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`s bossRudolf Jaenisch, MD

Alex Meissner

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

• 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

MspI Recognition SiteInsensitive to mC

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

Recognizing site: 5’-C|CGG-3’ 3’-GGC|C-5’

Cleavage happens only when internal methylation occurs within recognizing site

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

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.

74 CpGs within sperm-specific DMR tiles

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 developmentLess methylated in oocyte pre-implantation stagesMore methylated in E6.5/7.5 and somatic cells

Summary• Does it mean retrotransposon is more active in pre-

implantation 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 DMRsCompare 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

Oocyte-contributed DMR Sperm-contributed DMR

Intergenic regions

Methylation pattern in Cpne7oocyte-contributed DMR

Promoters hypermethylated in oocytes

Intermediate through cleavage

HCP methylation structure

Mean methylation change

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