Take Home Problem Set

Due Tuesday, December 3, 2019

The questions in this problem set refer to “A dominant-negative effect drives selection of TP53
missense mutations in myeloid malignancies” by Boettcher et al., Science 365, 599–604
(2019), and the supplemental material available on line.

https://science-sciencemag-org.libezp.lib.lsu.edu/content/365/6453/599

 These questions are worth 50 points (5 points each). The answers must be in your own words
and TYPED and STAPLED. If you use a reference outside of this research article, cite the
reference at the end your answer for that question.  Internet references are OK, but NOT
Wikipedia.  This problem set is due at the beginning of class December 3.  There are severe
penalties for late problem sets. 
The answers must be your own work: please see  http://saa.lsu.edu/Plagiarism.html and
http://saa.lsu.edu/collaboration
Some answers will require you to look up techniques in the supplemental figures and Experimental
Procedures section in the Supplemental Material and look up the vendor information about the
plasmid or technique.
There will be questions about this paper on the final exam.

Gain-of-function (GOF) vs. dominant-negative effect (DNE), that is the question. TP53 is the most
frequently mutated gene in human cancer. In mouse models, p53missense (mutant protein made)
tumors have a higher oncogenic potential than p53null (no protein made) tumors. Is this due to
new interactions with transcription factors in the missense mutation p53 that give new tumor-
promoting transcription activity (gain-of-function), or is it due to the missense mutation in one
allele impairing the function of the remaining wild-type allele (dominant-negative effect)?

1. A. Briefly describe the function of p53 in normal cells. Do not just quote the paper.

P53 is a transcription factor coded by the TP53 gene. The general function of this protein is
to stop the formation of tumors. It does this by preventing the damaged cell from continuing along
the cell cycle. A damaged cell can be a result of “DNA damage, oxidative stress, and oncogenic
hyperproliferation” (Boettcher et al.) First, p53 binds to DNA to stimulate the transcription, then
translation, of a p21 protein. This protein forms a complex with cell division-stimulated protein
(cdk2). This prevents the cell from moving forward in the cell cycle.

National Center for Biotechnology Information (US). Genes and Disease [Internet]. Bethesda
(MD): National Center for Biotechnology Information (US); 1998-. The p53 tumor suppressor
protein. Available from: https://www.ncbi.nlm.nih.gov/books/NBK22268/
 B. Name the gene and its associated protein that the authors used as a surrogate marker for
the function of p53.
Cyclin dependent kinase inhibitor 1A (CDKN1A) AKA p21

C. What is the cellular function of the protein in part B?

P21 binds to cyclin dependent kinase 2 or 4 to inhibit it. This results in less cyclin
dependent kinase activity as well as a halt in G1 growth. Its transcription is stimulated by
the binding of p53 proteins on CKDN1A gene.

“Chapter 32- Prostate.” Modern Surgical Pathology: 2-Volume Set, Expert Consult - Online & Print, by
Noel Weidner et al., Elsevier Health Sciences, 2009, pp. 1121–1180.

2. For the experiment in Fig S2, explain in your own words:

A. What is TP53 Q136fs?

TP53 Q136fs is a mutated version of a normal gene that codes for the p53 protein
(TP53). It lacks wild type p53 activity. In this article, researchers repaired this
mutant gene via CRISPR homology directed repair and then introduced missense
and null mutations to compare to wild type TP53.

B. What is 4D-Nucleofector?
This is a machine sold by Lonza and is used for nucleofection. Nucleofection is a
process to introduce genes directly into the cell nucleus. The 4D-Nucleofector
achieves this by having set voltages and reagents for a specific cell. Each cell type
will have different voltage values and reagents.

“Electroporation and NucleofectorTM Technology.” Lonza,

bioscience.lonza.com/lonza_bs/CH/en/nucleofector-technology.

