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A novel screening approach to identify regulators of polycomb-dependent gene silencing

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A novel screening approach to identify regulators of polycomb-dependent gene silencing

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A Novel Screening Approach to Identify Regulators of Polycomb-Dependent Gene Silencing

By

Bailey Richardson

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR MEDICINE)

August 2023
ii
Acknowledgements
I would like to give special thanks to my mentor, Dr. Oliver Bell for his invaluable guidance and
support throughout my journey in the lab. His expertise, patience, and dedication have been
instrumental in shaping my academic and research experiences. I extend my deepest appreciation
to Dr. Bell for his instrumental role in my professional life, and for instilling in me a passion for
scientific inquiry that will continue to guide my future endeavors.

I would also like to thank all of my lab members in the Bell Lab, past and present, for their never-
ending support and guidance, including Eda Atmaca, Jasmine Martinez, Daniel Bsteh, Matthew
Lowe, Shweta Mendiratta, Bo Cheng, Roy Bian. and honorary Bell Lab member Ben Weekley.

I would also like to thank my committee members, Dr. Unmesh Jadhav and Dr. Yali Dou for their
insightful guidance throughout my research project.

Thank you to everyone in the MSSR lab at UCLA, including Dr. Robert Damoiseaux and Brandon
Han

Finally, I would like to thank my partner, Andrea for her unwavering support through my early
mornings and late nights studying and working in the lab the past two years.

iii
Table of Contents
Acknowledgements………………………………………………………………………..………ii
List of Figures…………………………………………………………………………………….iv
Abstract…………………………………………………………………………………………....v
Chapter 1: Introduction…………………..………………………………………………………..1
Chapter 2: Methods…..……………………………………………………………………………6
Chapter 3: Results
Selection of endogenous polycomb target for tagging…………………………………..10
Development and clone selection of endogenous polycomb reporter…………………...12
Characterization of endogenous polycomb reporter mESCs…………………………….15
Polycomb reporter sensitivity to CRISPR deletion…………………………………...…18
Polycomb reporter is sensitive to RNAi knockdowns……………………………..….…19
RNA knockdown can be scaled to HTS format………………………………………….22
Chapter 4: Discussion………..………………………………………………………………..…26
References………………………………………………………………………………………..30

iv
List of Figures
Figure 1: Schematic of Polycomb Group Protein interaction…………………………………….5
Figure 2: Selection of endogenous polycomb target for tagging……………………………...…11
Figure 3: Development and clone selection of endogenous polycomb
reporter in mouse embryonic stem cells with vPRC loss of function……...………………….....14
Figure 4: characterization of endogenous polycomb reporter mESCs……………………….…...17
Figure 5: Polycomb reporter is sensitive to CRISPR deletion of polycomb genes………………..19
Figure 6: Polycomb reporter is sensitive to RNAi knockdown of polycomb genes………...…….21
Figure 7: RNAi knockdown in polycomb reporter mESCs can be scaled
down to a high-throughput screening format………………………………………………….....24

v
Abstract
Polycomb group (PcG) proteins are highly conserved transcriptional repressors that are
critical for maintenance of cell identity. Mutations and dysregulation of genes encoding PcG
proteins are frequently associated with human cancer, motivating the development of novel
therapies. Originally identified in Drosophila melanogaster, PcG proteins assemble into two main
families of repressors, Polycomb repressive complex 1 (PRC1) and PRC2, which enforce gene
silencing through catalytic and non-catalytic modifications of chromatin structure. PRC1 is
thought to exert gene silencing activity by catalyzing monoubiquitination of histone H2A at lysine
119 (H2AK119ub1) and mediating chromatin compaction whereas PRC2 catalyzes methylation of
histone H3 at lysine 27 (H3K27me1, 2 and 3). By recognizing each other’s histone modifications,
PRC1 and PRC2 engage in a feedback loop that stabilizes and enhances the formation of
transcriptionally repressive chromatin domains. In mammals, PcG protein paralogues have
massively expanded and diversified giving rise to six distinct canonical and variant PRC1
subcomplexes with different compositions and functions. To dissect the mechanisms of canonical
PRC1-dependent gene silencing, we developed a novel endogenous reporter system in mouse
embryonic stem cells (mESCs). During my MSc thesis, I characterized nature and dependence of
this reporter and showed that it has great sensitivity and selectivity to genetic and pharmacological
perturbations of canonical PRC1 and PRC2. I subsequently worked to adapt the reporter system to
facilitate systematic, comprehensive screening using RNAi to identify novel genetic dependencies
in mammals. In the future, we hope to use this genetic screen to compliment small molecule
screening also being performed with this reporter.

1
Introduction
Epigenetics encompasses modifications or marks that can be added to the DNA molecule
or its associated proteins, exerting regulatory control over gene expression without altering the
DNA sequence itself.
1
These modifications have a profound impact on the activation or silencing
of genes, thereby playing a pivotal role in determining their functional outcomes. Epigenetics not
only enhances our understanding of gene regulation, development, aging, and disease origins, but
also underscores the dynamic nature of gene expression. It reveals how external factors can
influence our genetic inheritance beyond the DNA sequence alone. While DNA-binding
transcription factors are fundamental in regulating gene expression, the significance of post-
translational modifications of histones and chromatin cannot be overstated. These crucial
processes contribute to the intricate orchestration of gene regulation in multicellular organisms.
2

The discovery of Polycomb group (PcG) proteins has greatly advanced our understanding
of gene regulation and epigenetic control. These proteins were initially identified in Drosophila
melanogaster as chromatin modifiers involved in the regulation of homeotic (Hox) genes, which
play a crucial role in determining body segment identity and development.
3
This finding in fruit
flies paved the way for subsequent investigations that revealed the conservation of PcG proteins
across species, including mammals, highlighting their fundamental importance in diverse
biological processes.
4

