Nicki Jariwala
Princy Shah
Case Study 1
Part 1:
https://learn.genetics.utah.edu/content/epigenetics/inheritance/
Epigenetics is changes in gene expression that is not related to changes in the DNA itself, but rather the regulation of the genes. In most cases, these changes are not passed to the offspring because the “tags” are reprogrammed so that the new cells can specialize into anything they need to be. However, a small number of cells remain tagged with the modification, and they can carry on onto the offspring.
Info from lecture
One mechanism of epigenetics is the chemical modification of histone tails, which is also known as histone code. One way this can occur is through histone acetylation. Histone acetylation (or deacetylation) is facilitated by HATs (or HDACS). Acetylation of lysine residues at the histone tail gets rid of the positive charge on the amino acid residue, which decreases the attraction it has with negative DNA, loosening the chromatin material and creating easy access to DNA. The acetyl then attracts TAFs (TATA binding Protein Associated Factors) and the TAFs bring in the promoter and transcription factors, and then transcription begins. In deacetylation of histones, the acetyls groups are removed by HDACs, which tightens the chromatin due to the restored attraction between positive lysine and negative DNA. The second mechanism of epigenetics is the methylation of DNA, which is also known as 5’-CpG-3’. There are CG sequences found outside the gene before the promoter regions of DNA. These regions can be methylated by methyltransferases, which cause the methylated group to protrude into major grooves of the DNA. This, in turn, interferes with transcription factors and RNA polymerase and recruits repressor complexes that prevent acetylation of Lysine histones. This increased methylation hence turns transcription off. The third mechanism of epigenetics is post-transcriptional controlled gene expression. After the mRNA is made, the mRNA can still be destroyed to prevent protein synthesis. microRNAs have specificity to particular genes that need to turn off or on. The microRNAs first bind to Argonaute family proteins. The microRNAs then guide the Argonaute to the mRNA substrate and form stable complexes with the mRNA. Argonaute has an endonuclease component, which then can cleave the mRNA in the center. This forms two mRNA segments, one with the 5’ cap and the other with the 3’ poly-A tail, but both are needed to protect the mRNA, so without one, the mRNA can easily be degraded by cell Rnases. Once the mRNA is degraded, protein synthesis cannot occur.
Info from lecture
The mechanism that most likely played a role in the development of Wiggle’s cancer is the chemical modification of the histones. Histone deacetylases inhibitors (HDACi) were suggested as the treatment for Wiggle’s lymphoma. Histones are proteins that pack the DNA close together due to the positive charge on the histones attracting to the negative charge of the DNA. Histone deacetylases (HDAC) remove the acetyl group (that were added to remove the positive charge on the histone to uncoil the chromatin) from the amino acid lysine on the histone tails, making it positive again. This causes the DNA and histones to pack together again because of the attraction of the opposite charges. Since the DNA and histones are tightly bound, transcription factors can’t get in, preventing transcription of particular genes. This can also cause cancer because genes that inhibit the cell cycle regulation and induce apoptosis are turned off, which allows the cancer cells to grow unchecked. However, if HDACi are given, these genes can be turned back on and cancer cells can stop proliferating.
http://www.biochemistry.ucla.edu/Faculty/Feigon/153bh/2003/Philip_Rosen/mol_master.html
A nucleosome core particle contains the histone octamer and a small, 145 base pair segment of DNA wrapped around it. The nucleosome core packs DNA, which reduces the length from 680 Å to 110 Å.
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/histone-code
The histone code hypothesis says that the chemical modifications to histone proteins regulate DNA transcription. DNA wraps around histone octamers to form nucleosomes, which then form chromatin. Chromatin is a complex that includes all of the DNA and its histones. The histones are usually small and basic, meaning they have a positive charge (examples are Arg and Lys). Histones are globular with an N-terminus, which is also known as the tail. These tails protrude from the nucleosome and are flexible. These tails can be modified in many different ways to regulate gene expression. The main four examples of different ways to modify histones include histone acetylation, histone methylation, histone phosphorylation, and histone ubiquitination.
Info from lecture
The histone code hypothesis relates to oncogenesis because cancer cells are a proliferation of cells that have cell death mechanisms such as apoptosis turned off. This allows the cancer to continue to grow with no checks. One way this happens is the deacetylation of the gene that controls apoptosis. If the gene is deacetylated, then it isn’t transcribed. This means that any damaged cells will not die and continue to harm the body.
