DNA Modifications Regulate Chromatin
DNA methylation
DNA methylation involves the addition of a methyl group to carbon 5 of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring. The cytosines targeted for methylation are frequently followed by a guanosine, forming the repetitive unit of CpG dinucleotide islands. This methylated cytosine is referred to as 5mC. DNA (CpG) methylation affects DNA accessibility to proteins that bind DNA or chromatin. This ultimately establishes as an epigenetic silencing mechanism that silences at regions of constitutive heterochromatin, and suppresses the expression of viral genes and other deleterious elements that have been incorporated into the genome of the host over time.
DNA methylation deposition: Intro to the 'writers'
DNA methyltransferases (DNMTs) methylate target cytosines. In mammalian cells, DNA methylation occurs mainly at the C5 position of CpG dinucleotides and is carried out by two general classes of enzymatic activities – de novo methylation and maintenance methylation.
de novo DNA methylation
DNMT3a and DNMT3b serve as de novo methyltransferases that establish DNA methylation patterns early in development. DNMT3L is a protein that is homologous to the other DNMT3s but has no catalytic activity. Instead, DNMT3L assists the de novo methyltransferases by increasing their ability to bind to DNA and stimulating their activity. Finally, DNMT2 (TRDMT1) has been identified as a DNA methyltransferase homolog, containing all 10 sequence motifs common to all DNA methyltransferases; however, DNMT2 (TRDMT1) does not methylate DNA but instead methylates cytosine-38 in the anticodon loop of aspartic acid transfer RNA.
maintenance of DNA methylation
Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle since replication machinery alone would produce unmethylated daughter strands that gradually result in passive demethylation. DNMT1 binds 'hemi-methylated' DNA to copy DNA methylation patterns to the daughter strands during DNA replication. The UHRF1 (aka NP95?) cofactor recognizes 'hemi-methylated' DNA at replication forks, and subsequently recruits DNMT1. Mouse models with both copies of DNMT1 deleted are embryonic lethal at approximately day 9, due to the requirement of DNMT1 activity for development in mammalian cells.
Removal of DNA methylation
Passive DNA demethylation is the inevitable consequence of repeated genome replication in the absence of functional DNMT1. Active DNA methylation is catalyzed by the ten eleven translocation (TET) family of Fe(II) and α-ketoglutarate–dependent dioxygenases that catalyze sequential oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), then subsequent generation of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) which is excised by the thymine-DNA glycosylase (TDG)-mediated base excision repair (BER) pathway.
Anjana Rao 2019:
The oxidized methylcytosines (oxi-mC) generated by TET proteins are intermediates in at least 2 pathways of DNA demethylation: 1) replication-dependent loss of methylation, reflecting inability of the DNMT1/UHRF1 complex to methylate unmodified CpGs on newly replicated DNA strands if an oxi-mC (rather than 5mC) is present on the template strand, and 2) a replication-independent process in which thymine DNA glycosylase (TDG) excises 5fC and 5caC, which are then replaced with unmodified cytosine through base excision repair (4).
TETs in Cancer Anjana Rao 2019:
TET loss of function and low 5hmC levels are strongly associated with cancer (5). TET2 mutations are frequent in diverse hematopoietic malignancies, including myelodysplastic syndromes, acute myeloid leukemias, and peripheral T cell lymphomas (6⇓–8). However, both solid tumors and hematopoietic malignancies display TET loss of function without TET coding region mutations, as a result of TET promoter methylation, increased degradation of TET proteins, or aberrant microRNA expression (9⇓–11). In addition, hypoxia and a variety of metabolic alterations impair the enzymatic activity of TET and other dioxygenases, by decreasing the levels of the substrates α-ketoglutarate and molecular oxygen or increasing the levels of the inhibitor 2-hydroxyglutarate (10, 11).
DNA methylation during development
Global versus imprinted DNA methylation: Imprinted - DNA methylation can regulate expression of clusters of genes via imprinted control regions (ICRs) or gDMRs. Imprinted gDMRs acquire allele-specific methylation during gametogenesis. Three waves of DNA methylation reprograming occur during gamete formation and preimplantation development.
