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Přírodovědecká fakulta UK
EPIGENETIKA MB150P85
Petr Svoboda
mail:
tel:
[email protected]
241063147
EPIGENETIC
REPROGRAMMING
DURING THE MAMMALIAN
LIFE CYCLE
Mammalian “life cycle” of
genetic and epigenetic information
1C
MII
GV
2C
FEMALE GC
blastocyst
GERMLINE
SOMATIC CELLS
MALE GC
CONCEPT OF REPROGAMMING
no transcription
resumption
of meiosis
fully-grown
GV oocyte
(prophase I)
= differentiated cell
14-18 hours
mature
oocyte
metaphase II
oocyte
fertilization
20-22 hours
1-cell embryo
MINOR
ZGA
cleavage
MAJOR
ZGA
= pluripotent cell
2-cell embryo
2-cell
1-cell
4-cell
morula
8-cell
blastocyst
S
S
mitosis
100
92
84
76
68
60
52
44
36
28
20
12
0
fertilization
MII egg
S
mitosis
compaction
differentiation
hCG injection
Zygotic transcription
Transcriptional repression
Enhancer requirement
ZGA
Translation of zygotic mRNA
The timescale is based on Forlani et al. (1998), Development 125, 3153-3166
Morgan 2005
Li 2002
Morgan 2005
EMBRYO
REPROGRAMMING
DNA METHYLATION
Li 2002
Lucifero 2004
Variable timing of maternal imprinted marks
De novo DNMTs, DNMT3L
Li 2002
DNA demethylation mechanisms
passive DNA demethylation
• inhibition of DNA methylase activity (Li, 1992)
• replication dependent
• does not require transcription (Matsuo, 1998)
active DNA demethylation
• erythroleukemia cells (Razin, 1986)
• early embryo (Kafri, 1993; Oswald 2000; Mayer, 2000)
• 2 models: - direct DNA demethylase activity
- DNA repair mechanism
Morgan 2005
somatic cells
methylation sperm
sperm
oocyte
oocyte
PGC
time
fertilization
1st cleavage
blastocyst
CpG methylation during development
1-cell 6h
1-cell 8h
2-cell 22h
4-cell 45h
Adapted from Mayer et al. (2000). Nature 403(6769):501-2
4-cell asymmetrical staining
8-cell weak labeling, rare asymmetrical chr.
Adapted from Rougier et al. (1998). Genes Dev 12(14):2108 -13
CpG methylation during development
Santos 2004
Adapted from Rougier et al. (1998). Genes Dev 12(14):2108 -13
Anti-5-methylcytosine (MeC) immunofluorescence
b) 6 h (>10)
c) 8 h (>20)
d) aphidicolin-treated (>20).
Controls. Anti-DNA immunofluorescence
g) 6 h (>5)
h) 8 h (>5)
i) Aphidicolin treatment (>5)
Adapted from Mayer er al. (2000). Nature 403(6769):501-2.
MBD2 and 4 were possible candidates for active demethylation mechanisms
• MBD2 is a putative demethylase (Bhattacharya et al., 1999)
• MBD4 is methyl-CpG-binding endonuclease (Bellacosa et al, 1999)
BOTH RULED OUT! (see Santos 2004)
gal
confocal
1-638
1-259
Li 2002
IAP
L1
CHROMATIN
CONCEPT OF REPROGAMMING
no transcription
resumption
of meiosis
fully-grown
GV oocyte
(prophase I)
= differentiated cell
14-18 hours
mature
oocyte
metaphase II
oocyte
fertilization
20-22 hours
1-cell embryo
MINOR
ZGA
cleavage
MAJOR
ZGA
= pluripotent cell
2-cell embryo
De La Fuente 2006
OOCYTE
Santos 2005
ZYGOTE
Santos 2005
ZYGOTE
Santos 2005
ZYGOTE
Morgan 2005
ZYGOTE
Morgan 2005
PREIMPLANTATION EMBRYO
DIFFERENTIATION
TRANSCRIPTIONAL
SILENCING OF Oct-4
Surani 2007
Loh 2006
Pluripotency factors
• Irreversibility of Oct-3/4 silencing is mediated by recruiting of DNMT3a/b by G9a,
rather then by direct H3K9 methylation by G9a.
PGC REPROGRAMMING
Surani 2007
Surani 2007
Germ cell specification
Hajkova 2002
Hajkova 2002
Kimmins 2005
EPIGENETICS OF CANCER
… anything’s possible
Robertson 2005
Weber 2005
RNA SILENCING
RNAi
0.5 - 1.0x106 dsRNA molecules per each gonad arm
uninjected parent
mex-3 in situ hybridization
not stained
antisense injected
dsRNA injected
SUMMARY
• post-transcriptional effect
• reduction of mRNA level
• interference in both the injected animals and their progeny
-> sounds EPIGENETIC!
• only a few molecules of injected double-stranded RNA were required
per affected cell
• spreading across cellular boundaries
• unknown mechanism, called RNA interference
RNAi in mammals?
dsRNA >30bp
2’,5’-OAS
interferon
PKR
active
activation
2’,5’ A
It can’t work!
RNaseL
eIF2
active
general mRNA
degradation
P
inhibition of
translation
APOPTOSIS
SEQUENCE INDEPENDENT
GV oocyte
oocyte isolation
CZB+IBMX
5% CO2
microinjection of dsRNA
CZB+IBMX
5% CO2
GV oocyte
in vitro culture 20 hours
RT-PCR analysis
IBMX removal
CZB
5% CO2
MII egg
maturation in vitro for ~ 18 hours
phenotype evaluation
(e.g. MAP and H1 kinase assay)
DECIPHERING THE MECHANISM
General approaches
- genetic
- biochemical
- W.E.G. = wild educated guess
- benefits and limits of individual approaches
- different solutions in different model systems
e.g. compare mutant screens in mice and Drosophila
- different approaches yield different data
integration of information from
different model systems and
different approaches.
Biochemical approach
Biochemical approach
Evolution of RNAi models ...
Montgomery et al., 1998
Mary K. Montgomery, SiQun Xu, and Andrew Fire,
RNA as a target of double-stranded RNA-mediated
genetic interference in Caenorhabditis elegans
PNAS. 1998 December 22; 95(26): 15502–15507
Zamore et al., 2000
Elbashir et al., 2001
Genetic approach
MUTAGENIZE -> BACK-CROSS -> FIND RIGHT PHENOTYPES -> MAP GENETICALLY
Bag of worms
Genetic approach
Verify, sort out linkage groups, test allele types, genetic pathways ...
Fine mapping and gene identification
-> integrate with
biochemical data
Wild educated guess
Genetic approach
Evolution of RNAi models ...
2002 (Hutvagner)
How about the RISC cleavage factor?
Stay tuned ...