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Elucidating the p53 Signalling Network by Reverse Genetics

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  • Reiyyan Tariq Nizami

Aim

In my research project I am working with four different strains Caenorhabditis elegans. I have been performing a double mutant screen using an RNAi knockout library. Cep-1 is a protein that causes apoptosis due to DNA damage in the worm. The cep-1 protein is an ortholog of the human tumor suppressor protein p53, which is found mutated in many cancerous cells. The aim of my experiment is to find genes that are regulators of cep-1 which cause lethality if mutated with cep-1. Alternatively mutants which are lethal as single mutants but survive as double mutants in combination with a cep-1 mutant are also potential genes of interest. These mutants have the potential to be regulated by drugs/proteins to change the levels of cep-1 and induce/prevent apoptosis in cells.

Background

Cancer is a disease of uncontrolled cell growth in our body. Under normal conditions a cell has regulatory proteins and check points that make sure that a cell is growing at the right pace and if for any reason the cell starts to grow irregularly then these proteins stop the cell growth and cause apoptosis. Apoptosis is the programmed death of a cell, as you can imagine it is tightly regulated and loss of regulation can result in catastrophe for the cell and the organism. When apoptosis is over active, healthy cells will die and this is often seen in diseases such as neurodegenerative diseases, hematologic diseases and many other tissue damage diseases. Loss of apoptosis in turn results in cancers, autoimmune diseases and inflammatory diseases.

P53 is a tumor suppressor protein that was discovered in 1979. Since then a lot of research has been done into p53 and its functions. P53 is a tumor suppressor protein that is involved in defense of the cell. It is activated when a cell undergoes many various kinds of stress, such as DNA damage, hypoxia, metabolic stress and oncogene activation. It is one of the most important barriers to cancer in many mammals. P53 works in many various pathways, primarily p53 is involved in binding to transcription factors that then activate pathways involved in cellular defense, such as preventing angiogenesis and cellular growth.

Malfunctioning p53 is one of the greatest hallmarks of cancer. The majority of mutations found in p53 are due to single nucleotide substitutions in the amino acids of the DNA binding domain of the protein. Similar cancerous phenotypes are observed when either p53 loses function due to a loss of function mutation or when negative regulators of p53 are overactive or positive regulators of p53 lose their function. However p53 is quite unique amongst tumor suppressor proteins because different missense mutations in the protein can cause different levels of p53 activity and hence have varying effects on the host. The effect of the mutation is further modified depending on the genetic background of the person with the mutation.

Due to the different effects of various amino acid substitutions and the varying genetic background of patients it is quite difficult to study large populations. This is because high-throughput sequencing and genome wide single nucleotide polymorphism maps are expensive to obtain. The prices are going down as new technologies are becoming available however until now there have been no larger scale studies relating different mutations to varying levels of risks for different types of cancers. It is also hard to perform molecular studies on entire humans and so research is done on cell lines, mice and on cep-1 the Caenorhabditis elegans ortholog of p53.

Caenorhabditis elegans is a worm in the nematodes phylum. They are a very well-studied organism due to their ease of study. C. Elegans are one of the simplest organisms that have a nervous system and that make them a very good model organism for neuronal studies. They are transparent creatures and so many molecular and cellular processes especially those involved in development have been thoroughly researched. They have a short generation time and are very cheap to maintain. They are an extremely good model organism for performing screens because any of their nonessential genes can be knocked out easily by using RNAi.

The cep-1/p53 pathway is highly conserved throughout evolution as it is an extremely important pathway that is essential for cellular survival over time.

The cep-1 protein works through the following pathway to cause apoptosis in cells suffering DNA damage.

Cep-1  Egl-1/Ced-13 –| Ced-9 –| Ced-4  Ced-3  Apoptosis

A similar pathway is observed in humans

p53  BH3 –| Bcl2 –| Apaf1  Caspase  Apoptosis

A majority of these proteins are orthologs to one another. It was hoped that through my screen further proteins which interact with Cep-1 would be found and then their orthologs in humans could be found and then researched and targeted to regulate p53.