The conservation of PcG proteins across species, from flies to mammals, underscores their
fundamental and evolutionary significance in orchestrating cellular identities, tissue
differentiation, and the maintenance of cellular states. Furthermore, the discoveries surrounding
PcG proteins have broadened our understanding of epigenetic regulation, highlighting the dynamic
interaction between DNA sequence and chromatin modifications. This intricate interplay,
2
governed by PcG proteins and other chromatin modifiers, enables the precise control of gene
expression patterns, allowing cells to respond to developmental cues and environmental stimuli.
PcG proteins operate within multiprotein complexes known as polycomb repressive
complexes (PRCs). These groups can be broadly broken up into PRC1 and PRC2 and play a
significant role in establishing polycomb chromatin domains, which are believed to counteract
transcriptional activity. PRC2 functions as a methyltransferase responsible for the methylation of
lysine 27 on histone H3 (H3K27), encompassing mono-, di-, and trimethylation (H3K27me1/2/3).
The catalytic activity of PRC2 relies on three core subunits: embryonic ectoderm development
(EED), suppressor of zeste 12 (SUZ12), and either enhancer of zeste homologue 2 (EZH2) or its
paralogue EZH1 (Fig. 1).
5
These subunits collaborate to deposit H3K27me3 marks, predominantly
found in facultative heterochromatin regions.
6
On the other hand, PRC1 functions as an E3
ubiquitin ligase that mono-ubiquitylates lysine 119 on histone H2A (H2AK119ub1).
7
It can be
further subclassified into canonical PRC1 (cPRC1) and variant PRC1 (vPRC1). In cPRC1,
chromobox domain-containing (CBX) proteins are recruited to H3K27me3 marks deposited by
PRC2, enabling the catalysis of H2AK119ub1, and thereby contributing to transcriptional
repression (Fig. 1). In contrast, vPRC1 has demonstrated the ability to catalyze H2AK119ub1
independently of H3K27me3 recruitment, functioning autonomously in gene silencing.
8
Both
cPRC1 and vPRC1 share the presence of the protein really interesting new gene 1A (RING1A) or
its paralogue RING1B, which acts as the catalytic subunit of PRC1 (fig. 1).
While the involvement of polycomb in gene repression is well-established, unraveling the
precise molecular mechanisms underlying this process remains a pivotal and unresolved question
in the field. Recent investigations have shed light on the diverse range of mechanisms through
which polycomb repressive complexes are recognized and recruited, involving transcription
3
factors, CpG islands elements, and non-coding RNAs, revealing a diverse repertoire of factors that
contribute to the recruitment of polycomb complexes.
9
Moreover, as our understanding of the
polycomb system expands, it has become increasingly evident that the interplay between PRC1
and PRC2 is far more intricate than initially acknowledged. This heightened awareness emphasizes
the critical need for a comprehensive comprehension of the polycomb system's dynamics.
Furthermore, the significance of this pursuit is underscored by the prevalence of mutations
in genes encoding chromatin structure and DNA modification regulators, including PRC1 and
PRC2, in the majority of human cancers.
10
The dysregulation of PcG proteins has been implicated
in various cancer types, spanning lymphomas, leukemias, bladder cancer, melanoma, and
pancreatic cancer.
6,11,12
Recent studies have highlighted the pivotal roles of PcG proteins in the
development and progression of cancer. The deregulation and dysfunction of PcG proteins often
result in the aberrant activation or suppression of developmental pathways, promoting
uncontrolled cellular proliferation, suppressing apoptosis, and fostering the expansion of cancer
stem cell populations.
11
These findings illuminate the impact of PcG proteins in cancer
pathogenesis, presenting them as promising targets for therapeutic intervention and further
underscoring the significance of unraveling their precise biological mechanisms.
Due to their implication in cancer development and progression, PRC1 and PRC2 have
emerged as promising therapeutic targets. Consequently, several small molecule inhibitors have
been discovered, targeting key components such as Ring1B, Cbx7, and the catalytic activity of
EZH2.
13,14,15
Notably, certain EZH2 inhibitors have been recently approved for the treatment of
specific polycomb-mutated cancers.
16
However, as EZH2 is ubiquitously expressed and has been
found to possess tumor suppressor activity, there are concerns regarding the potential risk of
secondary cancer formation associated with EZH2 inhibitor treatment.
17
Furthermore, the
4
development of resistance to EZH2 inhibitors has been observed as tumors acquire mutations
17
,
highlighting the need for alternative therapeutic strategies that target polycomb-dependent cancers.
Studying polycomb biology presents inherent challenges due to its intricate nature and the
mechanisms involved in gene regulation. One of the primary difficulties arises from the repressive
nature of polycomb complexes. Polycomb-mediated gene silencing leads to the suppression of
target genes, resulting in low expression levels. Consequently, detecting the upregulation of these
genes upon the removal of polycomb becomes challenging because of the modest increase in
expression, impeding the comprehensive understanding of polycomb function. Furthermore, the
multifaceted interactions and context-dependent roles of polycomb complexes contribute to the
difficulty of studying their biology. Polycomb proteins operate in intricate networks, interacting
with numerous cofactors and other regulatory elements. Additionally, the diverse functions of
polycomb complexes across different cellular contexts and developmental stages further
complicate their study, necessitating the use of novel strategies for research.
Previously, our laboratory has developed an exogenous system to recapitulate regulation
of specific PRC1 or PRC2 complexes and investigate the underlying mechanisms. We utilize
TetOFF to reversibly tether PRC1 or PRC2 to an ectopic reporter locus in mouse embryonic stem
cells (mESCs).
19
Through fusion with the Tet repressor (TetR) DNA binding domain, which binds
to a genomic Tet operator (TetO) site, tethering of specific subunits effectively nucleates PRC1 or
PRC2 complexes at the ectopic reporter locus. Furthermore, addition of Doxycycline reverses TetR
interaction with TetO, allow to examine the capacity to establish epigenetic modifications that
promote heritable silencing in the absence of the initial stimulus. Using this approach, we
demonstrated that canonical PRC1 but not variant PRC1 is able to promote long-term silencing.
19

5
While our synthetic system has proven effective to simplify complex regulation, there are
concerns about the artificial nature of this approach. Therefore, we aimed to establish a polycomb
reporter system using endogenous polycomb genes in mESCs. This system disrupts the major
vPRC1 complex, shifting gene silencing dependence to the canonical cPRC1/PRC2 pathway. We
validated the system using an auxin-based degron, observing increased expression of endogenous
polycomb genes. Gene expression changes were quantified using a fluorescence readout.
Studying polycomb biology is crucial for understanding gene regulation, development, and
the origins of diseases. Here, we present a novel endogenous polycomb reporter cell line that we
hope will serve as a powerful tool for high-throughput screening in both genetic and small
molecule experiments. Through our genetic screen, we aim to uncover unknown aspects of
polycomb-dependent silencing, providing insights into the mechanisms underlying gene
regulation. This approach is useful as polycomb-mediated gene silencing is complex, and
comprehensive knowledge of its biology is necessary for developing alternative therapeutics for
polycomb-mutated cancers.

