Group 6
Xiaopeng Ma, Weiru Liu, Zhirui Hu, Weilong Guo
About author
• Alexander Meissner
– Harvard University Department of
Stem Cell and Regenerative
Biology, Broad Institute
– The epigentic mechanism of stem
cell, ips
Alex Meissner
Alex Meissner`s boss
Rudolf Jaenisch, MD
Functions of DNA methylation
• Promoter/enhancer repression
• Retrotransposon silencing
• Imprinting patterns
Background
DNA methylation dynamic in lifecycle
Smallwood et al. Trends in Genetics, January 2012, Vol. 28, No. 1
RRBS: Reduced Representation
Bisulfite Sequencing
MspI Recognition Site
Insensitive to mC
• Pros:
– low cost
– Low cell numbers
– high enrich in CpG island
• Cons:
– low coverage of genome(1%)
– Possible low C->U conversion rate.
– Can not distinguish mC and hmC
Hongcang Gu et al, 468|VOL.6 NO.4 |2011 |nature protocols
Genomic coverage of typical RRBS
libraries
Hongcang Gu et al, 468|VOL.6 NO.4 |2011 |nature protocols
reduced representation bisulphite sequencing (RRBS) library
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Recognizing site: 5’-C|CGG-3’
3’-GGC|C-5’
Cleavage happens only when
internal methylation occurs
within recognizing site
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Result: obtained the methylation status of 1,062,216 CpGs for comparative analysis
1. Murine embryogenesis
BDF1 （♀） ×
129X1（♂）
•Oocyte methylation levels more closely resembles those of early embryonic time
points than the levels in sperm, post implantation embryos, of adult tissues.
•from oocytes to the early ICM: gradual increase in the fraction of tiles that exhibit
intermediate an low methylation values, which is consistent with loss of methylation
over mutiple cleavage divisions
CpG density vs methylation levels
Pre-implantation development represents a unique developmental period where methylation
is defferentially positioned and regulated before being restored in a somatic fashion
Substantial methylation changed in regional DNA
37%
66%
Most stable/
increased
slightly
Differentially methylated regions (DMRs): differential methylated tiles between 2 gametes
74 CpGs within sperm-specific DMR tiles
The regions that are significantly
hypermethylated in oocyte compared
to sperm exhibit intermediate values
in the zygote
Suggestion: the oocyte methylome, rather than the sperm methylome, seems
to be more reflective of patterns in the early embryo.
Retroelement methylation &
Gametes contributed DMRs
Compare between sperm and zygote
methylation
• Original view:
– Paternal genome actively depleted methylation at
fertilization
• 96% tiles hypermethylated in sperm, less
methylated in zygote
• Already low methylation in oocyte
• Where these regions locate?
Retroelement dynamics at fertilization
• LINEs: Most extreme changes in sperm to
zygote transition, binomal
• LTR: similar demethylation but not binomal
• SINEs: less methylated in sperm than other
repeats and thus less change, not binomal
Mean methylation level for
Retroelements
All retrotransposons follow the same path in early development
Less methylated in oocyte pre-implantation stages
More methylated in E6.5/7.5 and somatic cells
Summary
• Does it mean retrotransposon is more active in preimplantation stages?
– LINE-1 retrotransposon is required for early embryo
preimplantation development (Beraldi R. et al. 2006)
– Associates with earliest transcriptional events during
zygotic genome activation
– Not address how retrotransposon methytion related to
transcription
• Doesn’t mean paternal de-methylation in these regions
– distinguish paternal and maternal methylation
• Remember the bias of RRBS (CpG rich regions)
• Can’t address whether mC first convert to hmCs
Gametes confer DMRs
Compare between sperm and oocyte methylation
• Some allele-specific methylation pattern is
maintained (e.g. ICR)
• CpG methylation was lower overall in mature
oocytes than sperm; methylation in a CGI context
was markedly lower in sperm (Smallwood,S.A. et
al. 2011)
• DMRs contributed from either gamete
– DMR: differential methylated tiles between 2 gametes
– Different allelic methylation: Linear regression for
DMR with zygote methylation level half-way
oocyte-contributed DMRs vs.
sperm-contributed DMRs
• Differ in CpG densities
• 376 oocyte-contributed DMRs
– Enriched in HCP, no functional enrichment, including
Dnmt1,Dmnt3b, Cpne7 (DMR near promoter)
– intermediate level from zygote to ICM and then
hypomethylation (expected for HCP)
• 4894 sperm-contributed DMRs
– Intergenic regions
– Intermediate level to ICM and then hypermethylated
(typical in somatic)
DMRs vs genomic features
Intergenic regions
Oocyte-contributed DMR
Sperm-contributed DMR
Methylation pattern in Cpne7
oocyte-contributed DMR
Promoters
hypermethyla
ted in oocytes
Mean methylation change
Intermediate
through
cleavage
HCP
methylation
structure
Non-CpG
• Non-CpG inherited by oocyte alleles but lost quickly
– Highest mCpA in oocytes and decrease ~50% in zygote
Summary
• Defining oocyte/sperm contributed DMR by
linear regression is indirect
• Can’t cover much non-CpGs
• Why oocyte-contributed DMR in HCP but
sperm-contributed DMR in intergenic regions?
A model for DNA methylation dynamics
during early embryogenesis