Materials and Methods

The experiment in itself was an extremely simple but time consuming experiment. Screens were performed with mutant worm strains which were then fed RNAi, through Escherichia coli (E. coli), to silence the gene of interest and create double mutants.

To begin with a liquid screen is preferred over a solid screen. This is because with a liquid screen you can screen a larger sample of double mutants more easily than a solid screen.

Liquid Screen

The following strains of bacteria and worms were utilized, E. Coli – OP50, C. Elegans – N2, C. Elegans – GK138, C. Elegans – LG12501.

  • E. Coli – OP50: Food source for C. Elegans
  • C. Elegans – N2: Wild type worms
  • C. Elegans – GK138: Cep-1 Mutant worms
  • C. Elegans – LG12501: Cep-1 Mutant worms
  • RNAi Knockout library for C. Elegans Chromosome 1

Day 1:

  1. Grow worms on Nematode Growth Medium (NGM) plates with OP50 as a food source for the worms.

Day 3:

Bleach worms that were plated on day 1 so as to only have eggs remaining on plates.

  1. Bend glass rod into L shape using a high temperature flame (Bunsen burner)
  2. Pipette bleach onto plates with worms on them and gently scrape the surface to mix worms and eggs with bleach
  3. Pipette fluid into Eppendorf tube and centrifuge at max speed for 3 minutes
  4. Aspirate most of the fluid and keep as much of the pellet as possible
  5. Suspend pellet with bleach
  6. Repeat steps 3 and 4
  7. Suspend pellet using M9 solution
  8. Centrifuge at high speed for 1 minute
  9. Aspirate most of the fluid and keep as much of the pellet as possible
  10. Repeat 7 and 8
  11. Suspend pellet using M9 solution and vortex Eppendorf tube at low speed to mix solution
  12. Place Eppendorf tubes in slow rocker in a 20° fridge overnight to allow eggs to hatch

Day 4:

  1. Replicate bacteria containing RNAi from knockout library using a sterile 96 pin replication tool into a 96 well containing Liquid Broth (LB) with Ampicillin and allow the bacteria to replicate overnight at 37°C in an incubator
  2. Induce transcription of RNAi using adding 0.1 Molar IPTG into wells and place in a shaker for 1 hour.
  3. Pellet bacteria by centrifuging in a cold centrifuge at 5°C for 5 minutes at 2,500 g
  4. Remove the supernatant by flipping over the well quickly but carefully so as to keep pelleted bacteria in the wells
  5. Suspend bacterial pellet in wells using NGM
  6. Pipette worms into wells and place in 37°C shaker

Day 8:

  1. Remove worms from shaker gently making sure not to tilt the 96 well plates
  2. Observe and record phenotype of the worms
  3. Compare phenotype between the 3 different strains of worms
  4. Leave worms in 20°C fridge overnight

Day 9:

  1. Remove worms from fridge gently making sure not to tilt the 96 well plates
  2. Observe and record phenotype of the worms
  3. Compare phenotype from previous day
  4. Compare phenotype between the 3 different strains of worms

Sequencing

After potential hits were found in the liquid screen the RNAi from these bacteria were sequenced to ensure that the sequence of the RNAi was correct and hadn’t randomly mutated over time. RNAi was prepared by using a Qiagen miniprep spin kit.

  1. Suspend bacteria in 250 µl Buffer P1 and place in a microcentrifuge tube
  2. Mix 250 µl Buffer P2 and shake the mixture by flipping tube over a few times
  3. To the mixture add 350 µl N3 buffer, mix well quickly
  4. Centrifuge at 13000 rpm for ten minutes
  5. Aspirate supernatant into new tube
  6. Centrifuge again for a roughly 1 minute and discard the flow through
  7. Wash spin column with 0.5 ml PB buffer and centrifuge for 1 minute, discard flow through
  8. Wash spin column with 0.75 ml PE buffer and centrifuge for 1 minute
  9. Discard flow through and centrifuge at maximum speed for 1 minute
  10. Place prep column in a sterilized 1.5 ml microcentrifuge tube
  11. Add 50 μl of water to prep spin column and allow to rest for 1 minute after which centrifuge for 1 minute

After the Qiagen miniprep is complete the tubes were sent for sequencing to The Centre for Applied Genomics where it was sequenced and results were obtained within a week.