Figure 1: Schematic of Polycomb Group Protein interaction
Visual representation of the catalytic activities of vPRC1, cPRC1, and PRC2 complexes. PRC2
deposits H3K27me3 marks which is recognized by Cbx7 subunit of cPRC1 to engage gene
silencing, while vPRC1 is H3K27me3 independent.
6
Chapter 2: Methods
Culture conditions and treatments of mESCs
Pcgf1 KO dSuz12 mESCs were cultivated in high-glucose-DMEM supplemented with 13.5% fetal
bovine serum (sigma), 10mM HEPES pH 7.4, 2mM GlutaMAX (Gibco), 1mM Sodium pyruvate
(Sigma), penicillin/streptomycin (Sigma), 1x non-essential amino acids (Sigma), 50uM beta-
mercaptoethanol (Gibco), and recombinant LIF at 37 °C, 5% CO2. HEK293T cells cultured in
same conditions with media not containing LIF. For treatments with auxin and inhibitors, cells
were plated in solid white, clear bottom 96-well tissue culture plates (Corning 3903). 250uM auxin
(IAA Sigma), 5uM Tazemetostat, or 20uM UNC4976 was added to cells 24 hours after plating. 72
hours after treatment, luminescence was measured using the Nano-Glo HiBiT Lytic Assay.

HiBiT Luminescence assays
2,000 cells were plated in solid white, clear bottom 96-well tissue culture plates (Corning 3903) in
100ul growth medium. 24 hours after seeding, cells were treated with compounds or virus added
for CRISPR knockouts. 72 hours after treatments, HiBiT was detected using the Nano-Glo HiBiT
Lytic Detection System (Promega) following manufacturer’s instructions. 100ul of Nano-Glo
HiBiT Lytic Detection Reagent was added directly to the cells and placed on an orbital shaker for
5 minutes. After, cells were incubated at room temperature for 10 minutes before recording
luminescence on the CLARIOstar Plus Microplate Reader. For 384-well experiments, 2500 cells
were plated in solid white, clear bottom 384-well tissue culture plates (Greiner 781080). Prior to
HiBiT reagent dispensing, all but 15ul of medium was aspirated from each well using the EL406
washer dispenser for BioTek. 15ul of Diluted (1:2) HiBiT was added to cells and luminescence
was read on the EnVision Multimode Plate Reader.
7

Flow cytometry analysis
All Flow cytometry analyses were conducted on an Attune NxT Flow Cytometer (Thermo
Scientific). Cells were grown in 6-well tissue culture plates (Costar) until confluent. Prior to flow
cytometry analysis, cells were trypsinized for 5 minutes at 37 °C, 5% CO2 and resuspended in
growth medium at 2M cells/mL. 1mL of cells were transferred to 5mL polystyrene round-bottom
tubes with cell-strainer caps (Falcon 352235).

Western blot
Nuclear extract from 2 x 10
7
mESCs were obtained using nuclear lysis buffer (50mM HEPES-
KOH pH 7.3, 200mM KCL, 3.2mM MgCl2, 0.25% Triton X-100, 0.25% NP-40, 0.1% Na-
deoxycholate, 1mM DTT, 1x Complete Mini Protease Inhibitor) followed by collection in RIPA
buffer (150mM NaCl, 1% Triton, 0.5% sodium deoxy-cholate, 0.1% SDS, 50mM Tris pH 8.0).
Concentration of nuclear extracts were determined by Bradford assay (Biorad 23200). 30ug/lane
of protein was run on Novex Life Technology NuPAGE 4-12% Bis-Tris gels (Thermo Fisher
NP0336BOX) in Invitrigen NuPAGE MES SDS Running Buffer (G-Biosciences 786-531) and
transferred on a Merck Chemicals Immobilon-P Membrane PVDF 45um (Sigma-Aldrich
IPFL00010). The membrane was blocked (5% non-fat dry milk in 1x PBS and 0.1% Tween 20)
and incubated in 5% non-fat dry milk in 1x PBS and 0.1% Tween 20 with primary antibodies.
Following this, the membrane was incubated with corresponding secondary antibodies in 5% non-
fat dry milk, 1x PBS, and 0.1% Tween 20. Blots were imaged using the Odyssey CLx Imaging
System (LI-COR Biosciences).

8
CRISPR/Cas9 editing in mESCs
Knockout mESCs were generated using CRISPR/Cas9. CRISPR guide RNAs were designed
previously in the Bell Lab using the online tool of the Zhang lab (http://crispr.mit.edu, Zhang, MIT
2015). 300K HEK293T cells were seeded in 6-well plates (Costar). 24 hours later, 1ug desired
plasmid DNA was added with packaging vectors 90.91ng VSV-G (Addgene #8454) and 454.54ng
psPAX2 (Addgene #12260) along with Lipofectamine 3000 Transfection Reagent (Invitrogen
L3000001). 48-72 hours later, virus was collected and added to mESCs in the presence of 8ug/mL
polybrene (Santa Cruz Biotechnology, SACSC-134220). mESCs were incubated for 48 hours
before selection using 5ug/mL blasticidin for 5 days.

