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PRMT1 controls subcellular localization of p53 via PARP1
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PRMT1 controls subcellular localization of p53 via PARP1
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PRMT1 CONTROLS SUBCELLULAR LOCALIZATION OF P53 VIA PARP1
by
Wuyue Hua
August 2023
Copyright 2023 Wuyue Hua
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)
ii
Acknowledgements
I would like to give special thanks to my mentor, Dr. Jian Xu. Dr. Xu is an outstanding scientist
as well as a very responsible and caring mentor. Living aboard alone is very hard for a man who
is attached to his native land. But Dr. Xu helped me a lot in both academic studies and my future
direction since I joined the lab. It is my great honor to learn experiment techniques and
biochemical analysis methods in Xu lab. Although I won’t pursue a research-focused career, this
year is my precious experience and the lab experience broadens my horizon, which will
positively impact my future teaching career.
I would also like to thank all my fellow lab members for their help, including Jiang (Julia) Qian,
Mehrnaz Zarinfar, Prerna Sehgal, Julia Raulino Lima, Reza Vatankhah, Tal Rosen, Yidan (Cloris)
Zhang and Greg Park. My fellow lab members always have my back, creating a happy study
environment together in the daily life. I would like to give special thanks to Julia Qian, Mehrnaz and
Prerna for training me experimental techniques and skills. I really thank to all the help I received
during my stay in the lab.
I would like to give gratitude to my committee members, Dr. Young-Kwon Hong and Dr. Ching-
Ling (Ellen) Lien, for their helpful suggestions for improving my project and thesis.
Specially, I would like to thank the musicians of Paradox Interactive. Their music inspired me
keep moving when I tried to make an excuse to flee away from working. Lastly, I want to thank
my family, who encouraged me to go further, explore further and study further. I would never
come to here without their support.
iii
Table of Contents
Acknowledgements ......................................................................................................................... ii
List of Tables ................................................................................................................................... iv
List of Figures ................................................................................................................................. v
List of Abbreviations ....................................................................................................................... vi
Abstract ......................................................................................................................................... vii
Chapter 1 Introduction ...................................................................................................................... 1
1.1 PRMT1 influences multiple cellular pathways ....................................................................... 1
1.2 The function and dynamic of p53 ........................................................................................... 3
1.2.1 Brief introduction: the function of p53 ......................................................................... 3
1.2.2 The dynamic of p53 ...................................................................................................... 6
1.3 PRMT1 regulates p53 ............................................................................................................. 8
1.3.1 PRMT1 regulates the level of p53 ................................................................................ 8
1.3.2 Does PRMT1 control the distribution of p53? ............................................................ 11
Chapter 2 Materials and Methods ................................................................................................... 15
2.1 Materials ............................................................................................................................... 15
2.2 Methods ................................................................................................................................ 16
2.2.1 Cell culture ................................................................................................................... 16
2.2.2 Transfection ................................................................................................................... 16
2.2.3 Immunofluorescent staining .......................................................................................... 17
2.2.4 Western blotting ........................................................................................................... 17
Chapter 3 Results ............................................................................................................................ 19
3.1 PRMT1 controls the subcellular localization of p53 ............................................................. 19
3.2 PRMT1 controls the expression level of PARP1 .................................................................. 21
3.3 PARP1 knockdown led to nuclear export of p53. .................................................................. 23
3.4 Overexpression of PARP1 reverses the nuclear export of p53 in PRMT1
Depleted cells… ........................................................................................................................ 24
Chapter 4 Discussion and Future Directions ................................................................................... 29
Experimental method restrictions ............................................................................................... 29
Future Directions ........................................................................................................................ 31
Are there other proteins involved in the PRMT1-p53 pathway? ............................................... 31
How does PRMT1 regulate PARP1? ........................................................................................ 31
What is the function of the cytosolic p53? ................................................................................ 32
What are the dynamics of cytosolic p53 in PRMT1 knockdown cells? ..................................... 34
References ..................................................................................................................................... 35
iv
List of Tables
Table 2.1 Materials used in the experiments ..................................................................................15
Table 2.2 siRNA transfection compound concentration ................................................................. 16
Table 2.3 DNA transfection compound concentration ...................................................................16
v
List of Figures
Figure 1.1 PRMTs can be classified into 3 types ...............................................................................1
Figure 1.2 The function of p53 is a network ......................................................................................5
Figure 1.3 Regulation of p53 controls the fate of cells ...................................................................... 7
Figure 1.4 PRMT1 controls the degradation by regulating the alternative splicing
of MDM4 through PRMT1-p53 pathway .........................................................................................9
Figure 1.5 p53 is translocated to the cytoplasm through CRM1-p53 export system .......................12
Figure 1.6 PARP1 participates in DNA damage repair ...................................................................13
Figure 3.1 PRMT1 knockdown altered p53 subcellular localization ..............................................20
Figure 3.2 PRMT1 knockdown downregulated the level of PARP1 ...............................................22
Figure 3.3 PARP1 knockdown alters the cellular p53 distribution .................................................23
Figure 3.4 Overexpression of PARP1 in PRMT1 knockdown cells rescued the nuclear
export of p53 ................................................................................................................................ 26
Figure 4.1 PRMT1 controls the distribution of p53 via PARP1 ...................................................... 29
Figure 4.2 The histogram based on the original data of PARP1 knockdown group shows
that this group of data didn’t conform to the normal distribution ...................................................30
Figure 4.3 Cytosolic p53 triggers apoptosis ....................................................................................33
vi
List of Abbreviations
Cas9 CRISPR-associated protein-9 nuclease
CRISPR Clustered regularly interspaced short palindromic repeats
DBD DNA binding domain
EMT Epithelial-to-mesenchymal transition
IF Immunofluorescence
KD Knockdown
MDM2 MDM2 proto-oncogene
MDM4 MDM4 regulator of p53
MOMP Mitochondrial outer membrane permeabilization
mTOR Mechanistic target of rapamycin kinase
OE Overexpression
PARP1 Poly(ADP-ribose) polymerase 1
PRMT Protein arginine methyltransferase
PUMA p53 upregulated modulator of apoptosis
siRNA Small (or short) interfering RNA
WB Western blot
vii
Abstract
As a well-known tumor suppressor, p53 is a transcription factor that functions in the nuclear and
transactivates genes involved in diverse cellular stresses to regulate expression of target genes,
such as the cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism.
