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DNA demethylation
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DNA demethylation
For molecular biology in mammals, DNA demethylation causes replacement of 5-methylcytosine (5mC) in a DNA sequence by cytosine (C) (see figure of 5mC and C). DNA demethylation can occur by an active process at the site of a 5mC in a DNA sequence or, in replicating cells, by preventing addition of methyl groups to DNA so that the replicated DNA will largely have cytosine in the DNA sequence (5mC will be diluted out).
Methylated cytosine is frequently present in the linear DNA sequence where a cytosine is followed by a guanine in a 5' → 3' direction (a CpG site). In mammals, DNA methyltransferases (which add methyl groups to DNA bases) exhibit a strong sequence preference for cytosines at CpG sites. There appear to be more than 20 million CpG dinucleotides in the human genome (see genomic distribution). In mammals, on average, 70% to 80% of CpG cytosines are methylated, though the level of methylation varies with different tissues. Methylated cytosines often occur in groups or CpG islands within the promoter regions of genes, where such methylation may reduce or silence gene expression (see gene expression). Methylated cytosines in the gene body, however, are positively correlated with expression.
Almost 100% DNA demethylation occurs by a combination of passive dilution and active enzymatic removal during the reprogramming that occurs in early embryogenesis and in gametogenesis. Another large demethylation, of about 3% of all genes, can occur by active demethylation in neurons during formation of a strong memory. After surgery, demethylations are found in peripheral blood mononuclear cells at sites annotated to immune system genes. Demethylations also occur during the formation of cancers. During global DNA hypomethylation of tumor genomes, there is a minor to moderate reduction of the number of methylated cytosines (5mC) amounting to a loss of about 5% to 20% on average of the 5mC bases.
The mouse sperm genome is 80–90% methylated at its CpG sites in DNA, amounting to about 20 million methylated sites.[citation needed] After fertilization, the paternal chromosome is almost completely demethylated in six hours by an active process, before DNA replication (blue line in Figure).
Demethylation of the maternal genome occurs by a different process. In the mature oocyte, about 40% of its CpG sites in DNA are methylated. While somatic cells of mammals have three main DNA methyltransferases (which add methyl groups to cytosines at CpG sites), DNMT1, DNMT3A, and DNMT3B, in the pre-implantation embryo up to the blastocyst stage (see Figure), the only methyltransferase present is an isoform of DNMT1 designated DNMT1o. DNMT1o has an alternative oocyte-specific promoter and first exon (exon 1o) located 5' of the somatic and spermatocyte promoters. As reviewed by Howell et al., DNMT1o is sequestered in the cytoplasm of mature oocytes and in 2-cell and 4-cell embryos, but at the 8-cell stage is only present in the nucleus. At the 16 cell stage (the morula) DNMT1o is again found only in the cytoplasm. It appears that demethylation of the maternal chromosomes largely takes place by blockage of the methylating enzyme DNMT1o from entering the nucleus except briefly at the 8 cell stage. The maternal-origin DNA thus undergoes passive demethylation by dilution of the methylated maternal DNA during replication (red line in Figure). The morula (at the 16 cell stage), has only a small amount of DNA methylation (black line in Figure).
DNMT3b begins to be expressed in the blastocyst. Methylation begins to increase at 3.5 days after fertilization in the blastocyst, and a large wave of methylation then occurs on days 4.5 to 5.5 in the epiblast, going from 12% to 62% methylation, and reaching maximum level after implantation in the uterus. By day seven after fertilization, the newly formed primordial germ cells (PGC) in the implanted embryo segregate from the remaining somatic cells. At this point the PGCs have about the same level of methylation as the somatic cells.
The newly formed primordial germ cells (PGC) in the implanted embryo devolve from the somatic cells. At this point the PGCs have high levels of methylation. These cells migrate from the epiblast toward the gonadal ridge. As reviewed by Messerschmidt et al., the majority of PGCs are arrested in the G2 phase of the cell cycle, while they migrate toward the hindgut during embryo days 7.5 to 8.5. Then demethylation of the PGCs takes place in two waves. At day 9.5 the primordial germ cells begin to rapidly replicate going from about 200 PGCs at embryo day 9.5 to about 10,000 PGCs at day 12.5. During days 9.5 to 12.5 DNMT3a and DNMT3b are repressed and DNMT1 is present in the nucleus at a high level. But DNMT1 is unable to methylate cytosines during days 9.5 to 12.5 because the UHRF1 gene (also known as NP95) is repressed and UHRF1 is an essential protein needed to recruit DNMT1 to replication foci where maintenance DNA methylation takes place. This is a passive, dilution form of demethylation.
In addition, from embryo day 9.5 to 13.5 there is an active form of demethylation. As indicated below in "Molecular stages of active reprogramming," two enzymes are central to active demethylation. These are a ten-eleven translocation methylcytosine dioxygenase (TET) and thymine-DNA glycosylase (TDG). One particular TET enzyme, TET1, and TDG are present at high levels from embryo day 9.5 to 13.5, and are employed in active demethylation during gametogenesis. PGC genomes display the lowest levels of DNA methylation of any cells in the entire life cycle of the mouse at embryonic day 13.5.
