DNA Methylation I

epigenome is composed of two main components

1. histones which are associated with DNA

2. DNA methylation which is covalently bound to the genome and thus a stable long-term signal

history of DNA methylation

Rollin Hotchkiss (1911 – 2004) first discovered methylated DNA in 1948.
He found that DNA from certain sources contained, in addition to the standard four bases, a fifth: 5-methyl cytosine.
It took almost three decades to find a role for it.
In the mid-1970s, Harold Weintraub and his colleagues noticed that active genes are low in methyl groups or under methylated.
Therefore, a relationship between under methylation and gene activity seemed likely, as if methylation helped repress genes.

DNA methylation key concepts

chemical modification of DNA

can be inherited without sequence change

common in plants (30%), vertebrates (10%), and most fungi

absent or rare in yeast, flies, nematodes

occurs predominantly at 5’-CG-3’ (CpG) positions

in mammals, 60-90% of CpG sites have this

CpG islands

high frequency (>50%) of CG dinucleotides

typically 300-3,000 base pairs in length and hypomethylated

near approximately 70% human promoters

methylation correlated with tissue-specific gene expression

which enzyme methylates cytosine?

DNA methyltransferase (DMNT)

C to 5-methylcytosine (5mC)

SAM to SAH (substrate)

cytosine methylation maintains

inactive-condensed chromatin state, heterochromatic regions

DNA methylation and histone modifications help to

compartmentalize the genome into domains of different transcriptional potentials

euchromatin and modifications

high histone acetylation

low DNA methylation

H3-K4 methylation

heterochromatin and epigenetics

low histone acetylation

dense DNA methylation

H3-K9 methylation

how many active DNMTs have been identified in mammals?

three

DNMT3L

a protein closely related to DNMT3A and DNMT3B in structure and critical for DNA methylation, but appears to be inactive on its own

establishment and maintenance of DNA methylation requires

distinct enzymatic machinery

maintenance methylation (DNMT1)

methylation of newly synthesized DNA strand at positions opposite methylated sites on the parent strand (occurs after DNA replication)

De novo methylation (DNMT3A, 3B)

methylation of totally new positions

changes the pattern of methylation in a localized region of the genome during gametogenesis and early development

sodium bisulfite sequencing

method of DNA methylation analysis

methylated cytosine is unaffected

converts unmethylated cytosine to uracil

during PCR (which converts any uracil base to thymine) and subsequent sequencing, the ratio of cytosine and thymine present at each CpG site is quantified, reflects the methylation level of that site in genomic DNA

biological functions of DNA methylation

transcriptional regulation of cellular genes, role in mammalian development, including imprinting

heterochromatin formation

some DNMTs are

essential, including DNMT1, DNMT3A, and DNMT3B

NuRD complex

nucleosome remodeling deacetylase

mediator of methylation induced gene silencing

methyl CpG binding proteins are

repressive

MeCP2

has a methyl CpG binding domain and a transcriptional repression domain

interacts with the mSin3 co-repressor complex which associates with HDAC to repress transcription

knockout mouse also embryonic lethal

transcription factors are _______ while DNA methylation is _______

transient, not

How does DNA methylation repress gene transcription?

unmethylated (or hypomethylated) promotor allows gene transcription

direct mechanism- methylated CpGs block binding of TFs

indirection mechanism- Me-CpG binding proteins also preclude TF binding to the promoter region

indirect mechanism of DNA methylation gene repression

crosstalk between DNA methylation and chromatin modification, resulting in transcriptional repression

methylation of the CpG island upstream of a gene provides recognition signals for the MeCP components of a histone deacetylase complex (HDAC)

the HDAC modifies chromatin in the region of CpG island and hence inactivates the gene

trichostatin A (TSA)

blocks histone deacetylase activity, prevent DNA methylation dependent repression

sodium butyrate

mimics histone acetylation, used to loosen up chromatin

chromatin conformation, transcriptionally inactive

closed, highly condensed conformation

DNA CpG methylation, transcriptionally inactive

methylated, including at promoter regions

histone modification, transcriptionally inactive

deacetylated, methylated H3-K9me

chromatin conformation, transcriptionally active

open, extended conformation

DNA CpG methylation, transcriptionally active

relatively unmethylated, especially at promoter regions

histone modification, transcriptionally active

acetylated, methylated (H3-K4me3, R17me2)

the steady state methylation pattern is a dynamic equilibrium between

demethylation (DNA deMetase, HATs) and methylation (HDACs, DNMTs)

essential roles of cytosine methylation in mammals

gene expression, chromosomal stability, cell differentiation, imprinting, X-inactivation, carcinogenesis, aging

differentiated cells become

more restricted in their potential

stages of nuclear transfer

nucleus is removed from an egg 

replaced by a nucleus from a donor cell

who is dolly?