However, it also plays an important role in the cytoplasm, where it triggers apoptosis and inhibits
autophagy under stress. We previously uncovered a PRMT1-p53 pathway, in which PRMT1
increases the turnover of p53. In this thesis project, we further show that PRMT1 plays an
important role in nuclear export of p53. PRMT1 knockdown significantly altered the subcellular
distribution of p53. Using immunostaining and western blotting, we showed that p53 transferred
to the cytoplasm from the nuclei in PRMT1 knockdown mouse epicardial cells (MEC1).
Poly(ADP-ribose) polymerase 1 (PARP1) is potentially a key component of this pathway.
PRMT1 downregulated the expression level of PARP1, causing p53 to translocate to the
cytoplasm. Our results showed that both PRMT1 and PARP1 knockdown significantly increased
cytosolic distribution of p53Altogether, our study discovered a PRMT1-p53 translocation
pathway via PARP1.
1
Chapter 1 Introduction
1.1 PRMT1 influences multiple cellular pathways
Protein arginine methyltransferases (PRMTs) are a class of enzymes that methylate arginine
residues on histones and non-histone proteins. The family of the nine sequence-related enzymes,
PRMT1–9, modifies various of proteins using the cofactor S-adenosylmethionine (AdoMet),
resulting in three different types of methyl-arginines. (Figure 1.1) (Xu and Richard, 2021).
Figure 1.1. PRMTs can be classified into 3 types.
(A) There are 3 types of methyl-arginines. Our PRMT1 is type 1. (B) The panel shows the
preferences of arginine motifs for the PRMTs. PRMT1 has a preference for the arginines with
neighboring glycines within RGG/RG motifs. Figure is taken from Xu and Richard (2021)
2
PRMT1 is the major PRMT, which is responsible for more than 75% methylation activities in
human cells, and is documented as a key regulator of cellular signal transduction, epigenetic
regulation, and DNA repair (Blanc and Richard, 2017b; Xu et al., 2013). PRMT1 plays multiple
regulatory roles in processes including transcription regulating, DNA damage response signaling
and immune signaling. Arginine methylation of DNA damage proteins by PRMT1 is essential to
maintain genome stability and ensure homologous recombination and non-homologous end
joining (NHEJ) (Xu and Richard, 2021). PRMT1 also regulates cancer immunity. Using a
murine colon adenocarcinoma cell line, previous researchers found that CRISPR-Cas9 knockout
of PRMT1 in MC38 improved anti-PD-1 immunotherapy effectiveness when administered to
syngeneic C57BL/6 mice (Hou et al., 2021).
One of the most well-characterized roles of PRMT1 is its epigenetic functions. PRMT1 strongly
prefers the intrinsically disordered sequences; namely, the RGG/RG motif commonly found in
RNA binding proteins (RBPs) (Thandapani et al., 2013). Methylation on these RBPs regulates
protein-RNA binding and interactions between proteins (Guccione and Richard, 2019). PRMT1
has multiple substrates, especially in the pathways mediated by DNA damage, growth factors,
metabolites, and the immune response. One of the substrates of PRMT1 is histone. PRMT1
methylates H4 on H4R3me2a, which serves in the activation of gene expression (Wang et al.,
2001). PRMT1-mediated H4R3me2a facilitates the recruitment of histone acetyltransferases,
including p300, and subsequent lysine acetylation on H3 and H4 (An et al., 2004). Also, for the
proliferation and migration of normal and cancer cells, the PRMT1-mediated H4R3me2a mark is
critical (Blanc and Richard, 2017a; Gou et al., 2018; Hashimoto et al., 2021). For examples,
3
H4R3me2a can be recognized by the Tudor domain-containing protein TDRD3, which recruits
methyl-USP9X to promote cell proliferation (Narayanan et al., 2017; Yang et al., 2010). Yao et
al. also identified SMARCA4, an ATPase subunit of the SWI/SNF (switch/sucrose non-
fermentable) chromatin remodeling complex, as a reader of PRMT1-mediated H4R3me2a for
epidermal growth factor receptor expression in colorectal cancer (Yao et al., 2021). The cells
treated with type 1 PRMT inhibitor lack histone activating marks, which inhibits the expression
of cell cycle genes. Type I PRMT inhibitors inhibit the expression of cell cycle genes because of
lack of histone-activating marks, which halts cell proliferation and interestingly manifests as
hallmarks of DNA damage (Yu et al., 2009).