Solid Screen

Solid screens were performed on genes which were found to have increased lethality with cep-1 deletion or increased survivability with cep-1 deletion.

The following strains of bacteria and worms were utilized, E. coli – OP50, C. Elegans – N2, C. Elegans – TG12

  • E. Coli – OP50: Food source for C. Elegans
  • C. Elegans – N2: Wild type worms
  • C. Elegans – TG12: Cep-1 Fluorescent tagged (GFP) worms
  • C. Elegans – Ned-8: Positive control
  • C. Elegans – HT115: Negative control
  • RNAi Knockout library for C. Elegans Chromosome 1

Day 1)

  1. Streak RNAi bacteria of interest from RNAi Library to obtain single colonies. Allow them to grow overnight in 37°C incubator

Day 2)

  1. Pick and grow a single colony in 5 ml of LB + Amp + Tet overnight in a 37°C shaker

Day 3)

  1. Add 0.1M IPTG for 4 hours to induce RNAi
  1. Plate 100 µl of induced bacteria on RNAi plates & incubate at 37°C incubator overnight

Day 4)

  1. Pick 5 worms at the same stage and plate onto RNAi plates
  2. Allow to grow over 4 days at 20°C

Day 8)

  1. Score phenotypes and compared between different strains

Day 9)

  1. Score phenotypes again on next day and compare between different strains and the previous day

Fluorescent Microscopy

We used a Differential interference contrast (DIC) microscopy to observe localization of cep-1 in TG12 worms. Worms were mounted using the following method

  1. Place a drop or two of hot liquid agarose onto microscope slide
  2. Immediately place a second slide perpendicularly on top of the agarose
  3. Allow agarose to settle for one to three minutes
  4. Gently slide off second slide so as to leave an agarose patch behind
  5. Add a drop of 1mM Levamisole to paralyze worms and prevent their movement
  6. Pick worms and place them on the slide
  7. Slowly place coverslip on top of worms, be very gentle

Once prepared the slides were observed using a DIC microscope to find any irregularities in the localization or amount of cep-1 in the worms, especially in the germline and the eggs.

Results

The aim of this experiment was to find genes that cause lethality as double mutants with cep-1 mutants but not in wild type worms, these genes would be positive regulators of cep-1 and cause apoptosis through cep-1. The screen was also designed to find worms that cause lethality in wild type worms but not in cep-1 mutants, these would be negative regulators of cep-1 and cause apoptosis through cep-1.

The results were gathered and tabulated to allow for an easier and better analysis of data.

Note: There is a lot of data and so only data that is relevant is shown.

Liquid Screen Results

Legend

L = Synthetic LethalE = Embryonic LethalG = Slow GrowthB = Egg laying abnormal R = No RNAi C = Contamination A = Larval Arrest S = Sterile V = Variable Morphology P = Lethal progeny W = No Worms

Cells of interest are highlighted

Chromosome 1 – Plate 1

N2

1

3

5

7

9

11

13

15

17

19

21

23

A

 

R

G

         

G/L

L

   

C

       

A/C

 

A/C

         

E

 

R

       

E/S

         

G

       

R

           

R

I

   

S/L

S

 

R

R

R

R

     

K

             

R

R

R

 

R

M

R

       

R

R

 

R

   

S

O

 

R

 

R

   

S/E

R

       

cep-1 (gk)

1

3

5

7

9

11

13

15

17

19

21

23

A

 

R

L

         

C/L

C/L

 

G/E

C

       

G/S

 

G/L

 

C/L

     

E

 

R

       

E/S

         

G

       

R

           

R

I

   

E/S

E/S

 

R

R

R

R

     

K

             

R

R

R

 

R

M

R

G/E

     

R

R

 

R

   

/G/S

O

 

R

 

R

   

