Investigating the Functions of PLK4 in Epigenetic Regulation

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Necky Tran University of Windsor

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Investigating the Functions of PLK4 in Epigenetic Regulation

Necky Tran

A Thesis

Submitted to the Faculty of Graduate Studies

through Biological Sciences

in Partial Fulfillment of the Requirements for

the Degree of Master of Science

at the University of Windsor

Windsor, Ontario, Canada

2020

Investigating the Functions of PLK4 in Epigenetic Regulation

Necky Tran

APPROVED BY:

______

S. Ananvoranich

Department of Chemistry and Biochemistry

______

P. Karpowicz

Department of Biomedical Sciences

______

J. Hudson, Advisor

Department of Biomedical Sciences

October 26, 2020

DECLARATION OF ORIGINALITY

I hereby certify that I am the sole author of this thesis and that no part of this thesis has been published or submitted for publication.

I certify that, to the best of my knowledge, my thesis does not infringe upon anyone’s copyright nor violate any proprietary rights and that any ideas, techniques, quotations, or any other material from the work of other people included in my thesis, published or otherwise, are fully acknowledged in accordance with the standard referencing practices. Furthermore, to the extent that

I have included copyrighted material that surpasses the bounds of fair dealing within the meaning of the Canada Copyright Act, I certify that I have obtained a written permission from the copyright owner(s) to include such material(s) in my thesis and have included copies of such copyright clearances to my appendix.

I declare that this is a true copy of my thesis, including any final revisions, as approved by my thesis committee and the Graduate Studies office, and that this thesis has not been submitted for a higher degree to any other University or

Institution.

iii

ABSTRACT

PLK4 is a serine/threonine kinase known to be involved in cell cycle regulation and centrosome duplication. Its protein levels are tightly regulated in order to prevent centrosome irregularities that would lead to aneuploidy and eventual oncogenesis.

Previous studies have shown that PLK4 is under epigenetic control and could also be involved in regulating epigenetic mechanisms. In this study, centrinone-B (a PLK4 inhibitor) was used to determine the functional mechanisms of PLK4 in epigenetic regulation. We determined that centrinone-B was able to change the localization patterns of de-novo methyltransferases DNMT3a and DNMT3b in mouse fibroblasts but did not change their overall protein levels. Furthermore, application of centrinone-B could affect global methylation levels in the same cells, which we speculate is due to the localization changes. Although we could not observe a direct kinase interaction between PLK4 and

DNMT3a, these findings suggest that PLK4 may regulate epigenetic mechanisms by affecting the localization of these de-novo methyltransferases.

DEDICATION

I dedicate this work to my family, friends, and to anyone who has ever encouraged me to pursue a higher education. Your support and advice has given me the strength to overcome my struggles and allowed me persevere through difficult times. Thank you.

ACKNOWLEDGMENTS

I would first like to acknowledge my supervisor and mentor, Dr. Hudson, for providing me with a research opportunity in his lab. His guidance was important in directing my work and I am thankful for his support and patience.

I would also like to acknowledge Brayden and Natalie of the Hudson Lab. They were essential to my success throughout my Master’s. All techniques and methodologies I have learned in this lab are thanks to them. Our coffee runs and weekly lunch outings are memories that I will never forget and I am grateful for their support and friendship.

Finally, I would like to acknowledge everyone who had come to work in the lab while I was there. To Giordano, Dominika, Patrick and Danny, you are amazing lab-mates and I wish you the best with your future endeavours.

TABLE OF CONTENTS

DECLARATION OF ORIGINALITY ...... iii

ABSTRACT ...... iv

DEDICATION ...... v

ACKNOWLEDGMENTS ...... vi

LIST OF TABLES ...... ix

LIST OF FIGURES ...... x

LIST OF ABBREVIATIONS/SYMBOLS ...... xi

CHAPTER 1 Introduction ...... 1 Polo-like Kinase Family Overview ...... 1 PLK4 Structure ...... 3 PLK4 Functions ...... 7 Centriole Biogenesis / Centriole Duplication ...... 7 Cell Cycle Regulation ...... 8 PLK4 Mouse Model ...... 11 Tumorigenesis ...... 11 Epigenetics ...... 13 DNA Methylation ...... 13 Histone Modifications ...... 14 Cancer Epigenetics ...... 17 Epigenetics of the Polo-like Kinases ...... 18 Project Outline ...... 20

CHAPTER 2 Materials and Methods ...... 21 Cell Culture ...... 21 Immunocytochemistry/Immunofluorescence ...... 21 Transient Transfections ...... 21 Flow Cytometry ...... 22

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Western Blot ...... 22 Expression and Purification of GST-Fusion Protein ...... 22 Invitro Kinase Assay ...... 23 Endogenous Coimmunoprecipitation ...... 23 Global Methylation Assay ...... 24 Perinuclear Cell Counts ...... 24

CHAPTER 3 Results ...... 25 PLK4 interaction with DNMT3a ...... 25 Localization of DNA-Methyltransferase proteins in response to Centrinone-B ...... 29 Localization of DNA-Methyltransferase proteins in response to shRNA-PLK4 ...... 32 Localization of DNA-Methyltransferase proteins in PLK4+/- MEFs ...... 32 Centrinone-B cell cycle profiles ...... 37 Centrinone-B affects global methylation in NIH-3T3 Cells ...... 40

CHAPTER 4 Discussion ...... 43 DNMT3a is not a substrate of PLK4 ...... 43 Centrinone-B affects DNMT3a and DNMT3b localization ...... 44 Centrinone-B affects global DNA methylation ...... 46 Concluding Remarks ...... 48 Future Directions ...... 49

REFERENCES/BIBLIOGRAPHY ...... 53

APPENDICES ...... 61 Appendix A: BioID Project ...... 61 Appendix B: Plasmid Maps ...... 62 Appendix C: Table of Primers ...... 66

VITA AUCTORIS ...... 67

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LIST OF TABLES

Table 1: Phosphorylation Motif Sequences

LIST OF FIGURES

Figure 1: Polo-like Kinases Protein Structure and Domains

Figure 2: The Centrosome Cycle

Figure 3: Epigenetic Modifications of DNA and Histones

Figure 4: DNMT3a is not a PLK4 substrate

Figure 5: Centrinone-B affects localization of the DNA-Methyltransferase proteins

Figure 6: Knockdown of PLK4 affects localization of DNMT3a and DNMT3b

Figure 7: PLK4 haploinsufficiency does not affect localization of DNMT3a and

DNMT3b

Figure 8: Nocodazole does not affect localization of DNMT3a

Figure 9: Analysis of global methylation levels in NIH/3T3 cells

Figure 10: Model for PLK4-mediated regulation of DNA methylation through PRMT5

LIST OF ABBREVIATIONS/SYMBOLS

5mC – 5-methylcytosine

AML – Acute Myeloid Leukemia

CA – Centrosome amplification

CEP152 – Centrosomal Protein 152

CEP192 – Centrosomal Protein 192

DNA – Deoxyribonucleic Acid

DNMT – DNA-Methyltransferase

ELIZA – Enzyme-linked Immunosorbant Assay

Flag-G95L – Inhibitor Resistant PLK4

Flag-KA – Kinase Active PLK4

Flag-KD – Kinase Dead PLK4

G0/G1 – Phase 1 of mitosis

G2 – Phase 2 of mitosis

HCC – Hepatocellular carcinoma

HDAC – Histone Deacetylase

MDS – Myelodysplastic Syndrome

MEF – Murine Embryonic Fibroblast

NIH/3T3 – Mouse Fibroblast Cell line

P53 – Tumor Protein P53

PBD – Polobox Domain

PLK – Polo-like Kinase

ShPLK4 – Short-hairpin PLK4

ShSCR – Short-hairpin Scrambled

WT- Wild type

CHAPTER 1

Introduction

Polo-like Kinase Family Overview

The polo-like kinases (collectively, PLKs) are a family of serine-threonine kinases that have important roles in cell cycle regulation and centrosome duplication. They were first discovered in D. melanogaster as the gene polo, named after an abnormal spindle pole phenotype [1]. Since then, they have been identified in a wide range of organisms including S. cerevisiae (Cdc5), X. laevis (Plx), and C. elegans (Zyg-1), highlighting their conservation across species and importance in mitotic pathways [2]. In mammals, there are five known members (PLK1-5) that are each uniquely regulated with separate functions in mitosis.

PLK1 is the best-characterized member of the family with roles primarily centralized in mitosis.

It localizes to multiple structures during mitosis and has been shown to be required for proper cell division. During late G2, PLK1 localizes to the centrosomes and regulates mitotic entry by phosphorylating CDC25C, which allows nuclear translocation of CDC25C and subsequent dephosphorylation of cyclin B-bound CDK1 [3,4]. It has also been shown to regulate nuclear envelope breakdown during prophase by phosphorylating p150glued-dynactin complexes [5]. PLK1 also negatively regulates PCR1 (protein regulator of cytokinesis) in order to prevent premature cytokinesis [6].

Overexpression of PLK1 increases cellular susceptibility to cancer formation through aberrant mitosis and increased sensitivity to IR and UV radiation [7]. This is due to the decreased functionality of the DNA damage response pathway; proteins such as ATM and Chk2 were suppressed [7]. Coincidentally, upregulation of PLK1 is also commonly found in a wide-range of cancers [8].

PLK2 is predominantly known for its interaction with DNA damage response proteins and involvement in cell cycle regulation. PLK2 mRNA levels are upregulated in response to cellular stress and it has been shown to directly interact with CHK1, CHK2, and p53 through in vitro binding assays [9].

PLK2 also localizes to the centrosomes during S phase and participates in the centrosome duplication

process by promoting procentriole formation [10]. Cells deficient in PLK2 have been shown to be susceptible to genotoxic stresses and have increased frequencies apoptosis, which indicate potential tumor suppressive roles [11]. Respectively, PLK2 is also predominantly transcriptionally silenced in B-cell malignancies [12].

Similar to PLK2, PLK3 is most known for its activities in the DNA damage response pathway and its tumor suppressive roles. Studies have shown that PLK3 acts downstream of Chk2 and phosphorylates p53 at Ser20 after ionizing radiation treatments which leads to increased expression of p21 [13,14]. PLK3 is also responsible for phosphorylating and stabilizing tumor suppressor PTEN under hypoxia, which negatively regulates the AKT1 pathway for cell growth and proliferation [15].

Investigations into aged PLK3 null mice reveal weight gain and tumor formation in multiple organs [16].

Furthermore, downregulation of PLK3 expression has been reported in multiple cancers including lung and ovary, suggesting that lower levels of PLK3 are associated with tumor development [17,18].

PLK4 is known mainly for centrosome regulation but has other roles in mitotic progression and ciliogenesis [19]. PLK4 directly phosphorylates centrosome proteins CEP152 and CEP192 at the centriole base, which is required for centriole duplication [20,21]. The localization of Ect2 to the cleavage furrow for the completion of cytokinesis is also dependent on PLK4 phosphorylation [22]. Studies investigating primary cilia formation have found that PLK4 plays a major role in the formation of new cilium.

Dysregulation of PLK4 in this context reduces the total number of cilia in keratinocytes and contributes to defective cilium function, which indicates that PLK4 may be a biomarker for ciliary dyskinesia

[19,23,24].

PLK5 is the newest member of the family, as such, not much is known about it. Unlike the other

PLKs, PLK5 has a truncated kinase domain but still retains some phosphorylating function [25]. It has been implicated in the DNA damage response much like PLK3 and PLK2. PLK5 mRNA is upregulated in response to DNA damage and microtubule disruption, suggesting that it is a stress-induced protein involved in maintaining genomic stability. Furthermore, overexpression of PLK5 in HEK293 or NIH/3T3 cells leads to cell cycle arrest and apoptosis [26]. Depletion of PLK5 in neuronal cells has been shown to

inhibit axon growth and neurite formation, which may suggest that it is involved in neurodevelopment.

Interestingly, PLK5 is transcriptionally silenced by promoter methylation in glioblastomas and astrocytomas [25].

PLK4 Structure

The mammalian PLK family members likely all diverged from an ancestral PLK1-like protein.

Each member shares a conserved catalytic N-terminal kinase domain (PLK5 kinase domain is truncated), which is responsible for phosphorylating protein substrates. The non-catalytic C-terminal end contains a polo-box domain (PBD) that is unique to the PLKs (Figure 1). The polo-box domain consists of two highly conserved polo-box (PB) motifs of about 70-80 amino acids each and a linker region between them. It regulates subcellular localization of the protein, substrate binding and autoinhibition of the kinase domain [27].

PLK4 is the most divergent member of the family. It possesses a kinase domain as well, however, unlike the kinase domain of PLK1, it has a unique phosphorylation motif. This may imply that it can target a wider range of substrates [28]. Also, PLK4 contains 3 polo-box motifs instead of the usual 2 found in PLK1-3. PB1 and PB2 act as one unit and mediate the homodimerization of PLK4 and also its interaction with Asl (Asterless/CEP152) [29]. PB3 is required for PLK4 binding to STIL, which leads stabilization at the centriole and is a crucial step for centriole duplication [30]. When all three Polo-boxes are removed PLK4 loses its capability to localize to the centrosome [29].

