allele WITHOUT
DNA methylation
Sanli and Feil, Int. J. Biochem. Cell Biol (2015)
Perurbed DNA methylation and
human disease
Environmental Factors
Environmental Factors,
Intrinsic Factors
Intrinsic Factors
GENOME
EPIGENOME(s)
EPIGENOME(s)
Developmental
establishment of
epigenetic marks
Somatic
maintenance of
epigenetic marks
PHENOTYPE
PHENOTYPE
Feil R, Mutation Research 2006
The epigenome(s) undergoes changes
during development and postnatal life
ENVIRONMENT
STOCHASTIC
EVENTS
Many chromosomal regions show differential DNA
methylation between ‘old’ mono-zygotic twins
Fraga MF et al. PNAS USA (2005)
‘Epigenetic drift’ at ICRs gives rise to
imprinting-related diseases in Humans.
Silver-Russell Syndrome (SRS)
• Intra-uterine growth
restriction (IUGR)
• Postnatal growth deficiency
• Learning disabilities
• Mostly sporadic
ICR
Maternal
ICR
Paternal
IGF2
Silver-Russell Syndrome
(40% of cases)
ICR
Maternal
ICR
Paternal
IGF2
Beckwith-Wiedemann Syndrome (BWS)
• Foetal overgrowth
• Large internal organs,
large tongue
• Predisposition to Wilms’
tumour of the kidney
• Mostly sporadic
ICR
CDKN1C
ICR
M
ICR
P
IGF2
ICR
Beckwith-Wiedemann Syndrome
(foetal overgrowth)
CANCER
ICR
M
CDKN1C
ICR
M
ICR
P
IGF2
ICR
P
Hypomethylation occurs often in concert
at multiple imprinted loci
in BWS,
SRS,
TNDM &
Pseudohypoparathyroidism-1B
Mackay DJ et al. 2008. Nature Genetics
Bliek J et al. 2009. Eur J Hum Genet
Azzi S et al. 2010. Epigenetics
Court F et al. 2013. Hum. Mutation
‘Epimutation’
‘ heritable change in gene expression
without change in DNA sequence ’
(epigenetic diseases, cancer)
Environmental Effects
Minor effects of nutrition on methylation
imprints in human and animal studies
Endocrine disruptors:
Minor ‘long-term’ effects on methylation imprints
Frequent major perturbation of methylation
imprints upon in vitro culture and manipulation
Increased occurrence of different
‘Imprinting Disorders’
in cohorts of ‘Assisted Reproduction’ babies
Beckwith-Wiedemann Syndrome (BWS)
Silver-Russell Syndrome (SRS)
Angelman Syndrome (AS)
Transient Neonatal Diabetes Mellitus (TNDM)
Causal mechanism(s) unclear: ART or ‘predisposing infertility’?
Dias and Maher, Epigenomics (2013).
Sato T et al. Reproduction Med. Biol. (2014).
Faugue P Fertil Sterility (2013).
Lim et al. Hum Reprod (2009).
Sutcliffe et al; Human Reprod. (2006).
Cox et al. Am. J Hum Genet (2002).
Endocrine disruptors:
* Long-term effects on reproduction
* MINOR effects on DNA methylation imprints
• Vinclozolin (50mg/kg, E10-18) and methoxychlor (10 mg/kg
E10-18) administration to females during pregnancy:
Altered methylation in sperm of F1, F2 & F3 males:
* Slight reductions in DNA methylation at ‘paternal ICRs’
* Gains in DNA methylation at ‘maternal ICRs’
Stouder et al, Reproduction (2010) (2011)
Kang et al, Epigenetics (2011)
Somm et al., Reprod Toxicol (2013)
Perturbed sperm DNA methylation imprints
in oligozoospermia
normal
spermatogenesis
ICR
ICR
oligozoospermia
ICR
Marques et al. 2004, 2008
Kobayashi et al. 2007
Boissonnas et al. 2010
ICR
Endocrine Disruptors:
Stouder et al. 2010, 2011
Question time…..
How do ICRs bring about
imprinted gene expression ?
Free Review:
Sanli and Feil (2015)
Int J Biochem Cell Biol 67, 13-147.