C. What is Recombinant Cas9-3NLS?

NLS means nuclear localization signal. The Recombinant Cas9-3NLS is a version of the
CRISPR Cas9 system engineered to be more efficient in gene editing (high fidelity). This
variation of CRISPR Cas9 keeps the protein from cutting at other unintended locations
while staying just as efficient as other Cas9 nucleases.

Packer, H. (2019). Improve your genome editing with the Alt-R S.p. Cas9 Nuclease 3NLS and modified
crRNAs. [online] Idtdna.com. Available at:
https://www.idtdna.com/pages/education/decoded/article/improve-your-genome-
editing-with-the alt-r-s.p.-cas9-nuclease-3nls-and-modified-crrnas [Accessed 25 Nov. 2019].
Prediger, E. (2017). A recombinant Cas9 enzyme that drastically reduces CRISPR off-target effects.
[online] Idtdna.com. Available at:
https://www.idtdna.com/pages/education/decoded/article/a recombinant-cas9-enzyme-
that-drastically-reduces-crispr-off-target-effects [Accessed 25 Nov. 2019].

D. What is a ssODN HDR template?

ssODN- single-stranded oligodeoxynucleotide.
ssODN is used to introduce mutations or repairs in a gene after the CRISPR Cas9
complex makes a double stranded cut in the sequence of interest. It serves as the
template for the sequence we want to add to repair. It is especially useful for point
mutations.

Harmsen, Tim et al. “DNA mismatch repair and oligonucleotide end-protection promote base-pair
substitution distal from a CRISPR/Cas9-induced DNA break.” Nucleic acids research vol. 46,6
(2018): 2945-2955. doi:10.1093/nar/gky076
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5888797/

3. Explain the experiment in Fig. S2B. What exactly is being measured? What is ACTB? What is
K562 (TP53Q136fs)? What is K562 (TP53wt)

ACTB is the gene responsible for coding for the β-actin protein. β-actin is one of the types of
actin. One well-known place that actin can be found is in the myofibril of skeletal muscle. β-actin,
however, is found in the cytoplasm of many other cells (including non-muscular).
K562, in general, is an immortal cell line first taken from a patient with myelogenous
leukemia. This is an overproduction of white blood cells in the bone marrow. The TP53 gene is the
cell’s version of that gene. If it is TP53Q136fs, then that cell has a framshift mutation in that gene in
which normal p53 activity is not present. “Wt,” or wild type, has normal p53 activity.
In the experiment referred to in Fig. S2B, researchers tested for the amount of TP53 and
CDKN1A made in the cell. They used RT-qPCR to determine how much of the transcript they had,
and then normalized the data to ACTB for easy comparison. Researchers exposed two types of K562
cells to increasing levels of gamma-irradiation. The two types were K562 cells that carried the wild
type TP52 gene and K562 cells with a frameshift mutated version of the TP52 gene. The researchers
made a graph showing percent of actin increase or TP53 + CDKN1A transcripts made vs DNA
mutation induced by gamma-irradiation.

https://www.atcc.org/products/all/CCL-243.aspx#characteristics
https://www.molbiolcell.org/doi/full/10.1091/mbc.e11-06-0582
4. Explain RT-qPCR (as performed in this paper for Fig. S2B) in detail.
RT stands for reverse transcription, which is when RNA is converted to cDNA. The “qPCR”
stands for quantitative PCR. Together, RNA can be converted to cDNA and then amplified normally
through PCR. During each cycle, the amount of DNA is measured using a fluorescent signal. There
are many ways to make a fluorescent signal, but New England BioLabs highlights two popular ways.
The first way is to use a double-stranded DNA binding dye such as SYBR Green. Hydrolysis probs
such as TaqMan is another way to detect the amount of DNA during a PCR cycle.
RT-qPCR can be performed in either one step or two steps. The one-step reaction will have
all the reagents and buffers so that reverse transcription and amplification occur in that tube. The
two step reactions will separate the RT and amplification step (do them in separate tubes).
Generally, the two-step reaction will be better at optimizing the reactions to their ideal conditions
but present a greater risk of DNA contamination (Because there are more steps involved). The one
step reaction is less time consuming and does not have a big risk of DNA contamination.

https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-
biology/molecular-biology-learning-center/molecular-biology-resource-library/spotlight-
articles/basic-principles-rt-qpcr.html

https://www.neb.com/applications/dna-amplification-pcr-and-qpcr/qpcr-and-rt-qpcr

5. How did the authors carry out CRISPR-HDR of TP53 Q136fs?

The authors created the template needed to repair the gene with the protocol established by
Richardson et al.

