1. Running head: THE ROLE OF PARC/CUL9 1
Research Internship Final Paper: PARC/Cul9
Leigh Anne Kline
Gama Lab

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Research Internship Final Paper: PARC/Cul9
During my semester in the 3860 Research Internship curriculum, I gained experience in
both research and lab functions. My research project involved understanding the function of the
recently discovered E3 ubiquitin ligase, called PARC/Cul 9, and its role in determining cell fate.
The Gama Lab’s primary investigator first discovered PARC/Cul 9 in her time at the University
of North Carolina. This project has been continued here at Vanderbilt, in order to expand upon
these findings. This research of PARC/Cul 9 is fascinating because it could provide a better
understanding of how stem cells regulate pluripotency and self-renewal. Results in the lab also
provide evidence for a role of PARC in cancer and in promoting the survival of nonrenewable
cells, such as neurons.

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Background
Apoptosis: The Big Picture
Apoptosis is a crucial step for organism development, neuron pruning, embryogenesis,
and for maintaining tissue homeostasis. It is a highly regulated process which can be triggered by
external or intrinsic cellular signals (Green, Llambi, 2015). When the apoptotic pathway is
dysregulated, it can cause improperly timed cell death (for example in neurodegeneration), or
absence of cell death leading to unregulated tumor growth (like seen in cancer).
Apoptosis is also known as the intrinsic mitochondrial pathway because the proteins
mediating this process converge at the outer mitochondrial membrane. Any kind of stress to the
cell due to a change in pH, temperature or DNA damage triggers the activation of the BH3-only
proteins. These BH3-only proteins bind directly to Bax and activate it. Bax is the main effector
of the apoptotic pathway. Antiapoptotic proteins, such as Bcl-2, Bcl-xl and Mcl-1, can bind to
Bax, however, and deactivate Bax. If no antiapoptotic Bcl-2 family proteins are present to
intercept this step, then the Bax protein will translocate and become attached to the outer
mitochondrial membrane. Bax then oligomerizes, ultimately forming pores in the mitochondrial
membrane through which the pro-apoptotic cytochrome c, is then released into the surrounding
cytosol (Walensky, 2012). Once cytochrome c is released into the cytosol, the cell is on its death
march. Cytochrome c then binds to Apaf-1, triggering a chain where initiator caspases, a family
of degrading enzymes, activate effector caspases. These effector caspases are responsible for the
physical degradation of the cytoskeleton and organelles, that ultimately lead the cell death
(Lopez and Tait, 2014).
PARC/Cul9, is an E3 ubiquitin ligase, which aids in attaching ubiquitin to the target
substrate. Once the substrate is tagged with ubiquitin, it is then recognized as being marked for

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degradation. The Gama lab found that the specific role of PARC/Cul 9 is to ubiquitinate
cytochrome c once released from the mitochondria in both differentiated neurons and brain
tumors, targeting it to the proteasome for degradation. Once cytochrome c is degraded, Apaf1
and caspases cannot be activated, and the cell does not engage the apoptotic program (Gama et
al, 2014). This mechanism provides both neurons and brain tumor cells increased resistance to
cell death.
The role of PARC/Cul9 in Human Embryonic Stem (hES) cells
The Gama Lab is primarily interested in researching the role of PARC/Cul 9 in human
embryonic stem cells. PARC/Cul 9 has been found to be highly expressed in hES cells. However,
PARC/Cul 9 does not degrade cytochrome c in hES cells. The main purpose of my project is to
help elucidating the function of PARC/Cul9 in hES cells.
In hES cells, it has been found that Bax is already activated without a BH3-only protein
trigger (Raff, 1998 and Dumitru, Gama et al, 2014). Though hES cells are responsible for much
cell growth and renewal, hES cells also are rather susceptible to damage and die easily. Since
Bax is already activated, it is ready to trigger cell death, even without the direct activation via
BH3-only proteins. This is likely due to the fact stem cells are the cells of origin for most tissues
in an organism and it is crucial to stop any mutations early that would otherwise occur and be
deleterious for the organism.
Human embryonic stem cells are unique in their pluripotency, or ability to differentiate
into any other type of human cell, and also in their self-renewing capabilities. Our hypothesis is
that PARC/Cul 9 is regulating one or both of these stem cell-specific functions since it is so
highly expressed under basal conditions (Figure1). Interestingly, PARC/Cul9 levels are further