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DNA demethylation
For molecular biology in mammals, DNA demethylation causes replacement of 5-methylcytosine (5mC) in a DNA sequence by cytosine (C) (see figure of 5mC and C). DNA demethylation can occur by an active process at the site of a 5mC in a DNA sequence or, in replicating cells, by preventing addition of methyl groups to DNA so that the replicated DNA will largely have cytosine in the DNA sequence (5mC will be diluted out).
Methylated cytosine is frequently present in the linear DNA sequence where a cytosine is followed by a guanine in a 5' → 3' direction (a CpG site). In mammals, DNA methyltransferases (which add methyl groups to DNA bases) exhibit a strong sequence preference for cytosines at CpG sites. There appear to be more than 20 million CpG dinucleotides in the human genome (see genomic distribution). In mammals, on average, 70% to 80% of CpG cytosines are methylated, though the level of methylation varies with different tissues. Methylated cytosines often occur in groups or CpG islands within the promoter regions of genes, where such methylation may reduce or silence gene expression (see gene expression). Methylated cytosines in the gene body, however, are positively correlated with expression.
Almost 100% DNA demethylation occurs by a combination of passive dilution and active enzymatic removal during the reprogramming that occurs in early embryogenesis and in gametogenesis. Another large demethylation, of about 3% of all genes, can occur by active demethylation in neurons during formation of a strong memory. After surgery, demethylations are found in peripheral blood mononuclear cells at sites annotated to immune system genes. Demethylations also occur during the formation of cancers. During global DNA hypomethylation of tumor genomes, there is a minor to moderate reduction of the number of methylated cytosines (5mC) amounting to a loss of about 5% to 20% on average of the 5mC bases.
The mouse sperm genome is 80–90% methylated at its CpG sites in DNA, amounting to about 20 million methylated sites.[citation needed] After fertilization, the paternal chromosome is almost completely demethylated in six hours by an active process, before DNA replication (blue line in Figure).
Demethylation of the maternal genome occurs by a different process. In the mature oocyte, about 40% of its CpG sites in DNA are methylated. While somatic cells of mammals have three main DNA methyltransferases (which add methyl groups to cytosines at CpG sites), DNMT1, DNMT3A, and DNMT3B, in the pre-implantation embryo up to the blastocyst stage (see Figure), the only methyltransferase present is an isoform of DNMT1 designated DNMT1o. DNMT1o has an alternative oocyte-specific promoter and first exon (exon 1o) located 5' of the somatic and spermatocyte promoters. As reviewed by Howell et al., DNMT1o is sequestered in the cytoplasm of mature oocytes and in 2-cell and 4-cell embryos, but at the 8-cell stage is only present in the nucleus. At the 16 cell stage (the morula) DNMT1o is again found only in the cytoplasm. It appears that demethylation of the maternal chromosomes largely takes place by blockage of the methylating enzyme DNMT1o from entering the nucleus except briefly at the 8 cell stage. The maternal-origin DNA thus undergoes passive demethylation by dilution of the methylated maternal DNA during replication (red line in Figure). The morula (at the 16 cell stage), has only a small amount of DNA methylation (black line in Figure).
DNMT3b begins to be expressed in the blastocyst. Methylation begins to increase at 3.5 days after fertilization in the blastocyst, and a large wave of methylation then occurs on days 4.5 to 5.5 in the epiblast, going from 12% to 62% methylation, and reaching maximum level after implantation in the uterus. By day seven after fertilization, the newly formed primordial germ cells (PGC) in the implanted embryo segregate from the remaining somatic cells. At this point the PGCs have about the same level of methylation as the somatic cells.
The newly formed primordial germ cells (PGC) in the implanted embryo devolve from the somatic cells. At this point the PGCs have high levels of methylation. These cells migrate from the epiblast toward the gonadal ridge. As reviewed by Messerschmidt et al., the majority of PGCs are arrested in the G2 phase of the cell cycle, while they migrate toward the hindgut during embryo days 7.5 to 8.5. Then demethylation of the PGCs takes place in two waves. At day 9.5 the primordial germ cells begin to rapidly replicate going from about 200 PGCs at embryo day 9.5 to about 10,000 PGCs at day 12.5. During days 9.5 to 12.5 DNMT3a and DNMT3b are repressed and DNMT1 is present in the nucleus at a high level. But DNMT1 is unable to methylate cytosines during days 9.5 to 12.5 because the UHRF1 gene (also known as NP95) is repressed and UHRF1 is an essential protein needed to recruit DNMT1 to replication foci where maintenance DNA methylation takes place. This is a passive, dilution form of demethylation.
In addition, from embryo day 9.5 to 13.5 there is an active form of demethylation. As indicated below in "Molecular stages of active reprogramming," two enzymes are central to active demethylation. These are a ten-eleven translocation methylcytosine dioxygenase (TET) and thymine-DNA glycosylase (TDG). One particular TET enzyme, TET1, and TDG are present at high levels from embryo day 9.5 to 13.5, and are employed in active demethylation during gametogenesis. PGC genomes display the lowest levels of DNA methylation of any cells in the entire life cycle of the mouse at embryonic day 13.5.
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