W4 - The Epigenome

Question 1

What is pharmacoepigentics?

Answer 1

1) Epigenetic regulation of genes: Involved in the absorption, distribution, metabolism and regulate of drugs

2) Epigenetic effects of drugs: Disease related.

There might also be drugs that cause epigenetic changes.

Question 2

Can we treat cancer by targeting epigenetic enzymes?

Answer 2

Global DNA methylation is altered in tumour cells
* Hypermethylation of tumour suppressor genes
* Hypomethylation of tumour activating genes

Epigenetic enzymes are often mutated in tumour cells.

Question 3

What are Pharmacoepigenetics drugs?

Answer 3

DNA Methyl Transferase Inhibitors
* 5-Azacytidine (Vidaza)
* Myelodysplastic syndrome

Histone Deacetylase Inhibitors
* Romidepsin (Istodax)
* Cutaneous T-cell lymphoma

Seven drugs FDA-approved so far

Question 4

What is the genome?

Answer 4

• The complete set of genetic material in a cell
• The DNA sequence that is present in a single full set of chromosomes

Question 5

What is the nucleosome?

Answer 5

• Histone proteins and DNA form the first level
  of packing – the nucleosome

DNA is associated with histones and pairs of histones form Histone October plus 147 bp of DNA. There is a Spacer DNA where histone 1 is attached to give higher-order structures fibre.

Question 6

What is the packing solution?

Answer 6

• Nucleosomes are wound up to form 30nm fibres
• Fibres are then wound up further with scaffold proteins to generate higher-order structures
• Chromosomes are the most densely packed form of genomic DNA

Question 7

What is Chromatin?

Answer 7

1) Euchromatin - gene-rich and transcriptionally active. Dispersed appearance (nucleosomes are far apart) with unique DNA sequence.

2) Heterochromatin - Gene-poor, less transcriptionally active, condensed appearance and repetitive DNA sequence

In an electron micrograph, the lighter areas are the euchromatin.

Question 8

What is the Epigenome?

Answer 8

• The sum of all the (heritable) changes in the genome that do not occur in the primary DNA sequence and that affect gene expression
• An epigenetic change results in “A change in
  phenotype but not in genotype”

Question 9

Why is the Epigenome useful?

Answer 9

• The epigenome is central to the regulation of gene expression
• DNA methylation and histone modification are mechanisms by which gene expression is regulated
• X-inactivation and imprinting are important epigenetic mechanisms for controlling expression from groups of genes
• Epigenetics is being used to identify novel drug targets

Question 10

What are the Epigenetic Mechanisms?

Answer 10

• DNA Methylation
• Histone modification
• X-inactivation
• Genomic Imprinting

Question 11

What is DNA Methylation?

Answer 11

• DNA methylation in humans is the addition of a methyl group in the 5’ position of a Cytosine
• This is catalysed by DNA methyltransferase enzymes: DNMT1, DNMT3a and DNMT3b
• It requires S-Adenosyl Methionine to provide the methyl group
• In differentiated cells it occurs in CpG dinucleotides
  The p stands for phosphates and if there are lots of these dinucleotides, they are referred to as CpG islands.

Question 12

How does methylation affect gene expression?

Answer 12

• In general, DNA Methylation turns transcription off by preventing the binding of transcription factors
• DNA methylation patterns change during development and are an important mechanism for controlling gene expression.

The CpG islands are often in the promoter region. When unmethylated, there is no marks and gene expression happens as normal. If methylated, transcription factors can’t bind and expression is repressed.

Question 13

What is histone modification?

Answer 13

• This is the addition of chemical groups to the proteins that make up the nucleosome
• There are a large number of known histone modifications (>100) and many are of unknown function
• Large range of enzymes catalyse modification

Question 14

What are the common modifications?

Answer 14

• Methylation
• Acetylation
• Phosphorylation
• Ubiquitination - cell signaling, apoptosis, protein processing, immune response, and DNA repair
• Many different amino acids can be modified and they may have 1-4 groups added
• This gives the large number of modifications
• Others are known but poorly understood

Question 15

What are histone modifiers?

Answer 15

Writers
* Histone Acetyltransferase - HAT1
* Histone Methyltransferase - EHMT1

Erasers
* Histone Deacetylase - HDAC1
* Histone Demethylase - KDM1

Readers
* Bromodomain and extra-terminal (BET) proteins – BRD2
* Chromodomain proteins – CBX1

The histone modification are named based on the histone, the amino acid and the actual modification.

Question 16

What are the roles of Histone modification?

Answer 16

• Histone acetylation at Lysine residues relaxes the chromatin structure, by reducing positive charge on the histones, and makes it accessible for transcription factors
• Histone methylation is more complex and can repress or activate transcription depending on where it occurs
• Histone modifications can occur concurrently (everything happens at the same time in the same nucleosome) and so their effects will interact

Question 17

What is X-inactivation?

Answer 17

• This is the inactivation of one of the two X chromosomes in every somatic cell in females
• This is needed as the Y chromosome has virtually no genes, so there is only one copy of each X chromosome gene in males (hemizygosity)
• X-inactivation ensures that every somatic cell in all humans has the same number of active copies of every gene

Xist gene is transcribed as long noncoding RNA from the X-inactivation centre and binds all over the X-chromosome. Histone acetylation is removed and DNA methylation occurs. This is then an inactive X-chromosome. It is heterochromatic - Barr body.

Tsix is derived by transcription in the opposite direction and antagonises Xist RNA to keep one X active.

Question 18

What is genome imprinting?

Answer 18

• Imprinting is the selective expression of genes related to the parental origin of the gene copy
• Every autosomal gene has one paternal and one maternal copy
• Imprinted genes tend to be found in clusters
• There are very few imprinted genes (~250)

It basically means either the maternal or paternal gene is repressed.

Question 19

What are imprinted genes?

Answer 19

Imprinting is mediated by imprinting control regions (ICRs)
* One copy is silenced by DNA methylation catalysed by DNMT3a and histone methylation leading to inactivation
* LncRNAs are essential to the process
* Imprinting patterns are reset during gamete formation

Question 20

What is DNA Demethylation?

Answer 20

Cytosine is catalysed with the DNA methyltransferase enzyme catalysing it. This is methylation.

Demethylation requires TET enzymes (Ten-Eleven Translocation enzymes) catalyses changes in that previously added methyl group to eventually result in it being removed.

The first change is to add an OH group.
Then it changes again with TET to a formyl group.
TET can act once more to make it a carboxyl group.

They can all be degraded at this point.
BUT there are active mechanisms that act on the formyl and the carboxyl groups catalysed by TDG (Thymine DNA Glycosylase), which removes those two based when they encounter them in the DNA.

BER (Base Excision Repair) is catalysed by TDG removing the cytosine, leaving behind the OH only. Then it will add back the cytosine that should be there.

It seems to be the case that 5’hydroxymethyl is an epigenetic signal and it does effect gene expression.