Questions & explanations
1. Compare the roles of CDK7 and CDK9 in CTD phosphorylation.
CDK7 and CDK9 are both kinases that phosphorylate the CTD, but they act at different stages of transcription. CDK7 is part of the TFIIH complex and phosphorylates Ser5 of the CTD during initiation. This modification helps the polymerase leave the promoter and recruit the capping enzyme. CDK9 is part of the P-TEFb complex and phosphorylates Ser2 during elongation. CDK9 also helps overcome pausing of the polymerase. While CDK7 works early to start transcription, CDK9 works later to keep transcription going efficiently. Both are essential: without CDK7, transcription cannot start properly; without CDK9, elongation is stalled. Their sequential action creates the CTD phosphorylation pattern that guides RNA processing.
2. Why do cancer cells often have high telomerase activity?
Cancer cells divide many more times than normal cells, so they need to maintain their telomere length to avoid senescence. Most cancer cells reactivate telomerase, the enzyme that adds telomeric repeats to chromosome ends. This allows them to keep their telomeres long and continue dividing indefinitely. In normal cells, telomerase is turned off in most adult tissues, which limits their lifespan. By upregulating telomerase, cancer cells bypass the normal telomere shortening that would stop growth. Some cancers use an alternative lengthening of telomeres (ALT) pathway instead. Targeting telomerase is a potential cancer therapy because it would cause telomere erosion in tumor cells.
3. Compare a riboswitch that turns off gene expression when bound to a metabolite versus one that turns it on. Give an example of each.
A riboswitch that turns off expression is like a brake: when the metabolite binds, the RNA folds to stop transcription or translation. For example, the TPP riboswitch in bacteria binds thiamine pyrophosphate and then forms a terminator, shutting down the thiamine synthesis genes. In contrast, a riboswitch that turns on expression is like an accelerator: binding the metabolite causes a shape change that allows gene expression. For instance, the glmS riboswitch in some bacteria binds glucosamine-6-phosphate and then cleaves itself, which actually activates a ribozyme to degrade the mRNA, but in other cases binding can expose the ribosome binding site to start translation.
4. Compare the role of DNA methylation in plants versus animals.
DNA methylation is found in both plants and animals, but it works a bit differently. In animals, methylation mostly happens on CG sequences (where a C is followed by a G) and is important for silencing genes and repetitive DNA. In plants, methylation can occur on CG, CHG, and CHH sequences (where H is any base except G). Plants use methylation to regulate genes in response to the environment, like during stress or development. For example, when a plant is attacked by a pest, it can change methylation on defense genes. Also, plants can pass epigenetic changes to their offspring more easily than animals. So, plants have a more flexible and diverse methylation system.
5. What are DNMT inhibitors?
DNMT inhibitors are drugs that block DNA methyltransferases, the enzymes that add methyl groups to DNA. By inhibiting these enzymes, the drugs reduce DNA methylation in cells. This can reactivate genes that were silenced by methylation, such as tumor suppressor genes in cancer. For example, azacitidine and decitabine are DNMT inhibitors used to treat certain blood cancers like myelodysplastic syndrome. They are taken up by cells and incorporated into DNA, trapping the DNMT enzymes and causing their degradation. This leads to lower methylation over time, allowing silenced genes to be expressed again. However, they can affect normal cells too, causing side effects.
6. Compare the roles of telomerase and shelterin in telomere maintenance.
Telomerase and shelterin both work on telomeres but have opposite jobs. Telomerase lengthens telomeres by adding repetitive DNA sequences to the ends, counteracting the shortening that happens during replication. Shelterin, on the other hand, protects the telomere ends and regulates access to telomerase. Shelterin prevents the telomere from being seen as damaged DNA and also controls how much telomerase can bind. Without shelterin, telomerase might add too many repeats or the ends might be wrongly repaired. Together, they keep telomeres at a healthy length and prevent genomic instability. In summary, telomerase adds DNA, while shelterin caps and guards the ends.