S

R

       

cep-1 (lg)

1

3

5

7

9

11

13

15

17

19

21

23

A

 

R

C/L

         

L

L

 

E/P

C

       

G/L

 

G

 

L

     

E

 

R

       

E/S

         

G

       

R

           

R

I

   

S/B

S/B

 

R

R

R

R

     

K

             

R

R

R

 

R

M

R

E/G

     

R

R

 

R

   

S

O

 

R

 

R

   

S/E

R

       

Chromosome 1 – Plate 5

N2

2

4

6

8

10

12

14

16

18

20

22

24

A

                       

C

                       

E

                       

G

 

E

                   

I

               

E

     

K

             

R

       

M

           

R

   

R

R

 

O

         

G

     

R

   

cep-1 (gk)

2

4

6

8

10

12

14

16

18

20

22

24

A

                       

C

                       

E

                       

G

                       

I

               

E

     

K

             

R

       

M

           

R

   

R

R

 

O

                 

R

   

cep-1 (lg)

2

4

6

8

10

12

14

16

18

20

22

24

A

                       

C

                       

E

                       

G

                       

I

               

E

     

K

             

R

       

M

           

R

   

R

R

 

O

                 

R

   

Key results of interest in liquid screen

Lethality in gk and lg but not in N2

Well

Gene

N2

gk

lg

A23

F53G12.5

-

G/E

E/P

M03

Y95B8A_85.h

-

G/E

E/G

Lethality in N2 but not in gk or lg

Well

Gene

N2

gk

lg

G04

F25H2.10

E

-

-

Solid Screen Results

3 hits of interest were obtained from all the plates that were screened. These 3 hits were then repeated multiple times on solid media to make sure the results were accurate.

 

N2

lg

gk

A23 -1

-

-

-

A23 -2

-

-

-

A23 -3

-

-

-

 

N2

lg

gk

M03 -1

E

-

-

M03 -2

E

-

-

M03 -3

-

-

-

 

N2

lg

gk

G04 -1

L

A

-

G04 -2

E

-

-

G04 -3

-

-

-

The M03 strain showed opposite results in the solid screen in comparison to the liquid screen. However since the results were consistent we decided to study both M03 and G04 under the microscope with fluorescent markers.

Fluorescent microscopy results

No major mislocalization of GFP tagged cep-1 was found and no extra or lack of GFP tagged cep-1 was observed.

Discussion

The finding of regulators of cep-1 that can be targeted by drugs and/or proteins is going to be extremely useful in cancer treatments and therapies. Cancers caused by p53, the human ortholog of cep-1 is mostly caused by down regulation of p53. Since p53 can no longer cause apoptosis in damaged cells the cells continue to replicate and eventually create a tumor which can later turn into a metastatic tumor and damage the entire body. If we were able to manipulate levels of a regulator using drugs/proteins we could potentially control the levels of p53 and so control apoptosis and increase its levels in cancerous tissues.

Until now we have been unsuccessful in finding a regulator of cep-1 in our experiment. This is to be expected with a screen. Although a screen is a good genetic technique for better understanding pathways and functions of proteins, it is a method that doesn’t guarantee results. It is possible to find results in the first screen you perform or after performing hundreds and thousands of screens. They are also a very time consuming technique depending on the model organism you are using. We are sure that screening is a good method of finding regulators of cep-1, however to get results we just have to continue going through the RNAi library.

There has been some inconsistency between our results in the liquid screen and the solid screen. We are unsure of any reasons behind this inconsistency. However it could be due to a variety of reasons. To begin with the environment in the liquid media is different from the environment in the solid media. The osmotic pressure created in the liquid media could put more stress on the worms and so may result in a different phenotype compared to the solid media.

The solid media is a friendlier medium for the worms to grow in compared to the liquid media and this may put the worms under less stress to develop, grow and mate. This may result in a better quality of life in the solid media and present us with a different phenotype compared to the liquid media. One way to combat this problem would be to perform all the screens on solid media, however this would be a very difficult and time consuming task and would slow down the process by a lot. Further studying the screening techniques will explain these different phenomena and help screen C. elegans in the future.