First, the authors mixed crRNA and tracrRNA with Cas9 buffer. They heated this mixture to 98C for
5 minutes and cooled down the mixture to room temperature to create chimeric RNA. Cas9-3NLS
was also diluted separately in the same buffer used before. These two mixtures were then slowly
mixed together and incubated at room temperature for 20 minutes. The purpose of this step was to
form RNP complexes (ribonucleoprotein complexes). This is the structure that include the
tracrRNA, crRNA, and Cas9 protein.

The next part involves preparing cells for nucleofection. First, the cells were harvest, centrifuged
down, and resuspended in SF nucleofection solution.

Both the cell solution and ribonucleoprotein complex solutions were placed in a nucleocuvette.
Then, the authors added the HDR templates that were designed by the protocol established by
Richardson et al. The 4D nucleofector carried out the electroporation to allow for CRISPR to go in
the nucleus and repair the mutated gene (HDR). These cells were then allowed to rest for 5-7 days.

6. Explain ultra-deep amplicon next-generation sequencing as used in this paper.

 Amplicon- This refers to amplified or copied DNA or RNA, which can be achieved through
PCR or normal cell replication.
Ultra-deep sequencing allows for scientists to search for genetic variation, or specific
gene(s), even if it is an extremely rare gene. Like normal PCR, the area of interest is flanked by two
oligonucleotide sequences. Scientists can then analyze the products (amplicons) for specific genes.
This is much more specific and less time-consuming than whole-genome sequencing.

https://www.qiagen.com/us/applications/ngs/ngs-life-sciences/dna-amplicon-sequencing/
https://www.abmgood.com/Amplicon-Sequencing-Service.html
https://www.illumina.com/techniques/sequencing/dna-sequencing/targeted-
resequencing/amplicon-sequencing.html

7. For Fig. 1C, name the technique and describe in detail the experimental procedure.

Scientists used immunoblotting (western blot) to test for p53, p21, and actin. They started
by lysing the cells with Pierce IP lysis buffer. Then, they used Pierce BCA Protein Assay Kit to
determine the concentration of the protein they recovered from the lysed cells. They used this
concentration to determine how much to load in the wells when running the polyacrylamide gel.
The bands were transferred to polyvinylidene difluoride (PVDF) and blotted. The p53, p21, and
actin proteins were blotted with their specific monoclonal antibodies. They then used specific
antibodies for the monoclonal antibodies to visualize the blot (detect amount of proteins
produced).

8. For Fig. 1C,

A. What is Dauno and what is its effect on cells in this experiment?

Dauno is just a short name for Daunorubicin, which is a drug used to treat cancers
such as leukemia. According to this experiment, Dauno can be responsible for DNA
damage and apoptosis. Dauno works by inhibiting DNA synthesis and/or transcription
(DNA-dependent DNA synthesis). Dauno achieves this by intercalating between base
pairs. This results in the DNA helix uncoiling and leads to inhibition of DNA synthesis
and transcription.

https://www.ncbi.nlm.nih.gov/pubmed/310906
http://www.bccancer.bc.ca/drug-database-site/Drug
%20Index/Daunorubicin_monograph.pdf

