Chapter 3: Gene expression (book) Flashcards
What are single-nucleotide polymorphisms (SNPs)?
A single-nucleotide polymorphism is a substitution of a single nucleotide at a specific position in the genome that is present in a sufficiently large fraction of the population
True/false: The majority of cancer-related SNPs are located in the coding regions of the genome
False, the majority is located in non-coding regions of the genome. This can be intergenic (regions between genes) or intronic (sequence of introns). Only a small number are found in coding regions
How can gene expression be modulated?
Through the regulation of transcription, chromatin structure and post-transcriptional mechanisms
Where do transcription factors (TF) bind to?
Gene promoters
What are the different independent domains TF can bind to?
- DNA-binding domains
- helix-turn-helix motif
- leucine zipper motif
- helix-loop-helix motif
- zinc finger motif,
- Transcriptional activation domains
- Dimerization domains
- Ligand binding domains
How do transactivation domains function?
By binding to other components of the transcriptional apparatus in order to induce transcription by RNA polymerase
Some transcription factors work in pairs. How is this called and how does this work
This pair is called a dimer. These transcription factors require a dimerization domain which facilitates protein-protein interactions between the two molecules
How can the activity of TF be regulated?
- Synthesis in particular cell types only
- Covalent modification such as phosphorylation
- Ligand binding
- Cell localization (if dimeric)
- Exchange of partner proteins
Which families of TF make up the AP-1 complex that plays a role in carcinogenesis?
The For and Jun family
Give an example of where the AP-1 complex plays a role in carcinogenesis
As TPA is a tumor promoter and the AP-1 complex binds to the TPA response element, an association of AP-1 with carcinogenesis was implicated early after this property was characterized. The first members of AP-1 identified, c-Jun and c-Fos were able to transform normal cells in culture to cancer cells and are frequently overexpressen in tumor cells
True/false: Steroid hormones only pass through the cell membrane and bind to their particular intracellular receptor in the cytoplasm to activate the TF
False, some don’t need to bind to a receptor and can directly enter the nucleus. Most, however, do need an intracellular receptor.
The retinoid acid receptor (RAR) is a member of the steroid hormone receptor family and is important during differentiation. RA is derived from vitamin A. Does the RAR act as a transcriptional activator or repressor in the absence of RA?
Repressor RAR acts as a transcriptional repressor in absence of RA.
What type of cancers are characteristic of aberrant forms of RARs?
Leukemias
Fill in: Somatic mutations upstream of the … oncogene, which codes for a basic helix-loop-helix transcription factor, have been shown to create a super-enhancer that upregulates the expression of this oncogene in T-cell leukemia
TAL1
What are super-enhancers?
Super-enhancers are clusters of regulatory DNA elements that recruit transcriptional and chromatin-modifying proteins and may be transcribed into RNA. Many are associated with key genes that control cell state
In a study was shown that DNA response elements for the MYB transcription factor are created by acquired insertional mutation. What does MYB then recruit?
MYB recruits important transcriptional (e.g. GATA3, Pol II) and epigenetic (e.g. histone lysine acetylene) regulators for strong expression of the TAL1 oncogene.
Note: a similar phenotype is exhibited when a deletion occurs that causes the TAL1 gene coding sequence to come under the regulation of a ubiquitously expressed promoter of the STIL gene
What is chromatin immunoprecipitation (ChIP) sequencing?
Chromatin immunoprecipitation (ChIP)-sequencing is a technique that is used to identify protein-DNA interactions at precise DNA sequences. The typical protocol involves cross-linking proteins to DNA, sonicating DNA to produce small fragments, adding bead-bound antibodies to capture your protein of interest for immunoprecipitation, unlinking protein from DNA, purifying DNA, and sequencing.
In a study by Mansour et al in 2014, a MYB-specific antibody was used to produce genomic maps of MYB binding in cells from the Jor-kat T-cell leukemia cell line and MOLT-3 cells that harbored insertional mutations of interest. What did the analysis show?
Precise alignment of MYB binding with other transcriptional and epigenetic regulators such as GATA3 and Pol II. This data suggests the formation of a super-enhancer.
What is the length of DNA when fully extended?