There are three PEST sequences within PLK4 that regulate stability and turnover rate of the kinase [31]. The first PEST sequence contains a degron motif that becomes a binding site for SCF

(Skp1/cullin/F-box) when phosphorylated. Upon binding, SCF ubiquitinates PLK4 and signals it for proteasomal degradation. Mutation of the degron motif to prevent phosphorylation increases the stability of PLK4 and leads to increased activity of wild-type PLK4 [32]. Phosphorylation of the degron motif is due to the autophosphorylation that occurs in trans during PLK4 homodimerization. Through this

mechanism, PLK4 is able to regulate its own destruction, which is a crucial factor in tightly regulating centrosome duplication [32,33].

Figure 1: Polo-like Kinase Protein Structure and Domains

A representation of the conserved domains and motifs that are unique to the PLK family. All PLK family members share a common protein structure: N-terminal kinase domains (KD) and C-terminal Polo-box motifs (PB). PLK4 uniquely has 3 Polo-box motifs that regulate substrate binding and subcellular localization. Numbers above boxes indicate the start and end of that domain/motif in the amino acid sequence.

PLK4 Functions

Centriole Biogenesis / Centriole Duplication

PLK4 is most known for regulating proper centriole duplication and centrosome number during cell division [34]. Centrosomes in mammalian cells act as microtubule organizing centers (MTOC), which are a major site for microtubule nucleation and localization. Proper organization of mitotic/meiotic spindles is necessary for the even distribution of chromosomes during cell division [35]. Centrosomes consist of two centrioles that are orthogonally arranged at their base (proximal end) and are surrounded by a protein matrix collectively named pericentriolar material (PCM). The PCM is a dynamic collection of proteins that include CEP192, CEP152, y-tubulin and many others which are required for the nucleation of new microtubules [36].

Centriole biogenesis occurs once per cell cycle and begins during the G1 phase. After mitosis, mother and daughter centrioles disengage through the regulation of PLK1 and Separase to allow formation of procentrioles on the proximal ends [37]. At the S phase transition, PLK4 is recruited to the centrosome by PCM components CEP152 and CEP192. Phosphorylation of CEP152 and other PCM components by PLK4 initiates centriole biogenesis. During S phase, STIL, SAS6 and CPAP are recruited to the procentriole location to form a cartwheel base where microtubules will dimerize and elongate to form a daughter centriole [38]. PLK4 is degraded by SCF during elongation to prevent centriole reduplication at other sites along the mother centriole. After duplication, both centrosomes recruit additional PCM and separate to form two mature centrosomes by the end of G2. These two centrosomes will act as MTOCs to preserve cellular polarity during mitosis [39].

The overall protein levels of PLK4 need to be tightly regulated in order to prevent centrosomal and mitotic abnormalities. Overexpression of PLK4 has been found to induce centriole amplification and de novo centrosome formation in drosophila [40]. Depletion of PLK4 inhibits centriole duplication and has been linked to cytokinesis failure [41]. Both overexpression and depletion of PLK4 lead to spindle disruptions during mitosis that ultimately produce aneuploid cells [42].

Cell Cycle Regulation

In addition to regulating centriole duplication, PLK4 has also been implicated in other cellular response pathways. Chk2 is a key protein involved in the DNA-damage signaling pathway. It plays a critical role in stabilizing p53 to induce cell cycle arrest or promote apoptosis in response to ionizing or

UV radiation [43]. In-vitro kinase assays have shown that Chk2 is a PLK4 substrate, which suggests that

PLK4 may be involved in maintaining genomic stability [44]. Furthermore, PLK4 has been shown to interact with p53 through its third Polo-box, however, the functional significance of this interaction is not understood [45]. Li et al. has suggested that PLK4 may be negatively regulated by p53 at a transcriptional level by recruitment of a histone deacetylase (HDAC) complex to the PLK4 promoter [46]. In the context of genotoxic stress, this response is needed to inhibit cell cycle progression to activate the DNA repair or apoptotic pathways. PLK4 could trigger a negative feedback loop where it phosphorylates Chk2 in order to stabilize p53 and ultimately repress itself. This model is supported by the observations of heterozygous

PLK4 embryonic fibroblasts (PLK4+/- MEFs) that have a reduced p53 response under oxidative stress

[47].

PLK4 is also involved in the progression of the cell cycle through its interactions with Cdc25C and Ect2. CDC25C is a phosphatase and plays a key role in mitotic entry. It is required to dephosphorylate Cyclin B-bound CKD1 to trigger entry into mitosis. CDC25C has been shown to localize to the centrosome and be a direct substrate of PLK4 [48]. The phosphorylation event has not been fully characterized but it is speculated that it could be involved in the nuclear localization of CDC25C. PLK1 and PLK3 are also shown to phosphorylate CDC25C and contribute to its nuclear accumulation; this suggests that PLK4 may have a similar overlapping role with the other PLK family members [49,50].

Furthermore, reciprocal co-immunoprecipitation and kinase assays have shown physical interactions between PLK4 and Ect2 [21]. Ect2 is a protein required for cytokinesis and its localization to the cleavage furrow during anaphase has been shown to be PLK4 dependent. PLK4+/- MEFs frequently fail to complete cytokinesis due to failed Ect2 localization which predisposes cells to genomic instability [21].

Figure 2: The Centrosome Cycle

An illustration that demonstrates the process of centriole duplication during different phases of the cell cycle. (1) Mother and daughter centrioles disengage in early G1 through the activity of PLK1 and

Separase. (2) PLK4 is recruited to the centriole base in late G1 and initiates procentriole formation with multiple interacting partners. (3) The procentriole elongates through the stabilization of alpha and beta tubulin dimers and completes at the end of the S-phase. (4) Two fully mature centrosomes form at G2 and separate to act as microtubule organizing centers (MTOC) during mitosis. (Figure adapted and modified from: Gilmore and Walsh 2013 [131])

PLK4 Mouse Model

PLK4 loss of function studies in mice has revealed that it is an essential protein for proper embryonic development. PLK4-null mice are embryonic lethal at day E7.5 after gastrulation due to late mitotic failure. These embryos displayed an increase in Cyclin-B1 positive cells in the late states of anaphase suggesting that PLK4 is needed for degradation of Cyclin-B1 and successive mitotic exit [51].

Interestingly, PLK4 heterozygous mice develop normally and are viable despite having only one functioning allele. However, elderly PLK4+/- mice had a greater incidence of liver and lung cancers compared to their wild-type littermates, which indicates a haploinsufficient effect for tumor suppression

[52]. Partial hepatectomy of PLK4+/- mice displayed tissue disorganization and spindle abnormalities due to mitotic defects associated with p53 and p21 suppression [51].

Studies investigating PLK4+/- embryonic fibroblasts have shown cellular irregularities and abnormal gene expression patterns. PLK4+/- MEFs have an increased incidence of uneven chromosome segregation and overall aneuploidy when compared to wild-type cells. This seems to be a consequence of an increased number of observed MTOCs during interphase [52]. Western-blot data has shown PLK4+/-

MEFs to have higher protein levels of p53, p21 and Chk2 when compared to wild-type cells which indicates genomic instability that is consistent with PLK4’s proposed role in the DNA damage response

[53].

Tumorigenesis

A review of PLK4’s substrates and its involvement in the centrosome cycle provide an attractive explanation for PLK4’s role in cancer formation. Theodor Boveri first proposed that centrosome amplification (CA) could be involved in generating cell malignancy by promoting chromosomal instability (CIN) and subsequent aneuploidy [54]. PLK4 overexpression is consistently used as a method to study centrosome amplification as well as the long-term consequences of cells having extra centrosomes [22,55]. For example, it has been reported that PLK4-overexpressing neural cells with supernumerary centrosomes could form tumors when injected into WT adult flies [56]. Additionally,

PLK4-overexpression in epithelial cells was found to disrupt cell-cell adhesion and promote malignant invasion [57]. Therefore, it is fitting that PLK4 is shown to be upregulated in a wide range of cancers such as gastric, breast and glioblastomas and that it is associated with poor prognostic outcomes

[58,59,60].

Contrarily, in hepatocellular carcinoma (HCC) tissues, PLK4 levels were recurrently downregulated [61]. Multiple HCC cell lines showed the same decrease in PLK4 expression and researchers determined that larger tumors were overall correlated with lower expression of PLK4. Furthermore, HCC patients with low PLK4 expression overall had a worse prognosis than those with higher levels of PLK4, which suggests that it may be a predictor for overall survival [61]. These findings may indicate dual roles for PLK4; it acts oncogenic in overexpressed conditions and as a tumor suppressor in haploinsufficient conditions.

Centrosome amplification has been reported in nearly all types of human cancer; however, there is some debate on whether it can be a true driver of tumorigenesis [62,63,64]. Studies in mice revealed that PLK4-induced centrosome amplification in epidermal cells caused spindle errors and chromosome segregation defects but failed to promote cell proliferation and carcinogenesis [65]. Additionally, centrosome amplification failed to enhance the growth of chemically induced skin tumors and did not cause spontaneous tumor formation in aging mice [66]. Other studies have found that centrosome amplification can accelerate tumorigenesis only in p53-deficient epidermal cells suggesting that supernumerary centrosomes are insufficient to drive tumor formation [67]. In contrast, Levine et al. demonstrated that centrosome amplification and aneuploidy in vivo could induce spontaneous formation of lymphomas and sarcomas in the presence of wild-type p53 [68]. Their mouse model drove overexpression of PLK4 at a low level that only led to the creation of one or two extra centrosomes per cell. This is similar to centrosome amplification found in human tumors [69]. Taken together, these contradicting studies exemplify the struggle to interpret the role of centrosome amplification in tumorigenesis.

Epigenetics

Epigenetics refers to the study of heritable changes in gene function that do not involve changes in DNA sequence [70]. These changes in gene function are due to the altered expression of genes through modified chromatin packaging. Over the past decade, a growing number of studies have shown how chronic exposure to environmental stresses and toxicants can promote tumorigenesis through epigenetic mechanisms [71,72]. Change of gene expression patterns in critical tumor suppressor or oncogenic genes through DNA methylation or histone modifications will increase the likelihood of carcinogenesis.

Luckily, epigenetic modifications are not permanent and many drugs exist today that specifically target epigenetic regulators [73].

DNA Methylation

One of the most extensively studied epigenetic mechanisms is cytosine methylation [74]. The fifth-carbon of a cytosine nucleotide can become methylated into 5-methylcytosine (5mC), which interferes with transcription factor binding and prevents gene expression. Methylated cytosines are primarily found on CpG dinucleotides and long stretches of DNA that contain greater proportions of CpG sites are known as CpG islands [75]. About 70% of human gene promoters contain CpG islands and hypermethylation of these areas is associated with transcriptional silencing [76,77].

The DNA-methyltransferase family of proteins are the enzymes that catalyze the methyl groups onto cytosine residues using S-adenosylmethionine (SAM) as the methyl donor [78]. There are five known members of the DNMT family but only DNMT1, DNMT3a and DNMT3b are known to contribute to the global patterns of DNA methylation. DNMT1 is the maintenance protein and is involved in the methylation of cytosine residues in newly synthesized daughter DNA strands to match the methylation patterns of the parental strands [79]. DNMT3a and DNMT3b are classified as de novo methylases that establish new methylation patterns during embryogenesis and cell differentiation [80].

Histone Modifications

In the nucleus, genomic DNA is packaged into nucleosome subunits that condense into chromatin. Each nucleosome consists of DNA wrapped around 4 core histone proteins (H2A, H2B, H3,

H4) and additional linker DNA stabilized by histone protein H1 [81]. Consecutive nucleosome subunits have been described as “beads-on-a-string” and can be categorized into two types of chromatin defined by their level of compaction. Euchromatin is loosely packed chromatin and typically represents transcriptionally active genetic regions. Heterochromatin is tightly packed chromatin and generally represents silenced genetic regions.

Chromatin conformation is dynamic and can switch to an active or inactive state by chemical modifications of histones. Histone methylation occurs on both arginine and lysine residues on the tails of histone proteins H3 and H4 [82]. Unlike DNA methylation, histone methylation is associated with both gene repression and activation. The H3K27me3 mark (tri-methylation of the 4th lysine residue of histone

H3) is associated with transcriptional repression while the H3K4me2 mark is associated with transcriptional activation [83,84]. Furthermore, lysine residues on histone tails can also be modified by acetylation and is strongly associated with transcriptional activation. Histone acetyltransferase proteins

(HATs) catalyze the addition of acetyl groups to lysine using coenzyme A as a cofactor and induce an open chromatin state, whereas histone deacetylase proteins (HDACs) remove acetyl groups and induce a closed chromatin state. Together, methylation and acetylation of histone tails play a key role in controlling gene expression by regulating chromatin structure.

