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AP609 - 2 > Epigentics > Flashcards

Epigentics Flashcards

1
Q

What are epigenetic marks ?

A

Changes to the genome which are inherited from one generation to the next which alter gene expression but which do not involve changes in the gene sequence

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2
Q

What is the principal methyl donation with explantion of the metabolic cycle involved?

A

Methyl groups for methylation reactions are derived from dietary methyl donors.

Within the Methionine metabolic cycle, Methionine is converted to S-Adenosyl Methionine (SAM; the principal methyl donor)

SAM is demethylated into S-Adenosylhomocysteine (SAH) which is hydrolyzed to homocysteine which is either catabolized or re-methylated to methionine

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3
Q

Explain how DNA becomes methylated?

A

Covalent addition of a methyl group to the 5′ position of the cytosine ring at CpG dinucleotide sites

DNA methyltransferase (DNMT) is an enzyme that catalyzes the transfer of a methyl group to the DNA molecule.

SAM is substrate, donating methyl group

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4
Q

How does methylation effect coding function?

A

Methylation changes the base itself but does not alter its coding function how Modulates gene expression

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5
Q

What catalyses Methylation?

A

DNA methyltransferase (DNMT) is an enzyme that catalyzes the transfer of a methyl group to the DNA molecule.

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6
Q

Describe the two types of DNA methyltransferases?

A

Type 1:
de novo (DNMT 3A and DNMT 3B)

can effectively methylate cytosines to 5-methylcytosines post-replicatively in un-methylated DNA

Type 2:
Maintenance DNMT 1 attaches a methyl group to hemi-methylated DNA during replication

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7
Q

What are the two types of chromatin

A

Euchromatin - decondensed and transcriptionally active

Heterochromatin - condensed and transcriptionally inactive

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8
Q

Describe the nucleosome sub unit?

A

consists of 146bp of DNA wrapped around an octamer of very basic histone proteins

Histone proteins Octamer consists of : two copies of H2A, H2B, H3, and H4

Each histone consist of globular C-terminal domain critical to nucleosome formation and a flexible N-terminal tail that extends from the nucleosome core

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9
Q

Explain the histone protein structure?

A

Each histone consist of globular C-terminal domain critical to nucleosome formation and a flexible N-terminal tail that extends from the nucleosome core

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10
Q

Explain the principle of Histone acetylation?

A

Acetylation is the process of adding an acetyl group (-COCH3) to the lysine amino acid residues on histones.

This modification can affect the structure and function of chromatin, Acetylation of histone lysine residues typically results in a more relaxed, “open” chromatin structure that allows for increased accessibility of the underlying DNA to the transcriptional machinery. This can lead to increased gene expression.

deacetylation of these residues typically results in a tighter, “closed” chromatin structure that can repress gene expression.

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11
Q

Which enzymes are responsible for Lysine Acetylation and what is thier effect?

A

Histone acetylation is mediated by enzymes called histone acetyltransferases (HATs), which add the acetyl group, and histone deacetylases (HDACs), which remove it.

The balance between HATs and HDACs determines the overall level of histone acetylation and thus the accessibility of DNA to the transcriptional machinery.

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12
Q

Why does Lysine Acetylation effect chomatin packaging?

A

DNA has negative charge, thus can be more tightly
packaged when histones are positively charged via
deacetylation

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13
Q

How does methylation recruit proteins involved in acetylation and what is the end effect?

A
  1. DNA methyl transferase 3 ( Dnmt3) methylates unmethylated DNA

2.Methylation of these CpG sites can recruit methyl-CpG binding proteins MeCP2 that bind to methylated DNA & recruits HDAC1 facilitating repression via histone deacetylation

  1. DNA replication
  2. MeCP2 recruits DNMT1 to facilitate methylation of hemimethylated DNA to become fully methylated
  3. Conformational change of MeCP2 recruits HDAC1

This leads to transcriptional repression by tighly packed (deacytelated) chromatin and DNA methylation

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14
Q

How many Methylation can Lysine receieve and what enzyme is this mediated by?

