Histone Modification: DNA Methylation and Acetylation

RECAP: What is epigenetics?

A mechanism for stable maintenance of gene expression that involves marking DNA or its proteins

RECAP: What is the function of epigenetics?

To make genotypical identical cels be phenotypically distinct

RECAP: What are the other control mechanisms?

DNA methylation and chromatin modification

RECAP: What 3 processes control epigenetics?

• RNA interference

• DNA methylation

• Histone modification

RECAP: How is DNA packaged in most cells?

Packaged with histone to from nucleosome in '“beads on a string” structure

RECAP: Chromatin regulation

What does it involve?

How can further regulation be achieved?

Which histones are prone to be post tanslational

Involves: High-order conformational changes

• Such as relaxation or tightening of the thread of DNA-histone complex

Further regulation achieved by: Assembling promotor-enxchanes compaxes through long range DNA looping

• Which can be blocked by specific DNA sequence

Histones: Subject to promoter-enhancer complex. Such as =

• Methylation

• Acetylation

Histone Modifications

What

Results

How can it change throughout lifespan

Role

What: Key epigenetic regulators that control chromatin structure and gene transcription

Results: Impacts phenotypes of similar genotypes

How can it change throughout lifespan: Because the abundance and localisation is responsive to environmental stimuli

Role:

• DNA transcription

• DNA repair

• DNA replication

What are 2 diseases that is caused by insufficient histone modification?

• Coffin-Lowry syndrome

• Rubinstein

Coffin-Lowry Syndrome

Characteristics

Caused by

Inherited as

Males or females suffer more?

Characteristics:

• Mental retardation

• Abnormalities of head and face

Caused by: Mutation in the RSK 2 gene (histone phosphorylation)

Inherited as: X linked dominant

Males or females suffer more: Males

Rubinestein-Taybi Syndrome

Characteristics

Caused by

Characteristics:

• Short

• Intellectual disapbility

• Distinctive factial features, board thumbs and free toes

Caused by: Mutations in CREB-biniding protein (histone actylation)

What is DNA methylation?

The addition of methyl group to C-5 position of cytosine residue at CpG dinucleotides

*Human genome is not methylated uniformly, containing regions of methylated and unmethylated

In what kind of organisms does DNA methylation occur in?

Occur in cells of fungi, plants, non-vertebrates and vertebrates

What is the percentage of methylated DNA cytosine in vertebrates and plants?

• Vertebrates = 3-6%

• Plants = 30%

In what organism does DNA methylation DOES NOT occur?

Many insects and single-celled eukaryotes

What is the function of DNA methylation?

• Turns off genes

• Inhibit transcription

What is DNA methylation a therapeutic target?

The change from DNA methylation is heritable and reversible

When is DNA methylation determined?

During embryogenesis and is passed over to differentiating cells and tissue

Where does methylation occur?

Occur at:

• In cytosine in sequence context 5’CG3’ that is immediately followed by guanine-CpG dinucleotide

• Non-coding regions

• Interspersed repetitive elements

DOESN’t occur at:

• CpG islands of active genes

When does methylation typically occur?

After DNA replication

What is the mechanism?

• Methyl groups are transferred from S-adenosyl methionine (SAM)

• In a reaction catalysed by a DMNT

• Causes SAM to be converted into SAH (S-adenosyl homocysteine)

What are the 3 types of methylation processes

Maintenance methylation: Refers to the preservation of existing DNA methylation patterns after DNA replication

• Requires a hemi-methylated DNA substrate

De Novo methylation: Refers to the addition of methyl groups to unmethylated DNA sequences, producing new methylation patterns

Demethylation: Refers to the removal of methyl groups from DNA

Control of DNA methylation

By what

What are the 5 classes of them?