Part 2:
https://www.genecards.org/cgi-bin/carddisp.pl?gene=CDKN1A
https://www.genecards.org/cgi-bin/carddisp.pl?gene=APAF1
The RNA polymerase that most likely transcribes p21 and Apaf-1 is RNA polymerase two because both genes code for proteins. p21 is a cyclin-dependent kinase inhibitor protein and Apaf-1 is an activator that initiates apoptosis. Since both are regulatory proteins, the genes would be transcribed by RNA polymerase two.
https://www.sciencedirect.com/topics/neuroscience/rna-polymerase-ii
One of the the three characteristics of an RNA polymerase ii core promoter are regions of DNA near the transcription start site (often these are AT-rich sequences called TATA boxes). There are also regions that bind to transcription factors, such as the TFIID recognition element, and downstream promoter and core elements.
http://www.csun.edu/~cmalone/pdf562/Chapter24.pdf
RNA polymerase ii transcribed gene enhancers are also short sequences, such as the CAAT sequence or GT box. These are acted upon by activators. The short sequences are spread out throughout the region before the promoter sequence. These upstream elements tend to increase transcription when acted upon by an activator.
Info from lectures
Histone acetylase transferases, or HATs, acetylate the lysine residues on the histone tails in the nucleosome core of the nucleosome. This neutralizes the positive charge of the lysine, which lessens the attraction to negative DNA and has an overall loosening effect on the DNA. The acetyls on the histone tails then attracts the TATA-Binding Associated factors, which help bring in TATA binding proteins and other transcription factors. This allows transcription to begin. In the case of the lymphoma, the opposite occurred. The HDAC removes the acetyls from the lysines, which increases the attraction between the histones and the negative DNA, preventing the transcription of essential cell cycle regulatory genes, such as p21 and Apaf-1.
Part 3:
Xyzi is more effective at inhibiting HDAC activity. There is lower HDAC activity associated with the Xyzi treatment than there is with the QB treatment.
At the same concentration (13 μM), there was much less HDAC activity associated with the Xyzi. Less HDAC activity suggests that there are more cells transcribing the important cell cycle proteins, p21 and Apaf-1, which are also associated with cell death. This indicates that more cancer cells are destroyed when compared to QB. This also means that a lower dose of the Xyzi will kill the same amount of cells as a higher dose of QB.
https://www.genecards.org/cgi-bin/carddisp.pl?gene=CDKN1A
P21 is a cyclin-dependent kinase inhibitor. The protein product of this gene plays a very important role in DNA replication and DNA repair in the S phase of the cell cycle. It also works with a group of enzymes called caspases, which helps initiate and control apoptosis. Overall, p21 is incredibly important in the regulation of the cell cycle and the suppression of cancer cells.
https://www.sepmag.eu/blog/northern-blot-protocol
https://bio-protocol.org/e1077#div_message
https://ocw.mit.edu/courses/biology/7-16-experimental-molecular-biology-biotechnology-ii-spring-2005/labs/rt_pcr_2step.pdf
In order to determine if there is a difference in p21 expression when the cells are treated with HDAC inhibitors Xyz1 and QB, a northern blot will be performed. Northern blots help show the relative amounts of a target transcription product, which in this case is the p21 mRNA. In this experiment, there will be four experimental groups in petri dishes that each contain a small amount of cancer cells. Each group will be treated with Apaf-1 inhibitor so there is no premature apoptosis when the groups are treated with the HDACi. The first group, which will be the negative control, will contain non-cancerous cells from somewhere else in the body. In these cells, the p21 gene should be fully expressed. This is to ensure that the northern blotting procedure works properly. If it does, there should be no tags on that lane. The second group is the positive control. It is composed of cancer cells but without any drug treatments. This gives a basis of comparison to show the “normal” expression of p21 mRNA without any drugs. The third group will have Xyzi in a concentration of 13 μM and the last will have QB in a concentration of 13 μM. Each group will be incubated for 30 minutes before the northern blot is performed. In general, the way a northern blot works is that the cell membranes are lysed and all the mRNA in the cell is extracted. Then, the mRNA is loaded onto an agarose gel and gel electrophoresis is performed. In gel electrophoresis, the RNA is loaded onto an agarose gel and the gel is placed in a chamber with a positive and negative electrode. Because RNA is negatively charged, it is loaded on the negative end of the gel and migrates toward the positive end. As it moves through the gel, it is separated by size. Then, the RNA (which is dyed as well) is transferred onto a membrane. The membrane is then treated with a cDNA probe with a tag on it. The presence and amount of this tag help determine if the gene in question, in this case p21, was transcribed and how much expression there was. If Xyzi is the more effective HDACi, then there will be increased expression of p21 RNA in the Xyzi treatment than in the QB treatment.
