Epigenetics, Gene Regulation and Disease

A set of flashcards covering key vocabulary and concepts related to epigenetics and gene regulation.

What is epigenetics?

The study of changes in gene expression that do not involve alterations to the underlying DNA sequence

What is gene regulation?

The process of turning genes on and off to control the timing, location, and level of gene expression

What are cis-regulatory elements?

Specific DNA sequences that control the expression of nearby genes

What is a core promoter?

The region of DNA located directly upstream of the start site of transcription which is essential for RNA polymerase binding

What is transcription?

The process of synthesising RNA from a DNA template

What are transcription factors

Proteins that bind to specific DNA sequences to regulate gene transcription

What are enhancers?

DNA elements that increase the likelihood of transcription of a particular gene from a distance

What are silencers?

DNA elements that repress the transcription of a gene

What is chromatin?

A complex of DNA and protein found in eukaryotic cells which packages DNA into a compact, dense shape

What is a nucleosome?

The fundamental unit of chromatin, consisting of a segment of DNA wrapped around a core of histone proteins

What is RNA polymerase II

An enzyme that synthesises mRNA from a DNA template during the process of transcription

What are post-translational modifications?

Chemical modifications made to a protein after its translation, affecting the protein’s structure and function

What is genome plasticity?

The capacity of the genome to change and adapt in response to environmental influences

What is immunoprecipitation?

A technique used to isolate a specific antigen out of a complex mixture, such as a protein extract

What is mRNA?

Messenger RNA, which carries genetic information from DNA to the ribosome for protein synthesis.

What are exons?

Portions of a gene that are retained in the final mRNA product after splicing

What are introns?

Non-coding segments of a gene that are removed during RNA splicing

What can RNA polymerase II not differentiate?

Between coding and non-coding regions on its own

What are the four mechanisms of transcriptional control?

• DNA sequences

• DNA structures

• Proteins

• Chromatin structure

How is gene transcription regulated?

All eukaryotic genes are modulated independently through
multiple levels of regulation - extremely controlled process

How many protein coding genes do eukaryotic genomes have?

Between ~6,000 and ~30,000

What components regulate gene transcription?

• Specific sequences on DNA itself

• Proteins that bind to those sequences

What are the features of cis-regulatory elements?

• Specific sequences located in the regulatory region

• Commonly regions of non-coding DNA

• Influence expression of relatively close genes

How many CREs is one gene typically controlled by?

Multiple CREs located at varying distances from the coding region

What are the features of core promoters?

• ~35bp Upstream and downstream of the TSS

• Essential for the assembly of RNA polymerase II machinery

How is RNA polymerase II machinery assembled?

• Pre-initiation complex

• Recruitment of RNA polymerase II

• Accurate position and direction of transcription

What are the most common elements in a core promoter?

TATA box, initiator and downstream core promoter elements (DPE)

What are the features of TATA boxes?

• 1st eukaryotic core promoter element identified

• Most common element in eukaryotic genes (24%

• Located 25bp upstream of TSS

• Consensus sequence TATAAA (10%)

What is the function of the TATA box?

Functions as the ‘landing site’ for TATA binding protein

What are the features of proximal promoter elements?

• Cooperate with core promoter to regulate transcription

• ~250bp Upstream of the TSS

• Binding sites for trans-acting regulatory elements

What are the functions of trans-acting regulatory elements?

• Modify the affinity the core promoter for RNA polymerase II

• Essential for optimal transcription

What are the common elements of proximal promoters?

• GC box

• CAAT box

• Site specific Transcription Factor

What are the features of the GC box?

• ~110bps Upstream of TSS

• Promoters of constitutively expressed genes

• Consensus sequence GGGCGG

What are the features of the CCAAT box?

• ~60-100bps Upstream of TSS

• Promoters of genes that are non constitutively expressed

• Genes required in specific circumstances, eg. cell type

What are the features of distal promoter features?

• Several Kbp away from TSS

• Binding sites for regulatory proteins

• Activate or repress transcription

What are the two types of distal promoter features?

Enhancer and silencer

What are the features of enhancers?

• ~50-1500bp in length

• Contain multiple protein binding sites

• Increase significantly the activity of the core promoter

• Can be located up to 1Mbp away from the target gene

Where are enhancers located?

In exons, introns, untranslated regions, gene deserts

Where can enhancers be located?

Upstream or downstream of TSS

How many genes can enhancers regulate?

Several

How many enhancers can one gene be regulated by?

Several

What is the mechanism of action for an enhancer?

Enhancers are brought to the vicinity of the promoter by direct interaction and looping out of the intervening chromatin region

What are the features of silencers?

• Reduces rate or prevents gene transcription

• Mostly located upstream of the core promoter

• One silencer can regulate several genes

• One gene can be regulated by several silencers

What are the features of general transcription factors?

• General machinery required for all transcribed genes

• Bind to the core promoter region

What do general transcription factors make up?

Transcription pre-initiation complex

What are the functions of general transcription factors?

• Recruit further general transcription factors

• Position RNA polymerase II in correct location and orientation

How are general transcription factors assembled into pre-initiation complexes?

1. Transcription Factor IID

2. Transcription factor TFIIA binds to TFIID

3. Transcription factor II B is recruited

4. Transcription factor II F and RNA polymerase II assemble

5. Transcription factor II E and Transcription factor II H are recruited

6. TFIIH binding completes the initiation complex

7. TFIIH causes DNA unwind

8. RNA pol II is released from the promote

9. Transcription starts

What is the structure of a transcription factor?

