1. Presentation people: Binzhi Xu and Peter Truong

2. What is known and not known Known: “epigenetic information is (erased and)reprogramed on genome wide scale in Primordial germ cells and early embryos.” Histone is modified(not the focus of this study) Timing: Globally the demethylation occurs early(E8.0-E8.5), but some regions such as DMRs occurs late(PCGs colonize gonad). Mechanisms: demethylation processes can be a combination of passive and active pathways.

3. What is known and not known Not known: The actual timing and mechanisms of demethylation. Purposes of reprogramming and outcomes: (1). related to pluripotency of PGCs? (2). Conservation of epigenetic information? (3). Effect on transposons?

4. DNA methylation Methyl group added to DNA, when on promoter, the transcription tends to be repressed(epigenetic mark) Important in genomic imprinting, and X chromosome inactivation(which this paper discusses) CpG dinucleotides can be methylated in eukaryotes(on 5’ cytosine of CpG dinucleotides) while Adenine methylation is restricted to prokaryotes Source: https://en.wikipedia.org/wiki/DNA_methylation

5. Bisulfate Sequencing (bs seq) Unmethylated Cytosine treated by HSO- becomes uracil Methylated cytosine are not affected Source: https://en.wikipedia.org/wiki/Bisulfite_sequencing

6. BS seq cont. Followed by PCR: on daughter strands unmethylated cytosine sites are now thymines, but on methylated cytosine sites they are still cytosines. Compare to PCR sample that is not treated by bisulfate: determine methylated cytosine content. Or tiling probes Source: https://upload.wikimedia.org/wikip edia/en/c/c9/Wiki_Bisulfite_seque ncing_Figure_1_small.png

7. Other techniques used Hairpin bs seq: bs seq but stick the original double strand together while treating the DNA with bisulfate. To detect the level of hemimethylation(vs. methylation vs. demethylation). RNA-seq: detect what is being transcribed, and compare to detected pieces of genes that are methylated. Immunofluorescence: detect 5-methylcytosine(5mC), Dnmt1(methylate DNA by some unknown pathway or regulate Np95 to methylate DNA) and Np95. Isolation of samples: embryonic cells from timed mating with C57BI/6J female mice, PGCs from timed mating with Oct4-Gfp expressing female mice under C57BI/6J back ground, J1 ESC…(see experimental procedures…) Illumina: yeah…

8. Gneeral Procedures (1). Isolate cell samples from E6.5, E10.5, E11.5, E13.5, E16.5(reasons on later slides) (2). Run Bs-seq and RNA-seq for cells above (3). Also run for J1 Embryonic Stem Cells(ESC) (4). Also use 5kb tiling to check bisulfate conversion rate(>60% complete conversion) (5). Run illumina for both bs-seq library and RNA-seq library

10. Findings : Demethylation phase 1 The timing of global demethylation is prior to E9.5, instead of accompanying the erasure of imprinting that starts on E11.5.

11. Findings: Demethylation phase 2 However, DMRs of imprinted genes, some CGIs(CpG island) on X chromosome, and some CGIs on promoters remained methylated until E11.5 (PGCs entry of genital ridges). Data shows(this one for promoters) that they are related to meiosis and gametes generation

12. Findings: passive pathway of demethylation Hairpin bs-seq shows that methylated CpGs are almost on the same strand, hemimethylation is rare. Indicate the major mechanism of demethylation is by dilution(passive pathway).

13. Findings: active pathway According to previous knowledge, Dnmt1 and Tet1 may play a role in maintaining methylation and demethylation. Minor number of strands that are hemimethylated shows the possibilities of the active pathway.

14. Findings: epigenetic reprograming and pluripotency Not entirely: They compare the complexity of RNA transcriptomes of in PGCs, ESCs, and somatic cell: no big difference Promoters become de novo methylated despite whether their genes are transcribed or not Only a few (clusters) of genes affect transcription that is correlate to their methylation level. Pluripotency network genes are activated by demethylation, but replaced by meiotic and germ-cell-function genes by E16.5(especially true in female)

16. Findings: transgenerational epigenetic inheritance IAPs: transposons, very active and needs to be silenced(methylated). Neighboring region tends to be silenced also. CGIs that are affected by IAPs are rare. VECs: CGIs that are not affected by IAPs, many of them remain methylated. Methylated VECs are like to be candidates for short term transgenerational epigenetic inheritance More of these found in sperms than oocytes, indicating sperms are more likely to escape reprogramming.