1.2 The function and dynamic of p53
1.2.1 Brief introduction: the function of p53
p53 was first discovered in 1970s as a 53 kD host protein bound to simian virus 40 large T
antigen in viral-transformed cells (Lane and Crawford, 1979; Linzer and Levine, 1979). p53 was
later identified to be a sequence-specific DNA binding protein that regulates transcription
(Laptenko and Prives, 2006)..
p53 promotes cell cycle arrest and apoptosis, which are its best understood functions. Hallmark
studies in 1990s proved that p53 is crucial for a reversible DNA damage-induced G1 phase
checkpoint (Kastan et al., 1991) that is mediated, partly, by its ability to activate the p21 cyclin-
dependent kinase inhibitor gene at the transcription level (el-Deiry et al., 1993) facilitating DNA
repair before cell division. In some cases, p53 triggers cellular senescence, a stable if not
permanent cell cycle arrest program (Serrano et al., 1997; Shay et al., 1991). p53 also promotes
4
apoptosis through the induction of pro-apoptotic BCL-2 family members whose action promotes
caspase activation and cell death (Miyashita et al., 1994), (Yonish-Rouach et al., 1991).
In traditional models, p53 plays the role of “guardian of the genome” to limit the deleterious
consequences of mutation (Lane, 1992). On one hand, its ability to arrest cells following DNA
damage indicates that it might prevent cancer by inhibiting the accumulation of oncogenic
mutations. In this model, p53 loss indirectly facilitates cancer by increasing the number of
mutations in daughter cells. On the other hand, the ability of p53 to halt the proliferation in
response to abnormal oncogene overexpression indicates a role in limiting the consequences of
oncogenic mutations. Here, p53 loss directly results in cancer development by allowing
oncogene-expressing cells to proliferate, which explains why TP53 mutations cooperate with
oncogenes in transformation. Researchers have been studying the function of p53 for decades.
However, this protein can always surprise us. The historic view provides a basic concept as to
why TP53 mutations are so common in tumors, while recent works paint a much more subtle
picture of p53 action that emphasizes on its context-dependent regulation and the diverse
consequences of its activation (Kastenhuber and Lowe, 2017).
Once DNA damage occurs, p53 is activated to facilitate the eradication or repair of damaged
cells, finally reducing the risk of propagating mutations. DNA damage response (DDR) kinases
phosphorylate p53, inducing cell-cycle arrest, senescence, and apoptosis (Williams and
Schumacher, 2016). Additionally, p53 promotes DNA repairing by activating target genes that
encode components of the DNA repair machinery (Williams and Schumacher, 2016). As if
maintaining genome integrity, cell cycle arrest, and apoptosis were not enough for onesingle
5
gene, an ever-growing body of work indicates that p53 controls additional programs that
contribute to its effects, as illustrated in the network below (Figure 1.2).
Figure 1.2 The function of p53 is a network
Various regulators control the activity of p53 (top). In turn, p53 controls many different
biological processes (bottom). Each line means an interaction. Direct p53 inputs are performed
as blue lines and direct p53 outputs are performed as red lines. Downstream pathways are highly
6
interconnected (gray lines). Interactions: positive (arrow), negative (T-bar), or modifying (solid
circle) Figure is taken from Kastenhuber and Lowe (2017).
1.2.2 The dynamic of p53.
In normal cells, because of fast degradation, p53 protein is maintained at relatively low levels by
a series of regulators including MDM2, which functions as a p53 ubiquitin ligase to facilitate its
degradation (Hafner et al., 2019). MDM2 promotes tumor formation by targeting tumor
suppressor proteins, such as p53, for proteasomal degradation. Also, MDM2 itself is
transcriptionally-regulated by p53 (Haupt et al., 1997; Kubbutat et al., 1997), thereby forming a
regulatory feedback loop. However, p53 can be stabilized and accumulated in response to
various cellular stresses, including DNA damage and replication stress produced by aberrant
oncogenes. Upon activation of the DNA damage response, p53 is phosphorylated, rendering it
insensitive to MDM2 (Shieh et al., 1997).
As we mentioned in the previous paragraph, p53 is crucial to maintaining the stability of
genome. In mammalian cells, based on the type and severity of DNA damage, the activation of
p53 pathway may lead to distinct result, cell-cycle arrest that allows repair of the damage or
alternatively, death of the cell through apoptosis (Chen et al., 2013). Not only the absolute levels
of p53 protein matter for the cellular fate in DNA damage response but the changes in p53 levels
over time (p53 dynamics) also controls cell fate (Figure 1.3) (Chen et al., 2013; Paek et al., 2016;
Purvis et al., 2012). For instant, ionizing radiation induces pulses of p53 protein levels, which
enable the cells to repair DNA damage and come back the cell cycle. However, converting the
pulses of p53 level into sustained p53 activation by a combination treatment of ionizing radiation
with MDM2 inhibition result in an irreversible cell cycle arrest and cellular senescence (Purvis et
al., 2012).
7
Figure 1.3 Regulation of p53 controls the fate of cells
Various mechanisms determine the activity of p53 together in response to DNA damage. The type
and extent of damage affect which post-translational modifications applied on p53 and the
dynamics of p53 expression. In addition, the sequence and chromatin structure at each site
influence the strength of p53 binding. Promoter-specific association of p53 with different cofactors
also have effects on the extent of gene activation. The level of p53-induced gene activation,
combined with the stability of the induced mRNAs, controls protein levels in the cell, which
ultimately lead the cell fate outcomes towards survival (DNA repair and cell cycle arrest) or cell
death. DDB2, DNA damage-binding protein 2. Figure is taken from Hafner et al., (2019)
8
1.3 PRMT1 regulates p53
1.3.1 PRMT1 regulates the level of p53
PRMT1 plays an important role in organ development. During heart development, epithelial-to-
mesenchymal transition (EMT) of epicardial cells has a decisive role in the coronary vessel and
chamber wall formation, where epicardial cells invade the developing muscle wall, giving rise to
the majority of cardiac fibroblasts, coronary vascular smooth muscle cells (cVSMCs), and
pericytes (von Gise and Pu, 2012).