• DNA binding domain

• Dimerization domain

• Regulatory domain

• Trans-activation domain

What is the primary function of a DNA binding domain?

To recognise cis-regulatory elements

What is the ultimate function of a trans-activation domain?

To activate transcription

What are transcription factors classified by?

Shape of their DNA binding domains

What are the different DNA binding domain shapes?

• Helix-turn-helix (HLH)

• Zinc finger

• Leucine zipper

What is the Estrogen Response Element?

A specific DNA sequence where the estrogen receptor (ER) binds which initiates the transcription of estrogen-responsive genes

What is the function of the dimerisation domain?

Many TFs bind DNA as homo or hetero dimers

What is the function of the ligand binding domain?

• Some TFs are activated through external signals

• Conformational change that either activates or inactivates TF

What is the function of the trans activation domain?

Binding site for other proteins

What is the structure of the estrogen receptor dimer?

• Activation region

• Ligand binding domain

• DNA binding

What are transcriptional activators?

Bind to proximal promoter elements and/or enhancers and lead to gene transcription

What are the mechanisms of transcriptional activators?

• Recruit general transcription factors to core promoter

• Recruit other regulatory proteins

What are the effects of transcriptional activators?

• Stabilize RNA polymer II to core promoter

• Trigger release of RNA polymer II to start transcription

• Alter chromatin structure

How many binding sites do enhancers have?

Multiple adjacent binding sites

What is an enhanceosome?

Multi-protein complexes

What does it mean that activators bind cooperatively?

Binding of one increases the binding affinity of another

What are transcriptional repressors?

Bind to silencers and reduce or prevent gene transcription

How do transcriptional repressors prevent the binding of RNA polymerase II?

• Overlap core promoter

• Alter chromatin structure

How do transcriptional repressors prevent the action of transcriptional activators?

• Overlap enhancers

• Disruption of enhanceosomes

What are transcriptional co-regulators?

Associate with other factors to regulate gene transcription

What do transcriptional co-regulators lack?

DNA binding domain

How do transcriptional co-regulators act?

Act via direct binding to transcription factors or in the context of multi-protein complexes

What is an example of indirect regulation?

Co-activators and/or co-repressors

How do co-regulators act as an additional layer of transcriptional control?

Induce highly specific expression of certain genes depending on cell type and developmental stage

Where do co-regulators bind?

To transcriptional activators

What do co-regulators function as?

Multi-protein complex scaffold

What is an example of a coregulator?

Histone methyltransferases (HMT) and histone acetyltransferases (HAT)

What is the function of histone acetyltransferases?

Alter chromatin structure to form euchromatin

What is the function of histone methyltransferases?

Alter chromatin structure to form heterochromatin

What is the mediator formed by?

Multi-protein complex of about 30 proteins with subunits varying under certain conditions

What is the function of the mediator?

Acts as bridge between transcription factors

What is an example of a co-activator?

Histone acetyltransferase

What is an example of a co-repressor?

Histone methyltransferases

What does estrogen promote?

Proliferation of cells in the breast and uterus

Where does breast cancer most often originate from?

Cells lining the milk ducts or lobules of the breast

What is the structure of steroids?

Non-polar lipids formed of a ring system of three cyclohexanes and one cyclopentane

What is estradiol?

• Predominant estrogen during reproductive years.

• Highest estrogenic activity

What is estrone?

Predominant circulating estrogen during menopause

What is the structure of nuclear receptors?

• A/B - N terminal domain

• C - DNA binding domain

• D - Hinge region

• E - Ligand binding domain

• F - C terminal domain

What are the features of luminal A breast cancer?

• ER+/PR+/HER2-

• Low levels of protein Ki-67

• Grow slowly, lower grade, most common

What are the features of luminal B breast cancer?

• ER+/PR+/HER2±

• High levels of protein Ki-67

• Faster growing, higher grade, less common

What are the features of HER2-enriched breast cancer?

• ER-/PR+/HER2+

• High levels of protein Ki-67

• Faster growing, higher grade, less common

• Treated with herceptin

What are the features of triple negative breast cancer?

• ER-/PR-/HER2-

• High levels of protein Ki-67

• Faster growing, higher grade, more common

How does tamoxifen work?

1. Binds to estrogen receptor

2. Receptor does not acquire changed shape

3. Receptor cannot bind to co-oactivators

Why does tamoxifen work?

Tamoxifen is larger than estrogen, and forces the activation loop out into an inactive conformation, blocking the signal to grow

Why are tamoxifen metabolites the active molecules?

1. Tamoxifen is a prodrug with relatively low affinity for ER.

2. Metabolised in liver by specific cytochrome P450s into the active metabolite 4-hydroxytamoxifen (4-OHT).

3. 4-OHT has ~50x higher affinity for ER than tamoxifen

4. Tamoxifen has 7% affinity of estradiol for the ERα, 4-OHT has 178% affinity

Why can tamoxifen be a cause of uterine cancer?

Estrogen receptor in uterine endometrial cell is activated by tamoxifen leading to endometrial cell proliferation and increased cancer risk

What is RAGE?

Receptor for Advanced Gylcation Endproducts

What is the function of RAGE?

Inducer of inflammation, tumour proliferation, chemoresistance and metastasis

Where is RAGE elevated?

Endometrial cancer patients

What are SERMs?

Selective Estrogen Receptor Modulators

Why is RAGE elevated in cancer?

• Sustains proliferative signalling

• Causes tumour-promoting inflammation

• Activates invasion and metastasis

What is RAGE regulated by?

Estrogen receptor ligands in endometrium