A
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15
Q

Explain Lysine methylasation and its importance?

A

There are three main types of lysine methylation: mono-methylation (one methyl group added), di-methylation (two methyl groups added) and tri-methylation (three methyl groups added). The specific type of methylation and the specific lysine residue that is methylated can lead to different effects on chromatin structure and gene expression.

Lysine methylation is mediated by enzymes called lysine methyltransferases (KMTs) which add methyl groups and lysine demethylases (KDMs) which remove them.

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16
Q

Explain Histone N-terminal tail role in modulating nucleosome structure and function.

A

The histone N-terminal tail is a small, unstructured region of the histone protein that protrudes from the nucleosome core.

This tail can be subject to various post-translational modifications, such as acetylation, methylation, and phosphorylation, which can alter the interactions between the histone tails and the DNA wrapped around the nucleosome.

These modifications can influence the accessibility of the DNA, thereby affecting gene expression and other chromatin-related processes.

Additionally, the histone N-terminal tail can interact with other proteins, such as chromatin remodeling complexes, to further modulate nucleosome structure and function.

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17
Q

Explain ubiquitination of histone proteins and thier potential roles?

A

Ubiquitination of histones can have different effects depending on the specific histone.

This process is mediated by enzymes called ubiquitin ligases, can be removed from histones by deubiquitinating enzymes

the ubiquitination of histone H2A on lysine 119 (H2A-K119ub) is associated with the formation of constitutive heterochromatin, which is tightly packed chromatin that is inaccessible to the transcriptional machinery.

On the other hand, ubiquitination of histone H2B on lysine 123 (H2B-K123ub) is associated with active gene expression.

Ubiquitination can also target specific residues on histone tails, which can lead to the recruitment of chromatin remodeling complexes and other proteins that can further alter the structure and function of chromatin.

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18
Q

explain histone phosphorylation

A

Histone phosphorylation is a process by which a phosphoryl group is covalently attached to specific residues on histones, typically on the histone N-terminal tail

phosphorylation are generally associated with active genes

Phosphorylation of histone H3 at serine 10 (H3S10ph) is associated with the formation of active chromatin, and it is often associated with the initiation of transcription. MOSTLY ACTIVATION

Phosphorylation of histone H3 at serine 28 (H3S28ph) is associated with the formation of repressed chromatin, and it is often associated with the repression of transcription.

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19
Q

What are the following histone modifications associated with in terms of gene expression?

methylation
acetylation
phosphorylation
ubiquitination

A

methylation - formation of compact, transcriptionally repressed chromatin. However can be gene acive associated

acetylation- formation of compact, transcriptionally repressed chromatin.

phosphorylation - Active genes expression

ubiquitination - Both active and repressed

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19
Q

What are the following histone modifications associated with in terms of gene expression?

methylation
acetylation
phosphorylation
ubiquitination

A

methylation - formation of compact, transcriptionally repressed chromatin. However can be gene acive associated

acetylation- formation of compact, transcriptionally repressed chromatin.

phosphorylation - Active genes expression

ubiquitination - Both active and repressed

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19
Q

What are the following histone modifications associated with in terms of gene expression?

methylation
acetylation
phosphorylation
ubiquitination

A

methylation - formation of compact, transcriptionally repressed chromatin. However can be gene acive associated

acetylation- formation of compact, transcriptionally repressed chromatin.

phosphorylation - Active genes expression

ubiquitination - Both active and repressed

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20
Q

What are CpG dinucleotides?

A

CpG dinucleotides are sequences of DNA that consist of a cytosine (C) base followed by a guanine (G) base. They are relatively rare in the genome, and are found in clusters called CpG islands.

CpG islands are typically found in the promoter regions of genes.

Methylation of CpG dinucleotides, a process called DNA methylation, is a key mechanism for regulating gene expression

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21
Q

How do Epigenetic marks determine tissue differentiation?

A

During early embryonic development all epigenetic marks are removed and as cells differentiate they are re-established.