siRNA knockdowns
For transfection, 4,000 mESCs were plated in solid white, clear bottom 96-well tissue culture
plates (Corning 3903). After 24 hours, 10uM siRNA was transfected using Lipofectamine
RNAiMAX Reagent per manufacturer’s instructions. Cells were incubated for 72 hours at 37 °C,
5% CO2 and luminescence was read using the HiBiT Lytic Detection System. For reverse
transfection, 1pmol of siRNA was plated in each well of solid white, clear bottom 384-well tissue
culture plates (Greiner 781080) using the Beckman Biomek FX automated liquid handler.
RNAiMAX lipofectamine (Thermo Scientific 13778500) diluted in Opti-MEM Reduced Serum
Medium (Thermo Scientific 31985070) were added on top of the siRNAs and left to incubate at
room temperature for 15 minutes. 1,250 reporter mESCs per well were then added on top of siRNA
and transfection reagent in 50ul medium. Transfection reagent and cell dispensing was done using
the Multidrop Combi Automated Reagent Dispenser from Thermo Scientific. mESCs were
incubated at 37 °C, 5% CO2 for 72h. Prior to HiBiT reagent dispensing, all but 15ul of medium
9
was aspirated from each well using the EL406 washer dispenser for BioTek. Diluted HiBiT was
added to cells and luminescence was read on the EnVision Multimode Plate Reader.

DAPI staining
Polycomb reporter mESCs were plated in cell numbers ranging from 250 cells/well to 2500
cells/well were seeded in solid white, clear bottom 384-well tissue culture plates (Greiner 781080)
in 25uL cell culture medium using the Multidrop Combi Automated Reagent Dispenser. Following
a 72-hour incubation (37 °C, 5% CO2), DAPI was diluted 1:2000 into mESC culture media and
25ul of this solution was plated onto previously seeded cells using the Multidrop Combi
Automated Reagent Dispenser. The plates were again incubated (37 °C, 5% CO2) for 1 hour. The
cell number was then read using the ImageXpress MicroXL microscope.

10
Chapter 3: Results
Selection of Endogenous polycomb target for tagging
To begin creating our endogenous polycomb reporter system in mESCs, we utilized the
natural colocalization of cPRC1 and vPRC1 at key developmental target genes. Since vPRC1 has
been seen to maintain silencing independently of the PRC2/cPRC1 pathway
8
, we have shown the
loss of the major vPRC1 complex by Pcgf1 knock-out (PCGF1 KO mESC) abolishes the PRC2-
independent, non-canonical deposition of H2AK119ub1.
19
As a result, the endogenous polycomb
target genes become solely dependent on the canonical cPRC1/PRC2 silencing pathway. Using
this system, we can disrupt cPRC1/PRC2 and see an increase in endogenous polycomb genes. As
a way to test how this system works, the Bell lab has utilized an auxin-based degron system
which enables the inducible, proteasome-mediated degradation of target proteins.
20
By placing
this degron on the Suz12 subunit of PRC2, we can easily disrupt polycomb with the addition of
auxin and look for upregulation in endogenous genes.
To identify endogenous Polycomb target genes whose silencing is dependent on
canonical PRC1 and PRC2, we performed RNA-seq analysis. Next, the top upregulated genes
were filtered for presence of PRC1, PRC2 and histone modifications (Fig. 2a). Finally, we used
treatment of EZH2 inhibitor Tazemetostat or the CBX7 inhibitor UNC4976 coupled to RT-qPCR
to validate the dependence of the top candidates on cPRC1/PRC2 activity (Fig. 2b). From these
results, we see Irf8 exhibited a modest response, however, Osr2 and Pitx1 demonstrated the
highest degree of upregulation (Fig. 2b). Based on these experiments, we selected Osr2 and
Pitx1 as our endogenous polycomb target genes to be used for our reporter cell lines.

11

Figure 2: Selection of endogenous polycomb target for tagging
a) RNA-Seq tracks of known polycomb targets Suz12, H3K27me3, Ring1B, and Cbx7 at potential
endogenous polycomb target genes in mESCs(top) and expression of these genes before and after
degradation of polycomb subunit Suz12 (bottom). b) RT-qPCR analysis of top genes expression
in untreated (black) and EZH2 inhibitor-treated (orange) or Cbx7 inhibitor-treated (blue) mESCs.
Displayed are expression changes in Log2 fold. Data in this figure generated by Oliver Bell in the
Bell Lab.

a)
b)
12
Development and clone selection of endogenous polycomb reporter in mESCs with vPRC
loss of function
With these results, we proceeded with the selection of Osr2 and Pitx1 as high-confident
polycomb targets for 5’ tagging to introduce a HiBiT fused to blue fluorescent protein (BFP) in-
frame with the endogenous genomic sequences of each gene. The Nano-Glo® HiBiT Lytic
Detection System from Promega enables the tagging of our endogenous polycomb genes. This
system utilizes an 11 amino acid peptide tag known as HiBiT, facilitating the rapid and highly
sensitive quantification of cellular proteins. By introducing the complementary polypeptide
LgBiT, the HiBiT and LgBiT molecules interact, leading to the reconstitution of the NanoBiT®
enzyme. This reconstituted enzyme emits a bright luminescent signal that directly correlates with
the amount of HiBiT-tagged protein present in the cell lysate. Consequently, the luminescence
readout obtained from the HiBiT system serves as a direct indicator of gene expression levels.
21

