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 boss Rudolf 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 Site Insensitive 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

Christoph Bock et al nature biotechnology

Comparison of genome-wide DNA methylation profiling technologies 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

Methylation for genomic feature annotations throughout pre-implantation development

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 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 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)

Oocyte- and sperm-DMR differed in CpG density

DMRs vs genomic features Oocyte-contributed DMR Sperm-contributed DMR Intergenic regions

Methylation pattern in Cpne7 oocyte-contributed DMR

Promoters hypermethyla ted 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