first mammal cloned from an adult cell by somatic cell nuclear transfer

out of 277 implants only she survived to birth

gave birth to 6 lambs herself, but died at age 6 to lung disease

nuclear equivalence

differentiated cells maintain the potential to generate an entire organism

induced pluripotent stem cells (iPS)

expression of 4 genes are sufficient to transform differentiated cells to “stem” cells

where are critical CpG sequences located?

in islands near promoters

DNA methylation roles in mammalian development

maintenance and inheritance of tissue-specific gene expression

inhibition of transposone (and other repetitive sequences) gene expression

genomic (parental) imprinting

inhibition of transposone gene expression

prevents transposition

inhibits DNA recombination between repetitive sequences

lower probability of genome rearrangements

genomic (parental) imprinting

inactivation by methylation of a gene on one of a pair of homologous chromosomes

relatively uncommon but important feature of mammalian chromosomes

• evolved in a common ancestor to marsupials (pouched) and eutherian (placental) mammals over 150 million years ago with advent of life birth (viviparity) and is thought to have evolved with the placenta and nutrient transfer from mother to embryo

• no evidence for this in monotremes (egg-laying) mammals

• although this evolved in therian mammals before the marsupial-eutherian split, the mechanisms have continued to evolve in each lineage to produce differences between the two groups in terms of the number and regulation of these genes

• most of these genes are expressed in the placenta, but also in the brain

differential expression of genes depending on parental inheritance

imprints are epigenetic instructions laid down in the parental germ cells

paternally expressed imprinted genes tend to promote growth while it is suppressed by those genes that are maternally expressed. thus, paternally expressed genes enhance the extraction of nutrients from the mother during pregnancy, whereas the maternal genome seeks to limit it

imprinting anomalies are often manifested as developmental and neurological disorders when they occur during early development, and as cancer when altered later in life

genomic imprinting and epigenetic inheritance mechanisms

involved epigenetic modifications that are erased and then reset during the creation of eggs and sperm

maternal methyl deficient diets during pregnancy can alter the expression of imprinted genes in the offspring. thus, imprinted genes likely epigenetic targets for environmental interactions with the genome

genomic regions with different methylation statuses among multiple samples (cells, tissues, individuals) are regarded as possible functional regions involved in gene transcription regulation: differentially methylated regions (DMRs) and imprinting centres or imprinting control regions/elements (ICs, ICR/E)

epigenetic imprinting

unequal expression of the maternal and paternal alleles of a gene

marked with their gametic (parental) origin

results in parent of origin dependent monoallelic expression, i.e. maternal and paternal genomes not functionally equivalent

example of epigenetic imprinting

insulator model for control of gene expression at the H19 and Igf2 imprinted locus

maternal chromosome produced MAS and H19 but not IGF2

paternal chromosome produced MAS and IGF2 but not H19

insulator model for control of gene expression at the H19 and IGF2 imprinted locus

in humans these genes are differentially expressed on chromosome 11p15.5 depending on parent of origin

in between these two genes lies an imprint control region (ICR)

the ICR is not methylated on the maternal allele. this allows the CTCD protein to bind to the ICR and prevent enhancing factors from activating expression of the IGF2 gene because CTCF is blocking access. the H19 gene is active on this allele

conversely, the ICR is methylated on the paternal allele. this prevents the CTCF protein from binding to the ICR and allows the enhancing factors to activate expression of the IGF2 gene because CTCF is not blocking access. the h19 gene is inactive on this allele

life cycle of an imprint

methylation ones are laid down in the germ line of parent

in offspring, differential methylation is erased in germ cells at an early stage of their development, and germ-line specific methylation imprints in DMRs are re-established (reset) around the time of birth according to sex of new organism

in somatic cells, imprints are maintained and modified during development

phenotypic effects of imprints

in utero effects, postnatal effects, genetic disorders

in utero effects of imprints

insulin and insulin-like growth factors, placental growth

postnatal effects of imprints

movement disorders, lactation, brain development and function

genetic disorders of imprints

epimutations, Prader-Willi syndrome, Angelman syndrome

Prader-Willi syndrome

deletion or inactivation of genes on the paternally inherited chromosome 15 while the maternal copy, which may be of normal sequence, is imprinted and therefore silenced

Angelman syndrome

deletion or inactivation of genes on the maternally inherited chromosome 15 while the paternal copy, which may be of normal sequence, is imprinted and therefore silenced