epigenetics in mammalian development, dna methylation

epigenetics

• environment → genes → phenotype

• stable propagation of phenotype without a change in genotype

stability

change is maintained (over time, cell divisions, generations) in the absence of the initiating event

epigenetics in maintains cellular identity during development

• in multipotent stem cells and primitive progenitor cells → genes associated with multipotency on, lineage-specific genes off

• in lineage committed and specialized cells → genes associated with multipotency off, lineage-specific genes on

• cells remember multipotency genes being turned off, despite initiating event being earlier in cell lineage

  • transmitted over cell divisions and generations

nucleosome

• fundamental unit of chromatin structure

• composed of DNA and histones

regulatory targets for chromatin states

• developmentally regulated genes

• repetitive DNA elements (transposons)

• large chromosomal domains

• whole chromosomes

constitutive heterochromatin

• in every cell, throughout development

• centromeres, telomeres, retrotransposons

• epigenetically stable

facultative heterochromatin

• varies with cell-type and stage of development

• developmental genes, imprinting, inactive X chromosome

• epigenetically stable

cellular mechanisms for altering chromatin structure

• nucleosome movement

• nucleosome assembly and disassembly

• large-scale movement of chromatin in nucleus

• covalent modification of chromatin components (DNA and/or histones)

DNA methylation

• 5-methylcytosine in CpG dinucleotides is present in DNA of all vertebrates and flowering plants, some invertebrates, protists, bacteria

• most CpGs in mammalian genome are methylated

• earliest discovered epigenetic mark (Holliday and Pugh in 1975)

• critical component of heterochromatin

DNA methylation and transposons

• ~40% of methylated CpGs reside in retrotransposons

• part of silencing mechanism that contributes to genome defense

• retrotransposons typically found in constitutive heterochromatin

• if retrotransposons are transcribed by RNAPII → mobilization → insertional mutagenesis and aberrant recombination, antisense transcription

DNA methylation and gene regulation

• ~70% of promoters of human genes contain CpG islands (CGIs)

  • > 55% C or G, enriched in CpG dinucleotides

• CpGs in CGIs are almost always (~95%) unmethylated, at both active and inactive promoters

• rare CGI methylation strongly correlated with repression of transcription

• exception: ICRs that control imprinted genes are commonly methylated

CpG island structure

• island is almost always unmethylated

• shore (area surrounding island) has cell-type specific methylation

• methylation at shores is more strongly associated with whether the gene is on or off

  • high methylation → low expression

  • low methylation → high expression

enhancers

• regulatory DNA elements that can act at large genomic distances

• are binding sites of transcription factors

• important for regulation of developmental genes

• high methylation is often anti-correlated with transcription factor binding and enhancer activity

mechanism of repression by DNA methylation

• methyl-DNA binding proteins (MeCP1, MeCP2, MBD family, UHRF1) function in combination with histone modifications to repress transcription

• block factor access to DNA, or DNA-binding protein can’t bind when target is methylated

bisulfite treatment to detect CpG methylation

• treatment of DNA with sodium bisulfite converts unmethylated cytosines to uracil through deamination

• sequence is amplified through PCR and sequenced

• all remaining cytosines after treatment are methylated

• thymines on complementary strand can be compared with wildtype sequence to determine if T is new (used to be C) or originally T

epigenomics

genome-wide mapping of chromatin modifications

DNA methyltransferases

• contain highly conserved catalytic domain (performs methylation) and variable targeting domain (for localization and protein interactions)

• DNMT1, DNMT3A/B, DNMT3L

DNMT1

• maintenance DNA methyltransferase

• involved in inheritance of DNA methylation

• methylates daughter strand of DNA during replication

• localizes to sites of DNA replication in S phase

DNMT3A/B

• de novo DNA methyltransferase → establishment of methylation

• DNMT3B is the major de novo methyltransferase in embryo

• DNMT3A essential for germline development and certain somatic tissues (blood cells)

• complex with DNMT3L in germ cells

• ADD and PWWP domains bind to chromatin

DNMT3L

• not catalytically active

• germline-specific

• forms a complex with DNMT3A/B in germ cells

• targeting function

dnmt mutant phenotypes

• reanimation of retrotransposons

• chromosome instability (loss of methylation in constitutive regions)

  • aneuploidy, compromised centromere function

• loss of imprinting

• defective expression of developmentally regulated genes

DNMT1 function in skin cell progenitor renewal

• wildtype keratinocytes (progenitors) or DNMT1 knockdowns are introduced into in vivo skin regeneration assay

• wildtype progenitors can restore graft, DNMT1 knockdown cells cannot

  • DNMT1 knockdown cells can’t self-renew

• DNMT1 knockdown progenitors show increased expression of differentiation genes

  • differential methylation of CGI shore regions of genes associated with skin cell differentiation

DNA demethylation

• passive: DNMT1 action prevented, methyl mark is diluted out over generations

• active: oxidation of 5mC by TET enzymes

5mC oxidation and hydroxylation by TET family enzymes

• 5mC → 5hmC (hydroxymethylcytosine) → 5fC (formylcytosine) → 5caC (carboxylcytosine)

• oxidation prevents DNMT1 from working (not DNMT1 targets)

• oxidized 5mC derivatives can be removed passively by replication or actively by DNA repair enzymes

  • base excision repair

  • recognized as instances of DNA damage (especially 5caC)