Figure 3: Epigenetic Modifications of DNA and Histones

(A) DNA hypermethylation of gene promoters by DNA-methyltransferase proteins (DNMT) can silence gene expression by inhibiting transcription factor binding. Cytosine methylation marks can be reversed by

TET2 enzymes. (B) Lysine and Arginine residues on histone tails can be modified by histone acetyltransferase (HAT) or histone methyltransferase (HMT) enzymes. These marks are dynamic and can be reversed by histone deacetylase (HDAC) and histone demethylase (HDM) enzymes. (Figure adapted and modified from: Roberti et al. 2019 [132])

Cancer Epigenetics

Global loss of DNA methylation was the first epigenetic change described in cancer. Feinberg and Vogelstein originally revealed that tumor tissues had a lower proportion of methylated cytosines when compared with normal tissues [85]. Later, studies using high-performance liquid chromatography confirmed that 5mC content was inversely associated with tumor progression [86]. Global hypomethylation of DNA is typically seen in intergenic repetitive regions of the genome and is correlated with elevated instability. Tandem repeats near centromeric DNA are required to keep DNA tightly packed into a heterochromatic state. Hypomethylation of these areas can lead to chromatin decondensation and the amplification of chromosome rearrangements and translocation events [87]. Frequent unbalanced translocation events increase the potential for disruptive gene deletions or carcinogenic chromosome fusions, for example, the Philadelphia chromosome found in leukemia [88]. Additionally, researchers have found that deleting DNMT1 in murine embryonic stem cells, which caused global hypomethylation, increased the incidence at which translocation events would occur [89].

Hypomethylation of normally silenced gene regions can contribute to tumorigenesis as well.

Long interspersed elements (LINE-1) are retrotransposons that make up about 17% of the human genome

[90]. In normal tissues these sequences are silenced by methylation but hypomethylation of these elements have been implicated in tumorigenesis. Activation of these retrotransposons can cause random insertional mutations or large gene inversions that can disrupt normal patterns of gene expression [91].

Hypomethylation of LINE-1 elements are regularly found in colorectal and hepatocellular cancers and play a key role in the formation of lung neoplasias [92,93,94]. Additionally, hypomethylation of imprinted regions can cause aberrant gene expression. Imprinted genes are normally mono-allelically expressed but reactivation of promoter regions in these genes lead to expression of both maternal and paternal alleles. Aberrant biallelic expression of IGF2 and H19 has been shown to be a significant risk factor for human colorectal cancer [94].

In contrast with hypomethylation of intergenic regions, hypermethylation of CpG islands can directly promote tumorigenesis by silencing functional genes. CpG islands in promoter regions of normal

tissues are typically unmethylated. However, hypermethylation of these sites is often an early event in cancer formation [95]. Tumor suppressor genes such as PTEN, a protein that inhibits proliferation, and p16, a cell-cycle regulator, are often found to be hypermethylated in multiple human cancers [96,97].

Inactivation of tumor suppressors directly promotes carcinogenesis due to lack of cell cycle controls.

Hypermethylation of transcription factors can also indirectly lead to tumorigenesis due to the uncontrollable downstream effects. GATA-4 and GATA-5 are transcription factors that are essential for development and differentiation of the gastrointestinal tract and both inactivated by promoter hypermethylation in colorectal cancers [98].

Furthermore, dysregulation of epigenetic modifying proteins is also associated with tumorigenesis. Overexpression of DNMT1/3a/3b is all commonly found in tumors and is speculated to be the cause of hypermethylation in CpG islands [99,100]. Mutations in TET2, a protein that catalyzes the first step of cytosine demethylation, have been observed in numerous hematological malignancies and is associated with poor prognosis in AML [101]. EZH2, a histone-lysine-methyltransferase, is overexpressed in prostate cancer and is linked to progressive malignancy [102]. Additionally, recurrent

DNMT3a loss-of-function mutations have also been reported in acute myelogenous leukemia [103]. Both overexpression and downregulation of epigenetic modifiers have a strong association with progressive cancer formation and poor patient prognosis.

Epigenetics of the Polo-like Kinases

Multiple members of the polo-like kinase family are epigenetically dysregulated in cancer. Ward and Hudson observed the CpG promoter of PLK1 to be methylated in normal human bone marrow aspirates [47]. However, when observing the aspirates of various blood neoplasms (B-cell lymphomas,

MDS and leukemias), the PLK1 promoter was hypomethylated, which corresponded with an increase in protein expression [104]. Interestingly, the opposite relationship is detected for PLK2 and PLK3; they are

both unmethylated in normal bone marrow tissue but hypermethylated in neoplastic tissues. Similar findings were noted when investigating the promoter methylation status of the PLKs in cell lines derived from blood neoplasms [104]. This signifies opposing roles for the PLKs in hematological malignancies:

PLK1 acting as an oncogene and PLK2/3 having tumor suppressive functions. Identical promoter methylation statuses for the PLKs have also been reported in human HCC cell lines [105]. These epigenetic studies are consistent with the overall literature in that PLK1 is regularly overexpressed in a multitude of malignant tissues and PLK2/3 are commonly suppressed [106,107,108].

The promoter of PLK4 is similarly observed to be epigenetically dysregulated in different human cancer types. In bone marrow aspirates of hematological neoplasms, PLK4 is hypermethylated, which also coincides with a decrease in PLK4 protein level. It is speculated that PLK4 levels may also be tethered to genetic biomarkers of hematological malignancies such as JAK2 because their expression is inversely correlated [104]. The PLK4 promoter is also hypermethylated in the liver tissues of aged PLK4+/- mice and it directly contributes to further deteriorated transcript levels, however, this effect is not as common with WT mice [104,109]. It seems that PLK4+/- mice are more susceptible to promoter hypermethylation and loss of PLK4 expression as aging occurs. Ward and Hudson observed that PLK4+/-

MEFs under exposure to alcohol and hypoxia are more prone to promoter methylation when compared to

WT MEFs [109,47]. This suggests that DNA methylation regulators in response to cellular stress may target the PLK4 promoter. Interestingly, there are some gender imbalances PLK4+/- mice; the PLK4 promoter is regularly methylated in the livers of male mice [109]. This coincides with the observation that

PLK4+/- male mice are more likely to develop liver tumors than their female counterparts. Furthermore,

DNMT3a protein levels are increased in PLK4+/- MEF under hypoxia and H202 treatment and global methylation levels in these cells are increased as well [47]. This result may imply a possible relationship between PLK4 and DNMT3a under cellular stress that contributes to increased global DNA methylation levels.

Project Outline

PLK4 is mainly known for cell cycle regulation and centrosome duplication, however, recent publications by Ward and Hudson point to a possible relationship between DNMT3a and PLK4 [47]. This study is aimed to elucidate the role of PLK4 in epigenetic regulation and its potential partnership with

DNMT3a as a substrate.

New therapeutic drugs aimed to target PLK4 specifically have been synthesized for possible chemotherapy. Centrinone-B is a unique drug that has shown high affinity and efficacy for PLK4 inhibition in vivo [110]. It specifically binds to the kinase domain of PLK4 and hinders its transition to an active state by forming hydrophobic contacts with PLK4’s activating DFG (Asp-Phe-Gly) motif [110].

Wong et al. has used centrinone-B to study the effects of prolonged centriole depletion. Treatment with centrinone-B will allow insight into the phenotypic cellular effects of PLK4 inhibition in regards to global

DNA methylation and localization of DNA methylation enzymes.

CHAPTER 2

Materials and Methods

Cell Culture

NIH/3T3 cells were cultured in Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with 10% fetal bovine serum (FBS). Murine embryonic fibroblasts (MEF) derived from PLK4+/- mice were cultured in DMEM supplemented with 20% FBS, 1% penicillin/streptomycin, 0.5% gentamicin and amphotericin

B. HEK293T cells were cultured in DMEM supplemented with 10% FBS. All cells were maintained at

37°C with 5% CO2.

Immunocytochemistry/Immunofluorescence

NIH/3T3 and MEF cells were grown on glass slides in 6-well plates at 60-80% confluence. NIH/3T3 cells were treated with DMSO or 300nM of centrinone-B for 16-20 Hours overnight. Cells were fixed with

3.7% PFA +0.1% triton X-100 and incubated with DNMT3a antibody (Sigma-Aldrich), DNMT3b antibody (Santa-cruz), or DNMT1 antibody (Cell-Signalling) at a 1:100 dilution for 16 hours at 4°C.

Secondary antibody Alexa Fluor 568 anti-rabbit (Invitrogen) was incubated at room temperature in a

1:500 dilution for 1 hour. Cells were then stained with Hoechst 33342 at a dilution of 1:10000 for 2 minutes at room temperature. Fluorescent images were taken using Northern Eclipse software.

Transient Transfections

Transient transfections of NIH/3T3 cells were performed with 1mg/ml of PEI (polyethylenimine- branched, Sigma-Aldrich) with 20g of purified plasmid DNA (PLK4 ShRNA) and supplemented with

300mM of NaCl. Cells were incubated with transfection reagents for 18-24 hours.

Transient transfections of HEK293T cells were performed with 1mg/ml of PEI with 10mg of purified plasmid DNA (Flag-PLK4 constructs). Cells were incubated with transfection reagents for 18-24 hours.

Flow Cytometry

NIH/3T3 cells were treated with DMSO, 300nM of centrinone-B or Nocodazole for 18 hours. Treated cells were collected and fixed in ice cold 80% ethanol for 1 hour. They were then washed in PBS and resuspended in a PBS solution supplemented with 0.5M EDTA and 0.1% Triton-X. Samples were incubated with propidium iodide overnight and cell cycle analysis was performed on a cytomic FC 500 flow cytometer.

Western Blot

Whole cell protein was extracted using a lysis buffer (50mM Tris-Cl, 150mM NaCl, 1% Triton-X, 0.1%

SDS supplemented with protease inhibitor). 20ug of total protein was used for Western Blot analysis.

Primary antibodies DNMT3a, DNMT3b, DNMT1, PLK4, and GAPDH were used. Secondary antibodies anti-rabbit HRP and anti-mouse HRP were used. Bands were visualized with ECL reagent (Thermo- fisher) and images obtained from ProteinSimple Fluorchem E system. Densitometry analysis was performed using ImageJ software.

Expression and Purification of GST-Fusion Protein

GST-DNMT3a (Cloned by Lana El-Osta) was expressed in BL21 cells by induction with 1mM of IPTG

(isopropyl-B-D-1-thiogalactopyranoside at 28°C for 4 hours. Bacterial pellets were lysed with a cell lysis buffer (50 mM Tris-HCl pH7.5, 100 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100, 2 mM DTT, and protease inhibitor pellet) and sonicated. After centrifugation at 13000 rpm for 30 mins, the supernatant was incubated with glutathione S-transferase beads in a column for 2 hours at 4°C. The beads were

washed with a cell lysis buffer and 2 times with a wash buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM DTT). Soluble fusion protein was then eluted from the column with an elution buffer (50mM Tris

HCl pH 7.5, 100 mM NaCl, 10 mM glutathione). Following purification, the eluted protein was concentrated using an Amicon Ultra centrifugal filter.

Invitro Kinase Assay

After 48 hour transfections, cells were lysed and whole cell lysate was collected and incubated with 1ug of anti-flag antibody (Sigma) for 1 hour at 4°C. The lysate was then incubated with 20ul of calibrated

Protein-G Sepharose (GE Healthcare Life Sciences) beads for 1 hour at 4°C. Immune Complexes were washed twice with a wash buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl,0.1% Triton X-100), twice with a second wash buffer supplemented with 500mM LiCl and once with a kinase buffer (60 mM Hepes pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 50 mM NaF,25 mM dithiothreitol, 125 µM cold ATP and Roche protease inhibitor pellet). Bacterial purified GST-DNMT3a was incubated with purified immune complexes of Flag-PLK4 constructs. For each kinase reaction, 10ug of GST-DNMT3a and 10 µCi of [γ-

32P] [Perkin Elmer] were supplemented to the kinase buffer. Kinase reactions incubated at 30°C for 30 mins. Protein samples were resolved through an SDS-PAGE and blotted onto PVDF membrane and subjected to autoradiography. Radioactive bands were visualized using Cyclone Plus Phosphor Imager

(Perkin Elmer) and Optiquant software.

Endogenous Coimmunoprecipitation

15ul per plate of protein-G sepharose beads were washed twice using a wash buffer (50mM Tris-Cl pH7.4, 150mM NaCl, and 0.025% Triton-X) and equilibrated with a lysis buffer (50mM Tris-Cl, 150mM

NaCl, 1% Triton-X, 0.1% SDS). Whole cell lysates were incubated with anti-PLK4 antibody for 1 hour at

4°C. The lysates were then incubated with the equilibrated beads for 1 hour at 4°C. Immune Complexes were washed with a wash buffer and resuspended in loading dye.