1) First, both global and imprinted DNA methylation patterns from previous generations are erased in primordial germ cells (PGCs) in two 'waves'.
i) Stage I erasure: E8.0-E9.0 Uhrf1/Np95 co-factor expression decreases to permit replication-dependent passive demethylation reducing 5mC levels to ~30%.
ii) Stage II erasure: E10.5-E13.5 TET1 and TET2-directed active demethylation (followed by BER pathway) further diminishes 5mC levels.
- By E13.5, 5mC declines to minimum levels to establish the epigenetic 'ground state' of germline genome.
2) Maternal and paternal-specific de novo DNA methylation and 'imprints' are acquired during oocyte and sperm development via DNMT3A/B and DNMT3L activity.
3) Imprints are maintained during preimplantation development when the rest of the genome becomes hypomethylated to establish totipotency of the early embryo.
i) paternal pronucleus: Following fertilization, active demethylation occurs globally through zygote to blastocyte stages of early embryogenesis via TET3 activity
i) maternal pronucleus: Following fertilization, replication-dependent passive demethylation occurs globally through zygote to blastocyte stages
Altered DNA methylation dynamics
Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis, there have been attempts to re-express these genes by inhibiting the DNMTs. 5-Aza-2'-deoxycytidine (decitabine) is a nucleoside analog that inhibits DNMTs by trapping them in a covalent complex on DNA by preventing the β-elimination step of catalysis, thus resulting in the enzymes' degradation. However, for decitabine to be active, it must be incorporated into the genome of the cell, which can cause mutations in the daughter cells if the cell does not die. In addition, decitabine is toxic to the bone marrow, which limits the size of its therapeutic window. These pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their mRNAs and preventing their translation. However, it is currently unclear whether targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation.
DNA methylation involves the addition of a methyl group to carbon 5 of the cytosine pyrimidine ring or the number 6 nitrogen of the adenine purine ring. The cytosines targeted for methylation are frequently followed by a guanosine, forming the repetitive unit of CpG dinucleotide islands. This methylated cytosine is referred to as 5mC. DNA (CpG) methylation affects DNA accessibility to proteins that bind DNA or chromatin. This ultimately establishes as an epigenetic silencing mechanism that silences at regions of constitutive heterochromatin, and suppresses the expression of viral genes and other deleterious elements that have been incorporated into the genome of the host over time.
DNA methylation deposition: Intro to the 'writers'
DNA methyltransferases (DNMTs) methylate target cytosines. In mammalian cells, DNA methylation occurs mainly at the C5 position of CpG dinucleotides and is carried out by two general classes of enzymatic activities – de novo methylation and maintenance methylation.
de novo DNA methylation
DNMT3a and DNMT3b serve as de novo methyltransferases that establish DNA methylation patterns early in development. DNMT3L is a protein that is homologous to the other DNMT3s but has no catalytic activity. Instead, DNMT3L assists the de novo methyltransferases by increasing their ability to bind to DNA and stimulating their activity. Finally, DNMT2 (TRDMT1) has been identified as a DNA methyltransferase homolog, containing all 10 sequence motifs common to all DNA methyltransferases; however, DNMT2 (TRDMT1) does not methylate DNA but instead methylates cytosine-38 in the anticodon loop of aspartic acid transfer RNA.
maintenance of DNA methylation
Maintenance methylation activity is necessary to preserve DNA methylation after every cellular DNA replication cycle since replication machinery alone would produce unmethylated daughter strands that gradually result in passive demethylation. DNMT1 binds 'hemi-methylated' DNA to copy DNA methylation patterns to the daughter strands during DNA replication. The UHRF1 (aka NP95?) cofactor recognizes 'hemi-methylated' DNA at replication forks, and subsequently recruits DNMT1. Mouse models with both copies of DNMT1 deleted are embryonic lethal at approximately day 9, due to the requirement of DNMT1 activity for development in mammalian cells.
