DISCOVERY and CHARACTERIZATION of PATHWAYS INVOLVED in FUS and TDP43-INDUCED TOXICITY in YEAST a Thesis Submitted in Partial

DISCOVERY AND CHARACTERIZATION OF PATHWAYS INVOLVED IN FUS AND

TDP43-INDUCED TOXICITY IN YEAST

A thesis submitted in partial fulfillment of the requirement for the degree of Master of Science

WESTON JOSEPH SHAW B.S., Wright State University, 2017

2020 Wright State University

WRIGHT STATE UNIVERSITY

GRADUATE SCHOOL

DATE OF DEFENSE 04 / 30 /2020

I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY WESTON JOSEPH SHAW ENTITLED DISCOVERY AND CHARACTERIZATION OF FUS AND TDP43-INDUCED TOXICITY IN YEAST BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF MASTER OF SCIENCE.

Shulin Ju, Ph.D. Thesis Director

Eric Bennett, Ph.D. Chair, Department of Neuroscience, Cell Biology and Physiology

Committee on Final Examination

Chris Wyatt, Ph.D.

Thomas Brown, Ph.D.

Barry Milligan, Ph.D. Interim Dean of the Graduate School

ABSTRACT

Shaw, Weston Joseph. M.S., Department of Neuroscience, Cell Biology, and Physiology, Wright State University, 2020. Discovery and characterization of pathways involved in FUS and TDP43-induced toxicity in yeast.

High-throughput genome-scale studies are becoming increasingly common as a means to discover genetic interactions. This methodology is particularly efficient when performed in the budding yeast Saccharomyces cerevisiae. Here, we (1) overexpress a large human gene library in yeast to assess how many of them are toxic, and (2) use the list of genes generated above to refine and analyze human genes previously identified to enhance toxicity of two ALS-associated proteins, FUS and TDP-43.

By introducing each of 13,500 human genes into yeast, we demonstrated that the majority of these genes (about 97%) are not toxic to yeast when overexpressed. These results indicated that toxicity of human genes, such as FUS and TDP-43, are very likely due to their interactions with specific cellular pathways, rather than simply a non-specific effect of their overexpression in yeast. This is supported by our analysis showing that the toxic human genes are enriched in RNA metabolic processes and DNA transcription, both of which are conserved between yeast and human. Our lab previously identified a preliminary list of 685 human genes that enhance toxicity of FUS and TDP-43 in yeast. However, none of these genes were tested for their own toxicity, so they were possibly false positives, and never were removed from the list. Using the toxic genes identified above, we refined the enhancers list from 685 to 358 genes, out of which 138 genes enhanced TDP-43 toxicity, 335 genes enhanced FUS toxicity, and 115 genes enhanced both. Interestingly, functional classification of the genes that enhance toxicity from both FUS and TDP-43 revealed a group of cell cycle

iii

regulators that have been linked to the DNA damage response (DDR) and repair. Given that

FUS and TDP-43 are RNA-binding proteins that have been implicated in DDR, these results suggest a possible mechanism of FUS and TDP-43 toxicity involving their abnormal activation of the cell cycle.