Over a meter (which is why it needs to be packaged tightly)
DNA is tightly packed around a nucleosome. What pattern does a row of nucleosomes show?
“beands-on-a-string”
The histone core is an October of histones. Which 4 histones (that are duplicated) are present there?
H2A, H2B, H3 and H4
Each histone contains domains. Can you name all the domains?
A domain for:
- histone-histone and histone-DNA interactions
- NH2-terminal lysine-rich
- COOH-terminal “tail” domains (which can be post-translationally modified)
What does H1 do?
It is a linker histone that binds to DNA outside the core
What is a secondary level of organization of DNA? (nucleosomes are primary)
Formation of 30 nm fibers
What tertiary structure can the 30 nm fibers form?
Radial loops. For an overview, see this figure
True/false: Histone modifications and DNA methylation can both be acquired or inherited
True
What post-translational modifications can occur on histone proteins?
Acetylation, methylation, phosphorylation and ubiquination
Fill in: Acetylation acts as a … (1) signal for the recruitment or the repulsion of … (2) factors.
1: docking 2: chromatin-modifying
Fill in: Acetylation of histones neutralizes the … charge on lysine residues and relaxes chromatin folding
Positive
Which enzyme adds, and which enzyme removes acetyl groups
HATs: add acetyl groups HDACs: remove acetyl groups
Transcriptional activators often recruit HATs and other chromatin-remodeling enzymes to the promoter region. Can you give an example of this?
The retinoblastoma tumor suppressor protein exerts is effects, in part, by recruiting HDACs to specific gene promoters. Thus a signaling network seems to underlie chromatin modeling.
Where, in DNA methylation, is the methyl group added?
To position 5 of cytosine, at the 5’ end of a guanine nucleotide (CpG) (3-4% of all cytosines are methylated)
Methylcytosine can spontaneously dominate. What does this result to?
A C -> T transition
In what promoter regions of repressed genes are CpG islands mainly found?
X-chromosome inactivated genes, imprinted genes, and some tissue-specific (methylation is heritem)
Which three DNA methyltransverases (DNMTs) are known
- DNMT1: involved in the conversion of hemi-methylated DNA to fully methylated DNA during replication. This mechanism allows methylation patterns to be inheritable.
- DNMT3a and DNMT3b: involved in de novo methyltransferase activity (methylation of new sites)
Fill in: It has been suggested that the mechanism by which methylation results in silencing is by recruiting methyl-binding domain (MBD) proteins, which have been shown to interact with HDACs and chromatin-remodeling enzymes. What does this mean?
That epigenetic regulation of transcription includes cross-talk between methylation, chromatin-remodeling enzymes and histone modification
Fill in: Areas in the chromatin that are relaxed and indicative of active transcription are associated with high/low mutation density, whereas condensed regions indicative of gene repression are associated with regions of high/low mutation density
Areas in the chromatin that are relaxed and indicative of active transcription are associated with low mutation density, whereas condensed regions indicative of gene repression are associated with regions of high mutation density
A gene that codes for HAT (EP300) has been found to be mutated in a specific type of cancer. Which one?
Epithelial cancers
(Besides epithelial cancers) Other frequent mutations of histone-modifying genes have been identified in a specific type of cancer. Which one?
Lymphoma
What is acute promyelocytic leukemia characterized by?
A chromosomal translocation that produces a fusion protein called PML-RAR.
What does PML-RAR do?
Recruit HDAC to the promoter region of RA target genes and represses the expression of these genes. The lack of activation of RAR target genes causes the block of differentiation that characterizes the leukemia
Note: also other tumors have been associated with aberrant recruitment of HDACs
In carcinogenesis, is there a general hypermethylation or hypomethylation of promoters?
Hypermethylation (so genes are inactivated)
There is strong evidence for epigenetic regulation of the estrogen receptor. What type of cancer does this lead to?
Breast and other cancers (yes these are the book’s words)
How many % of breast cancer patients are estrogen receptor-a-negative
30% (and the loss of expression is due to DNA hypermethylation in 41% of the cases)
BRCA1 is often mutated in a recessive manner in inherited breast cancer. However a mutation of BRCA1 is rarely observed in non-inherited breast cancer. However, is does play a role… how?