Global Methylation Assay

Genomic DNA was collected from cellular pellets using phenol/chloroform extraction. Genomic DNA from samples were subjected to global methylation analysis using the MethylFlash Global DNA

Methylation quantification kit (Epigentek). Experiments were carried out according to manufacturer’s recommendations using 100 ng of DNA. Colorimetric analysis was carried out on a Perkin Elmer Wallac

Victor3 1420 Multilabel Counter.

Perinuclear Cell Counts

NIH/3T3 cells were grown on glass slides in 6-well plates at 60-80% confluence. NIH/3T3 cells were transfected with a Flag-PLK4 or Flag-G95L plasmid and the next day treated with 300nM of centrinone-

B for 16-20 Hours overnight. Cells were fixed with 3.7% PFA +0.1% triton X-100 and incubated with

DNMT3a antibody (Sigma-Aldrich) at a 1:100 dilution for 16 hours at 4°C. Secondary antibody Alexa

Fluor 568 anti-rabbit (Invitrogen) was incubated at room temperature in a 1:500 dilution for 1 hour. Cells were then stained with Hoechst 33342 at a dilution of 1:10000 for 2 minutes at room temperature. Slides were observed under UV and >200 cells were manually counted to calculate the proportion of perinuclear stained cells. Results represent combined measurements from 3 independent experiments.

CHAPTER 3

Results

PLK4 interaction with DNMT3a

Ward and Hudson pointed to a possible substrate relationship between PLK4 and DNMT3a [47].

As an initial step towards understanding this relationship, a co-immunoprecipitation was performed to determine if a physical interaction between DNMT3a and PLK4 exists. HEK293T cells were lysed and a co-immunoprecipitation was performed with an anti-PLK4 antibody (cell signalling). Western blot analysis was then conducted and the membrane was probed with an anti-DNMT3a antibody (Santa-Cruz).

DNMT3a was shown to be present in the endogenous PLK4 pull-down (Figure 4b).

After the positive results from the co-IP, a protein pattern search was employed to find possible phosphorylation sites on DNMT3a. PLK4 binding motifs identified by 3 unique groups composed of

Leung et al. (2007), Johnson et al. (2007), and Silibourne et al. (2010) were used on the amino acid sequence of DNMT3a (Table 1). 2 sites on DNMT3a were found using the Leung sequence, 0 were found using the Johnson sequence and 5 were found using the Silibourne sequence. Of the 5 sites found in the

Sillibourne motif, 2 overlapped with the Leung motif, suggesting that serine 714 and serine 786 are potential sites of PLK4 phosphorylation (Figure 4a).

Subsequently, to determine if DNMT3a is a direct substrate of PLK4, an in vitro kinase assay was performed. Bacterially expressed GST-DNMT3a was purified and incubated with immunoprecipitated

Flag-PLK4 in a kinase buffer with ATP for 30 minutes. Kinase-dead and kinase-active mutants of PLK4 were used as controls for assessing phosphorylation status of DNMT3a. However, there were no signs of phosphorylation of GST-DNMT3a by PLK4 in vitro (Figure 4c).

Table 1: Phosphorylation Motif Sequences of PLK4.

Three different groups derived PLK4 phosphorylation motifs independently. These motifs were applied on the amino acid sequence of DNMT3a to find possible phosphorylation sites. Each column represents favourable amino acid residues in relation to the serine/threonine phosphorylation site at position 0. X denotes no amino acid preference.

Figure 4: DNMT3a is not a PLK4 substrate

(A) Positive results of PLK4 phosphorylation motif search for amino acid sequence of DNMT3a using a protein pattern search provided by gene infinity. Highlighted sequences display similar serine phosphorylation sites found by separate motifs. (B) Endogenous co-immunoprecipitation of PLK4 and

DNMT3a in HEK293T cells. (C) Kinase assays were performed to determine if PLK4 can phosphorylate

DNMT3a. HEK293T cells were transiently transfected with Flag-Empty and Flag-tagged PLK4 constructs, including wild-type (PLK), kinase active (KA, T170D), and kinase dead (KD, K41M) mutants. Constructs were immunoprecipitated with anti-FLAG antibody (Sigma) and incubated with bacterially expressed GST-DNMT3a in the presence of [ɣ-32P]. The assay was visualized by autoradiography (top image). Immuno-blots show loading of Flag-PLK4 constructs and purified GST-

DNMT3a protein (bottom image).

Localization of DNA-Methyltransferase proteins in response to Centrinone-B

Due to the physical interaction found between DNMT3a and PLK4 in the co-IP, it was of interest to further examine how PLK4 may indirectly affect methylation by examining the localization of DNA- methyltransferase proteins in response to PLK4 inhibition. Centrinone-B is a potent PLK4 inhibitor that can disrupt centriole assembly by binding to the kinase domain of PLK4 and preventing phosphorylation of bound substrates. NIH/3T3 cells grown on glass slides were treated with 300nM of centrinone-B or

DMSO (as a vehicle control). Cells were fixed with 3.7% PFA and incubated with DNMT1 (Cell-

Signalling), DNMT3a (Sigma-Aldrich), and DNMT3b (Santa-Cruz) antibody overnight and observed under immunofluorescence to visualize protein distribution. DNMT3a protein normally localizes to the nucleus as shown in the control samples. However, with centrinone-B treatment, the localization of

DNMT3a changes dramatically to a perinuclear pattern (Figure 5a). The same perinuclear pattern can be seen with DNMT3b in centrinone-B treated samples but not with DNMT1 (Figure 5b,c).

Due to the localization changes, it was important to examine if overall protein levels had been disrupted. To determine if DNA-methyltransferase protein levels changed under centrinone-B treatment the lysates from treated cells were extracted and Western blot analysis was performed. The blot was probed for DNMT3a, DNMT3b, DNMT1, and GAPDH was used as a loading control. Protein levels of the DNA-methyltransferases remained constant under centrinone-B despite the changes seen in localization (Figure 5d)

Figure 5: Centrinone-B affects localization of DNA-methyltransferase proteins

The localization of DNMT3a, DNMT3b and DNMT1 was examined in NIH/3T3 cells treated with

300nM of centrinone-B. DMSO was used as a vehicle control and DAPI (Hoechst 33342) was used to stain the nucleus. (A) Distribution of DNMT3a is dispersed throughout the nucleus but changes to a perinuclear localization with centrinone-B treatment. (B) Distribution of DNMT3b shows the same perinuclear staining as DNMT3a with centrinone-B. (C) Localization of DNMT1 is unaffected by centrinone-B. (D) Whole cell lysates (20ug) of centrinone-B treated cells were immunoblotted and probed for DNMT3a/DNMT3b/DNMT1. GAPDH-antibody was probed as a loading control.

Localization of DNA-Methyltransferase proteins in response to shRNA-PLK4

The perinuclear localization of DNMT3a/b under centrinone-B treatment was overall unexpected and it was of interest to try and confirm whether this was related to PLK4 levels. It was hypothesized that

PLK4 inhibition through shRNA knockdown might incur the same result. Thus, NIH/3T3 cells were transfected with an shRNA plasmid (shPLK4 or shSCR) and stained with DNMT3a and DNMT3b antibody to be monitored under immunofluorescence. The perinuclear localization of both DNMT3a and

DNMT3b was observed in shPLK4-transfected cells but not in the shSCR control (Figure 6a).

Densitometry was performed on 20ug of transfected cell lysates to determine PLK4 knockdown efficiency. GAPDH was used as a loading control and knockdown of PLK4 was calculated to be 56%

(Figure 6b).

Localization of DNA-Methyltransferase proteins in PLK4+/- MEFs

Acute inhibition of PLK4 in NIH/3T3 cells provoked a localization change in DNMT3a/b. To further elucidate the effects of PLK4 on the localization of these proteins the heterozygous mouse model was investigated. PLK4 haploinsufficiency has been known to cause improper mitotic division and centrosomal imbalances, which may have caused of disruption of DNMT3a/b localization. To determine if the perinuclear localization of DNMT3a/b was due to PLK4 haploinsufficiency, wild-type and heterozygous PLK4 MEFs were observed under immunofluorescence. There were no signs of perinuclear localization of DNMT3a or 3b in PLK4 wild-type or heterozygous MEFs (Figure 7).

Figure 6: Knockdown of PLK4 affects localization of DNMT3a and DNMT3b

The localization of DNMT3a and DNMT3b was examined in NIH/3T3 cells transiently transfected with an shPLK4 plasmid (MISSION shRNA, Sigma). A scrambled shRNA plasmid was used as a negative control (shSCR) and DAPI (Hoechst 33342) was used to stain the nucleus. (A) Distribution of DNMT3a and DNMT3b develop a perinuclear localization with PLK4 knockdown. (B) Immunoblot of whole-cell lysates (20ug) from shPLK4 transfected cells. Changes in PLK4 expression were quantified using densitometry. Error bars represent the standard deviation from three independent analyses.

Figure 7: PLK4 haploinsufficiency does not affect localization of DNMT3a and DNMT3b

The localization of DNMT3a and DNMT3b was examined in PLK4 heterozygous and wildtype murine embryonic fibroblasts (MEFs). Both proteins displayed normal phenotypes and their distribution was unaffected by PLK4 haploinsufficiency in this cell type.

Centrinone-B cell cycle profiles

PLK4 inhibition has many serious effects on a population of cells such as delayed mitotic progression and increased genome ploidy [42,52]. To establish whether the perinuclear staining of

DNMT3a was due to a cell cycle block, it was of interest to determine the cell cycle profile of centrinone- b treated cells. Treated cellular pellets were fixed in 80% ethanol and stained with propidium iodide overnight. Untreated and vehicle controls have similar cell cycle profiles while centrinone-B treated cells displayed a large proportion of cells in one peak at the G2/M stage indicating a mitotic block (Figure 8a).

Nocodazole is a mitotic inhibitor that disrupts polymerization of microtubules during mitosis and it produces a similar cell cycle profile to centrinone-B treated cells (Figure 7a). Because of the similar cellular effects of centrinone-B and nocodazole, it was important to investigate whether DNMT3a would have the same localization patterns under nocodazole as it did with centrinone-B. NIH/3T3 cells treated with nocodazole (Cell-signalling) and stained with an anti-DNMT3a antibody were monitored under immunofluorescence. There were no signs of perinuclear localization of DNMT3a after nocodazole treatment (Figure 8b). This indicates that the perinuclear staining was not due to a cell cycle block.

Figure 8: Nocodazole does not affect localization of DNMT3a

Cell cycle profiles of NIH/3T3 cells were obtained by flow cytometry. Cell samples were stained with propidium iodide to quantify DNA. An untreated sample was used to set up forward scatter, side scatter, and doublet discrimination for the subsequent samples. (A) Untreated and DMSO samples displayed regular cell cycle profiles with one peak at G1/G0 and a second peak at G2/M. Centrinone-B and nocodazole treated samples displayed one large peak at G2/M. (B) Localization of DNMT3a was examined in NIH/3T3 cells treated with nocodazole. The localization of DNMT3a was unaffected by nocodazole.

Centrinone-B affects global methylation in NIH-3T3 Cells

After determining that PLK4 inhibition caused disrupted localization of DNA-methyltransferase proteins, it was necessary to investigate PLK4’s impact on DNA methylation. A global DNA methylation assay was performed to assess the effects of centrinone-B. DNA was extracted from plated cells that were treated with centrinone-B, DMSO, or nocodazole by phenol/chloroform extraction. A methylation assay kit from epigentek was used to determine global methylation status of treated cells. The kit was followed as instructed by using 100ng of DNA per sample. Global methylation levels were compared relative to an untreated negative control sample. Global DNA methylation was overall decreased in centrinone-b treated samples when compared to the untreated or DMSO samples. Nocodazole treatments were utilized as a cell cycle control, the DNA methylation level of these samples were similar to the negative control samples (Figure 9a). A student’s t-test revealed no significant differences across sample means.

Due to the loss of global methylation levels after centrinone-b treatment, it was of interest to perform a rescue experiment. An inhibitor-resistant PLK4 mutant (G95L) has been shown to antagonize the effects of centrinone-b on centriole duplication. Hence, the PLK4 mutant (Flag-G95L) was transiently transfected in NIH/3T3 cells before centrinone-B treatment and observed under immunofluorescence.

Cells stained for DNMT3a were counted to assess the proportion of perinuclear stained nuclei in response to PLK4 transfection. Wild-type PLK4 (Flag-PLK4) was used as a control. The overall proportion of perinuclear stained cells with centrinone-B was determined to be 26%. Flag-G95L transfected cells displayed a small decrease in the proportion of perinuclear stained cells. Cells transfected with Flag-

PLK4 had a similar percentage of perinuclear cells to samples with just centrinone-B treatment (Figure

9b).

