Cancers are Genetic Diseases; Regulation of Genes

Question 1

What is a gene? How many copies are inherited from our parents?

Answer 1

• The functional unit of inherited information (DNA); where in mammals, one gene usually encodes one protein.
  »> Bits of DNA that have function, typically encoding proteins
• We have two copies of every gene; one from our mother, one from our father (hence X, Y chromosomes)

Question 2

What is the difference between oncogenes and proto-oncogenes?

Answer 2

• Oncogenes are aberrant (mutated) versions of proto-oncogenes
• Proto-oncogenes act in healthy cells to promote proliferation and survival
• Inappropriately activated/over-expressed proto-oncogene = become oncogenes
  “Stuck accelerator pedal”

Question 3

Why are tumour suppressor genes referred to as ‘pro-apoptotic?’?

Answer 3

• They put the brakes on proliferation
• The products of tumour suppressor genes protect against cancer initiation and progression
• Cancer is more likely when tumour suppressors are inactivated or repressed (e.g. mutation)

Question 4

What is the process of DNA translation to form a protein

Answer 4

• Gene encodes a protein (sequence of DNA; polynucleotide)
• Transcription of gene to pre-mRNA via RNA polymerase
• Pre-RNA has exons (coding sequences) and introns (non-coding)
• The introns and spliced out, to form mRNA (exon 1, 2, 3 etc) (above steps in nucleus)
• Translation to protein (protein synthesis in cytoplasm); mRNA exported to cytoplasm, decoded, make protein)

Question 5

Why are introns spliced out from pre-RNA to form mRNA?

Answer 5

• Introns are non-coding sequences

- If translated; would become nonsense = potentially harmful

Question 6

What are the steps involved in gene regulation?

Answer 6

0 - Chromatin remodelling
1 - Transcriptional control
2 - RNA processing control
3 - RNA transport and localisation control
4 - Translation control
5 - mRNA degradation control
6 - Protein activity control

Question 7

What are meant by epigenetic changes? Can these be inherited?

Answer 7

• Changes that affect gene expression, without changing the DNA base sequence
• Alters accessibility of DNA for transcription
• Can be inherited by daughter cells

Question 8

What are some examples of epigenetic changes?

Answer 8

Chromatin remodelling (Step 0 in gene regulation):

• Modification of histone tails (acetylation vs. methylation) determine how tightly packed DNA is
• The more tightly packed DNA is, the less accessible it is for transcription

DNA modifications
- Cytosine methylation = promoter repression

Question 9

What is chromatin?

Answer 9

• Each cell contains 2 metres of DNA

- Thus DNA is coiled around histone proteins to form chromatin

Question 10

What is the ideal conformation for chromatin domains to be in for ease of transcription, and how does this occur? What is the converse?

Answer 10

• ‘Open’ chromatin domains; acetylation of histone tails
  »> Allows RNA polymerase II complex to assemble for transcription when ‘free’ of histones
• ‘Closed’ chromatin domains; the converse, through methylation of histone tails - DNA harder to transcribe;.

Question 11

What are HDAC inhibitors, and how do they work? Give examples.

Answer 11

• HDACs (histone deacetylases) remove acetyl groups from histones; thus chromatin domain won’t be “open”
• E.g. Vorinostat, Romidepsin, Panobinostat.

Question 12

What are the effects of HDAC inhibitors on human cancer?

Answer 12

• Remove acetyl groups from histones (chromatin/DNA)
• HDAC induces protein p21
• p21 inhibits Cyclin D-CDK4 (CDK4), which normally phosphorylates (and thus inhibits) the tumour suppressor RB1
• Thus results in cell cycle arrest (differentiation)
• Apoptosis
• Anti-angiogenesis

Question 13

What is the function of transcription factors? What do they do?

Answer 13

• Transcription factors positively and negatively regulation how much mRNA is made for each gene
• They are proteins that binds to DNA (DNA binding proteins), which recognise DNA sequences in promoters and enhancers
• These transcription factors, along with mediator proteins, cooperate to direct RNA polymerase II where to start transcribing the gene
  »> Sequence of DNA is recognised by transcription factors, binds, thus recruits transcription machinery (mediator proteins, RNA polymerase II)

Question 14

Does the amount of transcription of a gene affect the amount of protein expressed?

Answer 14

Yes; more mRNA, more protein.

Question 15

What can the amount of transcription be affected by?