Several ICRs bring about allelic
‘chromatin loops’
‘Circular 3C’ unravels interactions at Igf2-H19 domain
Collaboration with
Dr Daan Noordermeer
I2BC,
Gif-sur-Yvette
Ectopic insertion of Igf2-H19 ICR into the
mouse Immunoglobulin Heavy chain locus (IgH)
VH1
VH2
VHn
D1-12
PDQ52
PIm
JH1-4
ICR
ICR
ICR/+
E m Sm
Cm
Cd
Cg1
Cg3
Sg3
+/ICR
Sg1
Ectopic insertion of Igf2-H19 ICR:
acquisition of imprinted DNA methylation
CTCF
Mat.
ICR
CTCF/Cohesin
CTCF
Pat.
ICR
Methylation
Igf2-H19 ICR insertion impairs VH-DJH recombination
ICR allele-specific PCR
WT allele-specific PCR
Pro-B cells
WT
DNA (ng)
36
12
ICR/+
4
36
12
ICR/
+/ICR
4
36
12
4
36
12
4
WT
36
12
ICR/+
4
36
12
ICR/
+/ICR
4
36
12
4
36
12
4
JH1
DH-JH
JH2
JH3
JH4
JH1
proximal VH-DJH
JH2
JH3
JH4
distal VH-DJH
normalization
JH2
JH
3
JH4
HS4
Nadine Puget/Ryutaro Hirasawa et al., MCB 2015
Other ICRs repress chromatin in cis…….
…..other ICRs activate gene expression in cis.
The evolution of genomic
imprinting
Imprinting Control Regions (ICRs):
Regulatory CpG islands ‘marked’
by germline-derived DNA methylation
CH3 CH3 CH3 CH3 CH3 CH3
Parental allele WITH
DNA methylation
Parental allele WITHOUT
DNA methylation
No genomic imprinting (?)
Genomic imprinting
Renfree & Kaneko-Ishino,
Phil. Trans. R. Soc. B 2013
Timing of imprinted CpG island emergence
Renfree & Kaneko-Ishino.
Phil. Trans. R. Soc. B 2013
Emergence and maintenance
of imprinted CpG islands
Evolutionary theories of imprinting
1.
Some non-equivalence of matrigenic allele and patrigenic
allele with respect to fitness
2.
Selection favors imprinted silencing of one of the alleles
Spencer & Wolf, Heredity 2014
Patten et al. Heredity 2014
Wolf, Oakey, Feil, Heredity 2014
Maternal-offspring co-adaptation theory
(Wolf & Hager 2006)
Patten et al. Heredity 2014
Kinship theory (Haig and Westoby, 1989)
Patten et al.
Heredity 2014
Kinship theory of genomic imprinting
(‘Parent-Offspring Conflict theory’)
the popular view……
….creates confusion with ‘Behavioural Imprinting’
An ‘imprinted gene network’
with multiple trans-regulatory links
Examples:
PLAGL1 / ZAC1 (TNDM)
IGF2-INS
signalling pathway
Imprinted ncRNAs
Evolutionary consequences: Patten M et al. Proceedings-B (2016)
oocyte
sperm
Genomic Imprinting:
~120 protein-coding genes
- hundreds of regulatory non-coding RNAs
(lncRNAs, miRNAs, snoRNAs)
genomic imprinting
Imprinted genes influence nutrient transfer
Placental development and function
Foetal growth control
Postnatal fitness
Postnatal behaviour
Placental expression levels of
imprinted genes correlate with birth weight
The zinc finger transcription factor PLAGL1 regulates a gene network
enriched in imprinted transcripts.
Levels of PLAGL1 are down-regulated in IUGR placentas.
(Iglesias-Platas et al., 2014).
PLAGL1 binds to IGF2-H19 enhancer regions
Hundreds of placenta-specific
maternally-methylated DMRs….
(A) Those that are unmethylated in somatic tissues (n= <500)
(B) Those that are methylated in somatic tissues (n=3)
All placenta-specific DMRs originate from germline DMRs that are methylated in oocytes
Sanchez-Delgado & Monk, 2015 & unpublished data
Hanna et al. (2016) Genome Res (Robinson/Kelsey)
Methylation levels at novel placenta-specific
DMRs vary between placentae
- This reveals that a low number of individuals are unmethylated at these DMRs,
- consistent with them being a polymorphic trait.
- Pyrosequencing of novel placenta-specific DMRs in 60 normal placenta samples
Unpublished observations Monk & Sanchez-Delgado
Hanna et al. (2016) Genome Res, Epub.
Patricia Cavelier
Claire Dupont
Satya Kota
Sébastien Lalevée
David Llères
Alice Marchand
Rakesh Pathak
Ildem Sanli