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induced in neuroprogenitor cells (Figure 2). We are using two strategies to validate our
hypothesis: 1) examining PARC binding proteins and 2) evaluating whether changing the levels
of PARC/Cul9 in hES cells affects cell cycle.
Methods
Stem Cell Culture
Human embryonic stem cells were maintained as undifferentiated colonies and used for
experiments between passages 15 and 40. Undifferentiated cells were mechanically passaged
from established colonies at day 4-5. Cells were seeded on plates coated with Matrigel
(Corning), maintained at 37ºC and 5% CO2. hES cells are grown and feed daily with mTeSR
(Stem Cell Technologies).
For differentiation of hES cells into neuro progenitors, day 4 old colonies of
undifferentiated hES cells were fed neuro induction media without FGF for 7 days.
Western Blot
Western blots are used to determine the presence and relative amounts of certain proteins
in the cell. Primary antibodies are used to bind to the specific protein being analyzed, and a
secondary antibody is used to bind to this first antibody for identification purposes. For the
PARC and APC7 Western Blots, the rabbit PARC Antibody (Bethyl Labs), APC7 Antibody
(Sigma), anti-beta-actin (Sigma), anti-APC7- conjugated (Sigma), and anti-PARC (Bethyl Labs
and Santa Cruz Biotechnology) were used. Anti- rabbit HRP-conjugated secondary antibodies
were purchased from Pierce Chemical Co. Western blots were developed with ECL-plus reagents
(Amersham Biosciences).

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Flow cytometry based- cell cycle analysis
Flow cytometry based cell cycle analysis is the measurement of DNA content in cells,
and the rapid identification of the cell phases including mitosis. This is useful to see how long
the cell is spending in each stage of the cycle. For instance, if just PARC is knocked down and
the cell then spends most of its time in one phase more than a normal cell, it can be concluded
that PARC has an effect on that transition. In order to use this technology, the cells must first be
stained with DAPI, which binds to minor grooves of the DNA helix in order to show where the
DNA is in which stage of the cycle.
Immunoprecipitation
PARC/Cul9 was pulled down via immunoprecipitation and Western Blot in order to see if
APC7 was binding to it. For the detection of PARC complexes: Cells were collected by scraping
and collected at 1000 rpm for 10 min at 4oC. Cell pellets were then lysed in 200 μL ice-cold 1%
Triton in phosphate-buffered saline containing protease inhibitors (protease inhibitor cocktail,
Sigma P8340, diluted 1:100, 1 mM phenylmethylsulfonyl fluoride (PMSF)). After pre-clearing
200 μL of the sample with 20 μL protein G-sepharose at 4°C for 1 h, immunoprecipitation was
performed by incubating 200 μL of the lysates with 2-4 μg of anti-PARC polyclonal antibody
(Bethyl Labs) at 4°C for 2 h. Immunocomplexes were precipitated with 20 μLprotein G-
sepharose. After extensive washing with buffer, beads were boiled in 30 μL Laemmli buffer, and
15 μL of the eluted proteins were analyzed by Western blotting with APC7 antibody. Mouse IgG
was used as negative control.

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Lentiviral Infection
For transductions, five E. coli clones expressing pLKO1 - shRNA Cul9 plasmids were
purchased from Open Biosystems. Viruses used to transduce stem cells were added to cells in 1:3
dilution of stock lentivirus, for 16 h. Cells were then cultured for 24 h in complete medium, and
then stable clones expressing the shRNA against PARC/Cul9, were selected using puromycin (1
ug/ml). To select the best shRNA clone against PARC/Cul9, samples were then analyzed by
Western blotting using PARC/Cul9 antibody.
Results
1. PARC binding proteins. Some potential binding proteins were identified by mass
spectrometry analysis, and the Gama Lab decided to focus on APC7 because of its role in mitosis
and potentially in self-renewal of stem cells. APC7 is a component of the anaphase promoting
complex/cyclosome (the APC complex), a cell cycle-regulated E3 ubiquitin-protein ligase
complex that controls progression through mitosis and the G1 phase of the cell cycle. In order to
validate the findings that PARC/Cul9 binds to APC7, we performed immunoprecipitation assays
These showed that both PARC/Cul9 and APC7 interact in hES cells (Figure 3). The results
proved that PARC/Cul9 does indeed bind to the APC7 molecule, suggesting PARC/Cul9 could
bind to this complex because it plays a role in mediating cell cycle stages.
2. Effect of downregulating PARC on APC7 levels. After performing specific Western
Blots wherein PARC was virally knocked down via infection with lentivirus, the amount of
APC7 present in the cell increased (Figure 4). This suggested that PARC is negatively affecting
or inhibiting the APC7 protein. When the PARC/Cul9 levels were lowered, then there was less
inhibition, so the APC7 levels rose. If the APC7 levels had stayed the same after this PARC/Cul9