Pcgf1 KO mESCs were electroporated with a CRISPR construct expressing Cas9 and
specific sgRNAs as a repair construct containing the HiBiT-BFP and was flanked by homology
arms for either Osr2 or Pitx1. Here, we created Pcgf1 KO mESCs that allow for both HiBiT lytic
detection of target protein de-repression as well as facilitate FACs analysis (fig. 3a). Cas9-BFP-
expressing cells were sorted using FACS, and from this population, we isolated 96 clones per
genotype. To confirm successful targeting, each clone was treated with auxin for 48 hours and
analyzed using flow cytometry to compare the BFP signal between untreated and auxin-treated
cells (Fig. 3b). When looking at histograms of the clones, we observe low sensitivity in
distinguishing between auxin-treated and untreated cells (Fig. 3c). This result is somewhat
expected, because one of the main reasons studying polycomb genes endogenously is difficult is
under normal conditions, they are repressed, and when polycomb is disrupted, they only show a
13
slight increase in expression. However, we have found when using the luminescent tag, we can
easily observe the increase in gene expression because of the sensitivity of the readout.
To test this HiBiT protocol, we selected the top 10 clones showing the most robust increase
in BFP expression and again treated them with auxin for 48 hours. Following the treatment, the
HiBiT Lytic Detection Reagent was added, and gene expression was measured by luminescence
(Fig. 3d). Utilizing the HiBiT system, we did observe a more substantial increase in gene
expression, allowing us to isolate the top clone from each reporter cell line. The top Osr2 clone
exhibited an approximate 8-fold increase in expression, while the Pitx1 clone showed a roughly 6-
fold increase (Fig. 3e). We then wanted to confirm that when using this auxin inducible degron
system, we actually see a loss of Suz12 as well as H3K27me3 deposited by PRC2. To test this, we
performed a Western blot and do observe the depletion of H3K27me3 as well as the successful
degradation of Suz12 (Fig. 3f).
We do see a notable increase in luminescent signal in both the Osr2 reporter as well as the
Pitx1 reporter. However, the Pitx1 clone shows an overall lower signal compared to Osr2. Because
of this, we have selected Osr2 as our primary reporter for further investigation, while utilizing the
Pitx1 reporter as a complementary counter-screen for confirmation of future experiments.
14

a)
- Aux +Aux
0
1000
2000
3000
4000
Median BFP
Osr2 BFP 96-well
-Aux +Aux
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2000
3000
4000
Pitx1 BFP 96-well
Median BFP
b)
-Aux
+Aux
Osr2 clone
Pitx1 clone
c)
-Aux +Aux
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2000
4000
6000
Pitx1 Top 10 Clones
Average Luminescence
-Aux +Aux
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6000
Average Luminescence
Osr2 Top 10 Clones
d)
DMSO
Auxin
0
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1000
1500
Luminescence
Pitx1 F3 Luciferase
reporter signal
DMSO
Auxin
0
1000
2000
3000
4000
Osr2 B4 Luciferase
reporter signal
Luminescence
e)
f)
DMSO
Aux 72h
H3K27me3
15
PARP
100
100
Suz12
H3
15
MW in kDa
BFP BFP
15
Figure 3: Development and clone selection of endogenous polycomb reporter in mouse
embryonic stem cells with vPRC loss of function
a) Schematic representation of the polycomb reporters created in vPRC loss of function mESCs
containing and the HiBiT-linker-BFP tag located on polycomb target genes Osr2 and Pix1. b) Flow
cytometry results of 96 clones picked Osr2 reporter (left) and Pitx1 reporter (right) untreated or
auxin (250uM, 48h) treated. c) Histograms of representative clones from flow cytometry results
measuring BFP in +aux/-aux groups. d) Top 10 clones from both Osr2 and Pitx1 polycomb reporter
mESCs analyzed using luminescence readout with HiBiT tag of -aux/+aux groups. e) HiBiT
luminescence detection of 72-hour DMSO or auxin treated for top clone of each polycomb reporter
(Osr2 and Pitx1). f) Western blot of DMSO-treated and auxin-treated Osr2 mESC reporter cells
showing levels of H3K27me3 and Suz12.

Characterization of endogenous polycomb reporter mESCs
We proceeded with the characterization of the Osr2 reporter mESC line and focused on
optimizing the HiBiT luminescent assay to achieve an optimal signal. We conducted experiments
involving varying cell numbers per well and performed a time course analysis to determine the
optimal treatment duration using auxin, aiming to maximize the robustness of the luminescent
signal. Through these optimizations, we consistently observed a substantial ~12-15-fold increase
in gene expression after 72 hours of auxin treatment utilizing the HiBiT detection system (Fig. 4a).
Next, we sought to study the kinetics of our polycomb reporter to assess the potential for
re-silencing of the polycomb gene locus upon the removal of treatment. We initiated a 72-hour
auxin treatment on the Osr2 reporter, followed by a washout procedure with fresh media on days
3, 4, and 5 of the assay (Fig. 4b, top). The results from this experiment revealed a complete
restoration of gene silencing in the reporter mESCs after 48 hours, indicating the dynamic nature
of the polycomb regulatory system (Fig. 4b, bottom).
Recognizing one of the objectives of utilizing this reporter system in high-throughput
screens for identifying potential small molecules targeting polycomb, we embarked on an
investigation to assess its pharmacological sensitivity using a panel of known polycomb small
molecule inhibitors. By subjecting the reporter cells to treatment with specific inhibitors targeting
16
Cbx7, Ezh2, and Ring1B, we aimed to gain insights into the differential responses and further
characterize our polycomb reporter cell line. This resulted in robust upregulation of Osr2 with the
Ezh2 inhibitor, less pronounced upregulation with the Cbx7 inhibitor, and slight increase observed
with the Ring1B inhibitor (fig. 4c).
Here, we have optimized the luminescent signal when treating with auxin, thereby
enhancing the sensitivity of our reporter system. We also have established its dependency on
polycomb by demonstrating its ability to re-silence upon auxin removal and PRC2 re-assembly.
Moreover, we demonstrated the system's sensitivity to pharmacological perturbation of polycomb,
highlighting its potential for identifying and evaluating therapeutic interventions targeting
polycomb-associated processes.