17. Other Conclusions PGCs have somatic methylation level to start with, because the methylation levels on earlier stages(E7.5-E8.5) are high or high at some locus. Genome wide methylation loss caused by a combination of downregulation of de novo methyltransferase and impairment of methylation maintenance factor Passive and active demethylation work together to resist epigenetic reprogramming LINE1 does not transcribe by E13.5, it is not responsible for transcriptional repression.

18. Problems to solve Whether the PGCs founder are converted to 5- hydroxymethylcytosine or 5-methylcytosine. (bs-seq cannot distinguish these two) How pluripotency net work is replaced by meiotic net work is unknown. Need further study of earlier embryo to confirm if there are more active way of demethylation then the passive one.(by positive feedback? ) The molecular mechanism of transgenerational epigenetic inheritance is unknown.

19. Take Home Genome wide demethylation occurs in two phases: (1).global demethylation starts prior to E9.5. (2). DMRs of imprinted genes, X- linked CGIs, and meiotic related promoter CGIs are demethylated starting around E10.5-E11.5 Both passive pathways(majority) and active pathways are involved in demethylation Epigenetic reprogramming is largely uncoupled with pluripotency. Methylation level is affected by IAPs.

20. Further Reading Complete version: http://www.cell.com/molecular- cell/abstract/S1097-2765(12)00932- X?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve% 2Fpii%2FS109727651200932X%3Fshowall%3Dtruehttp://www.cell.com/molecular- cell/abstract/S1097-2765(12)00932- X?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve% 2Fpii%2FS109727651200932X%3Fshowall%3Dtrue Isolation of cells:http://onlinelibrary.wiley.com/doi/10.1046/j.1440- 169x.1999.00474.x/full

21. References Seisenberger. S., Andrews S., et. al. 2012. The Dynamics of Genome- wide DNA Methylation Reprogramming in Mouse Primordial Germ Cells. Molecular Cell.48.849-862

22. Appendixes PGCs: “primordial germ cells are the common origins of spermatozoa and oocytes and thus represent the ancestors of the germline.”(www.embryology.ch/anglais/cgametogen/keimbahn01.ht ml)www.embryology.ch/anglais/cgametogen/keimbahn01.ht ml ICMs: the inner cell mass …is the mass of cells inside the primordial embryo that will eventually give rise to the definitive structures of the fetus(https://en.wikipedia.org/wiki/Inner_cell_mass)https://en.wikipedia.org/wiki/Inner_cell_mass Genomic imprinting: inherited epigenetic modification that silences some genes(https://en.wikipedia.org/wiki/Genomic_imprinting)

23. Appendixes X chromosome inactivation: one of the X chromosomes in female is silenced by packaged to heterochromatin (https://en.wikipedia.org/wiki/X- inactivation)https://en.wikipedia.org/wiki/X- inactivation Differentially methylated regions(DMRs): regions that are methylated differently among samples, related to imprinting. (https://en.wikipedia.org/wiki/Differentially_methylated_regions)https://en.wikipedia.org/wiki/Differentially_methylated_regions Passive pathway: lost of methylation due to dilution. (i.e. each time the DNA is replicated only one side the methylation marks stay) Active Pathway: lost of methylation due to methyltransferase (Dnmt3a, Dnmt3b, Np95) that targets Dnmt1(which maintain methylation level during mitosis) are suppressed, or demethylation pathways(Tet1, DNA repair mechanisms) are active.

24. Appendixes IAPs: intracisternal A particles, transposons, whoever get close to it shows resistant to demethylation. VECs: variably erased CGIs, CGIs that is not affected by IAPs shows variable state of methylation. Tet1: factor that is involved in active demethylation LINE1: retrotransposon

25. Some Significant stages In E6.5(epiblast), overall methylation level(71%) almost equals to that of J1 ESC(74%) In E9.5, the stage when PGCs start to migrate to where the gonad will be(start to becoming gametes), the methylation level drops to 30%. In E11.5, the stage when imprint erasure starts to occur, methylation level drops to 15%. In E13.5, the stage that male and female mice can be distinguished morphologically, the methylation level drops to 14% in male and 7% in female E16.5, meiotic prophase arrest(female)