Epigenetic mechanisms therefore shut down expression of specific genes in each cell type – once differentiated cells can’t be formed into a different cell type

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22
Q

Explain female X chromosome inactivation?

A

To avoid females getting a double dose of X chromosomes genes, one X chromosome is selected at random in each cell of the embryo and permanently switched off.

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23
Q

Give examples of disease caused by epigenetic marks

A

Cancer, insulin resistance ( type 2 diabetes) , cardiovascular diseases.

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24
Q

Explain Epigenetic Silencing of Tumour Suppressor and how this may lead to Cancer development?

A

Epigenetic silencing of tumor suppressor genes is a process by which certain genes that normally act to inhibit the development of cancer are turned off or suppressed.

Methylation of CpG dinucleotides in the promoter region of a tumor suppressor gene can prevent the binding of transcription factors, leading to the repression of transcription.

Histone modification, such as methylation, acetylation, and phosphorylation, can change the structure of chromatin and affect the accessibility of DNA to the transcriptional machinery

The methylation of the BRCA1 gene is common in breast cancer and can lead to the silencing of the gene and the progression of the disease.

p15INK4b prevents malignant
transformation (inhibits cyclin
dependent kinases)
No p15INK4b = Uncontrolled cell
division allowing
tumor formation

25
Q

Which Epigenetic therapies show anti-tumor effects?

A

DNA methyltransferase (DNMTi) and histone deacetylase (HDACi) inhibitors have FDA approval or under clinical trials

Histone acetyltransferase inhibitors (HATi) also show great promise

Cytidine Analogues - Cause hypomethylation when incorporated into DNA/RNA as methyl group cannot be added to the Nitrogen at position 5. these drugs are know to bind and inhibit DNA methyltransferase enzymes

26
Q

Explain the role of Cytidine Analogues?

A

Cause hypomethylation when incorporated into DNA/RNA as methyl group cannot be added to the Nitrogen at position 5.

Additionally, these drugs are know to bind and inhibit DNA methyltransferase enzymes

The hypomethylating agents (HMAs), decitabine and azacitidine, are valuable treatment Myelodysplastic syndrome & Acute myeloid leukemia

27
Q

Link Epigenetic mechanisms to Smoking and cardiovascular disease

A

Smoking associated with decreased methylation of the coagulation factor II receptor-like 3 gene (F2RL3)

Increased expression of the protease-activated receptor-4 (PAR4)

Induces platelet activation (aggregation)
Possible mechanism to explain smoking induced CVD

28
Q

How does ethnicity effect Epigenetics?

A

A large number of methylation sites differ by ethnic group.

British residents belonging to South Asian, African and African Caribbean race are at heightened risk of suffering from type 2 diabetes in comparison to those of European descent.

Methylation at 5 gene loci associated with ↑ diabetes risk

29
Q

Explain Genomic imprinting?

A

THE GENOMIC IMPRINTING PHENOMENON IS CHARACTERIZED BY MONO-ALLELIC EXPRESSION (I.E. ONE OF THE PARENTAL ALLELES IS SILENCED AND NOT EXPRESSED).

Paternally expressed imprinted genes tend to promote growth

Maternally expressed genes tend to supress growth

can make mutant alleles appear to behave as both dominant and recessive

30
Q

Explain the Epigentics of Prader-Willi and Angelman
Syndrome

A

Example of Genomic imprinting result of differential methylation and imprinting of two different genes in this region.

Prader-Willi and Angelman syndrome both caused by a deletion in the same region of chromosome 15 containing paternally expressed SNRPN and NDN and maternally expressed UBE3A

Maternally inherited deletion – Angelmans

Paternally inherited deletion – Prader-Willi

31
Q

Define Genome-wide vs Epigenome-wide vs Locus Specific Methylation?

A

Genome wide - across a range of unspecific CpGs
throughout the genome

Epigenome-wide - detailed analysis of specific
regions/CpGs across the genome

Gene/locus specific
Detailed analysis of specific regions of the genome

32
Q

Describe the Cytosine Extension Assay steps?

