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Group 6 Xiaopeng Ma, Weiru Liu, Zhirui Hu, Weilong Guo

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.

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Group 6 Xiaopeng Ma, Weiru Liu, Zhirui Hu, Weilong Guo

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  1. Group 6 Xiaopeng Ma, Weiru Liu, Zhirui Hu, Weilong Guo

  2. 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

  3. Functions of DNA methylation • Promoter/enhancer repression • Retrotransposon silencing • Imprinting patterns

  4. Background

  5. DNA methylation dynamic in lifecycle Smallwood et al. Trends in Genetics, January 2012, Vol. 28, No. 1

  6. 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

  7. Genomic coverage of typical RRBS libraries Hongcang Gu et al, 468|VOL.6 NO.4 |2011 |nature protocols

  8. 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   

  9.   Result: obtained the methylation status of 1,062,216 CpGs for comparative analysis

  10. 1. Murine embryogenesis

  11. BDF1 （♀） × 129X1（♂）

  12. 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

  13. 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

  14. Substantial methylation changed in regional DNA 37% 66% Most stable/ increased slightly

  15. 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.

  16. Retroelement methylation & Gametes contributed DMRs

  17. 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?

  18. 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

  19. 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

  20. 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 retrotransposonmethytion 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

  21. 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

  22. 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)

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

  24. Methylation pattern in Cpne7oocyte-contributed DMR

  25. Promoters hypermethylated in oocytes Mean methylation change Intermediate through cleavage HCP methylation structure

  26. Non-CpG • Non-CpG inherited by oocyte alleles but lost quickly • Highest mCpA in oocytes and decrease ~50% in zygote

  27. 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?

  28. A model for DNA methylation dynamics during early embryogenesis

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