TABLE OF CONTENTS

Page

I. INTRODUCTION ...... 1

Amyotrophic Lateral Sclerosis ...... 1

Epidemiology ...... 3

The Genetics Of ALS ...... 4

The Role of TDP-43 In ALS ...... 7

The Role of FUS In ALS ...... 10

The Cell Cycle And ALS ...... 12

A Yeast Model of FUS And TDP-43 ...... 15

Overexpression A Human Gene Library In Yeast ...... 18

Hypothesis ...... 19

Summary ...... 19

II. MATERIALS AND METHODS ...... 21

Yeast Strains And Media ...... 21

Human Gene Library ...... 21

Screening for Human Genes Toxic To Yeast ...... 23

Yeast Mating To Bring Plasmids Together ...... 25

Serial Dilution Growth Assay ...... 25

Fluorescence Microscopy ...... 25

Western Blot ...... 26

Gene Ontology Enrichment Analysis ...... 27

III. RESULTS ...... 28

Genetic Screen For Toxic Human Genes In Yeast ...... 28

Enrichment Analysis Of Toxic Human Genes ...... 31

Refining A Set Of FUS And TDP-43 Enhancers ...... 37

Confirming FUS And TDP-43 Enhancers ...... 38

Enrichment Analysis Of FUS And TDP-43 Enhancers ...... 40

Cell Cycle Regulators Enhance FUS And TDP-43 Toxicity In Yeast ...... 43

Hydroxyurea Enhances FUS and TDP-43 Induced Toxicity ...... 45

Cell Cycle Regulators Do NOT Change FUS and TDP-43 Aggregation Or

Localization ...... 47

Cell Cycle Regulators Do NOT Affect FUS and TDP-43 Protein Level ..... 47

IV. DISCUSSION ...... 50

Screening For Human Genes Toxic To Yeast ...... 50

Analyzing a set of FUS and TDP-43 enhancers ...... 52

Future experiments ...... 52

Conclusion ...... 55

V. REFERENCES ...... 56

LIST OF FIGURES

Figure Page

1. Motor Neuron Death And Muscle Atrophy In ALS ...... 2

2. ALS Gene Discovery By Year ...... 6

3. The RNA Recognition Motifs (RRMs) of TDP-43 Mediate Many Steps Of RNA

Processing ...... 9

4. Similar To TDP-43, The RRM Of FUS Mediates Many Steps of RNA Processing

...... 11

5. Schematic Of The Cell Cycle Phases And The Corresponding Phase-Specific

Promoting Factors ...... 13

6. The Overexpression Of Wild-type (WT) FUS And TDP-43 In Yeast Reduces

Yeast Cell Fitness And Produces Cytosolic Inclusions ...... 16

7. Plasmid Map Of pRS416Gal1-ccdB ...... 24

8. Mating-based Overexpression Screening of A Human Gene Library ...... 29

9. The Representative Glucose (Top) And Galactose Agar Plates (Bottom) From The

Human Gene Library Screen For Toxic Human Genes ...... 30

10. ~3% Human Genes Are Toxic To Yeast ...... 32

11. Distribution Profile Of FUS And TDP-43 Enhancers ...... 39

12. Cell Cycle Regulators Enhance FUS (A) And TDP-43 Induced Toxicity (B) .. 44

13. 10mM Hydroxyurea Enhances FUS (A) And TDP-43 (B) Induced Toxicity in

Yeast ...... 46

14. CDK2 Or CKS1 Does Not Alter FUS Or TDP-43 Aggregate Quantity And

Localization ...... 48

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15. FUS And TDP-43 Protein Level Does Not Change When Co-expressed With CDK2

And CKS1 ...... 49

16. Yeast Cellular And Nuclear Morphology Changes During Progression Through The

Stages Of The Cell Cycle ...... 54

viii

LIST OF TABLES

Table Page

1. List of Yeast Strains Used In This Study ...... 22

2. Gene Term Analysis For Toxic Human Genes ...... 33

3. Enrichment Analysis For GO Biological Process Terms Among The Human

Genetics Enhancers Of TDP-43 Induced Toxicity in Yeast ...... 41

ACKNOWLEDGEMENTS

For their support and encouragement, I would like to thank Dr. Larry Ream, Dr. Barbara

Kraszpulska, Dr. Kathrin Engisch, Dr. Laura Rouhana, Dr. Quan Zhong, and Bridgett Severt.

For their support and guidance, I would like to thank my committee members, Dr. Chris

Wyatt and Dr. Thomas Brown

For their support, friendship, and patience, I would like to thank the members of the JuZhong

labs (past and present), my parents, and my brother, Vincent.

I would like to extend a special thanks to Dr. Shulin Ju, without whom my personal and professional growth over the past two years would not have happened. Thank you for your

patience and generosity.

I: INTRODUCTION

AMYOTROPHIC LATERAL SCLEROSIS (ALS)

Amyotrophic lateral sclerosis (ALS) is a devastating progressive paralytic disease.

The disease attacks motor neurons in the brain and spinal cord, ultimately leading to muscular atrophy (wasting) and paralysis (Figure 1). ALS becomes fatal within 3-5 years, typically due to ventilatory paralysis (Brown et al, 2017). In healthy individuals, upper motor neurons originate in the motor cortex of the brain and extend to the brainstem and spinal cord to innervate lower motor neurons. Lower motor neurons extend from the spinal cord to make synaptic connections with skeletal muscles (voluntary), which contract upon stimulation.

Without stimulation, skeletal muscles will eventually undergo atrophy leading to paralysis.