Because it is hypermethylated!
Besides all the examples discussed, another example key target gene is the retinoblastoma (Rb) gene. What does this gene do?
It is the inhibitor of the cell cycle p16, INK4a, APC and pro-apoptotic death-associated protein kinase (DAPK)
What do sites of methylated DNA interact with (additional epigenetic regulatory proteins)
Histone-modifying enzymes, methyl cytosine-binding proteins and DNMTs (DNA methyltransferases)
True/false: Non-genotoxic carcinogens (agents that do not mutate genes) can also be epigenetic carcinogens by causing altered methylation
True! This is related to a tumor formation
Nutritional deficiencies (methionine, choline) seem to affect the cellular level of S-adenosylmethionine, an important methyl group donor. What does this suggest?
Perturbation of methylation can be produced through the diet
True/false: Epigenetic gene silencing is characteristic of normal stem cells and progenitor cells may be “locked in” during chronic injury and inflammation and contribute to carcinogenesis
True!
Overall, does the genome of a cancer cell have more or less methylation than a normal cell?
Less!!! About 20-60%. There is global hypomethylation (mainly in the coding regions of genes, transposable elements, and repetitive DNA sequences) at the same time as the hypermethylation of specific gene promoters
What mechanisms have been proposed as an explanation for the global hypomethylation?
activation of oncogenes and genes involved in metastasis, activation of transposable elements leading to genomic rearrangements , and alteration in the sequestering of transcription factors at repetitive repeats
Just a note
There are contrasting results in animal models regarding hypomethylation and cancer: some models show decreases in precancerous lesions, others show increases in tumors with increased hypomethylation
An example of a long non-coding RNA is IncRNA. Explain how this plays a role in cancer
Upon DNA damage, P53 binds to the promoter region of one IncRNA called lincRNA-p21. This IncRNA plays a role in tumor suppression by acting as a transcriptional repressor of genes known to be downregulated in the P53 response. HOTAIR and XIST are IncRNAs that guide chromatin-modifying factors to specific sites to facilitate epigenetic regulation. Misexpression of these IncRNAs has been linked to breast and female cancers, respectively
Why are microRNAs (miRNAs) powerful regulators for gene expression?
Because each miRNA can repress hundreds of gene targets post-transcriptionally (aka mRNAs)
Mature miRNAs require several steps of processing. Can you explain these steps?
- After they are transcribed by RNA polymerase II from intergenepic regions or from regions that code for introns, the primary transcript is processed by ribonucleases Drosha and DGCR8 in the nucleus.
- This processing produces pre-miRNAs, hairpin-shaped intermediates of 70-100 nucleotides.
- Exportin-5 transports pre-miRNAs into the cytoplasm where they are further processed by ribonuclease Dicer into a double-stranded miRNA.
- The strands separate, and a mature single-stranded molecule joins an RNA-induced silencing complex (RISC).
The repression of gene targets by miRNAs happens in one of two ways. Explain the two.
- Under the first scenario, the miRNA hybridizes perfectly to the 3’ untranslated region (UTR) of their target mRNA. The formation of this complex in the RISC leads to mRNA cleavage and subsequent degradation.
- Alternatively, miRNAs may bind to imperfect complementary sites in the 3’ UTR of their target mRNAs. The formation of this complex in the RISC blocks translation.
The net result for both scenarios is a decrease in the amount of the protein encoded by the gene from which the mRNA has been transcribed
How are miRNAs called that are oncogenic?
Oncomirs
Note: they can also act as tumor suppressors
True/false: mRNA profiles are more useful than miRN profiles fro cancer diagnosis and prognosis
False (miRNA are more useful than mRNA)
What is senescence?
Permanent growth arrest
What happens if a cell bypasses the cellular senescence, and the telomere become critically hosrt?
Apoptosis
What two domains does a telomerase consist of?
A human telomerase reverse transcriptase (hTERT) and a human telomerase RNA (hTR)
Why is it important that chromosome ends are distinguishable from DNA double-stranded breaks (by forming T-loops)?