A

Cytosine Extension Assay assesses methylation.

1.DNA is treated with a restriction enzyme Hpall to generate a specific fragment of DNA containing the region of interest. (methylated DNA will not be cut)

  1. Overhang is extended using Taq polymerase & radiolabelled cytosine.
  2. More radioactive cytosine = lower methylation
    Compare between samples and groups of samples
33
Q

Cytosine Extension Assay advantages?

A

-Quantification of methylation across genome

-Allows overall estimation of methylation between groups

-Relatively cheap

34
Q

Cytosine Extension Assay disadvantages?

A

-Limited to CpGs in HpaII sites

-Don’t know where in the genome methylation differences are

-Time consuming – only few samples at same time

-Uses radiolabelled cytosine

35
Q

Describe Methylation Sensitive Restriction Enzyme Sequencing (MRE-Seq)?

A

The process of MRE-Seq starts with a sample of DNA, which is split in half then treated with a set of restriction enzymes that are sensitive to methylation Hpa II and one that is not e.g Msp-1.

Both samples are put through adapter ligation and NSG (Illumina) is run.

Samples are compared to each other, non methylated DNA will be longer than methylated as restriction enzyme HPA-II wouldn’t be able to cut these.

36
Q

Advantages of Methylation Sensitive Restriction Enzyme Sequencing MRE-Seq?

A

-Quantification of methylation across genome

-Allows overall estimation of methylation between groups

-Sequencing method should allow for detecting where changes are in the genome

37
Q

Disadvatages of Methylation Sensitive Restriction Enzyme Sequencing MRE-seq?

A

Limited to CpGs in HpaII or other restriction sites
Expensive

38
Q

Explain the steps of Methylated DNA Immunoprecipitation (MeDIP)

A
  1. DNA fragmented using sonication then denatured using heat?
  2. Immunoprecipitation with 5-methylcytidine Ab taking only methylated DNA
    2.5- sequence DNA using Illumina
  3. Microarray with raw input sample vs immunoprecipitated sample after labelling with dye (Cy5). Hybridised to CpG Island and Promoter arrays
  4. Compare Input from MeDIP fractions for
    difference in methylation
39
Q

Advantages of Methylated DNA Immunoprecipitation (MeDIP)?

A

-Better knowledge of regions of the genome with methylation change

-Good genomic coverage (depending on array or depth of sequencing

40
Q

Disadvantages of Methylated DNA Immunoprecipitation (MeDIP)?

A

Can’t resolve single CpG methylation changes (tends to only capture large-scale regional changes)

Magnitude of methylation change not accurately assessed

41
Q

what is a cpg site?

A

CpG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. “CpG” stands for cytosine and guanine separated by a phosphate.

CpG islands are useful markers for genes in organisms containing 5-methylcytosine in their genomes

42
Q

Explain Bisulfite Modification of DNA?

A

Process of converting non-methylated cytosines to uracils, which are then converted to thymines, during DNA amplification by PCR, whereas methylated cytosines are protected from bisulfite modification.

43
Q

What is Illumina Infinium® MethylationEPIC BeadChips?

A

Essentially DNA methylation arrays that allow 850,000 CpG sites.

Highly reproducible across technical and biological replicates

Precise methylation quantification

44
Q

Explain Methylation Specific Primers?

A

Primer pairs are designed to be “methylated-specific” by including sequences complementing only unconverted 5-methylcytosines, or, on the converse, “unmethylated-specific”

This can be combined with PCR for semi-quantative analysis basically telling us which is methylated and which isn’t as the primer can only amplify one or the other.

45
Q

Compare Methylation specific primers vs Bisulfite sequencing?

A

MSP is cheap quick and doesnt require specialist training but also doesn’t give quantitative data.

BS is expensive, time-consuming and requires high skill but does give quantitative data.