Because of the observed decrease in perinuclear stained cells with Flag-IR, a global methylation assay was performed with similar cell treatments. Cells treated with centrinone-B show a similar decrease in methylation levels from previous experiments. Flag-PLK4 and Flag-G95L transfected cells both displayed a trending increase in methylation levels (Figure 9c)

Figure 9: Analysis of Global methylation levels in NIH/3T3 cells

(A) An ELISA-based global methylation assay was performed for the genomic DNA of NIH/3T3 cells treated with DMSO, centrinone-B and nocodazole. Histograms are representative of three independent experiments and normalized to the respective untreated samples. Error bars represent one standard deviation of the mean. (B) Perinuclear stained cells were counted in response to PLK4 transfection and centrinone-B treatment. The proportion of perinuclear stained cells were quantified by counting >200 cells. Error bars represent the standard deviation from three independent experiments. Immunoblot of whole-cell lysates of transfected NIH/3T3 cells display presence of Flag-PLK4 and Flag-G95L. (C) A global methylation assay was performed to determine global methylation levels of NIH/3T3 cells transfected with Flag-IR and treated with centrinone-B. Histograms are representative of three independent experiments and normalized to the respective untreated samples. Error bars represent one standard deviation of the mean.

CHAPTER 4

Discussion

DNMT3a is not a substrate of PLK4

Protein kinases are remarkable in that they can be involved in many different sub-cellular pathways due to the sheer number of possible phosphorylatable substrates within the cell at any given time. PLK4, thus far, is mainly noted for its roles in cell cycle regulation and centriole duplication. Ward and Hudson have alluded to PLK4 having a potential role in epigenetic regulation by affecting the protein levels of DNA-methyltransferase proteins [47]. Understanding PLK4’s involvement in this pathway may be useful in diagnosing and treating epigenetic related diseases. Therefore, the goal of this study was to determine the role of PLK4 in epigenetic regulation.

As an initial step we examined whether DNMT3a could be a direct substrate of PLK4. By utilizing co-IP experiments and in-vitro kinase assays we determined that there exists a physical interaction between the two proteins but did not identify a direct kinase interaction (Figure 4b,c). While we could not detect the phosphorylation of DNMT3a by PLK4 under these specific conditions, the co-IP results imply that both proteins could engage with a larger protein complex that may be composed of overlapping binding partners. Intriguingly, both proteins have been reported to physically interact with p53 in vitro [45,111]. The functional significance of the PLK4 and p53 interaction is not well understood but the binding of DNMT3 to p53 was shown to be inhibitory and able to repress p53-mediated transactivation of the p21 gene. This proposed complex involving PLK4, DNMT3a, p53 and other cell cycle regulatory proteins may be a major spotlight for future research.

By using the PLK4 phosphorylation consensus sequences, we found 2 possible phosphorylation sites for DNMT3a which were S714 and S786 (Figure 4a). Interestingly, S714 mutations have been reported to be a recurrent DNMT3a mutation involved in the development of myelodysplastic syndromes

[112]. This is a common missense mutation that turns serine into a non-phosphorylatable cysteine amino acid. Though we were not able to show a phosphorylation reaction between PLK4 and DNMT3a, this

finding may direct future studies to utilize other kinase assay methods. As we know, phosphorylation and dephosphorylation are vital signals that may alter the protein’s stability, activity, localization and affinity for other substrates. If this interaction exists, this may link PLK4 directly to an epigenetic mechanism that is related to disease progression. This is a promising finding because our lab has reported that PLK4 protein levels are commonly disrupted in MDS and other blood related malignancies and DNMT3a is known to be dysregulated in the same neoplasms [112].

Centrinone-B affects DNMT3a and DNMT3b localization

Most biological studies of gene function involve utilizing knockout and knockdown methods or small molecule inhibition to study phenotypic effects. Luckily for PLK4, much has been done in regards to knockout mouse models and functional assays. There are many selective PLK4 inhibitors that exist and have been employed in biological assays, some of which are in phase 1 clinical trials for the treatment of advanced solid tumors [113, 114]. Of the drugs that exist, centrinone-B has recently come into the spotlight as a potent and selective inhibitor for PLK4. Centrinone-B has been experimentally shown to directly target and inhibit PLK4 through its kinase domain and prevent centriole assembly in cultured cells [110]. Due to the specificity and availability of centrinone-B we decided to utilize it as a small molecule inhibitor to target PLK4 for epigenetic related experiments.

We examined if PLK4 inhibition could affect the localization of DNA-methyltransferase proteins and their overall abundance. To our surprise, we found that Centrinone-B treatments on mouse fibroblast cells could change the localization of DNMT3a and DNMT3b, two de-novo methyltransferases, without changing the overall levels of these proteins. Interestingly, the localization of DNMT1, the maintenance methyltransferase, remained unaffected by centrinone-B (Figure 5). This may be due to DNMT3a and 3b being similar in regards to protein structure, size, and amino acid sequence. DNMT1 is overall much larger than DNMT3a/b and has a larger number of defined, functional protein domains [115]. By affecting the de novo methylases, this may suggest that PLK4 plays a role in regulating differentiation.

There are numerous studies elucidating the functions of DNMT3a and DNMT3b in hematopoiesis and embryonic lethality is an outcome of DNMT3a/DNMT3b double knockouts in mice due to a differentiation block [116].

Interestingly, the overall protein level of PLK4 also affects the localization of the de-novo methyltransferases. With the ShRNA PLK4 construct, we observed that knockdown could cause the perinuclear localization of DNMT3a and DNMT3b (Figure 6). Centrinone-B does not affect the levels of

PLK4 but rather its kinase ability in vivo. Our findings here indicate that because the knockdown caused a similar effect, it must be a PLK4 specific mechanism that is causing the dysregulated localization patterns. This relationship is alluring because our lab has shown that the PLK4 gene promoter is commonly methylated in hematological cancer tissues and hematological cancer cell lines. Also, the

PLK4 promoter increasingly becomes methylated in blood neoplastic cell lines under hypoxia which means that lower levels of PLK4 protein could be induced under DNA damage and lead to changes in

DNA methylation [109]. This dysregulation of DNMT3a and DNMT3b localization could be an acute effect that may occur in response to cellular stresses where PLK4 becomes inhibited or downregulated.

The hypothesis that the perinuclear localization of the de-novo methylases is a short-term effect is supported by our own findings in PLK4+/- MEFS. When observed under immunofluorescence, there were no signs of disrupted localization of DNMT3a and DNMT3b (Figure 7). This signifies that the long-term haploinsufficient effects seen in PLK4 mouse models such as increased apoptosis or genomic instability was not the cause of the perinuclear localization. In fact, it only occurred under short time spans such as transient transfections with ShRNA or Centrinone-B treatment. Future studies could further investigate this relationship by finding other ways to acutely affect PLK4 levels such as using other well known

PLK4 inhibitors or using hypoxia treatment [109].

PLK4 has numerous roles in the cell cycle and inhibition causes drastic changes to the cellular environment. Most findings conclude that PLK4 inhibition and knockdown cause a mitotic block due to the incomplete duplication of centrosomes and causes an increase in the proportion of cells in the G2/M phase [117,118]. Due to these results, we investigated if the dysregulation of DNMT3a/b could be caused

by a cell cycle block. Our observations of Centrione-B treatments on the cell cycle agree with the overall literature; Centrione-B increases the proportion of cells in the G2/M phase (Figure 8). We sought to examine a similar population of cells using a different treatment to investigate whether the subcellular localization of DNMT3a/b is cell cycle dependent. Nocodazole is a microtubule inhibitor that has been used in many studies to effectively arrest cells in mitosis. It causes a similar cell cycle profile to centrione-B treated cells, however, we did not observe a perinuclear pattern of DNMT3a localization in cells with nocodazole treatment (Figure 8).

It is important to mention that while nocodazole produces a similar population of cells it does not inhibit the cell cycle in the same way. Centrinone-B causes centriole depletion causing cells to arrest in

G2 delaying entry into mitosis [110]. Nocodazole inhibits the polymerization of microtubules while in mitosis so that cells cannot properly divide. Nocodazole treated cells enter mitosis while centrinone-b treated cells do not, causing a similar cell cycle profile in regards to DNA content but a different state in regards to cell cycle phase. This differentiation is important because different states of the cell cycle will have different gene products activated during that time. At present, our results only suggest that DNA content does not affect the localization of DNMT3a.

Centrinone-B affects global DNA methylation

The subcellular localization of proteins is one mechanism by which proteins are regulated. For example, most proteins are created outside of the cell by ribosomes in the endoplasmic reticulum and need to be transported to the locations where they function. Many proteins that are known to work mainly in the nucleus must be transported inside through nuclear localization signals (NLS), which are programmed into their amino acid sequence. Studies that have mutated these NLS sites have found that without particular sequences, these proteins cannot enter the nucleus and they observe similar phenotypes to a full gene knockout [49,50]. Thus, the localization of a protein is important in its own functional abilities and we sought to analyze this with centrinone-B.

Due to the perceived localization changes to DNMT3a and DNMT3b found in response to centrinone-B, we investigated if this could affect global DNA methylation. Using an ELIZA-based assay, we determined that centrinone-B treatment decreased global DNA methylation within 3T3 cells (Figure

9a). In support of our other findings, this decrease was likely caused by the inhibited catalytic function of the de-novo methyltransferases due to dysregulated subcellular localization. The overall methylation changes were compared to an untreated control and the global methylation decrease was observed to be

~27%. While the relative change can be seen as a nominal amount, it may be important to note that many epigenetic studies have concluded that DNMT1, the maintenance methyltransferase, is the most important in regulating large changes in global DNA methylation. Individual knockouts of DNMT proteins reveal no large change in global methylation levels, however, double knockouts of DNMT1 along with

DNMT3a or DNMT3b have been shown to produce drastic differences in methylation [119,120,121]. We observed that DNMT1 was unaffected by centrinone-B which may explain why we did not observe a drastic change in global methylation levels and also why we did not find any significant difference using a student’s t-test. Furthermore, our nocodazole control did not show a decrease in global methylation indicating that the abundance of DNA did not change the proportion of methylated DNA. This was expected as nocodazole did not change the localization patterns of DNMT3a/b as we found in centrinone-

B treated cells.

The first group to investigate the effects of centrione-B on centriole duplication also found that a mutated form of PLK4 could rescue the effects [110]. The inhibitor resistant PLK4 (PLK4-G95L) has a mutation in the kinase domain that hindered centrinone-B binding. We used this same PLK4 mutant to see if it could rescue the effects of decreased DNA methylation. By transiently transfecting PLK4-G95L we observed that a lower proportion of cells displayed the perinuclear phenotype when treated with centrinone-B (Figure 9b). Furthermore, when we measured global DNA methylation, we saw a trending increase in global methylation (Figure 9c). Wild-type PLK4 was used as a control and we did not see a similar increasing trend in DNA methylation, it stayed relatively the same as centrinone-B treated cells.

Our results suggest that PLK4 plays a role in regulating global DNA methylation levels by governing the localization of DNMT3a and DNMT3b.

A student’s t-test revealed no significant difference between samples due to high variance. This is a common problem when working with a population of treated cells; not all cells will be expressing our

PLK4 plasmids due to transfection efficiencies. Replicative studies using a stable cell line expressing these plasmids should be considered for future experiments. We would still expect a similar outcome using a stable cell line, but with more pronounced differences when we measure DNA methylation.

Concluding Remarks

Our results suggest interesting roles for PLK4 in epigenetic regulation. Firstly, PLK4 inhibition and protein levels can regulate the localization of de-novo methyltransferases, which may indicate that

PLK4 is needed for cell differentiation. PLK4-/- mice are embryonic lethal at day 7.5 and morpholino studies in xenopus show that PLK4 is necessary for proper somite formation [122,123]. These phenotypes have been alluded to microtubule and cell polarity disruptions but our experiments show that PLK4- mediated epigenetic regulation may play a supporting role. Much of embryogenesis is controlled by large epigenetic reprogramming events through DNA methylation and the DNMT family members are crucial in this regard [124]. X-chromosome inactivation is one such event that completes by E6.5 in mammalian embryos and is carried out specifically through the de-novo methyltransferases [125]. PLK4-/- embryos may lack coordination of DNMTs to undergo this event and thus abort due to differentiation defects.

Additionally, other groups have determined that DNMT3a is especially important for proper somite and neural-tube formation in zebrafish and WT mouse embryos; structures that are morphologically absent in

PLK4-/- embryos [126,127,128]. We show that PLK4 inhibition can affect global DNA methylation by disrupting DNMT3a and DNMT3b localization and this may support the phenotypes observed in PLK4-/- embryos.

Interestingly, our lab has reported that hematological cancerous tissues and cell lines are subjected to PLK4 promoter methylation, which directly affects PLK4 protein levels [109]. PLK4 promoter methylation could also be induced by hypoxia, which may be an early step for cancer development. We observed that PLK4 levels directly affect DNMT3a/3b localization that may further increase potential for neoplastic development due to disrupted epigenetic pathways. Furthermore, we could not detect direct phosphorylation of DNMT3a by PLK4 with our methods but hypothesize that they could be involved in a larger cell cycle regulating protein complex that includes tumor suppressor p53.

