2. Cancer epigenomics

What are epigenetics?

Changes in the genome, often affecting gene expression, not due to changes in the DNA sequence.

Genetics vs Epigenetics example

• The dictionary itself is not changed, but your own interpretation can be added using the notes.

How can expression of genes be regulated?

• DNA methylation

• Histone modification

• miRNA

Genetic structure in our bodies

• Nucleus contains the DNA

• DNA is very long strand of information that has to fit in a very small space.

• Therefore the DNA is folded around certain proteins called histone proteins

• Those proteins stick together making it a very tightly packed DNA

DNA methylation and cancer

• During cancer the DNA methylation is changed

• Specifically, there is often hypermethylation, where certain genes are silenced such as tumor suppressor genes and DNA repair genes.

What is DNA methylation

• Adding of a methyl group to Cytosine

• Specifically the Cytosine that are next to a Guanine (CpG sites)

• This is done by DNA methyltransferases (DNMTs)

• High frequency of CpG sites (CpG islands)

Where do the methyl groups come from for DNA metylation

• Through a process known as one carbon metabolism

• Consists of two parts.

• Firstly: the folate pathway, folate is a vitamin

• Secondly: the methylation pathway.

  • Here methionine is converted to SAM which releases a methyl group that is picked up by an enzyme

Gene expression & DNA methylation

• When a gene is expressed in it transcribed into RNA and then translated into a protein.

• Gene transcription contains promoter regions and these often contain dense regions of CpG’s

• Hypermethylation of CpG islands in promoter regions prevents binding of transcription factors = silencing of gene expression

• DNA methylation can regulate gene expression.

Promoter regions and DNA methylation

• Promoter regions are regions before a gene that highlight where transcription should start

• These are often characterized by CpGs

• Therefore, the promoter region can be methylated if you want a gene to be silenced and not transcribed. Because if the methyl groups are there, the other enzymes that are needed for transcription cannot bind anymore.

Repetitive elements

• Sequences in the DNA that do not code for proteins

• These repetitive elements are methylated because they should be silenced and not transcribed as they do not code for a protein

• Methylation of these regions is very important for stability of DNA

• When there is not enough methyl groups, it is known as hypomethylation

Global DNA methylation

• DNA methylation can occur in the genes (gene-specific) or in the rest of the genome (global)

• A lack of methylation in global regions (e.g. repetitive elements) can result in genomic instability

• DNA methylation can regulate gene expression.

Meaning of low vs high methylation

• ‘Low methylation’ of promoter = expression

• ‘High methylation’ of promoter = hypermethylation = silencing

• Silencing of tumor suppressor genes may be a ‘hit’ for cancer development

Which genes can cause cancer when silenced?

• Tumor suppressor genes

• DNA repair genes (e.g. MGMT gene)

MGMT gene

Hypermethylation of MGMT promoter = silencing = mutation = cancer (epidriver)

DNA methylation and cancer

• Differs between global and gene-specific

How to measure DNA methylation

• Hard to do because there are many different cells that may be affected by DNA methylation differently. (e.g. breast cancer, there are different breast cells)

Genome-wide approaches of measuring DNA methylation

Can be done using array-based technology Illumina

• Infinium HumanMethylation450 BeadChip

• DNA methylation

• 500 ng DNA

• 12 samples

Gene-specific

• >450,000 CpG sites

• ~99% of RefSeq genes

What is chromatin?

• A complex of DNA and protein

• DNA wrapped around histone proteins

• 4 core histones (H2A, H2B, H3, H4)

• 1 linker histone (H1)

Histone modifications

• Modification on the tails of the histones (tails consist of amino acids) and affect chromatin structure (open/condensed)

• These modifications can be because of the addition of one of these groups:

  • Groups: methyl, acetyl, phosphate, ubiquitin

• Histone modifications may influence gene expression, DNA repair, cell cycle regulation, and genomic stability

• Aberrant histone modification patterns have been associated with many cancers

How to identify histone modification?

• Coding refers to residues and sites: e.g. lysine (K)

• Contains numbers to know where the groups are added

Why are histone modifications important?

• Determines if DNA is accessible (open or closed)

• Closed DNA is not accessible, and transcription cannot occur

• Open DNA is accessible, and transcription factors bind and induce gene expression.

Predicting the effect of histone modification

• Usually cannot be predicted

• However a few combinations and its effects are known:

  • Acetylation of lysine → transcriptional activation

  • Methylation of mono-, di- or trimethylation → repression and activation

How to read code: H3K27me3

• On histone number three

• Position 27 of the tail

• me 3 = three methyl groups there

What is miRNA?

• Non-coding RNA

• Small non-coding RNA fragments

• Regulatory function (regulation of gene expression)

• Aberrant expression in tumors

miRNA and cancer

• Post-transcriptional regulation of gene expression (stops messenger RNA from making proteins)

• Interaction with target mRNA

• Function:

  • Translational repression (protein)

  • Cleavage of mRNA

• Oncomirs function as oncogenes or tsp-genes

Folate vs folic acid

• Folate is natural form of vitamin B found in leafy greans or liver

• Folic acid is synthetic vitamin B, which is more stable.