Dr. Olan Jackson-Weaver et al. showed that PRMT1 drives epicardial invasion and
differentiation in the process of heart development through the PRMT1-p53 pathway(Jackson-
Weaver et al., 2020). In this study, PRMT1 knockout significantly impaired the formation of
chamber wall. He demonstrated that the level of p53 increase was caused by PRMT1 knockdown
via the PRMT1-p53 pathway. In this pathway, PRMT1 knockdown induces an alternative
splicing of MDM4, one of the most important regulators of p53 stability, leading to the
accumulation of p53 (Figure 1.4). PRMT1 knockdown didn’t alter the mRNA level but increased
the half-life of p53. There are 2 kinds of MDM proteins regulating the stability of p53, MDM2
and MDM4. MDM4 shows structural similarity to MDM2. Both proteins interact with the p53
tumor suppressor protein and inhibit its activity (Shvarts et al., 1996). MDM4 is important for
the effective degradation of p53—MDM4 stabilizes MDM2, and the ubiquitination of p53 is
upregulated and enhanced by the formation of the MDM2/MDM4 heterodimer (Gu et al., 2002;
Linares et al., 2003; Linke et al., 2008; Okamoto et al., 2009). Therefore, MDM4 can protect p53
protein from MDM2-targeted degradation, while maintaining the suppression of p53
transactivation and apoptotic functions. There are 2 isoforms of MDM4, long form and short
form. In PRMT1 knockdown cells, the functional long isoform of MDM4 was reduced while a
9
short isoform of MDM4 was increased. This short form of MDM4 is defective in p53 binding,
blocking the long form from binding to p53 in a dominant negative fashion and finally
stabilizing p53(Bardot et al., 2015).
Figure 1.4 PRMT1 controls the degradation by regulating the alternative splicing of
MDM4 through PRMT1-p53 pathway.
10
TGF-β is the activator of EMT process. However, the presence or absence of TGF-β only
changed the expression level of the proteins but have no effect on relative abundance. (A) Panel
shows the Graphic abstract of the PRMT1-p53 pathway. In mutant epicardial cells, PRMT1
knockdown induces alternative splicing of MDM4 that leads to accumulation of p53. (B) Cells
treated with PRMT1 siRNA had higher levels of p53. The cells treated with PRMT1 siRNA had
a significantly higher level of p53. (C) PRMT1 siRNA treatment didn't alter the level of mRNA.
There was no significant difference of PRMT1 mRNA level between the control and
experimental groups. (D&E) The half-life of p53 was increased when PRMT1 was depleted.
Compared with the cells treated with control siRNA, the cells treated with PRMT1 siRNA had a
significantly longer half-life time. (F) The panel shows the results of the western blot testing
MDM2 and MDM4, the 2 key regulators of the stability of p53. There was no significant
difference of MDM2 level between control and experimental groups. However, the result
showed that there were some differences of the MDM4 isoform between the cells under different
treatment. (G) The difference between MDM4-L and MDM4-S is MDM4 skips the exon 7.
MDM4-L is functional while the short form is nonfunctional. (H) and (I) The panel shows
relative expression level of the 2 MDM4 isoforms. In PRMT1 knockdown cells, the level of
MDM4-L was downregulated but the level of MDM4-S is upregulated. Figure is taken from
Jackson-Weaver et al., (2020).
11
1.3.2 Does PRMT1 control the distribution of p53?
Dr. Jackson-Weaver answered how the level of p53 was controlled by PRMT1-p53 pathway. He
showed that p53 accumulated in PRMT1 knockdown cells. However, where p53 accumulates
remains unknown. We are very curious about this. Then we designed a simple
immunofluorescent experiment to visualize the p53 in PRMT1 knockdown cells. Surprisingly we
discovered that PRMT1 also regulates the subcellular localization of p53. Our pilot assays
showed that PRMT1 knockdown increased cytosolic localization of p53, which are repeated and
validated in the results part.
To determine how PRMT1 regulates the localization of p53, we did a literature study on p53
translocation. The transcriptional activity of p53 is highly dependent on its localization to the
nucleus, which is regulated primarily by the CRM1 nuclear export pathway(Cai and Liu, 2008;
Freedman et al., 1998). CRM1 is a major nuclear export receptor that carries diverse cargoes,
including proteins, small nuclear RNAs, and ribosomal subunits, to the cytoplasm (Fornerod et
al., 1997). CRM1-dependent nuclear export of p53 is further controlled by poly(ADP-ribose)
polymerase 1(PARP1), illustrated by the graphic mechanism in the following figure (Kanai et
al., 2007), (Figure 1.5)
12
Figure 1.5 p53 is translocated to the cytoplasm through CRM1-p53 export system.
Normally, p53 interacts with CRM1, a kind of nuclear exportin, which carries p53 to the
cytoplasm through nuclear pore. However, when DNA is damaged, PARP1 is activated and
modifies p53. The modified p53 can no longer interact with CRM1 since the interacting domain
alters its structure after the modification. Afterwards, p53 is arrested in the nucleus and performs
its functions. Figure is taken from Kanai and Hanashiro, (2007)
PARP1 catalyzes the polymerization of ADP-ribose units and the attachment of linear or
branched PAR polymers to the substrate. The enzymatic activity of PARP1 is activated markedly
by stress including DNA damage (Ray Chaudhuri and Nussenzweig, 2017). PARP1 consists of 3
main domains shown in Figure 1.6.
