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 http://www.cell.com/trends/genetics/fulltext/S0168-9525(11)00158-2

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? https://pubs.niaaa.nih.gov/publications/arcr351/6-16.htm http://missinglink.ucsf.edu/lm/genes_and_genomes/methylation.html 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

Epigenetic Terminology CpG Islands Cytosine that is methylated and is almost always located next to a guanine nucleotide promoter https://en.wikipedia.org/wiki/CpG_site

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 http://learn.genetics.utah.edu/content/epigenetics/imprinting/ transgenerational sex-dependent inheritance pattern

Main Method Used -Bisulfite Sequencing (BS seq)- - Steps: Protection treatment: methylated cytosine do not get converted to uracil - Steps: DNA sonicated (300-500bp) DNA adaptor ligated DNA treated with sodium bisulfite Amplify DNA (PCR) Size selection: gel extraction for 200-250bp DNA https://www.diagenode.com/applications/dna-bisulfite-conversion

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

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 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

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)

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

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

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

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

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 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

Temporal Heatmap of Methylation CGI Containing Promoter Non-CGI Promoter Non-Promoter CGI Exon Intron Intergenic Region IAP LINE1 Maternal DMR 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 http://www.whatisepigenetics.com/dna-methylation/

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

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

Stagnant Transcriptional Profile Comparison

LINE transcription

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:

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/ This study only looked at mice-- this article looks at the epigenetics of human germline reprogramming

Questions?