If they were not, the DNA repair processes would produce chromosomal fusions and other aberrations in an attempt to repair the damage
Telomeres can be transcribed into a IncRNA that contains telomeric repeat-containing RNA (TERRA). That is, TERRA contains UUAGGG repeat sequences. What does TERRA play a role in? How does it do that?
TERRA plays a role in regulating the length of telomeres. It can form DNA-RNA hybrids with telomeric DNA, interact with heterochromatin proteins, and bind both telomerase RNA and TERT. There interactions stabilize telomere heterochromatin, inhibit telomerase activity and promote telomere shortening
How many % of tumors accomplish cell immortality and tumor growth by upregulating telomerase
90%
Fill in: Germline and recurrent somatic mutations in … (1) and other cancers have been identified in the TERT promoter. Also what do these mutations create? (2)
1) Melanoma 2) New transcription factor binding sites (Ets/TCF)
Which somatic mutations are seen in the TERT promoter that underlie melanoma?
C -> T, and CC -> TT tanden transitions that are characteristic of UV-induced carcinogenesis (hence melanoma)
Which transcription factor increases the expression of hTERT gene?
C-myc (I really don’t know if you’re supposed to know this, don’t think so)
How can there be acceleration of telomere shortening?
- Certain cells have few divisions, others have many divisions before they die
- Oxidative stress accelerates telomere shortening
- Unrepaired single-strand breaks (mechanism unclear)
- –> this suggests that telomeric DNA may act as a sensor for DNA Damage and may explain why there is great heterogeneity in the rate of telomeric shortening among cells.
What drugs can be used for epigenetic changes?
- DNA methylation inhibitors
- Histone deacetylases inhibitors
What are the two type of inhibitors that are used against DNA methylation?
There are two 5’-modified analogs of deoxcytidine, 5-azacytidine (5-azaC) and 5-aza-2’-deoxycytidine to target DNA methytransverases.
(remember DNA methylation occurs at position 5 on cytosine)
(also these have both been approved by the FDA)
How do DNA methylation inhibitors work?
These drugs are incorporated into DNA and/or RNA to covalently link with DNMTs and sequester its action such that there is significant demethylation after several rounds of replication. These drugs may result in DNA instability
What is a potential hindrance of DNA methylation inhibitors?
Aberrant methylation and gene repression return after treatment is stopped, dictating that administration of the drug must be prolonged
In what tumors do DNA methylation inhibitors work? (and which not?)
They show anti-leukemic activity in clinical trials, but were not successful in solid tumors. (this can be explained by the fact that leukemia patients often have mutations in genes involved in regulating DNA methylation patterns such as DNMT3A)
There are several classes of drugs that bind to the catalytic site of HDACs testes. Are these generally well tolerated?
Yes!! They can even be administered orally
True/false: HDACs inhibitors generally have lots of side effects because they also target normal cells
False! alteration of gene expression appears to be selective and have little/no effect on normal cells
What parameters need to be considered for the inhibition of telomerase as a cancer therapy?
- Effectiveness may depend on initial telomere length - Response may be slow, owing to the time needed for the telomeres of cancer cells to shorten enough to trigger senescence or opoptosis (long-term treatment) - Long-term treatment increases probability of drug resistance
Antisense oligonucleotides and ribozymes have been popular agents to target hTR. How do they work?
- Antisense oligonucleotides are complementary to part of the target RNA and hybridize by Watson-Crick base-pairing.
- Hybridization can inhibit function directly or trigger degradation by the recruitment of RNases.
- Hammer-head ribozymes contain antisense sequences for target recognition and an internal endonuclease activity that cleaves the target RNA.
Reverse transcriptase inhibitors against the catalytic domain of hTERT and nucleoside analogs have been investigated, but had problems. Which?
There were problems with solubility and bioavailability
One anti-telomerase drug candidate has entered Phase II clinical trials: imetylsat. What does this do?
Imetelsat, a modified 13-mer oligonucleotide, binds directly to the template region of the RNA component of hTR, which lies in the catalytic site of hTERT. Imetelstat binding to hTR results in direct, competitive inhibition of telomerase enzymatic activity. It is a promising pre-clinical study that may be effective agains pediatric brain tumors