The degeneration of these motor neurons is the hallmark feature of ALS; however, non- motor neuronal cells in the frontal and temporal lobes, as well as neuroglia, have also been shown to be affected by the disease (Brown et al, 2017; Taylor et al, 2016; Oskarsson et al,

2018). Clinical presentation of ALS is often heterogeneous in phenotype and pathology.

Most cases of ALS (65%) begin in the limbs, while a third (30%) show bulbar-onset, which presents as difficulty speaking and swallowing (Hardimann et al, 2011, Brown et al, 2017).

A small percentage of patients (5%) show respiratory-onset, which carries the worst prognosis. The site of symptom onset is determined by the populations of motor neurons that the disease affects; thus, ALS can also be classified into subtypes based on the site of neuronal degeneration. In primary lateral sclerosis (PLS), upper motor neurons are predominately affected, and patients often show clumsiness, brisk reflexes, and muscle stiffness. In progressive muscular atrophy (PMA), the dysfunctions of lower motors neurons predominates leading to muscle weakness and fasciculations (muscle twitching).

Figure 1. Motor neuron death and muscle atrophy in ALS. ALS most often targets upper and lower motor neurons of the human nervous systems. Death of these neurons causes muscle atrophy leading ultimately to paralysis. Image adapted from the ALS Foundation for Life

(www.alsfoundation.org/learn).

In progressive bulbar paralysis, the disease affects cranial nerves which control muscles that allow for chewing, swallowing, and speaking (Hardimann et al, 2011; Brown et al, 2017).

Additionally, 15-20% of cases also show progressive cognitive impairments such as executive function deficits, personality changes, poor insight, and late-stage dementia.

Furthermore, when these behavioral and cognitive changes correlate with evidence of neuronal degeneration in the frontal and temporal lobes, the condition is known as ALS with frontal temporal dementia, or ALS-FTD (Brown et al, 2017). It has been suggested that given its clinical and pathological heterogeneity, ALS can be considered a syndrome; that is, a collection of symptoms without a single underlying route of pathogenesis (Hardimann et al,

2011).

EPIDEMIOLOGY

ALS has been shown to occur worldwide across all ethnicities with an incidence rate of 1-2 new cases per year per 100,000 people and a prevalence of 3-5 cases per 100,000 people

(Brown et al, 2017; Marin et al, 2017). While some cases of ALS can be linked to mutations in single genes (familial ALS), most cases are considered to develop as a combination of genetic and environmental risk factors (sporadic ALS). However, the only consistently shown risk factors, apart from genetic mutations, are male gender and old age. In general,

ALS occurs more frequently in men than women, nearing a ratio of 2:1. Furthermore, the disease increases in incidence and prevalence from ages 50-75 (Hardimann et al, 2011). The presence of individuals with certain gene variants may predispose them to develop ALS many years earlier; as young as 11 has been described (Corcia et al, 2017; Conte et al, 2012).

Other environmental factors that have been investigated include military service, athleticism,

smoking, exposure to metals, and head trauma (Oskarsson et al, 2015). However, no single environmental factor has been definitively linked to ALS. The difficulty of identifying environmental risk factors lies in the large number of possible factors, as well as identifying the exposure limit.

There exist geographic clusters of increased incidence and prevalence, most notably in the Chamorro people of Guam. This group had, at one time, shown an incidence of 140 per

100,00 people of a distinct type of ALS which presents with Parkinson’s disease, and is known as ALS/Parkinson-dementia complex, or ALS/PDC (Oskarsson et al, 2015).

Interestingly, the incidence of ALS/PDC in this population quickly dropped in the latter half of the 20th century. Furthermore, the disease was also shown to develop in Filipino immigrants to this region, as well as Chamorro people who emigrated early in life

(Oskarsson et al, 2015). This suggests that there could be an acquired component to developing ALS/PDC. Investigations have revealed a high concentration of the neurotoxin, b-methylamino-L-alanine (BMAA), in the Chamorro diet. BMAA is produced in cycad plants (which was used for flour), as well as most cyanobacteria species. Furthermore, animal studies have shown that BMAA can cause motor neuron dysfunction at the right quantities in susceptible individuals (Chiu et al, 2011). The wider relevance of BMAA to neurodegenerative disease has yet to be determined.

THE GENETICS OF ALS

The development of ALS is known to be strongly linked to mutations in many genes.