13
Figure 1.6 PARP1 participates in DNA damage repair.
(a) PARP1 has a DNA-binding domain (DBD), containing three zinc finger motifs and a nuclear
localization signal; a central automodification domain; and a carboxy-terminal catalytic domain
that contains a signature of PARP proteins, the conserved domain (CD). (b) PARP1 detects DNA
damage site with DBD, and it is activated by catalyzing poly(ADP-ribose) (PAR) chains
formation (mainly on itself). (c) PARP1 recruits various proteins to the sites of DNA damage for
different aspects of DNA repair. Ade, adenosine; P, phosphate residue; Rib, ribose moiety;
ssDNA, single-stranded DNA. Figure is taken from Ray Chaudhuri and Nussenzweig, (2017)
14
There are evidence showing the co-effect of PRMT1 and PARP1. For examples, the inhibitors of
PRMT1 and PARP1 have synergistic effects against non-small cell lung cancer cells. Treatment
combining PARP inhibitors and type I PRMT inhibitors is a novel therapy to cancer cells
resistant to PARP inhibitors (Dominici et al., 2021). PRMT1 also coactivates NF-κB-dependent
gene expression synergistically with PARP1 (Hassa et al., 2008). But whether PRMT1 directly
regulates PARP1 is still unclear. Our hypothesis is that PRMT1 regulates the expression level of
PARP1 or the interaction between PARP1 and p53, leading to the nuclear export of p53.
15
Chapter 2 Materials and Methods
2.1 Materials
Table 2.1 Materials used in the experiments
REAGENT or RESOURCE SOURCE
Rabbit polyclonal anti-PRMT1 Cell Signaling Cat# 2449; RRID:AB_2237696
Mouse polyclonal anti-PARP1 Proteintech Cat# 66520-1-1g
Rabbit polyclonal anti-PARP1 Proteintech Cat# 13371-1-AP
Mouse monoclonal anti-p53 Cell Signaling Cat# 2524; RRID:AB_331743
Rabbit polyclonal anti-p53 Sana Cruz Cat# sc-6243; RRID:AB_653753
HRP secondary antibodies Jackson Immunoresearch various source
AlexaFluor secondary antibodies Invitrogen Cat#11029, #A11012
PRMT1 siRNA QIAGEN Cat# SI00441861 and SI02663493
PARP1 siRNA QIAGEN Cat# S102662996, S104433982 and
S104433989
Negative control siRNA QIAGEN QIAGEN Cat# 1022076
PARP1 overexpression plasmid Addgene
Triton X-100 Sigma Cat# X100
DAPI Sigma Cat# D9542
Paraformadehyde Sigma Cat# P6148
BioRad Protein Assay Bio-Rad Cat# 5000006
ECL developing reagent GE Life Sciences Cat# RPN2106
Lipofectamine RNAiMAX Thermo Fisher Cat# 13778500
Lipofectamine DNA Transfection
2000 Reagent
Invitrogen #1881534
MEC1 mouse epicardial cell line Li et al., 2011(Li et al., 2011)
16
2.2 Methods
2.2.1 Cell culture
MEC1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented
with 10% fetal bovine serum (FBS). Cells were subcultured when they reached 70-80%
confluence. The cells were kept at 37 ° C and 5% CO 2.
2.2.2 Transfection
For siRNA transfection assay, MEC1 cells were plated at 0.5~1 x 10^4 cells per well in 4-well
chambers, and transfected with the siRNA-RNAiMax (Invitrogen) complex following
manufacturer's protocol. Four to six hours after transfection, cells were moved to fresh medium
containing 10% FBS and cultured in 37° C for another 2 days. Detailed concentration and ratio
are shown in the following table. The concentration of siRNA is 20μM.
Table 2.2 siRNA transfection compound concentration
siRNA per well RNAiMax per well Total compound per well
Chamber 1 μl 2 μl 50 μl
6-well plate 5 μl 10 μl 200 μl
For DNA transfection, MEC1 cells were plated at 5 x 10^4 cells per well in 4-well chambers,
and transfected with 0.5μg plasmid. Detailed concentration and ratio are shown in the following
table.
Table 2.3 DNA transfection compound concentration
DNA per well Lipo 2000 per well Total compound per well
Chamber 0.5 μg 2 μl 25μl
6-well plate 2.5μg 10μl 100μl
17
2.2.3 Immunofluorescent staining
Cells cultured in 4-well chambers were washed gently and briefly for 3 times with room
temperature PBS, followed by fixation in 4% paraformaldehyde (PFA) at room temperature for
15 minutes. Cells were then blocked for 1 hour using 10% goat serum in PBS with 0.1% Triton
X-100. Next, cells were incubated with primary antibodies at 4° C overnight. On the next day,
cells were washed by PBS in room temperature for 3 times (5 minutes per time) and incubated
with second antibodies and DAPI (0.2μg/ml) for 1 hour in darkness at room temperature. Then
the mounting medium was added to the slides after 3 times PBS washing. The slides were sealed
by nail polish and stored at 4° C .
Antibodies and dilutions used were: PRMT1(1:100), p53 (1:200), PARP1 (1:100), fluorescence
secondary antibody (488 nm and 594 nm) (1:400). The stained cells were observed and imaged
on a Leica DMI 3000B microscope. The images taken by Leica 3000B were analyzed in ImageJ
and the quantification of immunofluorescent signal was performed in CellProfiler 4.2.5 using
meanintensity function.
