Editing Germline (section)

==Epigenetic alterations==

[[File:5 methylcytosine methyl highlight.png|thumb|300px|5 methylcytosine methyl highlight.  The image shows a cytosine single ring base and a methyl group added on to the 5 carbon. In mammals, DNA methylation occurs almost exclusively at a cytosine that is followed by a [[guanine]].]]
[[Epigenetics|Epigenetic alterations]] of DNA include modifications that affect gene expression, but are not caused by changes in the sequence of bases in DNA.  A well-studied example of such an alteration is the [[methylation]] of DNA cytosine to form [[5-methylcytosine]].  This usually occurs in the DNA sequence [[CpG site|CpG]], changing the DNA at the [[CpG site]] from CpG to 5-mCpG.  Methylation of cytosines in CpG sites in [[Promoter (genetics)|promoter]] regions of genes can reduce or silence gene expression.<ref name="pmid11782440">{{cite journal |vauthors=Bird A |title=DNA methylation patterns and epigenetic memory |journal=Genes Dev |volume=16 |issue=1 |pages=6–21 |date=January 2002 |pmid=11782440 |doi=10.1101/gad.947102 |url=|doi-access=free }}</ref>  About 28 million CpG dinucleotides occur in the human genome,<ref name="pmid26932361">{{cite journal |vauthors=Lövkvist C, Dodd IB, Sneppen K, Haerter JO |title=DNA methylation in human epigenomes depends on local topology of CpG sites |journal=Nucleic Acids Res |volume=44 |issue=11 |pages=5123–32 |date=June 2016 |pmid=26932361 |pmc=4914085 |doi=10.1093/nar/gkw124 |url=}}</ref> and about 24 million CpG sites in the mouse genome (which is 86% as large as the human genome<ref name="pmid16339371">{{cite journal |vauthors=Guénet JL |title=The mouse genome |journal=Genome Res |volume=15 |issue=12 |pages=1729–40 |date=December 2005 |pmid=16339371 |doi=10.1101/gr.3728305 |url=|doi-access=free }}</ref>).  In most tissues of mammals, on average, 70% to 80% of CpG cytosines are methylated (forming 5-mCpG).<ref name="pmid15177689">{{cite journal |vauthors=Jabbari K, Bernardi G |title=Cytosine methylation and CpG, TpG (CpA) and TpA frequencies |journal=Gene |volume=333 |issue= |pages=143–9 |date=May 2004 |pmid=15177689 |doi=10.1016/j.gene.2004.02.043 |url=}}</ref>

In the mouse, by days 6.25 to 7.25 after fertilization of an egg by a sperm, cells in the embryo are set aside as primordial germ cells (PGCs).  These PGCs will later give rise to germline sperm cells or egg cells.  At this point the PGCs have high typical levels of methylation.  Then primordial germ cells of the mouse undergo genome-wide DNA [[demethylation]], followed by subsequent new methylation to reset the [[epigenome]] in order to form an egg or sperm.<ref name=Zeng>{{cite journal |vauthors=Zeng Y, Chen T |title=DNA Methylation Reprogramming during Mammalian Development |journal=Genes (Basel) |volume=10 |issue=4 |date=March 2019 |page=257 |pmid=30934924 |pmc=6523607 |doi=10.3390/genes10040257 |url=|doi-access=free }}</ref>

In the mouse, PGCs undergo DNA demethylation in two phases. The first phase, starting at about embryonic day 8.5, occurs during PGC proliferation and migration, and it results in genome-wide loss of methylation, involving '''almost''' all genomic sequences.  This loss of methylation occurs through passive demethylation due to repression of the major components of the methylation machinery.<ref name=Zeng />  The second phase occurs during embryonic days 9.5 to 13.5 and causes demethylation of most remaining specific loci, including germline-specific and meiosis-specific genes.  This second phase of demethylation is mediated by the [[TET enzymes]] TET1 and TET2, which carry out the first step in demethylation by converting 5-mC to [[5-hydroxymethylcytosine]] (5-hmC) during embryonic days 9.5 to 10.5.  This is likely followed by replication-dependent dilution during embryonic days 11.5 to 13.5.<ref name=Yamaguchi>{{cite journal |vauthors=Yamaguchi S, Hong K, Liu R, Inoue A, Shen L, Zhang K, Zhang Y |title=Dynamics of 5-methylcytosine and 5-hydroxymethylcytosine during germ cell reprogramming |journal=Cell Res |volume=23 |issue=3 |pages=329–39 |date=March 2013 |pmid=23399596 |pmc=3587712 |doi=10.1038/cr.2013.22 |url=}}</ref>  At embryonic day 13.5, PGC genomes display the lowest level of global DNA methylation of all cells in the life cycle.<ref name=Zeng />

In the mouse, the great majority of differentially expressed genes in PGCs from embryonic day 9.5 to 13.5, when most genes are demethylated, are upregulated in both male and female PGCs.<ref name=Yamaguchi />

Following erasure of DNA methylation marks in mouse PGCs, male and female germ cells undergo new methylation at different time points during gametogenesis. While undergoing mitotic expansion in the developing gonad, the male germline starts the re-methylation process by embryonic day 14.5. The sperm-specific methylation pattern is maintained during mitotic expansion.  DNA methylation levels in primary oocytes before birth remain low, and re-methylation occurs after birth in the oocyte growth phase.<ref name=Zeng />