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knockdown, then that would have suggested that APC7 was not directly regulated by
PARC/Cul9.
3. Effect of PARC down regulation on hES cell cycle. This analysis was performed in
order to determine whether or not PARC/Cul9 has an effect or regulates a part of the cell cycle or
how a cell transitions in or out of certain stages. The analysis showed that the down regulation of
PARC/Cul9 did not have a significant effect on the time spent in the various cell cycle stages
(data not shown). Further research looking at an individual phases of the mitotic phase of the
cell’s cycle may provide more insight.
Discussion
In the near future, we want to better examine the role of PARC/Cul9 in regulating
mitosis. Since there was no significant effect on the overall cell cycle analysis of many cells, it
would be interesting to see how it affects the specific mechanisms of one individual cell’s cycle
using single cell detection methods.
Our data demonstrated that PARC/Cul9 and APC7 interact in hES cells. Our future
studies will characterize this interaction further. For example, does PARC/Cul9 target APC7 for
ubiquitination? If so, does it regulate the whole APC complex? Why is it that PARC/Cul9 is
induced in neural progenitors? Does it have a role in mitosis as well, or does it have a different
function once the cells are no longer pluripotent?
Since all of these discoveries are new in the world of stem cells, future directions include
a further understanding of the role of PARC/Cul9 in stem cells and how they differ from brain
tumor and nerve cells. The discovery that PARC/Cul9 is highly expressed in stem cells, but does
not have the same anti-apoptotic function, could suggest that PARC/Cul9 is involved in the

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specific hES cell function of self-renewal or pluripotency. If this function is better understood, it
could lead to many important applications regarding cancer and neurogenesis.

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References
Dumitru, R., Gama, V., Fagan, B. M., Bower, J. J., Swahari, V., Pevny, L. H., & Deshmukh, M.
(2013, June 8). Human Embryonic Stem Cells have Constitutively Active Bax at the
Golgi and are Primed to Undergo Rapid Apoptosis. Retrieved April 24, 2016, from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372694/
Gama, V., Swahari, V., Schafer, J., Kole, A. J., Evans, A., Huang, Y., . . . Deshmukh, M. (2014,
July 15). PARC/CUL9 Mediates the Degradation of Mitochondrial-released Cytochrome
c and Promotes Survival in Neurons and Cancer Cells. Retrieved February 21, 2016, from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4182917/
Green, D. R., & Llambi, F. (2015). Cell Death Signaling. Cold Spring Harbor Perspectives in
Biology, 1-17. Retrieved April 25, 2016.
Lopez, J., & Tait, S. (2014). Killing the Killer: PARC/CUL9 Promotes Cell Survival by
Destroying Cytochrome c. Science Signaling, 7(334), 1-2. Retrieved February 21, 2016,
from www.sciencesignaling.com.
Raff, M. (1998). Cell Suicide for Beginners. Nature, 396, 119-122. Retrieved February 21, 2016,
from www.nature.com.
Walenksy, L. (2012). Stemming Danger with Golgified BAX. Molecular Cell, 46, 554-555.
Retrieved March 21, 2016.

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Figures
Figure 1. hESCs (H9) and mouse ESC (mESCs) expressed high levels of PARC/Cul9 compared
to human fibroblasts (hFiB) and mouse embryonic fibroblasts (MEFs)
Figure 2. PARC/Cul9 is induced in Neural Progenitor cells (NP).
Figure 3. PARC/Cul9 and APC7 interact in hES cells.

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Figure 4. PARC/Cul9 down regulation causes the significant induction of APC7. Cul7 (other
Cul9 interacting protein) levels were not affected.