1. Breanna Perreault and Xiao Liu D145 Presentation

2. Background : Primordial Germ Cells
PGCs: cells that give rise to gametes
Genome-wide DNA methylation reprogramming occurs in mouse PGCs

3. What is Epigenetics? WHY is it important?
Changes in gene expression caused
by mechanisms other than changes
in the DNA sequence
WHY is it important?
Chromatin structure, Histone modification, acetylation, DNA methylation, histone methylation
The epigenetic modification that we’ll be mainly discussing today involves methylation of cytosine to convert
In the progress of methylation which usually occurs on CpG islands- genes are silenced

4. Epigenetic Terminology
CpG Islands
Cytosine that is methylated and is almost always located next to a guanine nucleotide
promoter

5. Epigenetic Terminology
Imprinting
epigenetic process that involves DNA methylation
and histone methylation
- transgenerational sex-dependent inheritance pattern
From dad -> reprogrammed in female offsprings-> to maternal pattern
From mom -> reprogrammed in male offsprings-> to paternal pattern
transgenerational sex-dependent inheritance pattern

6. Main Method Used -Bisulfite Sequencing (BS seq)- - Steps:
Protection treatment: methylated cytosine do not get
converted to uracil
- Steps:
DNA sonicated ( bp)
DNA adaptor ligated
DNA treated with sodium bisulfite
Amplify DNA (PCR)
Size selection: gel extraction for bp DNA

7. Main findings 2 main phases of demethylation in PGCs
Combination of passive + active maintenance of methylation during global demethylation
Global erasure does not mean indiscriminate transcription- some other mechanism controls this
VECs- potential carrier of short term transgenerational epigenetic inheritance by sustained methylation
Primordial germ cell development and methylation linked with pluripotency
DEMETHYLATION- expression

8. Timeline of demethylation in PGCs
-E6.5: ~40 PGCs arise in the epiblast
-E9.5: ~200 PGCs migrate through hindgut endoderm to reach the gonads by E
-E13.5 and E16.5 males and females were profiled separately
To give a little overview about what developmental processes are happening in concurrence to the PGCs getting demethylated
At day 13.5 gender is specified in PGCs from thereon out the genders are profiled separately
Whole genome bisulfite sequencing of each PGC they sampled and got the % CG methylation at each of these days of development

9. 2 main phases of demethylation:
Early phase (Global): E6.5
--networks related to pluripotency are activated
-affect promoters, CpG islands (CGIs), introns, exons, intergenic sequences
-retrotransposons (LINEs, SINEs)

10. Temporal Heatmap of Methylation
GI containing promoter because another mechanism (histones) are responsible for this

11. 2. Late phase: E10.5 -DMR of imprinted genes
-CpG Islands found on X chromosomes
-CpG Islands associated with germline specific genes

12. DMRs of Imprinted Genes
Imprinting not propagated by CpG methylation
promoter CGIs of DMRs imprinted genes that retained more than 25% methylation
Imprinting not propagated by CpG methylation

13. CpG Islands on X chromosome
Suggested Association with X-Chromosome Inactivation
It is noteworthy that CGIs on the X chromosome show elevated methylation levels in the E6.5 epiblast, as this is a pooled sample from male and female cells and thus includes a reduced but undefined number of inactivated X chromosomes contributed by female cells - highly demethylation contributed mostly from
PGCs inherit a randomly inactivated X chromosome -
methylation at CGIs on the X chromosome is actively maintained during global methylation loss and results in a slow and gradual demethylation pattern, which is consistent with the gradual reactivation of X-linked genes

14. CpGs associated with Germline Specific Genes
As most of the genome is demethylated - group of CGIs controlled for the activation of these germline specific genes retained >25% of their methylation meaning these are genes that are resistant to being expressed at this time in point

15. Reprogramming the Transcriptional Landscape of PGCs
- RNA Seq
-figures
-analysis

16. Methylated Regions IAPs- (Intracisternal A-Particles)
-retrotransposons
-can interrupt or enhance gene expression
VECs- (Variably Erased CpGIs)
occurs in male and female
not related to imprinted
Act as short term transgenerational carriers
Even though most of the genome are hypomethylated, few sequences where the methylation are still maintained
which as a result, act as transgenerational carriers because it is passed on usually to the next generation without any modifications

17. Temporal Heatmap of Methylation
CGI Containing Promoter
Non-CGI Promoter
Non-Promoter CGI
Exon
Intron
Intergenic Region
IAP
LINE1
Maternal DMR
Paternal DMR

18. IAP vs LINE Methylation Level

19. CPGI Methylation Decreases with IAP Proximity
DETAIL

20. Whole Genome GCI Promoter Demethylation
Note Male and Female Difference in VECs (13.5)

21. Global Demethylation mechanism
Passive: DNA methyltransferases
Dnmt1+ Np95
Active: DNA demethylase
Tet1

22. Hairpin Bisulfite Seq Alignable sequence data
Used to determine difference between double stranded and single stranded demethylation

23. Active and Passive Demethylation
Performed on LINEs, however this is global demethylation trend
Predominantly Passive

24. Stagnant Transcriptional Profile Comparison

25. LINE transcription

26. Conclusions: Global DNA methylation- passive + active demethylation
2 main phases of demethylation in PGCs
DMR
X chromosome
Germline specific
Global erasure does not mean indiscriminate transcription
Lines and transcription
Pluripotency and Meiosis
IAP
VECs- carrier of short term transgenerational epigenetic inheritance
Conclusions:

27. Critiques Couldn’t tell exactly mechanism of Global Demethylation
Too much detail to make a coherent point out of the study when not an expert in the field
Mentioned differences between male and female, but did not investigate why

28. Recommended Readings
This study only looked at mice-- this article looks at the epigenetics of human germline reprogramming

29. Questions?