2.2.4 Western blotting
Cells collected from 6-well plates were lysed in approximately 120 μl lysis buffer (50mM pH 7.5
Tris-HCl, 250mM NaCl, 2mM EDTA, 0.1% NP-40, 10% glycerol, supplemented with protease
inhibitor cocktail) per well. The concentration of protein was quantified using Bio-Rad protein
assay (Bio-Rad Laboratories), and 5-50 μg of protein (load 5-10 μg for PARP1) was separated
by SDS-PAGE on a 10% acrylamide gel run at 70-90V. The separated proteins were then
transferred to 0.45 mm PVDF membrane at 30V for 16 hours.
18
Membranes were shaking blocked in TBS, 0.1% Tween 20, and 5% powdered milk (blocking
solution) for 2 h, followed by overnight incubation with primary antibody diluted in TBS, 0.1%
Tween 20, and 3% BSA at optimal concentration: PRMT1 1:1000, PARP1 1:2000, and 1 h
incubation with HRP-conjugated secondary antibody diluted at 1:5,000. Immunoreactive protein
was detected using ECL (GE Healthcare) and HyBlot CL film (Denville Scientific)
19
Chapter 3 Results
Our hypothesis is that PRMT1 controls the subcellular localization of p53 via PARP1. Therefore,
our main approach is to knockdown the expression of the expected upstream protein and
visualize the distribution of p53 by immunofluorescent staining. In the future, subcellular
fractionation followed by western blot will be conducted to strengthen the conclusion.
3.1 PRMT1 controls the subcellular localization of p53.
We used an embryonic epicardial cell line, MEC1 cells, which was previous utilized in Dr.
Jackson-Weaver’s study for the identification of PRMT1-p53 pathway in epicardial invasion
(add reference). To determine whether PRMT1 regulates the nuclear localization of p53, we
transfected MEC1 cells with control siRNAs or two independent siRNAs that depleted PRMT1
(QIAGEN Cat# SI00441861), and performed immunofluorescent staining to assess the
subcellular localization of p53.
The figure showed that p53 localized mainly in the nuclei in cells treated with control siRNA,
indicated by the co-localization of p53 with DAPI (Figure 3.1A). In contrast, p53 was
predominantly in the cytosol in cells treated with PRMT1 siRNAs (Figure 3.1B) We further
quantified the nuclei/cytoplasm ratio of p53 and showed nuclear export of p53 in PRMT1
knockdown cells (Figure 3.1C). These findings demonstrated that PRMT1 regulates the
subcellular localization of p53. In addition, p53 had a higher level in PRMT1 knockdown cells,
which is consistent with Olan’s previous studies (Jackson-Weaver et al., 2020).
20
21
Figure 3.1 PRMT1 knockdown altered p53 subcellular localization.
The experiments validated the nuclear export of p53 in PRMT1 knockdown MEC1 cells.
PRMT1 and p53 were visualized by red and green fluorescence secondary antibodies. We see
very low red signal in panel B, which means PRMT1 knockdown is successful (A) p53 stayed in
the nuclei in the cells treated with control siRNA. The PRMT1 staining validated the relatively
high level of PRMT1. (B) Cells treated with PRMT1 siRNAs performed a different subcellular
distribution of p53. The cells whose p53 was mainly located in cytoplasm can be clearly
identified. (C) The panel shows the quantification result of the immunofluorescent pictures. The
cells treated with PRMT1 siRNAs had higher intensity of p53 (p-value < 0.001), which is
consistent with Dr. Jackson-weaver’s result that PRMT1 knockdown stabilize p53. On the other
hand, the cells treated with PRMT1 siRNAs had a significant lower p53 nuclei/cytoplasm ratio
(p-value < 0.001).
3.2 PRMT1 controls the expression level of PARP1
To test whether the expression level of PARP1 is controlled by PRMT1, we depleted PRMT1 in
MEC1 cells and assessed whether PARP1 expression was altered by immunostaining and
Western Blotting. First, we used two different PRMT1 siRNAs to knockdown PRMT1 and
performed immunostaining for visualizing PARP1. The protein level of PARP1 was significantly
reduced in the cells treated with PRMT1 siRNAs. Next, we conducted western blotting analysis
and confirmed that PARP1 protein expression was reduced by PRMT1 knockdown. Altogether,
these data suggested that PRMT1 knockdown led to a reduction in PARP1 protein expression.
22
Figure 3.2 PRMT1 knockdown downregulated the level of PARP1.
(A) and (B) PARP1 Immunostaining results show that the protein level of PARP1 was reduced
in PRMT1 knockdown cells. We see very low red signal in (B), which means PRMT1
23
knockdown was successful. (C) Panel shows the Western blotting results showing PARP1
expression level was decreased in the cells treated with PRMT1, which strengthens the
conclusion that PRMT1 knockdown downregulate the expression level of PARP1.
3.3 PARP1 knockdown led to nuclear export of p53.
Next, we determined whether PARP1 is involved in the regulation of p53 localization. We first
generated PARP1 knockdown MEC1 cells to examine whether PARP1 is required for p53
localization in the nucleus. We quantified the p53 nuclei/cytoplasm ratio in MEC1 cells
transfected with control siRNA or 3 different PARP1 siRNAs (QIAGEN Cat# S102662996 is the
best for PARP1 knockdown) and showed that p53 exhibited enhanced localization in the cytosol
in PARP1 knockdown cells
24
Figure 3.3 PARP1 knockdown alters the cellular p53 distribution.
The result of this group experiments suggests that PARP1 knockdown had a similar effect on the
distribution of cellular p53. (A) and (B) Both panels show a significantly different distribution of
p53 in the cells under different treatment. In contrast of the nuclear p53, p53 in the cells treated
with PARP1 siRNAs was not arrested in the nuclei. Panel (C) is the result of immunofluorescent
quantification. The cells under PARP1 siRNA treatment had a significantly lower p53
25
nuclei/cytoplasm ratio. The quantification results also strengthen the conclusion that PARP1 has
a similar effect on p53 distribution.