• Which 3 is the most important and their function

What controls DNA methylation: DNA methyltransferase (DNMTs)

5 classes:

• DNMT1

• DMNT2

• DMNT 3a and DMNT3b

• DMNT3L

Most important 3 and their functions:

• DNMT1: For maintenance methylase

• DMNT 3a and DMNT3b: For de novo DNA methylation

What kind of methylation process would occur in these situations =

• Pre-implantation

• After implantation of embryo and during carcinogenesis

• During replication

Pre-implantation: Genome undergoes demethylation

After implantation of embryo and during carcinogenesis: Novo methylation creates new methylation patterns

• By DMNT 3a and DMNT 3b

During replication: Maintenance methylation allows methylation patterns to be maintained

• DMNT1 methylates the hemimethylated DNA after strand synthesis

What are the 3 types of methylated bases in DNA?

• C5-methylcytosine(5-mc)

• N4-methylcytosine

• N6-methyladenine

What are the 5 effects of DNA methylation?

• Deactivaton of parasitic Transposons

• Somatic hyper-mutations at lg locus in B and T cells

• Embryonic development and growth

• Genomic imprinting

• X-chromosome inactivation

• Heterochromatin maintenance

• Tissue specific expression controls

• Silencing of repetitive elements

CpG islands

What

Methylated or unmethylated

Relationship between non-methylated regions and transcription

What occurs at promotor when methylation occurs

What occurs at non-promotor when methylation occurs

Amount in human genes

Present in what type of gene

What: Regions with high concentration of phosphate-linked cytosine-guanine pairs (cytosine nucleotide is followed by guanine nucleotide)

It can be: Both unmethylated or methylated

• Unmethylated at promotor regions

• Methylated at non-promotor regions

  • Correlates to transcriptional silencing due to the inhibition of transcription factor binding

Relationship between non-methylated regions and transcription:

• Allows binding of transcription factors

• Allowing transcription to occur

• HOWEVER there are cases where even though CpG island is unmethylated, the gene is still silent

What occurs at PROMOTOR when methylation occurs:

• Inhibition of TF binding

• Causes transcriptional silencing

What occurs at NON-PROMOTOR when methylation occurs

• Silence parasitic genetic elements

• Genomic stability

Amount in human genes: Half of all genes have CpG islands

Amount in genome: Low abundance

Present in:

• Housekeeping genes

• Genes with tissue-specific patterns of expression

Dysregulation of methylation process results in?

Many disorders like:

• Cancers (Deactivation of tumour suppressor genes

• Imprinting disorder:

  • BWS (Beckwith-Wiedemann syndrome)

  • PWS (Prader-Willi syndrome)

  • TNDM (Transient neonatal diabetes melllitus)

• Repeat-instability diseases

  • FRAXA (Fragile X syndrome)

  • Facioscapulohumeral muscular dystroph

• Defects of the methylation machinery

  • SLE (Systemic lupus erythemtosus)

  • ICF (Immunodeficiency, centromeric instability and facial abnormalities)

How can methylation imbalance contribute to tumor progression?

• Global HYPOmethylation

  • Activates oncogenes and genomic instability

• DNA HYPERmethylation

  • Inactivation of tumor suppressor genes

  • Inactivation of DNA repair genes

What can cause DNA hypermethylation? (risk factors)

Carcinogenes

• Because chronic exposure of human bronchial epithelial cells to tobacco-detrived carcinogens drives cells to tobacco-derived carcinogens

What can cause DNA HYPOmethylation? (risk factors)

Cigarette smoke

What are other 2 risk factors?

• Reactive oxygen species (ROS)

• Aging

What are the 5 ways DNA methylation can be detected?

• Sodium bisulfite conversion (SBC)

• SBC LC-MS-MS

• cDNA microarray

• Restriction landmark genomic sequencing

• CpG island microarray

Sodium bisulfite conversion (SBC)

Allows the highest degree of resolution of methylation status

Therefore what does it determine?

Involves?

How does it detect methylated regions?

What is done after

Therefore what does it determine: The positional CpG genotype

Involves: The chemical modification of DNA by bisulfite treatment

• Sodium bisulfite converts cytosine to uracil

How does it detect methylated regions: Methylated cytosine is resistant to this conversion

What is done after:

• PCR to amplify alleles

• Methylation-specific polymerase chain reaction is most useful