There are not many reasonable explanations for why no florescence was found in the TG12 strain of worms. The two reasonable explanations that we found were that either the mutant genes M03 and G04 cause lethality through a different pathway that is remotely related to cep-1 or that the results of both the liquid screen and the solid screen were false positives and redoing the screen multiple times on solid media will show the no lethality in C. elegans.

Our aim for the near future is that more screening needs to be done on what was thought to be a potential regulator and the rest of the chromosomes of C. elegans need to be screened in search of more potential regulators of cep-1. Research on screening techniques also needs to be done to find the most effective and accurate method to screen entire chromosomes.

Citations

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  2. Arum, Oge, and Thomas E. Johnson. "Reduced Expression of the Caenorhabditis Elegans P53 Ortholog Cep-1 Results in Increased Longevity." The Journals of Gerontology 62.9 (2007): 951-59
  3. Klug, Stefanie J., Meike Ressing, Jochem Koenig, Martin C. Abba, Theodoros Agorastos, Sylvia MF Brenna, Marco Ciotti, Br Das, Annarosa Del Mistro, and Aleksandra Dybikowska. "TP53 Codon 72 Polymorphism and Cervical Cancer: A Pooled Analysis of Individual Data from 49 Studies." The Lancet Oncology 10.8 (2009): 772-84.
  4. Shu Ito, Brent Derry. “Cell Non-autonomous Regulation of Death in C. elegans.” University of Toronto Research Repository (2011)
  5. Adrienne S. B. Rollie. “REGULATION OF CEP-l/p53 BY DVE-1, A C. elegans SATB FAMILY MEMBER” University of Toronto – Graduate Department of Molecular Genetics (2008)
  6. Anita K. Jolliffe “The C. elegans p53 Family Gene cep-1 and The Nondisjunction Gene him-5 Are Required for Meiotic Recombination ” University of Toronto (2011)
  7. Gao, M. X., E. H. Liao, B. Yu, Y. Wang, M. Zhen, and T. Borsello. "The SCFFSN-1 Ubiquitin Ligase Controls Germline Apoptosis through CEP-1/p53 in C. Elegans." Cell Death and Differentiation 15.6 (2008): 1054-062.
  8. G Matlashewski. “Isolation and characterization of a human p53 cDNA clone: expression of the human p53 gene.” EMBO 20; 3(13) (1984).
  9. Brenner, S. "The Genetics of Caenorhabditis Elegans." Genetics 77.1 (1974): 71-94.
  10. "Primary Information of P53 Gene." Www.bioinformatics.org. (Accessed March 24th)
  11. "TP53."Tumor Protein P53. http://ghr.nlm.nih.gov/gene/TP53 (Accessed March 24th)
  12. National Center for Biotechnology Information (US). Genes and Disease [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 1998-. The p53 tumor suppressor protein.
  13. “daf-16 Protein DAF-16 [ Caenorhabditis elegans ]” http://www.ncbi.nlm.nih.gov/gene/172981
  14. “Cancer in Canada” http://www.cancer.ca/~/media/cancer.ca/CW/publications/Canadian Cancer Statistics/canadian-cancer-statistics-2013-EN.pdf (Accessed March 20th)
  15. Hanahan, D. "The Hallmarks of Cancer."Cell100.1 (2000): 57-70.
  16. “TCAG Facilities” http://www.tcag.ca/facilities/dnaSequencingSynthesis.html (Accessed March 20th)
  17. Reeve, Eric. "The Nematode Caenorhabditis elegans. Edited by William B. Wood and the Community of C. elegans Researchers. New York: Cold Spring Harbor Laboratory."Genetical Research52 (1988): 587-606.
  18. Qiagen. “QIAprep® Miniprep Handbook.” 19-20.
  19. "Mounting C. Elegans for Imaging." <http://groups.molbiosci.northwestern.edu/morimoto/research/Protocols/IX. C. elegans/A. General/5. mounting for imaging.pdf>. (Accessed March 24th)

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