17
Figure 4: characterization of endogenous polycomb reporter mESCs
a) Western blot of DMSO-treated and auxin-treated Osr2 mESC reporter cells showing levels of
H3K27me3 and Suz12 (left). HiBiT luminescence detection of polycomb reporter of 72-hour auxin
and DMSO-treated cells (right). b) Schematic of kinetic experiment of auxin treatment withdraw
over a 48h time period on Osr2 B4 reporter cells (top). Luminescence readout of polycomb gene
expression after withdrawing auxin treatment 12h, 24h, and 48h before reading plate (bottom). c)
HiBiT luminescent detection of 72h treatments with DMSO treated, EZH2 inhibitor, CBX7
inhibitor, or RING1B inhibitor on Osr2 B4 reporter mESCs. Data represent the mean of triplicate
experiments with error bars indicating standard deviation.
DMSO
Auxin
0
2000
4000
6000
8000
10000
12000
14000
Luminescence
Osr2 B4 Luciferase signal
a)
DMSO
CBX7i
EZH2i
RING1Bi
0
5000
10000
15000
20000
Luminescence
Luciferase Signal:
small molecules
c)
Untreated
48h Aux withdraw
24h Aux withdraw
12h Aux withdraw
0h Aux withdraw
0
2000
4000
6000
8000
Total Luminescence
b)
18
Polycomb reporter is sensitive to CRISPR deletion of polycomb genes
Expanding on our research objectives, our primary focus with the polycomb reporter
system involves conducting a comprehensive genetic screen to gain insights into the genetic
determinants underlying polycomb silencing. To ensure the sensitivity of our reporter system to
genetic disruption, we employed CRISPR constructs expressing Cas9 and specific sgRNAs. By
transfecting these constructs into HEK293T cells, we generated viruses that would subsequently
be used to infect our reporter cells, allowing us to investigate the impact of genetic perturbations
on polycomb-mediated gene regulation (Fig. 5a). To test this, we selected sgRNAs targeting
various subunits of vPRC1, cPRC1, and PRC2 (Fig. 5b).
As expected, disruption of Suz12, a crucial subunit of PRC2, and Ring1B, a key component
of PRC1, resulted in the most robust increase in polycomb gene expression (Fig. 5c). This finding
further validates the essential role of Suz12 and Ring1B in maintaining polycomb-mediated
repression in our reporter mESCs. we observed minimal changes in gene expression upon
disrupting the vPRC1 subunits Bcor and Pcgf3, which can be attributed to the presence of Pcgf1
knockout in our reporter cell line, as it eliminates the primary vPRC1 component. However, we
did observe an increase in signal upon disrupting the Rybp subunit, suggesting that Rybp may have
a differing role in this context (Fig. 5c). On the other hand, we observed relatively low signal upon
disruption of cPRC1 subunits Cbx7 and Pcgf2, likely due to the existence of multiple Cbx and
Pcgf analogues that can compensate for their activity. This suggests the presence of functional
redundancy within these subunits and underscores the complexity of the polycomb regulatory
network (Fig. 5c).
Through these experiments, we have shown that our reporter cell line is sensitive to genetic
disruption of polycomb genes.
19
Figure 5: Polycomb reporter is sensitive to CRISPR deletion of polycomb genes
a) Scheme of transfection method for CRISPR deletion of polycomb genes in mESC reporter
system. b) Representation of the cPRC1, vPRC1, and PRC2 subunits being targeted using
CRISPR/Cas9. c) Graph depicting fold change of luminescent signal of CRISPR targets and auxin
treatment in polycomb reporter cells of various PRC1 and PRC2 subunits when compared to
untreated control cells.

Polycomb reporter is sensitive to RNAi knockdown of polycomb genes
Through our CRISPR targeting experiments, we have demonstrated the sensitivity of our
reporter to polycomb gene disruption, validating its utility for genetic screening. The genetic
screen we will be conducting with this reporter involves an RNAi knockdown-based approach to
uncover previously unknown dependencies in polycomb gene repression. We have decided RNAi
Auxin
SUZ12
EED
EZH2
CBX7
RING1B
PCGF2
RYBP
BCOR
PCGF3
0
5
10
15
20
Fold Change
CRISPR Targeting of polycomb genes and auxin treatment
a)
c)
b)
20
knockdown screening is the best system for our luciferase reporter, as the assay is well-based and
is not set up to do pooled screening, such as a pooled CRISPR screen.
Building upon the insights gained from the CRISPR targeting experiments, our subsequent
objective was to establish its responsiveness to RNAi knockdown. The RNAi knockdown
procedure involved transfection of siRNAs into pre-seeded polycomb reporter cells, followed by
incubation for 72 hours to allow for efficient knockdown (Fig. 6a). To evaluate the transfection
efficiency, we initially transfected our reporter cells with a control siRNA. This control siRNA
contained TYE563, a bright red fluorescent dye that facilitated measurement using flow cytometry.
The analysis of this control siRNA revealed an 80% transfection efficiency, indicating the
reliability and efficacy of our transfection protocol (Fig. 6b).
To investigate the impact of specific gene knockdown on polycomb gene expression, we
selected Ring1B and Suz12 for our initial siRNA tests. Considering that disruption of these genes
using CRISPR resulted in the most substantial increase in gene expression, we procured three
siRNAs for each gene and performed the transfection protocol employed for the positive control.
Subsequently, we measured gene expression by measuring luminescence. The results obtained
with the Ring1B siRNAs demonstrated a notable increase in gene expression for two out of the
three siRNAs tested (Fig. 6c). These findings are consistent with the observations from our earlier
CRISPR experiments, further validating our polycomb reporter is sensitive to genetic disruption.
However, to our surprise, none of the three Suz12 siRNAs resulted in a substantial increase in gene
expression (Fig. 6c).
We have chosen a specialized siRNA library specifically designed to target "druggable"
mouse genes for our genetic screening. This siRNA library encompasses approximately 7,000
siRNAs against a wide range of potential drug targets, including kinases, methyltransferases, and
21
metabolites, among others. Because all siRNAs druggable targets in this library, we hope to
leverage our hits from this with any hits from the small molecule screen also being conducted with
our reporter system.