Overall, we examined the effects of PLK4 inhibition on DNA methylation. We show that

Centrinone-B, a potent PLK4 inhibitor, can disrupt the localization of DNMT3a and DNMT3b and cause a global loss of DNA methylation in mouse fibroblast cells without modifying protein levels.

Dysregulated localization of DNMT3a/3b can also be achieved with ShRNA knockdown of PLK4 indicating that it must be a PLK4-specific mechanism. Our results highlight a potential mechanism for

PLK4-mediated epigenetic regulation that may be important in tissue differentiation. PLK4 mainly regulates centrosomes but could also act in multiple pathways during development and embryogenesis when tissue differentiation is crucial to the formation of highly complex structures. Understanding

PLK4’s various roles in the cell will provide insight into its role in cancer progression and embryogenesis.

Future Directions

Our results indicate that DNMT3a is not phosphorylated by PLK4 in our specific in-vitro conditions. The potential phosphorylation sites found for DNMT3a in the motif search are still compelling because both proteins are involved in the progression of blood neoplasms [104,112]. Due to serine 714 in DNMT3a being a recurring mutated amino acid linked to MDS we think that other known in vivo kinase assay methods could be employed to determine if this interaction exists. There is also the possibility of a secondary effect in which PLK4 phosphorylates another protein that then regulates the

localization of DNMT3a; this could involve p53. Whether or not DNMT3a is a PLK4 substrate, it would be interesting to further elucidate their interactions with p53 because it is a common binding partner for both proteins. Future experiments could consist of co-IP or mass spectrometry analysis of PLK4 pull- down samples to determine the presence of both DNMT3a and p53 in tandem. Furthermore, since PLK4 and p53 activity can be induced by DNA damage, functional assays may investigate how DNMT3a might be involved in the same pathways that may include: localization, substrate binding, and catalytic activity.

Experiments to assess overexpression effects of each protein during embryogenesis or DNA damage should also be considered.

The results using nocodazole as a cell cycle inhibitor should be more refined. Nocodazole does not inhibit the cell cycle the same way PLK4 does. In this regard, I would suggest using a CDK1 inhibitor or an additional but different PLK4 inhibitor to arrest cells in G2. These treatments would be used to further assess DNMT3a/b localization and global methylation levels.

Further experiments that revolve around PLK4 and epigenetic regulation should also consider its interaction with PRMT5, a histone methyltransferase. Our lab has identified PRMT5 as a PLK4 substrate that localizes to the centrosomes and is dysregulated in PLK4+/- MEFS [129]. Experiments involving

PLK4 inhibition or knockdown with PRMT5 may further clarify PLK4’s role in epigenetic regulation because histone-methyltransferases frequently associate with DNA-methyltransferases in larger protein complexes [130]. PLK4 could possibly regulate localization and function of DNMT3a through the phosphorylation of PRMT5 (Figure 10).

Figure 10: Model for PLK4-mediated regulation of DNA methylation through PRMT5

PRMT5 is a PLK4 substrate that also localizes to the centrosome. After phosphorylation by PLK4,

PRMT5 could localize to its histone targets (H4R3) and methylate the arginine tails. Methylation of histones can also promote the recruitment of DNMT3a to further methylate nearby CpG sites. Catalytic inhibition of PLK4 would disrupt PRMT5’s function and subsequent DNA methylation by DNMT3a.

REFERENCES/BIBLIOGRAPHY

1. Sunkel, C. E., & Glover, D. M. (1988). polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. Journal of cell science, 89(1), 25-38. 2. Archambault, V., & Glover, D. M. (2009). Polo-like kinases: conservation and divergence in their functions and regulation. Nature reviews Molecular cell biology, 10(4), 265- 275. 3. Lindqvist, A., Rodríguez-Bravo, V., & Medema, R. H. (2009). The decision to enter mitosis: feedback and redundancy in the mitotic entry network. Journal of Cell Biology, 185(2), 193-202. 4. Schmidt, M., Rohe, A., Platzer, C., Najjar, A., Erdmann, F., & Sippl, W. (2017). Regulation of G2/M transition by inhibition of WEE1 and PKMYT1 kinases. Molecules, 22(12), 2045. 5. Li, H., Liu, X. S., Yang, X., Song, B., Wang, Y., & Liu, X. (2010). Polo-like kinase 1 phosphorylation of p150Glued facilitates nuclear envelope breakdown during prophase. Proceedings of the National Academy of Sciences, 107(33), 14633-14638. 6. Hu, C. K., Özlü, N., Coughlin, M., Steen, J. J., & Mitchison, T. J. (2012). Plk1 negatively regulates PRC1 to prevent premature midzone formation before cytokinesis. Molecular biology of the cell, 23(14), 2702-2711. 7. Li, Z., Liu, J., Li, J., Kong, Y., Sandusky, G., Rao, X., ... & Liu, X. (2017). Polo-like kinase 1 (Plk1) overexpression enhances ionizing radiation-induced cancer formation in mice. Journal of Biological Chemistry, 292(42), 17461-17472. 8. Takahashi, T., Sano, B., Nagata, T., Kato, H., Sugiyama, Y., Kunieda, K., ... & Saji, S. (2003). Polo‐like kinase 1 (PLK1) is overexpressed in primary colorectal cancers. Cancer science, 94(2), 148-152. 9. Matthew, E. M., Yen, T. J., Dicker, D. T., Dorsey, J. F., Yang, W., Navaraj, A., & El- Deiry, W. S. (2007). Replication stress, defective S-phase checkpoint and increased death in Plk2-deficient human cancer cells. Cell cycle, 6(20), 2571-2578. 10. Chang, J., Cizmecioglu, O., Hoffmann, I., & Rhee, K. (2010). PLK2 phosphorylation is critical for CPAP function in procentriole formation during the centrosome cycle. The EMBO journal, 29(14), 2395-2406. 11. Burns, T. F., Fei, P., Scata, K. A., Dicker, D. T., & El-Deiry, W. S. (2003). Silencing of the novel p53 target gene Snk/Plk2 leads to mitotic catastrophe in paclitaxel (taxol)-exposed cells. Molecular and cellular biology, 23(16), 5556-5571. 12. Syed, N., Smith, P., Sullivan, A., Spender, L. C., Dyer, M., Karran, L., ... & Griffin, B. (2006). Transcriptional silencing of Polo-like kinase 2 (SNK/PLK2) is a frequent event in B-cell malignancies. Blood, 107(1), 250-256. 13. Xie, S., Wu, H., Wang, Q., Cogswell, J. P., Husain, I., Conn, C., ... & Dai, W. (2001). Plk3 functionally links DNA damage to cell cycle arrest and apoptosis at least in part via the p53 pathway. Journal of Biological Chemistry, 276(46), 43305-43312. 14. Xie, S., Wu, H., Wang, Q., Kunicki, J., Thomas, R. O., Hollingsworth, R. E., ... & Dai, W. (2002). Genotoxic stress-induced activation of Plk3 is partly mediated by Chk2. Cell Cycle, 1(6), 424-429. 15. Xu, D., Yao, Y., Jiang, X., Lu, L., & Dai, W. (2010). Regulation of PTEN stability and activity by Plk3. Journal of Biological Chemistry, 285(51), 39935-39942. 16. Yang, Y., Bai, J., Shen, R., Brown, S. A., Komissarova, E., Huang, Y., ... & Winkles, J. A. (2008). Polo-like kinase 3 functions as a tumor suppressor and is a negative regulator of hypoxia-inducible factor-1α under hypoxic conditions. Cancer research, 68(11), 4077-4085.

17. Li, B., Ouyang, B., Pan, H., Reissmann, P. T., Slamon, D. J., Arceci, R., ... & Dai, W. (1996). Prk, a cytokine-inducible human protein serine/threonine kinase whose expression appears to be down-regulated in lung carcinomas. Journal of Biological Chemistry, 271(32), 19402-19408. 18. Weichert, W., Denkert, C., Schmidt, M., Gekeler, V., Wolf, G., Köbel, M., ... & Hauptmann, S. (2004). Polo-like kinase isoform expression is a prognostic factor in ovarian carcinoma. British journal of cancer, 90(4), 815-821. 19. Hori, A., Barnouin, K., Snijders, A. P., & Toda, T. (2016). A non‐canonical function of Plk4 in centriolar satellite integrity and ciliogenesis through PCM1 phosphorylation. EMBO reports, 17(3), 326-337. 20. Dzhindzhev, N. S., Tzolovsky, G., Lipinszki, Z., Schneider, S., Lattao, R., Fu, J., ... & Glover, D. M. (2014). Plk4 phosphorylates Ana2 to trigger Sas6 recruitment and procentriole formation. Current Biology, 24(21), 2526-2532. 21. Hatch, E. M., Kulukian, A., Holland, A. J., Cleveland, D. W., & Stearns, T. (2010). Cep152 interacts with Plk4 and is required for centriole duplication. Journal of Cell Biology, 191(4), 721-729. 22. Rosario, C. O., Ko, M. A., Haffani, Y. Z., Gladdy, R. A., Paderova, J., Pollett, A., ... & Swallow, C. J. (2010). Plk4 is required for cytokinesis and maintenance of chromosomal stability. Proceedings of the National Academy of Sciences, 107(15), 6888-6893. 23. Coelho, P. A., Bury, L., Shahbazi, M. N., Liakath-Ali, K., Tate, P. H., Wormald, S., ... & Zernicka-Goetz, M. (2015). Over-expression of Plk4 induces centrosome amplification, loss of primary cilia and associated tissue hyperplasia in the mouse. Open biology, 5(12), 150209. 24. Nanjundappa, R., Kong, D., Shim, K., Stearns, T., Brody, S. L., Loncarek, J., & Mahjoub, M. R. (2019). Regulation of cilia abundance in multiciliated cells. Elife, 8, e44039. 25. de Cárcer, G., Escobar, B., Higuero, A. M., García, L., Ansón, A., Pérez, G., ... & Malumbres, M. (2011). Plk5, a polo box domain-only protein with specific roles in neuron differentiation and glioblastoma suppression. Molecular and cellular biology, 31(6), 1225-1239. 26. Andrysik, Z., Bernstein, W. Z., Deng, L., Myer, D. L., Li, Y. Q., Tischfield, J. A., ... & Bahassi, E. M. (2010). The novel mouse Polo-like kinase 5 responds to DNA damage and localizes in the nucleolus. Nucleic acids research, 38(9), 2931-2943. 27. Elia, A. E., Rellos, P., Haire, L. F., Chao, J. W., Ivins, F. J., Hoepker, K., ... & Yaffe, M. B. (2003). The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell, 115(1), 83-95. 28. Leung, G. C., Ho, C. S., Blasutig, I. M., Murphy, J. M., & Sicheri, F. (2007). Determination of the Plk4/Sak consensus phosphorylation motif using peptide spots arrays. FEBS letters, 581(1), 77-83. 29. Slevin, L. K., Nye, J., Pinkerton, D. C., Buster, D. W., Rogers, G. C., & Slep, K. C. (2012). The structure of the plk4 cryptic polo box reveals two tandem polo boxes required for centriole duplication. Structure, 20(11), 1905-1917. 30. Arquint, C., Gabryjonczyk, A. M., Imseng, S., Böhm, R., Sauer, E., Hiller, S., ... & Maier, T. (2015). STIL binding to Polo-box 3 of PLK4 regulates centriole duplication. Elife, 4, e07888. 31. Rechsteiner, M., & Rogers, S. W. (1996). PEST sequences and regulation by proteolysis. Trends in biochemical sciences, 21(7), 267-271. 32. Cunha-Ferreira, I., Rodrigues-Martins, A., Bento, I., Riparbelli, M., Zhang, W., Laue, E., ... & Bettencourt-Dias, M. (2009). The SCF/Slimb ubiquitin ligase limits centrosome amplification through degradation of SAK/PLK4. Current Biology, 19(1), 43-49. 33. Guderian, G., Westendorf, J., Uldschmid, A., & Nigg, E. A. (2010). Plk4 trans- autophosphorylation regulates centriole number by controlling βTrCP-mediated degradation. Journal of cell science, 123(13), 2163-2169.