7. What are the challenges of using dCas9 epigenome editing in humans?
One challenge is delivering the dCas9 complex into the right cells in the body. Current methods use viruses, which can cause immune reactions or insert into the genome. Another challenge is off-target effects: dCas9 might bind to similar sequences elsewhere, causing unintended epigenetic changes. The effects might also be temporary if the epigenetic marks are not maintained. Additionally, long-term safety is unknown; changing the epigenome could have unpredictable consequences. Finally, ethical issues arise when editing the human germline. Despite these hurdles, dCas9 tools hold promise for treating diseases caused by epigenetic silencing, like some cancers.
8. What would happen if the CTD of RNA polymerase II were missing?
If the CTD were missing, RNA polymerase II would still be able to synthesize RNA, but it would not properly coordinate RNA processing. The enzyme would fail to recruit the capping enzyme, so the RNA would not get a 5' cap, making it unstable and untranslatable. Splicing would be inefficient because splicing factors need to bind the CTD. Polyadenylation and cleavage at the 3' end would also be disrupted. Additionally, the polymerase might not respond correctly to transcription elongation factors. Essentially, the cell would produce defective mRNAs that cannot be used to make proteins. The CTD is therefore critical for linking transcription to RNA maturation.
9. How does DNA methylation silence tumor suppressor genes in cancer?
DNA methylation adds a methyl group (a small chemical tag) to cytosine bases in CpG islands, which are regions rich in CG repeats near gene promoters. In normal cells, these CpG islands are usually unmethylated, allowing the gene to be active. In cancer cells, enzymes called DNA methyltransferases add methyl groups to these islands, especially in tumor suppressor genes. The methylated DNA attracts proteins that compact the chromatin, making the gene inaccessible to transcription factors. This prevents the gene from being expressed, so the tumor suppressor protein is not made. As a result, the cell loses its ability to control growth, contributing to cancer.
10. Why might combining DNMT and HDAC inhibitors be more effective than using one alone?
Combining both drugs can synergistically reactivate silenced genes because they target different epigenetic layers. DNA methylation and histone deacetylation often work together to silence genes. DNMT inhibitors reduce methylation, but if histones are still deacetylated, the gene may not fully activate. Adding an HDAC inhibitor increases acetylation, opening chromatin further. This dual action can lead to stronger re-expression of tumor suppressor genes. In studies, the combination has shown better anti-cancer effects in some cancers. However, it also increases side effects, so careful dosing is needed. The approach is still being tested in clinical trials.
11. How does phosphorylation of the CTD change as RNA polymerase II moves from the promoter to the end of a gene?
At the start of transcription, when RNA polymerase II is at the promoter, the CTD is mostly unphosphorylated. After initiation, the kinase CDK7 (part of TFIIH) phosphorylates Ser5 of the CTD repeats. This helps the polymerase escape the promoter and recruit the capping enzyme. As the polymerase moves into elongation, CDK9 (part of P-TEFb) phosphorylates Ser2. Ser2 phosphorylation increases and stays high through the gene body, recruiting splicing factors and the polyadenylation machinery. Near the end of the gene, Ser5 phosphorylation decreases. This phosphorylation cycle ensures that each step of transcription and RNA processing happens at the right time.
12. What are the main side effects of DNMT inhibitors and HDAC inhibitors?
DNMT inhibitors like azacitidine often cause low blood cell counts (neutropenia, thrombocytopenia), leading to infection or bleeding risk. They can also cause nausea, fatigue, and injection site reactions. HDAC inhibitors like vorinostat may cause fatigue, diarrhea, and heart rhythm changes (QT prolongation). Both drugs can affect normal cells that divide quickly, like those in bone marrow and gut. Side effects are usually manageable with supportive care. Because these drugs are not specific to cancer cells, they can also cause epigenetic changes in healthy cells, which may lead to long-term risks. Doctors monitor patients closely during treatment.