Figure 6: Polycomb reporter is sensitive to RNAi knockdown of polycomb genes
a) Scheme of transfection method for RNAi knockdown of polycomb subunits in a 96-well format.
HiBiT Lytic Detection Reagent is added prior to luminescence readout. b) Flow cytometry
histograms showing transfection efficiency in polycomb reporter mESCs using TYE563-labeled
siRNA comparing untransfected (left) to transfected (right). c) Graph depicting polycomb gene
activation upon RNAi knockdown of PRC1 subunit RING1B and PRC2 subunit SUZ12. Three
siRNAs were tested for both RING1B and SUZ12. Data represent the mean of triplicate
experiments with error bars indicating standard deviation.
RFP
Untransfected
Transfected with
TYE563-labeled siRNA
DMSO
Auxin
1
2
3
1
2
3
0
15000
30000
45000
60000
Luminescence
Luciferase reporter signal
with RNAi and auxin treatment
RING1B siRNA SUZ12 siRNA
RFP
a)
c)
b)
22
Knockdown of polycomb genes in our reporter can be scaled down to a high-throughput
screening format.
The next step in the utilization of this polycomb reporter is to ensure its performance can
be effectively translated into a 384-well high-throughput screening format. To accomplish this, our
initial objective was to determine the optimal cell seeding density for the assay. We seeded a range
of cell numbers, from 250 to 2500 cells per well, in a 384-well plate and incubated them for the
duration of the assay. Subsequently, we stained the cells with DAPI to visualize their nuclei and
assess their viability under different density conditions (fig. 7a). Upon examination of the well
images, we hypothesized that seeding 1250 cells per well provided the most favorable cellular
density for optimal assay performance. To validate this finding, we seeded cells at densities of 625
cells/well, 1250 cells/well, and 2500 cells/well, and treated them with auxin to evaluate the signal
intensity at each density. We observed an increase in signal for all three cell densities, with the
1250 cells/well density demonstrating a slightly higher signal above the background noise (fig.
7b).
During the pilot assays, we observed some cell death over the course of the 72 hours. Our
hypothesis was that this might be attributed to the cells being plated in a low volume of 25 μl of
medium per well, as it was necessary to leave space to introduce the HiBiT reagent prior to taking
the luminescent reading. To address this issue, we tested an alternative approach by seeding the
cells in a higher volume of medium (50 μl) and subsequently aspirating all but 15 μl before adding
fresh medium and the HiBiT reagent. To verify that no cells were being aspirated during this
process, we conducted a cell adherence and aspiration test, which confirmed the absence of cell
loss before and after aspiration (Fig. 7c).
23
For siRNA transfections in the 384-well plate format, we had to adapt our protocol from
the 96-well format to a reverse transfection method, which is better suited for high-throughput
screening. This involved spotting individual siRNAs onto each well of the plates, followed by the
addition of transfection reagents and cells (Fig. 7d). In the initial trial, we observed a pattern of
signal similar to that seen in the 96-well plates but with an overall lower signal intensity (Fig. 7e).
After careful analysis of each step in the protocol, we were able to optimize the procedure further,
leading to a subsequent pilot test that yielded close to double the signal intensity (Fig. 7f).
These optimizations allowed us to transition from 96-well format to 384-well format, better
positioning the polycomb reporter system for our next step; efficient high-throughput screening
applications.

24

250 cells/well 1250 cells/well 2500 cells/well
Post-aspiration Pre-aspiration
mESC media
625 cells
1250 cells
2500 cells
0
2000
4000
6000
8000
10000
Luciferase response to auxin treatment
Luminescence
-Aux
+Aux
Auxin 1 2 3 1 2 3
0
1
2
3
4
5
Fold Change
384-well RNAi Pilot 1
plate 1
Plate 2
RING1B siRNA SUZ12 siRNA
Auxin 1 2 3 1 2 3
0
2
4
6
8
10
384-well RNAi Pilot 2
Fold Change
plate 1
Plate 2
RING1B siRNA SUZ12 siRNA
a)
c) b)
d)
e) f)
cells/well
250
500
750
100
1250
1500
2000
2500
25
Figure 7: RNAi knockdown in polycomb reporter mESCs can be scaled down to a high-
throughput screening format
a) Representative heat map depicting cell number using DAPI staining following a 72-hour
incubation. Cells were plated in varying cell numbers in 2 parallel 384-well plates (top).
Representative images of lowest cell number plated (250/well), mid-level (1250/well) and high
number (2500/well) following a 72-hour incubation and DAPI staining (bottom). b) Luminescent
signal of auxin-treated cells plated in a 384-well plate at varying cell numbers with -aux/+aux
treatment. Data represent the mean of 32 replicate experiments with error bars indicating standard
deviation. c) Representative images depicting cells before and after aspirating all but 15ul medium
from each well of a 384-well plate d) Schematic of reverse transfection method to be used for
high-throughput screening on 384-well plates. e) Bar graph depicting the first pilot experiment
testing RNAi knockdown and auxin treatment in high-throughput format. Data represent the fold
change of the average of 8 replicate experiments when compared to the average of untreated cells.
f) Bar graph depicting the second pilot experiment testing RNAi knockdown and auxin treatment
in high-throughput format. Data represent the fold change of the average of 8 replicate experiments
when compared to the average of untreated cells.