3.4 Overexpression of PARP1 reverse the nuclear export of p53 in PRMT1-depleted cells.
To determine whether PRMT1 regulated p53 localization via PARP1, we designed a group of
rescue experiments — overexpressing PARP1 in the PRMT1 knockdown cells.
We transfected MEC1 cells with control or PRMT1 siRNAs followed by transfection of DNA
plasmid expressing empty vector or PARP1. Then p53 localization was analyzed by
immunostaining. In our negative control groups (treated with control siRNA and empty vector),
the co-staining result showed that p53 mainly localized in nuclei (Figure 3.4A). But in our
positive control groups, we found that the difference in fluorescence signal intensity between the
nucleus and cytoplasm was significantly smaller (Figure 3.4B), and the level of PARP1 was
significantly reduced. When we transfected the cells with both PRMT1 siRNA and PARP1
overexpression plasmid (Figure 3.4C), the level of PARP1 was restored and p53 mainly
localized in nuclei again. Furthermore, the quantification of the IF results suggested that the
nuclei/cytoplasm ratio of p53 follows the same trend as the level of PARP1 and the nuclear
export of p53 in PRMT1 knockdown cells could be reversed by PARP1 overexpression (Figure
3.4D and E).
26
27
Figure 3.4 Overexpression of PARP1 in PRMT1 knockdown cells rescued the nuclear
export of p53.
Compared with previous siRNA knockdown experiments, double transfection experiments need
more transfection reagent, which is toxic to the cells. Therefore, the density of the cells is lower
28
and the cells are under some degree of stress. (A) Similar to Figure 3.1A, these negative control
cells (treated with control siRNAs and plasmid) kept p53 in their nuclei and showed strong
PARP1 signal. (B) We can neither see the boundary of the nucleus nor see a strong red
fluorescence signal in the cells treated with PRMT1 siRNA and control plasmid. (C) Some
PRMT1 knockdown cells treated with PARP1 overexpression plasmid regain the high level of
p53 in nuclei. (D) The quantification of the p53 immunofluorescent staining. (E) The
quantification of PARP1 level.
We also noticed that the nuclear export of p53 in this group of double transfection experiments
was not as significant as in the previous transfection group, potentially because of toxicity from
transfection reagents.
29
Chapter 4 Discussion and Future Directions
In summary (Figure 4.1), we discovered that PRMT1 regulates the subcellular localization of
p53 and provide evidence that PARP1 is an important component of this pathway. PRMT1
knockdown downregulates the expression level of PARP1, which inhibits the nuclear export of
p53.
Figure 4.1 PRMT1 controls the distribution of p53 via PARP1.
In PRMT1 knockdown cells, the expression level of PARP1 is downregulated. The modification
on p53 then is weakened. The unmodified p53 can pass through the nuclear pore and move to the
cytoplasm.
Experimental method restrictions
However, we still have an unsolved problem about experiment condition. We observed a mass
cell death in double transfection experiment, potentially because of the harsh environment
caused by excessive transfection reagent. How to overcome this issue? One possible way is to
create PARP1 or PRMT1 inducible conditional knockout cell lines using Crispr-Cas9 system,
30
and conduct single transfection using these knockdown cells. However, this approach takes a
long time that expands beyond the project period.
In our original expectation, we analyzed immunofluorescent images by quantifying fluorescent
intensity. However, the method also has restrictions. In some cells, the fluorescent signal
intensity of some proteins, such as p53,is too weak to be detected. But the strong signals were
always captured. This eventually leads to some data not conforming to the normal distribution,
like shown in Figure 4.2.
Figure 4.2. The histogram based on the original data of PARP1 knockdown group shows
that this group of data didn’t conform to the normal distribution.
We guess the reason is that the data in the left tail of the normal distribution is out of detective
range of the immunofluorescent microscope or the camera. We could try a longer exposure time
or lower the minimum threshold, but these 2 approaches will defiantly cause worse accuracy.
31
One reasonable way is to try another detecting method such as western blot (though it would be
very hard because the half-life time of p53 is too short, only about 4 minutes).
Future Directions
Are there other proteins involved in the PRMT1-p53 pathway?
MDM2/4 are candidates. MDM2 binds to PARP1 and activates ubiquitination to enhance DNA
replication fork progression (Giansanti et al., 2022). Though the level of MDM2 doesn’t alter in
PRMT1 knockdown cells, MDM4 responds to PRMT1 knockdown (Bardot et al., 2015). MDM4
is an important regulator of MDM2 and itself shares a similar structure with MDM2 (Shvarts et
al., 1996). MDM4 is likely to play a role in the MDM2-PARP1 regulation, or MDM4 itself may
regulate PARP1 since it shares structural similarity with MDM2. Therefore, the role of MDM4
or MDM4-S in the regulation of PARP1 awaits further investigation.
How does PRMT1 regulate PARP1?
Our findings raised a follow-up questions:how does PRMT1 regulate PARP1? There are 2
potential ways, regulating the level of PARP1by facilitating expression or suppressing the
degradation of PARP1, or regulating PARP1 activity by binding its interaction domain. To
answer the question, we could quantify the mRNA level or the half-life time of PARP1 and do
Co-IP analysis.