For many of these genes, mutations have been shown to be directly responsible for a heritable type of ALS, known as familial ALS (fALS), which represents up to 10% of all

ALS cases. The other 90% of cases, denoted as sporadic ALS (sALS), do not show a pattern of inheritance and are thought to result as a mixture of genetic and environmental risk factors

(Mejzini et al, 2019). However, the genetic profile underlying most of these cases remains unknown. Over 25 genes have been shown to cause fALS and many are also implicated in sALS (Figure 2). These genes can be grouped into three, often overlapping, cellular functions: (1) protein homeostasis, (2) RNA homeostasis, and (3) cytoskeletal dynamics. Of all fALS cases, most can be linked to mutations in four genes; SOD1, C9ORF72, TDP43, and FUS. All other fALS genes make up <1% of cases (Brown et al, 2017).

The SOD1 gene was the first to be linked to causing ALS (Rosen et al, 1993). This

- gene encodes Cu/Zn superoxide dismutase which catalyzes the conversion of toxic O2 to O2 and H2O2 (the Fenton reaction). SOD1 protein forms stable homodimers which reside in the cytosol and intermembrane species to provide defense against antioxidants (Pansarasa et al,

2018). Over 185 variants in the SOD1 genes have been identified to cause ALS. Most are missense mutations which have been shown to result in the loss of SOD1 function. However,

SOD1-knockout studies in mice have not demonstrated ALS phenotypes, suggesting that

SOD1-ALS may be due to toxic gain-of-function. While no definitive mechanism of SOD1- induced toxicity has been identified; mutant SOD1 has been implicated in glial cell toxicity, induction of ER stress, and dysregulation of axonal transport (Taylor et al, 2016; Pansarasa et al, 2018; Mejzini et al, 2019).

In 2011, two groups independently identified the most common genetic cause of fALS, as well as FTD, to be hexanucleotide (GGGGCC) repeat expansion in the gene

C9ORF72 (DeJesus Hernandez et al, 2011; Renton et al, 2011). While healthy individuals have less than 30 repeats, ALS/FTD-affected individuals will often have hundreds or

Figure 2. ALS gene discovery by year. The accompanying graph illustrates the discovery of genes linked to ALS since the first gene discovered, SOD1, in 1991. Each circle represents a single ALS-linked gene, with the size of the circle representing the number of cases with which the gene has been linked. The color of the circle represents the form of ALS cases (red as sporadic and blue as familial) with which the gene has been linked. The color of the gene name represents if functions of the genes are involved in protein homeostasis (green), RNA homeostasis (red), or cytoskeletal dynamics (blue). Adapted from Brown et al, 2017.

thousands of repeats. The hexanucleotide expansion results in the formation of three types of cytosolic aggregates; TDP-43 positive inclusions, p62-positive inclusions, and dipeptide repeat protein inclusions, which are made through unconventional translation of the repeat expansion (Taylor et al, 2016). Interestingly, C9-ALS individuals also develop nuclear structures which contain these expanded RNA transcripts and are known as RNA foci

(DeJesus Hernandez et al, 2011). Similar RNA foci produced in fragile X tremor ataxia syndrome and myotonic dystrophy types 1 and 2, have been shown to cluster with RNA- binding proteins leading to RNA processing defects (Taylor et al, 2016). This suggests

C9ORF72 RNA foci could provide a similar mechanism of pathology in motor neurons.

Of the four major fALS genes, TDP-43 and FUS (discussed below) each account for about 5% of cases (Brown et al, 2017). Interestingly, both genes encode RNA binding proteins which aggregate in mutant-dependent cytosolic inclusions during ALS. Furthermore, non-mutant TDP-43 is known to aggregate in over 90% of ALS cases, regardless of sporadic or familial. Taken together, the plethora of RNA pathway-related proteins involved in sporadic and familial ALS suggest that RNA pathway dysregulation could play a significant role in the development of the disease. Moreover, the direct involvement of TDP-43 and FUS in multiple RNA pathways make them prime targets for investigation of ALS pathological mechanisms.

THE ROLE OF TDP-43 IN ALS

The TARDP gene encodes the 414 amino acid TAR DNA-binding protein, or TDP-

43. This protein contains a nuclear localization signal and a nuclear export signal, thereby allowing it to shuttle in and out of the nucleus. TDP-43 also contain a c-terminal Gly-rich

region, which is thought to function in protein-protein interactions (Mejzini et al, 2019).