26
Chapter 4: Discussion
Through our research efforts, we have developed a novel polycomb reporter system that
utilizes an endogenous polycomb gene as its readout in mouse embryonic stem cells. This
innovative reporter system has enabled us to not only create a valuable tool for studying polycomb
biology but also to gain insights into its functional characteristics. The design of this reporter
system allows us to quantitatively measure the expression of endogenous polycomb genes using
two different readout methods: fluorescence-activated cell sorting (FACS) and luminescence. In
our initial experiments utilizing FACS as the readout method, we employed blue fluorescent
protein (BFP) as the indicator of polycomb gene expression. With this approach, we observed only
minor changes in BFP levels before and after auxin treatment, suggesting that this particular
readout may not provide a robust signal for detecting variations in polycomb gene expression.
However, upon utilizing the HiBiT Lytic Detection System, which enables luminescent readout,
we discovered a substantial increase in polycomb gene expression. This observation indicates that
the luminescence-based readout offers greater sensitivity and serves as a more suitable approach
for accurately assessing the activity of our polycomb reporter system.
When testing the pharmacological effects of using known polycomb inhibitors, we
surprisingly saw very little increase in gene expression when using the published Ring1B inhibitor,
RB-3. It is possible that compensation from RING1A, another subunit of vPRC1, occurs when the
Ring1B inhibitor is employed. On the other hand, we observed a notable increase in polycomb
gene expression when using the EZH2 inhibitor. EZH2 is a key catalytic subunit of PRC2, which
likely accounts for the observed effect. In the case of the Cbx7 inhibitor, we observed a slight but
consistent increase in gene expression. Considering the presence of multiple Cbx proteins
associated with polycomb, it is reasonable to assume that other Cbx proteins may compensate for
27
the loss of Cbx7 activity. Overall, these experiments demonstrate the sensitivity of our polycomb
reporter system to small molecule inhibition of polycomb, providing evidence of its potential as a
tool for drug screening and identifying compounds targeting polycomb-mediated gene regulation.
The ultimate goal of this reporter will be to conduct high-throughput screening in the
context of small molecules as well as an RNAi genetic screen. To achieve this, we have established
collaborators at Sanford Burnham Prebys (SBP), who are currently using our endogenous
polycomb reporter mESCs for a small molecule screen aimed at identifying novel inhibitors of
polycomb. Simultaneously, we have undertaken the RNAi genetic screen ourselves, utilizing the
resources provided by the Molecular Screening Shared Resource lab at UCLA. To ensure the
success of our genetic screen, we have dedicated efforts to optimize the protocol for RNAi
knockdown in high-throughput screening conditions.
As a first step, we selected two genes, Ring1B and Suz12, for validation as positive controls
due to their robust upregulation in our polycomb reporter when targeted using CRISPR. We
obtained two sets of siRNAs, consisting of three different siRNAs targeting Ring1B and three
different siRNAs targeting Suz12. In subsequent pilot tests using these siRNAs, we consistently
observed a substantial upregulation in gene expression with the majority of the Ring1B siRNAs,
as anticipated. However, intriguingly, we did not observe any increase in gene expression when
using any of the three siRNAs targeting Suz12. This outcome raises the possibility that the
effectiveness of Suz12 knockdown alone may be limited due to the nature of RNAi targeting,
which reduces gene expression but does not result in a complete knockout as achieved through
CRISPR. One plausible explanation for the lack of response to Suz12 knockdown is that RNAi
targeting, while reducing gene expression, does not completely eliminate the protein's presence in
the cells. It is conceivable that even with reduced levels of Suz12, the residual amount of the
28
protein might be sufficient to initiate gene repression by facilitating the deposition of H3K27me3.
This observation suggests that Suz12 may function with remarkable efficiency, where only a
minimal quantity of the protein is required for the establishment of repressive chromatin marks
and subsequent gene silencing.
Our RNAi genetic screen, utilizing the "druggable" siRNA library, holds great potential in
complementing the small molecule screen currently being conducted at SBP with our polycomb
reporter cell lines. By identifying potential overlap between the targets of small molecule hits and
genetic hits from our screen, we can strengthen our understanding of polycomb biology. This
approach also provides a streamlined pathway to validate small molecules hits and further down
the line, investigate their effects on cancer cells known to be influenced by polycomb mechanisms.
By building our polycomb reporter cell line in mouse embryonic stem cells (mESCs), we
have developed a novel screening approach of which has successfully provided a direct readout
of endogenous polycomb gene expression changes. Through characterization of our reporter, we
have established its kinetic activity and validated its sensitivity to both genetic disruption and
pharmacological interventions targeting known polycomb subunits. The successful establishment
of positive controls for our luminescent readout further confirms the reliability and reproducibility
of our polycomb reporter system. These positive controls serve as a benchmark for detecting
variations in polycomb gene expression and lay the foundation for our subsequent high-throughput
screens. Our ongoing RNAi genetic screen, utilizing a specialized siRNA library targeting
druggable mouse genes, holds great promise in uncovering novel dependencies and unraveling
previously unknown biology surrounding polycomb gene repression. This screen, combined with
the parallel small molecule screen being conducted by our collaborators, will provide a
29
comprehensive assessment of potential regulators and therapeutic targets associated with
polycomb activity.
Through these combined efforts, we hope to shed light on the intricate molecular
mechanisms underlying polycomb-mediated gene silencing and contribute to the identification of
innovative therapeutic strategies for targeting polycomb in various diseases. By leveraging the
power of our polycomb reporter system and the insights gained from our high-throughput screens,
we aim to pave the way for the development of novel therapies aimed at modulating polycomb
function and restoring aberrant gene expression patterns.

30
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Abstract (if available)

Abstract Polycomb group (PcG) proteins are highly conserved transcriptional repressors that are critical for maintenance of cell identity. Mutations and dysregulation of genes encoding PcG proteins are frequently associated with human cancer, motivating the development of novel therapies. Originally identified in Drosophila melanogaster, PcG proteins assemble into two main families of repressors, Polycomb repressive complex 1 (PRC1) and PRC2, which enforce gene silencing through catalytic and non-catalytic modifications of chromatin structure. PRC1 is thought to exert gene silencing activity by catalyzing monoubiquitination of histone H2A at lysine 119 (H2AK119ub1) and mediating chromatin compaction whereas PRC2 catalyzes methylation of histone H3 at lysine 27 (H3K27me1, 2 and 3). By recognizing each other’s histone modifications, PRC1 and PRC2 engage in a feedback loop that stabilizes and enhances the formation of transcriptionally repressive chromatin domains. In mammals, PcG protein paralogues have massively expanded and diversified giving rise to six distinct canonical and variant PRC1 subcomplexes with different compositions and functions. To dissect the mechanisms of canonical PRC1-dependent gene silencing, we developed a novel endogenous reporter system in mouse embryonic stem cells (mESCs). During my MSc thesis, I characterized nature and dependence of this reporter and showed that it has great sensitivity and selectivity to genetic and pharmacological perturbations of canonical PRC1 and PRC2. I subsequently worked to adapt the reporter system to facilitate systematic, comprehensive screening using RNAi to identify novel genetic dependencies in mammals. In the future, we hope to use this genetic screen to compliment small molecule screening also being performed with this reporter.

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Asset Metadata

Creator Richardson, Bailey (author)

Core Title A novel screening approach to identify regulators of polycomb-dependent gene silencing

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Medicine

Degree Conferral Date 2023-08

Publication Date 07/11/2023

Defense Date 06/26/2023

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biochemistry,Biology,cell culture,epigenetics,OAI-PMH Harvest,screening

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Advisor Bell, Oliver (committee chair), Dou, Yali (committee member), Jadhav, Unmesh (committee member)

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