Removal of DNA methylation
Passive DNA demethylation is the inevitable consequence of repeated genome replication in the absence of functional DNMT1. Active DNA methylation is catalyzed by the ten eleven translocation (TET) family of Fe(II) and α-ketoglutarate–dependent dioxygenases that catalyze sequential oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), then subsequent generation of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) which is excised by the thymine-DNA glycosylase (TDG)-mediated base excision repair (BER) pathway.
Anjana Rao 2019:
The oxidized methylcytosines (oxi-mC) generated by TET proteins are intermediates in at least 2 pathways of DNA demethylation: 1) replication-dependent loss of methylation, reflecting inability of the DNMT1/UHRF1 complex to methylate unmodified CpGs on newly replicated DNA strands if an oxi-mC (rather than 5mC) is present on the template strand, and 2) a replication-independent process in which thymine DNA glycosylase (TDG) excises 5fC and 5caC, which are then replaced with unmodified cytosine through base excision repair (4).
TETs in Cancer Anjana Rao 2019:
TET loss of function and low 5hmC levels are strongly associated with cancer (5). TET2 mutations are frequent in diverse hematopoietic malignancies, including myelodysplastic syndromes, acute myeloid leukemias, and peripheral T cell lymphomas (6⇓–8). However, both solid tumors and hematopoietic malignancies display TET loss of function without TET coding region mutations, as a result of TET promoter methylation, increased degradation of TET proteins, or aberrant microRNA expression (9⇓–11). In addition, hypoxia and a variety of metabolic alterations impair the enzymatic activity of TET and other dioxygenases, by decreasing the levels of the substrates α-ketoglutarate and molecular oxygen or increasing the levels of the inhibitor 2-hydroxyglutarate (10, 11).
DNA methylation during development
Global versus imprinted DNA methylation: Imprinted - DNA methylation can regulate expression of clusters of genes via imprinted control regions (ICRs) or gDMRs. Imprinted gDMRs acquire allele-specific methylation during gametogenesis. Three waves of DNA methylation reprograming occur during gamete formation and preimplantation development.
1) First, both global and imprinted DNA methylation patterns from previous generations are erased in primordial germ cells (PGCs) in two 'waves'.
i) Stage I erasure: E8.0-E9.0 Uhrf1/Np95 co-factor expression decreases to permit replication-dependent passive demethylation reducing 5mC levels to ~30%.
ii) Stage II erasure: E10.5-E13.5 TET1 and TET2-directed active demethylation (followed by BER pathway) further diminishes 5mC levels.
- By E13.5, 5mC declines to minimum levels to establish the epigenetic 'ground state' of germline genome.
2) Maternal and paternal-specific de novo DNA methylation and 'imprints' are acquired during oocyte and sperm development via DNMT3A/B and DNMT3L activity.
3) Imprints are maintained during preimplantation development when the rest of the genome becomes hypomethylated to establish totipotency of the early embryo.
i) paternal pronucleus: Following fertilization, active demethylation occurs globally through zygote to blastocyte stages of early embryogenesis via TET3 activity
i) maternal pronucleus: Following fertilization, replication-dependent passive demethylation occurs globally through zygote to blastocyte stages
Altered DNA methylation dynamics
Since many tumor suppressor genes are silenced by DNA methylation during carcinogenesis, there have been attempts to re-express these genes by inhibiting the DNMTs. 5-Aza-2'-deoxycytidine (decitabine) is a nucleoside analog that inhibits DNMTs by trapping them in a covalent complex on DNA by preventing the β-elimination step of catalysis, thus resulting in the enzymes' degradation. However, for decitabine to be active, it must be incorporated into the genome of the cell, which can cause mutations in the daughter cells if the cell does not die. In addition, decitabine is toxic to the bone marrow, which limits the size of its therapeutic window. These pitfalls have led to the development of antisense RNA therapies that target the DNMTs by degrading their mRNAs and preventing their translation. However, it is currently unclear whether targeting DNMT1 alone is sufficient to reactivate tumor suppressor genes silenced by DNA methylation.