7) Epigenetics, chromatin & DNA methylation – Maike Stam
All cells in a body carry the same DNA but do not look the same → transcription factors and
chromatin modifications.
Heritable change in gene expression or genome function is determined by modifications of the DNA
or chromatin. Heritable can mean within one generation → mitosis (brain/liver). Heritable can also
mean through meiosis, to the next generation. Epigenetic traits can be stably transmitted through
many cell divisions but can potentially be reversed (some easier than others).
• If epigenetic regulation goes wrong: Disturbed development of an organism, cancer, (trans)
gene silencing, faster aging, cysts (somewhere in your belly with hair and teeth).
• Behaviour of a rat has a heritable effect on its progeny. If the mother licks and grooms the
pups in the proper manner → DNA methylation level at the glucocorticoid receptor gene
decreases → increased expression of the gene → pups grow up less stressed (they lick and
groom their own pups). When a rat does not lick and groom → DNA methylation stays →
higher stress level (they do not lick and groom their own pups).
DNA is present within chromatin in the cell nucleus. Nucleosome consists of histones + DNA + other
proteins. DNA is wrapped around histone proteins to form nucleosomes, packing affects DNA
accessibility and thereby gene expression.
Nucleosome consists of 2 H2A, H2B, H3, H4 and
DNA.
Accessible chromatin: There is more space
between the nucleosomes, DNA is easier to
reach by proteins.
Inaccessible chromatin: Densely packed, not
accessible for proteins.
• Euchromatin: Contains genes and can be active or inactive. Active genes have H3ac, H3K4me
and activator/trithorax proteins. Inactive genes have repressor/polycomb proteins and
H3K27me. Facultative heterochromatin is another name for inactive euchromatin.
o You can switch between the two states of euchromatin. This happens all the time
during cell differentiation.
• Heterochromatin: Consists of repetitive DNA sequences, transposons among others. Some
genes can be present in heterochromatin but they are not necessarily active. Inactive genes
have H3K9me together with DNA methylation.
, A transposon is a DNA sequence that can change
position in the genome. The majority of DNA
methylation is present in transposons or other
repetitive DNA. About 45% of the human (70% of
maize) genome consists of transposons.
Transposons threaten genome function and
stability (image kernels). Transposon activity has
to be inactivated to circumvent insertion
mutagenesis → DNA methylation.
Model systems with small genomes are often
used to study chromatin based and epigenetic
mechanisms. Advantages (they all have H3K4):
• S. cerevisiae: Fast life cycle, small genome, homologous recombination.
• S. pombe: Fast life cycle, small genome, homologous recombination, RNAi (silencing of
genes), H3K9me.
• C. elegans: Fast life cycle, small genome, cell lineage well known, H3K27me/polycomb
silencing, RNAi, H3K9me, multicellular.
• D. melanogaster: Fast life cycle, small genome, lots of mutants, H3K27me/polycomb
silencing, RNAi, H3K9me, multicellular, you can see morphological changes.
• A. thaliana: Relatively fast life cycle, small genome, lots of mutants, H3K27me/polycomb
silencing, RNAi, H3K9me, multicellular, DNA methylation.
Mutations that are lethal in organisms with large genomes are often viable in organisms with small
genomes.
Disadvantages model organisms:
• S. cerevisiae: Lacks DNA methylation, H3K9 methylation & H3K27 methylation, no RNAi,
unicellular.
• S. pombe: lacks DNA methylation & H3K27 methylation, unicellular.
• C. elegans: Lacks DNA methylation.
• D. melanogaster: Lacks DNA methylation.
• A. thaliana: Not as many transposable element as organisms with large genome (true for all
the organisms).
DNA methylation
Covalent DNA modification on the cytosines (5 methylcytosine,
5mC). Not GCs but CGs are the main target (due to recognition by
methyl transferases).
• 5-hydrocymethylcytosine (5hmC) is recently discovered.
Result of DNA methylation: Transcription levels go down (repressed
chromatin/transcriptional silencing). 5mC affects transcription by recruiting DNA methyl-binding
proteins (MBPs) and by blocking the binding of transcription factors. 5-methyl cytosine is present in
the major groove of the DNA. Transcription factors generally bind here, so they are blocked. Binding
of repressor proteins is also stimulated. As a result, RNA polymerase cannot initiate transcription.
Examples:
, • Inhibition of binding transcription factors.
• Methyl-binding proteins bind 5mC & recruit repressor proteins (H3K9 methyltransferase) to
repress transcription.
• DNA methyltransferases (DNMTs) are bound to histone deacetylases (HDAC) and histone
methyltransferases (HMTs), resulting in transcription inactivation.
Typical DNA methylation landscape:
• Transposable elements are methylated.
• CpG rich island are unmethylated (higher
concentrations of C’s followed by G’s).
• Coding regions have DNA methylation.
There are several methods to study DNA methylation:
• Specific regions
o DNA blot analysis (methylation sensitive restriction enzymes).
o PCR analyses of DNA digested with methylation sensitive enzymes
o Bisulfite sequencing (<1kb, all cytosines).
• Genome wide
o Immunolabeling with antibodies against 5-methyl C.
o IP using an 5mC antibody, combined with microarray or sequencing.
o Sequencing of DNA methylation enriched libraries.
o Genome wide bisulfite sequencing (BS-seq).
DNA blot analysis: DNA is cut by a restriction enzymes sensitive to methylation.
• Alu1: Cuts AGCT only if its unmethylated.
• EcoRI: Cuts GAATTC no matter if cytosine is methylated.
➔ Lane 1 is unmethylated (you only see small sizes so both Alu1 and EcoRi
cut), 2 is methylated (you see larger fragments so only the EcoRI cuts).
You can easily examine many samples but information is limited
because it depends on restriction enzymes.
Bisulphite sequencing: Unmethylated cytosines are changed into a uracil (C → U). If the C is
methylated it stays a C. After sequencing you can see which ones were methylated. You look at it in a
strand specific way. It is combined with PCR to examine small DNA fragments or high throughput
sequencing to examine the entire genome.
➔ You have to denature the strand → treat with
bisulphite → primers to amplify bottom OR
top strand (different tubes) → clone &
sequence. The two sequences are not the
same because the unmethylated C changed
into a U → A in the other strand (so G → A).
If the promotor of tumour suppressor gene gets DNA methylated, transcription goes down→
increasing chance of tumorigenesis. In breast cancer samples there is more 5mC. DNA methylation
can be used as a biomarker for particular diseases.
Immunoprecipitation: You use an antibody that recognises methyl groups → purify → whole
sample and the purified methylated sample with different fluorophores on a microarray → colour