Previously we have discussed about MDM4, by which PRMT1 may regulate the degradation of
PARP1. Meanwhile, we have a pilot result indicating that PRMT1 may disturb the interaction
between PAPR1 and p53. One interesting question is that who is responsible for the less
interaction between PARP1 and p53? The downregulated level of PARP1 or weakened
32
interacting ability? This specific mechanism is an interesting research direction. Our guess is that
the interaction between of p53 and PARP1 might be disturbed. In normal cells, due to the fast
degradation, p53 is at a very low level (Hafner et al., 2019), which means PARP1 is in excess
compared to p53. ,
What is the function of the cytosolic p53?
Previous researches unraveled the function p53 in cytoplasm, where it triggers apoptosis by
MOMP (Figure 4.3) and inhibits autophagy, exerting control on cell fate (Green and Kroemer,
2009). Does cytosolic p53 in PRMT1 knockdown cells triggers apoptosis? Does cytosolic p53 in
PRMT1 knockdown cells inhibit autophagy? In Dr. Jackson-weaver’s research, we discovered
the heart chamber walls of PRMT1 knockout mice were significantly thinner (Figure 1.4A).
PRMT1 knockout upregulated the level of p53 and thus inhibited the EMT process, finally
results in heart development deficiency (Jackson-Weaver et al., 2020). According to our results,
PRMT1 knockdown facilitated the nuclear export of p53, which may play a role in heart
development deficiency. It’s interesting to study the if there is upregulated apoptosis during the
EMT process in the PRMT1 knockdown/out cells.
33
Figure 4.3 Cytosolic p53 triggers apoptosis.
The expression of MDM2 is induced by nuclear p53 and MDM2 ubiquitinates p53 through a
feedback inhibition. However, when the cell is under stress, p53 would be phosphorylated, which
is insensitive to MDM2 inhibition. Finally, p53 accumulates and p53 upregulated modulator of
apoptosis (PUMA) are thus upregulated. In cytoplasm, p53 originally is sequestered by anti-
apoptotic Bcl2 proteins such as Bcl-XL and therefore won’t trigger apoptosis. But the upregulated
PUMA disrupts the Bcl-XL–p53 interaction. The released p53 now can trigger apoptosis through
mitochondrial outer membrane permeabilization (MOMP). Figure adapted from Green and
Kroemer (2009).
Cytoplasmic p53 activates mammalian target of rapamycin (mTOR, also known as Frap1), a
negative regulator of autophagy and also inhibits the AMP-dependent kinase, a positive regulator
of autophagy (Tasdemir et al., 2008). To answer the 2 questions above, researchers may study
the interaction between PRMT1 and these important proteins in apoptosis and autophagy, such as
34
Bcl-XL and mTOR and AMP- dependent kinase. In addition, comparing the difference between
PRMT1 knockdown cells and p53 overexpression cells may also help demonstrating the role that
PRMT1 plays in apoptosis and autophagy.
What are the dynamics of cytosolic p53 in PRMT1 knockdown cells?
As we mentioned previously, both the expression level and dynamic of p53 control cell fate
(Hafner et al., 2019). Researchers discovered that both surviving and dying cells reach similar
levels of p53, suggesting that cell death is not determined by a fixed p53 threshold, but depends
on the time and levels of p53 (dynamic). For instance, in human colon cancer cell research, the
apoptotic cells accumulated p53 earlier and faster than surviving cells (Paek et al., 2016). In
addition, as we mentioned in introduction part, converting the pulses of p53 level into sustained
p53 activation by a combination treatment of ionizing radiation with MDM2 inhibition result in
an irreversible cell cycle arrest and cellular senescence (Purvis et al., 2012). So, what is the
dynamics of the cellular p53 after PRMT1 knockdown? Does p53 accumulates fast? What about
the degradation speed? Answering these questions could be very helpful in understanding the
fate of those PRMT1 knockdown cells and interpreting biological function of the PRMT1-p53
pathway.
35
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Abstract (if available)
Abstract
As a well-known tumor suppressor, p53 is a transcription factor that functions in the nuclear and transactivates genes involved in diverse cellular stresses to regulate expression of target genes, such as the cell cycle arrest, apoptosis, senescence, DNA repair, or changes in metabolism. However, it also plays an important role in the cytoplasm, where it triggers apoptosis and inhibits autophagy under stress. We previously uncovered a PRMT1-p53 pathway, in which PRMT1 increases the turnover of p53. In this thesis project, we further show that PRMT1 plays an important role in nuclear export of p53. PRMT1 knockdown significantly altered the subcellular distribution of p53. Using immunostaining and western blotting, we showed that p53 transferred to the cytoplasm from the nuclei in PRMT1 knockdown mouse epicardial cells (MEC1). Poly(ADP-ribose) polymerase 1 (PARP1) is potentially a key component of this pathway. PRMT1 downregulated the expression level of PARP1, causing p53 to translocate to the cytoplasm. Our results showed that both PRMT1 and PARP1 knockdown significantly increased cytosolic distribution of p53Altogether, our study discovered a PRMT1-p53 translocation pathway via PARP1.
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Hua, Wuyue
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Core Title
PRMT1 controls subcellular localization of p53 via PARP1
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Keck School of Medicine
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Master of Science
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Biochemistry and Molecular Medicine
Degree Conferral Date
2023-08
Publication Date
06/02/2024
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05/11/2023
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airlace1997@gmail.com,wuyuehua@usc.edu
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(batch),
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the author, as the original true and official version of the work, but does not grant the reader permission to use the work if the desired use is covered by copyright. It is the author, as rights holder, who must provide use permission if such use is covered by copyright.
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Repository Email
cisadmin@lib.usc.edu
Tags
localization
p53
PARP1
PRMT1