34. Habedanck, R., Stierhof, Y. D., Wilkinson, C. J., & Nigg, E. A. (2005). The Polo kinase Plk4 functions in centriole duplication. Nature cell biology, 7(11), 1140-1146. 35. Ganem, N. J., Godinho, S. A., & Pellman, D. (2009). A mechanism linking extra centrosomes to chromosomal instability. Nature, 460(7252), 278-282. 36. Woodruff, J. B., Wueseke, O., & Hyman, A. A. (2014). Pericentriolar material structure and dynamics. Philosophical Transactions of the Royal Society B: Biological Sciences, 369(1650), 20130459. 37. Tsou, M. F. B., Wang, W. J., George, K. A., Uryu, K., Stearns, T., & Jallepalli, P. V. (2009). Polo kinase and separase regulate the mitotic licensing of centriole duplication in human cells. Developmental cell, 17(3), 344-354. 38. Dzhindzhev, N. S., Tzolovsky, G., Lipinszki, Z., Abdelaziz, M., Debski, J., Dadlez, M., & Glover, D. M. (2017). Two-step phosphorylation of Ana2 by Plk4 is required for the sequential loading of Ana2 and Sas6 to initiate procentriole formation. Open biology, 7(12), 170247. 39. Wang, G., Jiang, Q., & Zhang, C. (2014). The role of mitotic kinases in coupling the centrosome cycle with the assembly of the mitotic spindle. J Cell Sci, 127(19), 4111-4122. 40. Peel, N., Stevens, N. R., Basto, R., & Raff, J. W. (2007). Overexpressing centriole- replication proteins in vivo induces centriole overduplication and de novo formation. Current Biology, 17(10), 834-843. 41. Holland, A. J., Fachinetti, D., Da Cruz, S., Zhu, Q., Vitre, B., Lince-Faria, M., ... & Cleveland, D. W. (2012). Polo-like kinase 4 controls centriole duplication but does not directly regulate cytokinesis. Molecular biology of the cell, 23(10), 1838-1845. 42. Maiato, H., & Logarinho, E. (2014). Mitotic spindle multipolarity without centrosome amplification. Nature cell biology, 16(5), 386-394.. 43. Hirao, A., Kong, Y. Y., Matsuoka, S., Wakeham, A., Ruland, J., Yoshida, H., ... & Mak, T. W. (2000). DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science, 287(5459), 1824-1827. 44. Petrinac, S., Ganuelas, M. L., Bonni, S., Nantais, J., & Hudson, J. W. (2009). Polo-like kinase 4 phosphorylates Chk2. Cell Cycle, 8(2), 327-329. 45. Swallow, C. J., Ko, M. A., Siddiqui, N. U., Hudson, J. W., & Dennis, J. W. (2005). Sak/Plk4 and mitotic fidelity. Oncogene, 24(2), 306-312. 46. Li, J., Tan, M., Li, L., Pamarthy, D., Lawrence, T. S., & Sun, Y. (2005). SAK, a new polo-like kinase, is transcriptionally repressed by p53 and induces apoptosis upon RNAi silencing. Neoplasia, 7(4), 312-323. 47. Ward, A., & Hudson, J. W. (2014). p53-Dependent and cell specific epigenetic regulation of the polo-like kinases under oxidative stress. PloS one, 9(1). 48. Bonni, S., Ganuelas, M. L., Petrinac, S., & Hudson, J. W. (2008). Human plk4 phosphorylates cdc25c. Cell Cycle, 7(4), 545-547. 49. Toyoshima‐Morimoto, F., Taniguchi, E., & Nishida, E. (2002). Plk1 promotes nuclear translocation of human Cdc25C during prophase. EMBO reports, 3(4), 341-348. 50. Bahassi, E. M., Hennigan, R. F., Myer, D. L., & Stambrook, P. J. (2004). Cdc25C phosphorylation on serine 191 by Plk3 promotes its nuclear translocation. Oncogene, 23(15), 2658-2663. 51. Hudson, J. W., Kozarova, A., Cheung, P., Macmillan, J. C., Swallow, C. J., Cross, J. C., & Dennis, J. W. (2001). Late mitotic failure in mice lacking Sak, a polo-like kinase. Current Biology, 11(6), 441-446. 52. Ko, M. A., Rosario, C. O., Hudson, J. W., Kulkarni, S., Pollett, A., Dennis, J. W., & Swallow, C. J. (2005). Plk4 haploinsufficiency causes mitotic infidelity and carcinogenesis. Nature genetics, 37(8), 883-888. 53. Morettin, A., Ward, A., Nantais, J., & Hudson, J. W. (2009). Gene expression patterns in heterozygous Plk4 murine embryonic fibroblasts. BMC genomics, 10(1), 319.

54. Boveri, T. (2008). Concerning the origin of malignant tumours by Theodor Boveri. Translated and annotated by Henry Harris. Journal of cell science, 121(Supplement 1), 1-84. 55. Denu, R. A., Zasadil, L. M., Kanugh, C., Laffin, J., Weaver, B. A., & Burkard, M. E. (2016). Centrosome amplification induces high grade features and is prognostic of worse outcomes in breast cancer. BMC cancer, 16(1), 47. 56. Basto, R., Brunk, K., Vinadogrova, T., Peel, N., Franz, A., Khodjakov, A., & Raff, J. W. (2008). Centrosome amplification can initiate tumorigenesis in flies. Cell, 133(6), 1032-1042. 57. Godinho, S. A., Picone, R., Burute, M., Dagher, R., Su, Y., Leung, C. T., ... & Pellman, D. (2014). Oncogene-like induction of cellular invasion from centrosome amplification. Nature, 510(7503), 167-171. 58. Shinmura, K., Kurabe, N., Goto, M., Yamada, H., Natsume, H., Konno, H., & Sugimura, H. (2014). PLK4 overexpression and its effect on centrosome regulation and chromosome stability in human gastric cancer. Molecular biology reports, 41(10), 6635-6644. 59. Li, Z., Dai, K., Wang, C., Song, Y., Gu, F., Liu, F., & Fu, L. (2016). Expression of polo- like kinase 4 (PLK4) in breast cancer and its response to taxane-based neoadjuvant chemotherapy. Journal of Cancer, 7(9), 1125. 60. Wang, J., Zuo, J., Wang, M., Ma, X., Gao, K., Bai, X., ... & Liu, H. (2019). Polo-like kinase 4 promotes tumorigenesis and induces resistance to radiotherapy in glioblastoma. Oncology reports, 41(4), 2159-2167. 61. Liu, L., Zhang, C. Z., Cai, M., Fu, J., Chen, G. G., & Yun, J. (2012). Downregulation of polo-like kinase 4 in hepatocellular carcinoma associates with poor prognosis. PloS one, 7(7). 62. Chan, J. Y. (2011). A clinical overview of centrosome amplification in human cancers. International journal of biological sciences, 7(8), 1122. 63. Godinho, S. A., & Pellman, D. (2014). Causes and consequences of centrosome abnormalities in cancer. Philosophical Transactions of the Royal Society B: Biological Sciences, 369(1650), 20130467. 64. Zyss, D., & Gergely, F. (2009). Centrosome function in cancer: guilty or innocent?. Trends in cell biology, 19(7), 334-346. 65. Kulukian, A., Holland, A. J., Vitre, B., Naik, S., Cleveland, D. W., & Fuchs, E. (2015). Epidermal development, growth control, and homeostasis in the face of centrosome amplification. Proceedings of the National Academy of Sciences, 112(46), E6311-E6320. 66. Vitre, B., Holland, A. J., Kulukian, A., Shoshani, O., Hirai, M., Wang, Y., ... & Tessarollo, L. (2015). Chronic centrosome amplification without tumorigenesis. Proceedings of the National Academy of Sciences, 112(46), E6321-E6330. 67. Serçin, Ö., Larsimont, J. C., Karambelas, A. E., Marthiens, V., Moers, V., Boeckx, B., ... & Blanpain, C. (2016). Transient PLK4 overexpression accelerates tumorigenesis in p53- deficient epidermis. Nature cell biology, 18(1), 100-110. 68. Levine, M. S., Bakker, B., Boeckx, B., Moyett, J., Lu, J., Vitre, B., ... & Foijer, F. (2017). Centrosome amplification is sufficient to promote spontaneous tumorigenesis in mammals. Developmental cell, 40(3), 313-322. 69. Kayser, G., Gerlach, U., Walch, A., Nitschke, R., Haxelmans, S., Kayser, K., ... & Lassmann, S. (2005). Numerical and structural centrosome aberrations are an early and stable event in the adenoma–carcinoma sequence of colorectal carcinomas. Virchows Archiv, 447(1), 61-65. 70. Berger, S. L., Kouzarides, T., Shiekhattar, R., & Shilatifard, A. (2009). An operational definition of epigenetics. Genes & development, 23(7), 781-783 71. Sharma, S., Kelly, T. K., & Jones, P. A. (2010). Epigenetics in cancer. Carcinogenesis, 31(1), 27-36. 72. Feinberg, A. P., & Tycko, B. (2004). The history of cancer epigenetics. Nature Reviews Cancer, 4(2), 143-153.

73. Robertson, K. D. (2005). DNA methylation and human disease. Nature Reviews Genetics, 6(8), 597-610. 74. Jang, H. S., Shin, W. J., Lee, J. E., & Do, J. T. (2017). CpG and non-CpG methylation in epigenetic gene regulation and brain function. Genes, 8(6), 148. 75. Saxonov, S., Berg, P., & Brutlag, D. L. (2006). A genome-wide analysis of CpG dinucleotides in the human genome distinguishes two distinct classes of promoters. Proceedings of the National Academy of Sciences, 103(5), 1412-1417. 76. Deaton, A. M., & Bird, A. (2011). CpG islands and the regulation of transcription. Genes & development, 25(10), 1010-1022. 77. Castillo-Aguilera, O., Depreux, P., Halby, L., Arimondo, P. B., & Goossens, L. (2017). DNA methylation targeting: the DNMT/HMT crosstalk challenge. Biomolecules, 7(1), 3. 78. Sen, G. L., Reuter, J. A., Webster, D. E., Zhu, L., & Khavari, P. A. (2010). DNMT1 maintains progenitor function in self-renewing somatic tissue. Nature, 463(7280), 563-567. 79. Okano, M., Bell, D. W., Haber, D. A., & Li, E. (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 99(3), 247- 257. 80. Kouzarides, T. (2007). Chromatin modifications and their function. Cell, 128(4), 693- 705. 81. Wang, G. G., Allis, C. D., & Chi, P. (2007). Chromatin remodeling and cancer, Part I: Covalent histone modifications. Trends in molecular medicine, 13(9), 363-372. 82. Hansen, K. H., Bracken, A. P., Pasini, D., Dietrich, N., Gehani, S. S., Monrad, A., ... & Helin, K. (2008). A model for transmission of the H3K27me3 epigenetic mark. Nature cell biology, 10(11), 1291-1300. 83. Lee, J. E., Wang, C., Xu, S., Cho, Y. W., Wang, L., Feng, X., ... & Ge, K. (2013). H3K4 mono-and di-methyltransferase MLL4 is required for enhancer activation during cell differentiation. Elife, 2, e01503. 84. Feinberg, A. P., & Vogelstein, B. (1983). Hypomethylation of ras oncogenes in primary human cancers. Biochemical and biophysical research communications, 111(1), 47-54. 85. Gama-Sosa, M. A., Slagel, V. A., Trewyn, R. W., Oxenhandler, R., Kuo, K. C., Gehrke, C. W., & Ehrlich, M. (1983). The 5-methylcytosine content of DNA from human tumors. Nucleic acids research, 11(19), 6883-6894. 86. Ehrlich, M. (2009). DNA hypomethylation in cancer cells. Epigenomics, 1(2), 239-259. 87. An, X., Tiwari, A. K., Sun, Y., Ding, P. R., Ashby Jr, C. R., & Chen, Z. S. (2010). BCR- ABL tyrosine kinase inhibitors in the treatment of Philadelphia chromosome positive chronic myeloid leukemia: a review. Leukemia research, 34(10), 1255-1268. 88. Chen, R. Z., Pettersson, U., Beard, C., Jackson-Grusby, L., & Jaenisch, R. (1998). DNA hypomethylation leads to elevated mutation rates. Nature, 395(6697), 89-93. 89. Lander, E. S., Linton, L. M., Birren, B., Nusbaum, C., Zody, M. C., Baldwin, J., ... & Funke, R. (2001). Initial sequencing and analysis of the human genome. 90. Ardeljan, D., Taylor, M. S., Ting, D. T., & Burns, K. H. (2017). The human long interspersed element-1 retrotransposon: an emerging biomarker of neoplasia. Clinical chemistry, 63(4), 816-822. 91. Sunami, E., De Maat, M., Vu, A., Turner, R. R., & Hoon, D. S. (2011). LINE-1 hypomethylation during primary colon cancer progression. PloS one, 6(4). 92. Tangkijvanich, P., Hourpai, N., Rattanatanyong, P., Wisedopas, N., Mahachai, V., & Mutirangura, A. (2007). Serum LINE-1 hypomethylation as a potential prognostic marker for hepatocellular carcinoma. Clinica chimica acta, 379(1-2), 127-133. 93. Saito, K., Kawakami, K., Matsumoto, I., Oda, M., Watanabe, G., & Minamoto, T. (2010). Long interspersed nuclear element 1 hypomethylation is a marker of poor prognosis in stage IA non–small cell lung cancer. Clinical cancer research, 16(8), 2418-2426.