B. What is the purpose of Actin in this experiment?

The purpose of Actin in experiment Fig 1C is to use it as a control. When running the
western blot, scientists need to make sure that each lane has the same number of cells to accurately
show how much of a gene product is collectively made. If the actin band is present and equally dark
to the other bands, then the number of cells is relatively the same (actin concentration does not
vary with the experimental procedure and will stay constant). When all actin bands are the same,
the scientist can accurately compare the gene products of many cell lines with confidence that the
number of cells is all the same.

https://www.novusbio.com/antibody-news/antibodies/the-importance-of-beta-actin-and-gapdh-
loading-controls

https://www.origene.com/products/antibodies/primary-antibodies/loading-control-
antibodies/beta-actin-loading-control

C. What exactly is R282W/- ?

This means that the cell line has a mutation in which one allele has a R282W
mutation while the other is a null allele. The null allele has no function. The R282W allele has an
arginine mutated to a tryptophan. This results in a p53 protein product with DNA promoter binding
activity similar to wild type with neomorphic (different) binding sites. This results in insufficient
production of its target genes.

E. Explain the results for R282W/- , -/-, and +/+ lanes with and without Dauno.
In the R282W/- lane, the mutant gene makes p53 regardless of Dauno, but increases
production in the presence of Dauno. However, it fails to make a meaningful amount of
p21. In the -/- lane, the TP53 gene is completely off and cannot make p53 or p21
regardless of Dauno. The +/+ lane is a normal-functioning gene and will only make p53
and p21 in the presence of Dauno AKA induced DNA damage.

9. For Fig. 2A, the authors compared how wild-type p53 and missense mutant p53 bind to
promoter regions compared to the p53 null state control. Most missense mutants had decreased or
no binding; the R282W mutant had increased binding.

The authors measured protein binding to the promoters using ChIP. Describe in your own words
the ChIP procedure used by the authors.

In general, ChIP is the process in which scientists can precipitate out any protein that may
be bound to DNA (p53) using antibodies against that protein. They can then sequence that protein
and see if there are any mutations present.
First, the cells were treated with formaldehyde to crosslink the DNA-binding protein with
the DNA. Then, the scientists used a mixture of Tris-HCl, NaCl, and EDTA for 10 minutes at room
temperature to separate the nucleus from the cytoplasm. The cell nuclei were then resuspended
and then the scientists sheared the DNA into smaller fragments. Using anti-p53 antibodies,
immunoprecipitation was performed followed by sequencing.

https://www.illumina.com/techniques/sequencing/dna-sequencing/chip-seq.html
10. Gain-of-function (GOF) vs. dominant-negative effect (DNE), that is the question.

A. In Fig. 3B, how does Annexin V measure apoptosis? That is, what is it measuring in
terms of biochemistry?
Annexin V is a marker used to bind and mark cells that went through apoptosis. This
method of measuring apoptosis is known as annexin v staining. According to
ThermoFisher, Annexin V is an anticoagulant present in humans and has a high affinity
for the phospholipid, phosphatidylserine. Normally, “PS” is present in the lipid bilayer
inner leaflet. After apoptosis, PS can be seen on the outer leaflet of the cell membrane.
Annexin V can then bind to the phospholipid and be detected via fluorescence.

https://www.thermofisher.com/us/en/home/life-science/cell-analysis/cell-
viability-and-regulation/apoptosis/annexin-v-staining.html

B. Which cell line in Fig. 3B indicates that missense mutations exert a dominant-negative
effect on the cell response to daunorubicin treatment? Explain your answer.

The cell lines with (-/-) and (R248Q/ + or -) are the cell lines that did not have the
expected amount of apoptosis. These results show that R248Q exerts a dominant-negative effect on
the cell when exposed to daunorubicin whether or not it had a wild type or null allele. It also
showed that having one R248Q allele is nearly identical to having two null alleles (in regards to
apoptosis). This is supported with Fig. 3B. In Fig. 3B, these three cell lines have considerably less
Annexin V than the (+/+ and +/-) cell types. Annexin V measured how many cells went through
apoptosis, which is an indicator of how well P53 and P21 were working to take the cell out of the
cell cycle.