Additionally, the protein contains two RNA recognition motifs (RRMs). These motifs allow the protein to function in a wide variety of RNA processing steps, including transcription, splicing, transport, stability, and translation (Figure 3) (Ratti et al, 2016). Recent research is also supporting a role for TDP-43 in DNA damage response (DDR) and DNA repair

(Kawaguchi et al, 2020, Mitra et al, 2019). In 2019, Mitra et al. reported a novel role of

TDP-43 in the repair of double-stranded DNA breaks (DSBs) through regulating nonhomologous end joining (NHEJ). In their study, they reported that a loss of TDP-43 in iPSC resulted in inhibited NHEJ, an accumulation of un-repaired DSBs, and activation of

DNA damage response (DDR) factors.

In 2006, TDP-43 was discovered to be a major component of the characteristic cytosolic inclusions in ALS and FTLD (Arai et al, 2006, Neumann et al, 2006). Furthermore, the majority of all ALS cases (sporadic and familial) contain TDP43-positive inclusions

(Mejzini et al, 2019). However, TDP-43 was not appreciated as a direct cause for neuronal toxicity until mutations in TARDP were found to cause a subtype of fALS (accounting for about 5% of all fALS cases) (Ratti et al, 2016). To date, over 50 ALS-causing mutations in

TARDP have been recognized, the majority of which are missense mutations and located in the c-terminal Gly-rich region (Ratti et al, 2016). How these mutations affect the normal function of TDP-43 remains to be determined.

Whether a mutation is present or not, TDP43-related cases of ALS show a concentration of cytosolic TDP-43 with the concomitant loss of nuclear TDP-43 (Mejzini et al, 2019). This could represent a potential gain-of-function in the cytosol or loss-of-function in the nucleus.

Figure 3. The RNA recognition motifs (RRMs) of TDP-43 mediate many steps of RNA processing. In the nucleus, TDP-43 will bind nascent pre-mRNA transcripts as they exit

RNA polymerase II and coordinate factors that regulate alternative splicing. TDP-43 also regulates the transcription of micro RNAs and noncoding RNAs (ncRNAs and long- ncRNAs). In the cytosol, TDP-43 predominantly interacts with RNA by binding to the transcripts 3’ untranslated regions (3’UTRs). This allows for regulation of RNA stability, transport, and translation. Adapted from Ratti et al, 2016.

Given the extensive involvement of TDP-43 in RNA processing, much research has focused on how it might cause RNA dysregulation in neurons. Notably, yeast models of TDP-43 have demonstrated that the protein’s RRM is essential for toxicity (Johnson et al, 2008). With the recent discovery of TDP-43’s involvement in DNA damage repair, it has been suggested that loss of TDP-43 from the nucleus could lead to accumulation of DNA damage leading to cell death pathways (Mitra et al, 2019). This is particularly relevant for non-dividing neurons, which have been suggested to primarily rely on NHEJ for DNA repair (Iyama et al, 2013).

THE ROLE OF FUS IN ALS

The gene fused in sarcoma/translocated in liposarcoma (FUS/TLS; hereafter referred to as FUS) encodes a 526 amino acid RNA/DNA-binding protein (Mejzini et al, 2019). The

FUS protein is very similar to TDP-43 both in structure and function. Like TDP-43, FUS contains nuclear import and export signals which allow it to shuttle in and out of the nucleus.

FUS also contains a glutamine-glycine-serine-tyrosine-rich (QGSY-rich) region and zinc finger domain that are utilized for protein-protein interactions, as well as an RRM that mediates RNA interactions (Ratti et al, 2016). It is through RRM that FUS plays a significant role in many RNA processes similar to TDP-43, including transcription, splicing, RNA transport, and protein translation (Figure 4) (Ratti et al, 2016). However, despite their involvement in the same RNA processes, FUS and TDP-43 are suspected to bind mostly different RNA transcripts and have been shown to interact with different proteins (Mejzini et al, 2019, Kawaguchi et al, 2020). Additionally, FUS is known to be involved in DNA damage repair through homologous recombination and NHEJ (Ratti et al, 2016).

Figure 4. Similar to TDP-43, the RRM of FUS mediates many steps of RNA processing.

This includes binding to nascent pre-mRNA transcripts and the formation of splicing complexes. FUS also similarly regulates the splicing of miRNAs. FUS is also known to regulate transcription in the nucleus via affecting the phosphorylation of RNA polymerase II.