Answer 15

• Changes in DNA structure e.g. epigenetic changes; histones (chromatin remodelling, #0), DNA methylation
• Changes in protein levels (e.g. transcription factors)
• Changes in DNA sequence (e.g. mutation of transcription factor binding sites; sequence not recognised, transcription factor does not bind, RNA polymerase II not recruited to start transcription)

Question 16

Describe p53’s actions as a transcription factor.

Answer 16

(Transcriptional Control, #1)

• Transcription factor, tetramer.
• Tumour suppressor
• Binds to target promoters to regulate transcription
• Detects when things are wrong and prevents proliferation (e.g. DNA damage; switching on/off genes accordingly, repair/apoptosis)

Question 17

Where does p53 bind? What does it trigger?

Answer 17

• p53 tetramers bind (in concert/along with other proteins) to specific DNA sequences in the promoters of many genes involved in proliferation, DNA repair, apoptosis.
• Upregulates other tumour suppressors e.g. P21 (cell cycle arrest; same one upregulated by HDAC inhibitors), BAX (apoptosis), P53R2 (DNA repair)
• Downregulates proto-oncogenes e.g. MYC, VEGF

Question 18

How can the tumour suppressor p53 become oncogenic?

Answer 18

Mutations in the TP53 gene, promoters of targets (where p53 binds).
= Oncogenic

Question 19

What is a common splicing mutation in the tumour suppressor, Rb?

Answer 19

• Retinoblastoma
• Normally inhibits transcription factors necessary for cell cycle progression
• But a single base mutation of G to C (guanine to cytosine) in intron 22 causes skipping of coding exon 22
• Mutation at intro-exon boundary stops exon 22 being recognised by splicing factors; exon 22 is skipped
• Thus mature mRNA is without exon 22; giving rise to non-functioning protein
• RB1 rendered ineffective

Question 20

What are MicroRNAs? (miRNAs, miRs)

Where do they bind?

Answer 20

• Short 21 nucleotide dsRNA molecules (don’t encode)
• Bind to (almost) complementary sequences in the 3’ UTRs of target mRNAs
• Inhibition of translation (protein synthesis); recognises and sticks to these sequences, switching off genes (inhibiting gene expression)
• mRNA degradation
• Important regulating molecule
  #5 mRNA degradation control

Question 21

How much of the human genome is regulated by miRs?

Answer 21

Greater than 50% of human genes; v. important regulator.

Question 22

What are oncomiRs/tumour suppressor miRs?

Answer 22

• OncomiRs; miRNAs that inhibit tumour suppressors

- Tumour suppressor miRs; miRNAs that inhibit oncogenes.

Question 23

Where can mutations occur that interfere with microRNA regulation? Give an example.

Answer 23

• Mutations in the miRs or the binding sites of target genes
  > Prevent regulation
  »> E.g. mutation in BRCA1 3’ UTR changes miR-320 binding site; BRCA1 expression disrupted.

Question 24

How is translational control (#4) achieved? How are these mutated in cancers? Give an example.

Answer 24

• Regulatory sequences in mRNA control translation rates
• Can be mutated in cancers:
  E.g. mutation in 5’ UTR of proto-oncogene MYC increases MYC synthesis (more MYC protein) in multiple myeloma

Question 25

What do oncogenic mutations of regulatory sequence in mRNA for translational control result in? Give an example.

Answer 25

• Increase the activity and abundance of ribosomes and translation factors
  E.g. via the MTORC pathway downstream of EGFR; increased ribosome recruitment (site of protein synthesis) leads to increased protein synthesis
  »> Greater translation rate = growth advantage

Question 26

What is the single base mutation that leads to multiple myeloma? Where does this occur?

Answer 26

• C to T mutation in the 5’ UTR of MYC gene
  (mutation occurs in the DNA)
  (transcribed to mRNA)
  (increased ribosome recruitment downstream)
• Increases ribosome recruitment, leads to increased synthesis of the oncoprotein MYC

Question 27

What post-translational modifications can influence whether a gene is expressed?

Answer 27

6 - Protein activity control

• Protein e.g. p53 function will be affected by changes in PTMs
• Phosphate, acetyl, SUMO peptide, ubiquitin, methyl groups all can affect the protein
• E.g. When p53 is ubiquiylated (by MDM2 ubiquitin ligase), it is targeted for degradation

Question 28

So, what levels is gene expression regulated at?

Answer 28

• Chromatin remodelling (changing structure of DNA/histones, #0)
• Transcription (mutations in transcription factor binding sites etc., #1)
• mRNA processing (splicing, stability; likelihood of degradation #2)
• microRNAs (#5)
• Translation (protein synthesis, #4)
• Post translational modifications (#6)