Northern Blot Protocol:
Incubate cells in petri dishes with the appropriate medium. In this case, we will incubate for 30 minutes, like in the first experiment.
Extract RNA
Use PBS buffer and a lysing reagent such as TriZol.
Centrifuge and rinse with ethanol and water. This is usually done in multiple steps to ensure maximum possible RNA purity.
Create Probes
Use RT-PCR
Add reverse transcriptase, buffer, dNTPs and template in a test tube, mix and wait.
The resulting product is a cDNA probe. This can be tagged if needed.
Run gel electrophoresis
Load dyed mRNA from lysed cells into each lane of the gel.
Place gel in a chamber filled with buffer and with negative and positive electrodes. The mRNA will be in the negative end of the chamber and migrate toward the positive end because mRNA is negatively charged.
The mRNA will be separated by size.
Transfer onto membrane
Stain gel.
Rinse gel and place on membrane.
Add tagged probes to membranes. If the tags are UV tags such as digoxigenin, place the membrane under UV light to visualise the p21 mRNA.
Predicted results if the hypothesis is supported. If the hypothesis is supported, there will be more mRNA in the Xyzi lane than in the QB lane.
Part 4:
https://canna-pet.com/lymphoma-in-dogs/
A dog of any age can be diagnosed with lymphoma, but the median age is usually between 6-9 years (middle aged dogs). The life expectancy of a golden retriever is about 10-12 years. This means that the dog has anywhere from 1 to 6 years left to live.
How Long Do Golden Retrievers Live – Golden Retriever Lifespan Guide
The treatment of lymphoma is to give histone deacetylase inhibitor (HDACi). Normally, histone acetylase transferease acetylate the lysine on the histone tails to neutralize the positive charge, making it bind to the negative DNA less and allow transcription factors to come in for transcription to occur. However, in those with lymphoma, the opposite occurs. Histone deacetylases (HDAC) come in and remove the aceylases from the lysines, causing the DNA and the histones to bind tightly. This decreases transcription of essential cell cycle regulatory genes (such as p21 and Apaf-1) since transcription factors cannot get in. If a patient with lymphoma was treated with HDACi though, this would inhibit the HDAC, so the lysines on the histone tails would remain acetylated, allowing for transcription of these cell cycle regulatory genes to occur and for cancer cell to stop proliferating.
http://clincancerres.aacrjournals.org/content/16/19/4832
In one study by Wittenburg et al., a HDACi was used in combination with another drug Doxorubicin in dogs. It was found that only 10% of the dogs had complete remission after the treatment, 14% had partial remission, 24% had no change, and 58% had the cancer worsen.
Persuasive Letter:
After doing research about the HDACi treatment, I think it may be best for Wiggles to not go through the treatment. The median age that dogs are usually diagnosed with lymphoma is 6-9 years old. Wiggles is already 10 years old. The life expectancy range for golden retrievers is between 10-12 years and so even without the cancer, he doesn’t have much time left and shouldn’t spend that last bit suffering through treatments and the side effects that come with it. In addition to that, the treatment itself doesn’t seem to be very effective. In one study by Wittenburg et al., it was found that when dogs were treated with a HDACi in combination with another drug Doxorubicin for cancer, only a 10% of the dogs at complete remission. The study also found that there were some adverse effects of the treatment on the dogs. It caused anorexia, lethargy, diarrhea, decreased appetite, and neutropenia. Although these effects may seem mild, it may not be pleasant for a dog that is already so old. Overall, I don’t think Wiggles should receive the treatment. Wiggles should just enjoy the last bit of time he has and not suffer through a treatment that most likely won’t even help him.