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Breanna Perreault and Xiao Liu
D145 Presentation
Background : Primordial Germ Cells
- PGCs: cells that give rise to gametes
- Genome-wide DNA methylation reprogramming occurs in
mouse PGCs
What is Epigenetics?
Changes in gene expression caused
by mechanisms other than changes
in the DNA sequence
WHY is it important?
Epigenetic Terminology
- CpG Islands
-
Cytosine that is methylated and is almost always located next to a
guanine nucleotide
-
promoter
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
Main Method Used
-Bisulfite Sequencing (BS seq)Protection treatment: methylated cytosine do not get
converted to uracil
- Steps:
1. DNA sonicated (300-500bp)
2. DNA adaptor ligated
3. DNA treated with sodium bisulfite
4. Amplify DNA (PCR)
5. Size selection: gel extraction for 200-250bp DNA
Main findings
1. 2 main phases of demethylation in PGCs
2. Combination of passive + active maintenance of methylation during global
demethylation
3. Global erasure does not mean indiscriminate transcription- some other mechanism
controls this
4. VECs- potential carrier of short term transgenerational epigenetic inheritance by
sustained methylation
5. Primordial germ cell development and methylation linked with pluripotency
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 E10.5-11.5
-E13.5 and E16.5 males and females were profiled separately
2 main phases of demethylation:
1. Early phase (Global): E6.5
--networks related to pluripotency are activated
-affect promoters, CpG islands (CGIs), introns, exons, intergenic
sequences
-retrotransposons (LINEs, SINEs)
Temporal Heatmap of Methylation
2. Late phase: E10.5
-DMR of imprinted genes
-CpG Islands found on X chromosomes
-CpG Islands associated with germline specific genes
DMRs of Imprinted Genes
Imprinting not propagated by CpG methylation
CpG Islands on X chromosome
Suggested Association with X-Chromosome Inactivation
CpGs associated with Germline Specific Genes
Reprogramming the Transcriptional Landscape
of PGCs
- RNA Seq
-figures
-analysis
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
Temporal Heatmap of Methylation
CGI Containing Promoter
Intergenic Region
Non-CGI Promoter
IAP
Non-Promoter CGI
LINE1
Exon
Maternal DMR
Intron
Paternal DMR
IAP vs LINE Methylation Level
CPGI Methylation Decreases with IAP Proximity
DETAIL
Whole Genome GCI Promoter Demethylation
Note Male and Female Difference in VECs (13.5)
Global Demethylation mechanism
Passive: DNA methyltransferases
Dnmt1+ Np95
Active: DNA demethylase
Tet1
Hairpin Bisulfite Seq
Alignable sequence data
Active and Passive Demethylation
Performed on LINEs, however this is global demethylation trend
Predominantly Passive
Stagnant Transcriptional Profile Comparison
LINE transcription
Conclusions:
1. Global DNA methylation- passive + active demethylation
2. 2 main phases of demethylation in PGCs
3. DMR
4. X chromosome
5. Germline specific
6. Global erasure does not mean indiscriminate transcription
7. Lines and transcription
8. Pluripotency and Meiosis
9. IAP
10. VECs- carrier of short term transgenerational epigenetic inheritance
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
Recommended Readings
http://www.nature.com/nrg/journal/v17/n10/pdf/nrg.2016.88.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3183171/
Questions?