94. Cui, H., Onyango, P., Brandenburg, S., Wu, Y., Hsieh, C. L., & Feinberg, A. P. (2002). Loss of imprinting in colorectal cancer linked to hypomethylation of H19 and IGF2. Cancer research, 62(22), 6442-6446. 95. Esteller, M. (2002). CpG island hypermethylation and tumor suppressor genes: a booming present, a brighter future. Oncogene, 21(35), 5427-5440. 96. Hino, R., Uozaki, H., Murakami, N., Ushiku, T., Shinozaki, A., Ishikawa, S., ... & Fukayama, M. (2009). Activation of DNA methyltransferase 1 by EBV latent membrane protein 2A leads to promoter hypermethylation of PTEN gene in gastric carcinoma. Cancer research, 69(7), 2766-2774. 97. Shim, Y. H., Kang, G. H., & Ro, J. Y. (2000). Correlation of p16 hypermethylation with p16 protein loss in sporadic gastric carcinomas. Laboratory investigation, 80(5), 689-695. 98. Hellebrekers, D. M., Lentjes, M. H., Van Den Bosch, S. M., Melotte, V., Wouters, K. A., Daenen, K. L., ... & Khalid-de Bakker, C. A. (2009). GATA4 and GATA5 are potential tumor suppressors and biomarkers in colorectal cancer. Clinical cancer research, 15(12), 3990-3997. 99. Lin, R. K., Hsu, H. S., Chang, J. W., Chen, C. Y., Chen, J. T., & Wang, Y. C. (2007). Alteration of DNA methyltransferases contributes to 5′ CpG methylation and poor prognosis in lung cancer. Lung cancer, 55(2), 205-213. 100. Girault, I., Tozlu, S., Lidereau, R., & Bièche, I. (2003). Expression analysis of DNA methyltransferases 1, 3A, and 3B in sporadic breast carcinomas. Clinical cancer research, 9(12), 4415-4422. 101. Delhommeau, F., Dupont, S., Valle, V. D., James, C., Trannoy, S., Masse, A., ... & Lecluse, Y. (2009). Mutation in TET2 in myeloid cancers. New England Journal of Medicine, 360(22), 2289-2301. 102. Varambally, S., Dhanasekaran, S. M., Zhou, M., Barrette, T. R., Kumar-Sinha, C., Sanda, M. G., ... & Rubin, M. A. (2002). The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature, 419(6907), 624-629. 103. Kim, S. J., Zhao, H., Hardikar, S., Singh, A. K., Goodell, M. A., & Chen, T. (2013). A DNMT3A mutation common in AML exhibits dominant-negative effects in murine ES cells. Blood, The Journal of the American Society of Hematology, 122(25), 4086-4089. 104. Ward, A., Sivakumar, G., Kanjeekal, S., Hamm, C., Labute, B. C., Shum, D., & Hudson, J. W. (2015). The deregulated promoter methylation of the Polo-like kinases as a potential biomarker in hematological malignancies. Leukemia & lymphoma, 56(7), 2123-2133. 105. Pellegrino, R., Calvisi, D. F., Ladu, S., Ehemann, V., Staniscia, T., Evert, M., ... & Longerich, T. (2010). Oncogenic and tumor suppressive roles of polo‐like kinases in human hepatocellular carcinoma. Hepatology, 51(3), 857-868. 106. Takahashi, T., Sano, B., Nagata, T., Kato, H., Sugiyama, Y., Kunieda, K., ... & Saji, S. (2003). Polo‐like kinase 1 (PLK1) is overexpressed in primary colorectal cancers. Cancer science, 94(2), 148-152. 107. Syed, N., Smith, P., Sullivan, A., Spender, L. C., Dyer, M., Karran, L., ... & Griffin, B. (2006). Transcriptional silencing of Polo-like kinase 2 (SNK/PLK2) is a frequent event in B-cell malignancies. Blood, 107(1), 250-256. 108. Helmke, C., Becker, S., & Strebhardt, K. (2016). The role of Plk3 in oncogenesis. Oncogene, 35(2), 135-147. 109. Ward, A., Morettin, A., Shum, D., & Hudson, J. W. (2011). Aberrant methylation of Polo-like kinase CpG islands in Plk4 heterozygous mice. BMC cancer, 11(1), 71. 110. Wong, Y. L., Anzola, J. V., Davis, R. L., Yoon, M., Motamedi, A., Kroll, A., ... & Mitchell, B. J. (2015). Reversible centriole depletion with an inhibitor of Polo-like kinase 4. Science, 348(6239), 1155-1160. 111. Wang, Y. A., Kamarova, Y., Shen, K. C., Jiang, Z., Hahn, M. J., Wang, Y., & Brooks, S. C. (2005). DNA methyltransferase-3a interacts with p53 and represses p53-mediated gene expression. Cancer biology & therapy, 4(10), 1138-1143

112. Walter, M. J., Ding, L., Shen, D., Shao, J., Grillot, M., McLellan, M., ... & Kandoth, C. (2011). Recurrent DNMT3A mutations in patients with myelodysplastic syndromes. Leukemia, 25(7), 1153-1158. 113. Holland, A. J., & Cleveland, D. W. (2014). Polo-like kinase 4 inhibition: a strategy for cancer therapy?. Cancer Cell, 26(2), 151-153. 114. Veitch, Z. W., Cescon, D. W., Denny, T., Yonemoto, L. M., Fletcher, G., Brokx, R., ... & Bray, M. R. (2019). Safety and tolerability of CFI-400945, a first-in-class, selective PLK4 inhibitor in advanced solid tumours: a phase 1 dose-escalation trial. British journal of cancer, 121(4), 318-324. 115. Lyko, F. (2018). The DNA methyltransferase family: a versatile toolkit for epigenetic regulation. Nature Reviews Genetics, 19(2), 81. 116. Challen, G. A., Sun, D., Mayle, A., Jeong, M., Luo, M., Rodriguez, B., ... & Cullen, S. (2014). Dnmt3a and Dnmt3b have overlapping and distinct functions in hematopoietic stem cells. Cell stem cell, 15(3), 350-364. 117. Kawakami, M., Mustachio, L. M., Zheng, L., Chen, Y., Rodriguez-Canales, J., Mino, B., ... & Silvester, J. (2018). Polo-like kinase 4 inhibition produces polyploidy and apoptotic death of lung cancers. Proceedings of the National Academy of Sciences, 115(8), 1913-1918. 118. Lei, Q., Xiong, L., Xia, Y., Feng, Z., Gao, T., Wei, W., ... & Li, Z. (2018). YLT-11, a novel PLK4 inhibitor, inhibits human breast cancer growth via inducing maladjusted centriole duplication and mitotic defect. Cell death & disease, 9(11), 1-14. 119. Bashtrykov, P., & Jeltsch, A. (2015). DNMT1-associated DNA methylation changes in cancer. 120. Shukla, V., Coumoul, X., Lahusen, T., Wang, R. H., Xu, X., Vassilopoulos, A., ... & Ouchi, T. (2010). BRCA1 affects global DNA methylation through regulation of DNMT1. Cell research, 20(11), 1201-1215. 121. Shen, L., Kondo, Y., Guo, Y., Zhang, J., Zhang, L., Ahmed, S., ... & Issa, J. P. J. (2007). Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters. PLoS Genet, 3(10), e181. 122. Rapchak, C. (2014). Exploring the developmental roles of plk4. 123. Sillibourne, J. E., & Bornens, M. (2010). Polo-like kinase 4: the odd one out of the family. Cell division, 5(1), 25. 124. Jenkins, T. G., & Carrell, D. T. (2011). The paternal epigenome and embryogenesis: poising mechanisms for development. Asian journal of andrology, 13(1), 76. 125. Rebuzzini, P., Zuccotti, M., & Garagna, S. (2020). X-Chromosome Inactivation during Preimplantation Development and in Pluripotent Stem Cells. Cytogenetic and genome research, 160(6), 283-294. 126. Hu, N., Strobl-Mazzulla, P., Sauka-Spengler, T., & Bronner, M. E. (2012). DNA methyltransferase3A as a molecular switch mediating the neural tube-to-neural crest fate transition. Genes & development, 26(21), 2380-2385. 127. Okano, M., Bell, D. W., Haber, D. A., & Li, E. (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell, 99(3), 247- 257. 128. Jessop, P., Ruzov, A., & Gering, M. (2018). Developmental functions of the dynamic dna methylome and hydroxymethylome in the mouse and zebrafish: similarities and differences. Frontiers in Cell and Developmental Biology, 6, 27. 129. Sivakumar, G. (2014). Polo-like kinase 4 at the nexus of epigenetic modifications and the DNA damage signaling network. 130. Castillo-Aguilera, O., Depreux, P., Halby, L., Arimondo, P. B., & Goossens, L. (2017). DNA methylation targeting: the DNMT/HMT crosstalk challenge. Biomolecules, 7(1), 3. 131. Gilmore, E. C., & Walsh, C. A. (2013). Genetic causes of microcephaly and lessons for neuronal development. Wiley Interdisciplinary Reviews: Developmental Biology, 2(4), 461-478.

132. Roberti, A., Valdes, A. F., Torrecillas, R., Fraga, M. F., & Fernandez, A. F. (2019). Epigenetics in cancer therapy and nanomedicine. Clinical epigenetics, 11(1), 1-18.

APPENDICES

Appendix A: BioID Project

Phosphorylation is the main post-translational modification that is involved in signal transduction

[1]. As a result, kinases and phosphatases are an important area of research to investigate inner cellular mechanisms [2]. By understanding these proteins and their substrates we can hypothesize how they function simultaneously or sequentially to affect the overall cellular architecture. Finding these substrates for kinases is an overall difficult task because of the sheer number of possible binding partners, however, many groups have identified that cellular localization and proximity can be indicative of potential substrate binding [3].

The BioID system is one technique that has been used to determine the interacting partners of proteins by employing a biotin ligase (BirA) fusion-protein [4]. In the presence of biotin, the BirA fusion- protein will biotinylate proximal proteins which can then be identified using LC/MS. This system has been used in mammalian cells to confirm PLK4 interacting with centrosomal proteins CEP152 and

CEP192 [5]. We developed a BioID-PLK4 plasmid to be expressed in xenopus laevis to confirm homologous gene interactions and to potentially find new binding partners. The plasmid was successfully expressed in HEK293T cells (Figure A1, A2).

1. Graves, J. D., & Krebs, E. G. (1999). Protein phosphorylation and signal transduction. Pharmacology & therapeutics, 82(2-3), 111-121. 2. Remenyi, A., Good, M. C., & Lim, W. A. (2006). Docking interactions in protein kinase and phosphatase networks. Current opinion in structural biology, 16(6), 676-685. 3. Litchfield, D. W. (2003). Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochemical Journal, 369(1), 1-15. 4. Roux, K. J., Kim, D. I., & Burke, B. (2013). BioID: a screen for protein‐protein interactions. Current protocols in protein science, 74(1), 19-23. 5. Firat-Karalar, E. N., Rauniyar, N., Yates III, J. R., & Stearns, T. (2014). Proximity interactions among centrosome components identify regulators of centriole duplication. Current biology, 24(6), 664-670.

Appendix B: Plasmid Maps

Figure A1: Myc-BIOID-PLK4 Mammalian

Depiction of a mammalian plasmid map containing full-length human PLK4 cDNA. Aaron Hagazi created this plasmid. Fully sequencing the plasmid revealed missense mutations at amino acid positions

637 and 652 in PLK4. These mutations were fixed using the primers found in table B1. The CMV promoter is depicted in green, the Myc-BirA fusion tag is depicted in red and the PLK4 cDNA is depicted in blue. The plasmid was successfully transfected and expressed in HEK293T as shown in the blot. Myc-

PRMT5 was used as a positive control.

Figure A2: Myc-BIOID-PLK4 Xenopus

Depiction of a Xenopus plasmid map containing full-length human PLK4 cDNA. This plasmid was created by using restriction enzymes Nru1 and Sma1 on the previous mammalian plasmid and blunt-end ligating the product into a new xenopus backbone. The CMV promoter is depicted in green, the Myc-

BirA fusion tag is depicted in red and the PLK4 cDNA is depicted in blue. This plasmid contains I-SceI mega-nuclease sites for the purposes of genomic integration into fertilized xenopus eggs. The plasmid was successfully transfected and expressed in HEK293T as shown in the blot and Myc-PRMT5 was used as a positive control.

Appendix C: Table of Primers

Primer Sequence 637 Mutation 5'-agaatatgtgaaagaagttcttcagatatctagtgatggaaatacga-3' sense 5'-tcgtatttccatcactagatatctgaagaacttctttcacatattct-3' anti-sense 652 Mutation 5'-gatggaaatacgatcactatttattatccaaatggtggtagaggtttt-3' sense 5'-aaaacctctaccaccatttggataataaatagtgatcgtatttccatc-3' anti-sense

Table B1: Primer sequences for site-directed mutagenesis.

VITA AUCTORIS

NAME: Necky Tran

PLACE OF BIRTH: Brantford, ON

YEAR OF BIRTH: 1993

EDUCATION: Walkerville Collegiate Institute, Windsor, ON, 2011

University of Windsor, B.Sc., Windsor, ON, 2016

University of Windsor, M.Sc., Windsor, ON, 2020