46
Q

Histone Modification Assays - nChIP

A

Chromatin immunoprecipitation (ChIP) assay assesses changes in chromatin structure

  1. Chromatin is digested by Mnase enzyme
  2. Fragment size is determined using SDS-gel
  3. Binding of an antibody specific for histone modifications in the human genome (methylation).
  4. Immunoprecipitation using A/G beads binding to the antibody
  5. Chromatin Immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) or qPRC / sequencing for analysis or

transcription factors, components of the transcription machinery, but needs additional cross linking step (using formaldehyde) to stabilize DNA-protein interaction.

47
Q

Explain DNA methylation?

A

DNA methylation is a process which involves methylating the DNA at cytosines that are located 5’ to guanosines within CpG dinucleotide regions. This process is mediated by three DNMTs that utilize SAM as a methyl donor group to transfer this moiety for covalent linkage to the cytosines

48
Q

Describe a nucleosome?

A

The basic molecular unit of DNA packaging is the nucleosome, a structure characterized by the wrapping of about 146bp of DNA around what is termed the “histone octamer” consisting of a tetramer of two histone H3, H4 dimers and a dimer of histone H2

49
Q

What is a histone code?

A

Post-translational modifications of key amino acid residues of the histones constitute what has been termed the “histone code” for gene expression regulation

50
Q

Explain the difference between closed and open chromatin?

A

“Closed chromatin” is characteristic of DNA regions that are transcriptionally silent vs. “open chromatin” which is characteristic of DNA regions where transcription is active

51
Q

Explain the effect of Acetylation compared to deacetylation on transcription?

A

Acetylation of key lysine residues by (HATs) usually specifies for transcriptionally active regions while deacetylation at these sites, mediated by HDACs, is usually associated with transcriptional repression

52
Q

What is the effect of Gene promoter hyper-methylation and hypo-acetylation?

A

well-known epigenetic mechanisms capable of triggering silencing of tumor suppressor genes

53
Q

What is the effect of global hypo-methylation and hyper-acetylation?

A

known to lead to the activation of proto-oncogenes

54
Q

What are the two commonly used techniques to analyze bisulfite-treated DNA ?

A

DNA sequencing - Methylation Sensitive Restriction Enzyme Sequencing (MRE-Seq)

methylation-sensitive primers (MSPs)

55
Q

How does nChIP prepare chromatin for anyalysis?

A

. nChIP uses micrococcal nuclease digestion to prepare the chromatin for analysis and so it allows for modifications of histones, such as methylation or acetylation, to be assessed more accurately than with formaldehyde fixation

nChIP does not allow for assessment of proteins with a weak binding affinity for DNA

56
Q

Explain the use of xCHIP?

A

xChIP uses formaldehyde to crosslink DNA and protein, followed by immunoprecipitation of DNA–protein complexes used to analyse transcription factors, components of the transcription machinery.

Recovered DNA can then be analyzed using PCR

57
Q

How does acetylation of lysine residues on histone tails influence chromosome packaging?

A

The acetyl groups result in a neutral charge on the lysine, relaxing the electrostatic attraction with negatively charged DNA resulting in loosely packaged DNA

58
Q

How does deacetylation of lysine residues on histone tails influence chromosome packaging?

A

The deacetylation result in a positive charge on the lysine, strengthening the electrostatic attraction with negatively charged DNA resulting more tightly packaged DNA

59
Q

Following DNA replication, what is role of themaintenance methyltransferase, DNMT1?

A

Copy the methylation pattern to the daughter strand, DNMT1 to facilitate methylation of hemimethylated DNA

60
Q

Global DNA hypomethylation is often observed in cancer. What might be the consequence of this?

A

Increased expression of genes with hypomethylated promotors

61
Q

You have obtained DNA from biopsies of CRC cancer tissueand normal mucosa. You are going to use a methylation-sensitive restriction enzyme assay todetermine the level of methylation in the samples. Compared to DNA from normal mucosa wouldyou expect the methylation sensitive restriction enzyme to digest the CRC DNA sample?

A

More as cancer shows less methylation than normal and Hpa-II restricts only methylated sites

AP609 - 2 (7 decks)

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