FUS has also been implicated in multiple steps of the DNA damage response pathway, including mediating homologous recombination and nonhomologous end joining. In the cytosol, FUS likely plays a role in mRNA stability, transport, and translation, by binding to the 3’UTR sequences of many transcripts. Adapted from Ratti et al, 2016.

Interestingly, Deng et al. (2014) demonstrated that, following induction of DNA damage in human cell cultures, FUS is mislocalized to the cytosol in a process regulated by phosphorylation. This mimics the aggregation of FUS in cytosolic inclusions in FUS-related

ALS, suggesting a possible mechanism of neurodegeneration in ALS neurons.

FUS was originally identified in liposarcoma tumors where it was translocated forming chimeric genes (Crozat et al, 1993). In 2009, the first mutations in FUS to cause a subtype of fALS were discovered (accounting for about 5% of all fALS) (Kwiatkowski et al,

2009, Vance et al, 2009). Much like TDP-43, over 50 disease-causing mutations in FUS have since been identified, with the majority being missense mutations and located in the proteins

NLS (Mejzini et al, 2019). This disruption in the NLS leads to the abnormal cytosolic localization of FUS protein characteristic of FUS-related ALS neurons. It is unclear whether mutant FUS results in a toxic gain- or loss-of-function. Both knockout and overexpression of

FUS in animal models has shown to cause neuromotor defects (Mejzini et al, 2019). Similar to TDP-43, much research has focused on how mutant FUS may cause dysregulation of RNA processing.

THE CELL CYCLE AND ALS

During mitotic division, cells progress through distinct developmental phase which are tightly regulated by a variety of cyclin-dependent kinase complexes (Figure 5). These complexes are composed of several integral proteins including, cyclin-dependent kinases

(CDKs), and often cyclin-dependent kinase regulatory subunits (CKSs), and cyclins

(Vermeulen et al, 2003, Harper, 2001). CDKs (including CDK1/2/4/6) are primarily known to phosphorylate and active substrates which allow for entrance into the next cell cycle stage.

Figure 5. Schematic of the cell cycle phases and the corresponding phase-specific promoting factors. The promoting factors include various types of cyclin-dependent kinases

(CDKs) and cyclins, as well as small regulatory subunits (CKSs; not shown). Adapted from

Vermeulen et al, 2003.

CDK protein levels will remain stable throughout the cell cycle, unlike the family of cyclins which increase and decrease throughout at different stages. Cyclins (including cyclin

A/B/D/E) function by binding and activating specific types of CDKs (Figure 5). For example, during G1, cyclin D levels rise to bind and activate CDK4 and CDK6 which will phosphorylate retinoblastoma protein (pRb) (Vermeulen et al, 2003). pRb subsequently activates the transcription factors, E2F-1 and DP-1, which promote passage through S-phase

(Vermeulen et al, 2003). CKSs (including CKS1 and CKS2) are small (9-18 kDa) regulatory proteins which are essential for the function of CDKs, specifically CDK1 and CDK2 (Harper et al, 2001).

Interestingly, several cell cycle proteins are known to interact with FUS. Following a

DNA damage event during the cell cycle, CDKs and cyclins are inhibited to prevent progression through the cell cycle (Vermeulen et al, 2003). However, evidence has also suggested that CDKs function in DDR (Satyanaraya et al, 2009; Cerqueira et al, 2009).

Additionally, FUS has been shown to inhibit cyclin D (CCND) to allow for DNA repair

(Wang et al, 2008). Moreover, in an overexpression screen for enhancers of FUS-induced toxicity in yeast, Sun et al. (2011) identified the yeast homolog of cyclin B (CCNB). Thus, the loss of FUS from the nucleus and abnormal cell cycle activity during DDR could suggest a mechanism in FUS-induced toxicity. Furthermore, immunoblot studies indicated that

CDK4 and CDK6, as well as their downstream targets, pRb, E2F-1 and DP-1, were upregulated in ALS-affected neurons (Ranganathan et al, 2003). This is unexpected given that neurons are non-dividing cells. Thus, it has been suggested that the pathology of ALS could involve a fatal attempt by neurons to re-enter the cell cycle (Ranganathan et al, 2003).

A YEAST MODEL OF FUS AND TDP-43

To study the biological processes of FUS and TDP-43, a yeast model of FUS and

TDP-43 expression has been developed. Yeast has long been the model of choice for biologists thanks to its rapid growth time, simple growth requirements, amenability to genetic manipulation, and more importantly, conservation of major cellular pathways. