Lecture 5 - Epigenetics

Question 1

Epigenetics:

Answer 1

Heritable modifcations of DNA or chromatin that doesn’t alter the primary nucleotide sequence

Question 2

4 main types of epigenetic regulations at the chromatin level:

Answer 2

1. DNA methylation
2. Histone Modifications
3. ATP-dependent chromatin remodelling
4. Long non-coding RNAs

NOTE: These processes don’t operate in isolation, but are co-ordinated to precisely regulate gene expression

Question 3

Hetereochromatin vs Euchromatin:

Answer 3

Heterochromatin:

• Condensed
• Transcriptionally silent
• High levels of DNA methylation
• Common in somatic cells

Euchromatin:

• Decondensed
• Actively Transcribed
• Lower levels of DNA methylation
• In the early embryo and ESCs, majority of chromatin is euchromatin

Question 4

4 main families of ATP-dependent chromatin remodelling complexes:

Answer 4

SWI/SNF
CHD
ISWI
INO80

Question 5

ATP-dependent chromatin remodelling complexes affect chromatin structure by:
[3 things]

Answer 5

Nucleosome sliding
Histone exchange
Histone eviction

Question 6

SWI/SNF family of ATP-dependent chromatin remodelling complexes:

Answer 6

Crucial for early development

Known as BAF complexes in mammals

Complex comprised of:

• ATPase subunit
• Core subunits that scaffold the complex on chromatin
• Lineage-specific subunits that affect target site selection

ESCs contain a unique BAF complex known as esBAF, regulates expression of pluripotency associated genes (Oct4, Sox2)

Question 7

Types of histone modifications:

Answer 7

Acetylation
Methylation
Ubiquitination
Sumoylation
Phosphorylation

Question 8

Histone Code:

Answer 8

Sum of all Post-translational modifications

Controlled by histone writers (e.g. HATs) and histone erases (e.g. HDACs)

Question 9

Acetylation of histone Lysine residues is associated with euchromatin/heterochromatin as it strengthens/weakens the charge attraction between histones and DNA, serving to decondense chromatin and facilitate gene transcription

Answer 9

Acetylation of histone Lysine residues is associated with EUCHROMATIN as it WEAKENS the charge attraction between histones and DNA, serving to decondense chromatin and facilitate gene transcription

Question 10

HATs activate gene expression via acetylation

TRUE OR FALSE

Answer 10

TRUE

Question 11

HDACs activate gene expression via deacetylation

TRUE OR FALSE

Answer 11

FALSE

HDACs silence genes via deacetylation

Question 12

2 types of HATs

Answer 12

Type A - Nucleus, chromatin remodellers

Type B - Cytoplasm, acetylates histones prior to nucleosome assembly

Question 13

4 classes of HDACs

Answer 13

I, II, III, IV

Question 14

Histone Methylation:

Answer 14

More complicated than acetylation, can be either activating or repressing depending on location of methylated residue.

Methylation of Lys20 of H4 is important to ensure genome integrity via DNA damage repair, DNA replication, and chromatin compaction.

Unlike acetylation, doesn’t affect histone charge, instead creates docking sites for other proteins.

Chromo-domains on recruited proteins recognise methylated Lys residues

The hallmark of transcriptionally active genes is H3K4me3 in the promoter region and H3K36me3 across the gene body.

H3K27me3 is central for gene repression

Question 15

Bivalent Domains:

Answer 15

Function to hold the promoters of key developmental genes in a state of minimal expression that is poised for full activation (following removal of H3K27me3) or definitive repression (following removal of H3K4me3)

Question 16

H2AK119Ub1 mark has been shown to repress gene expression

TRUE OR FALSE

Answer 16

TRUE
Mono-ubiquitination of H2A is a major modification in mammalian cells
Has been mapped to Lys119

Question 17

The Polycomb system:

Answer 17

The Polycomb system is an important gene regulatory system that negatively regulates gene expression during development and in ESCs.

Polycomb Group (PcG) proteins belong to 2 multiprotein complexes:

• Polycomb Repressive Complex (PRC1): E3 Ubiquitin Ligase -> H2AK119Ub1
• PRC2: Methyltransferase -> H3K27Me1/2/3

Mutations in PcG genes lead to developmental abnormalities.

Question 18

DNA methylation generally activates/represses gene expression.

Answer 18

DNA methylation generally REPRESSES gene expression.

Promotes binding of proteins that promote chromatin condensation, preventing access of transcription machinery.

Question 19

DNA is methylated on the Cytosine residue of CpG dinucleotides by BLANKS

Answer 19

DNA is methylated on the Cytosine residue of CpG dinucleotides by DNA methyltransferases

After fertilisation: DNA methylation erased in the embryo
After implantation: DNA methylation restored to high levels

Question 20

There are 3 DNA methyltransferases (DNMTs) in mammals:

Answer 20

DNMT1: Maintains DNA methylation, strong affinity for hemi-methylated sites implicates DNMT1 in epigenetic inheritence and DNA replication
DNMT3A: Important for de novo methylation
DNMT3B : Important for de novo methylation

Deletion of DNMT1 -> Cell Death (In human ESCs)
Deletion of DNMT3A/B -> Doesn’t Kill Cell (In human ESCs)

Question 21

DNA Methylation and Genomic Imprinting:

Answer 21

Small fraction of genes undergo imprinting, most frequently arranged in clusters which are co-regulated.

Most clusters feature a CpG rich differentially methylated imprinting control region (ICR)

ICR methylation occurs in gametes, but is maintained post-fertilisation

ICR methylation normally leads to repression of the imprinted cluster

Imprinted clusters contain several protein-coding genes and at least one lncRNA gene

Clusters are divided into 2 types:

• ICR methylated during oogenesis on maternally inherited chromosome
• ICR methylated during spermatogenesis on paternally inherited chromosome

Question 22

BLANK maintains genomic imprinting via strong affintiy for hemi-methylated DNA

Answer 22

DNMT1 maintains genomic imprinting via strong affintiy for hemi-methylated DNA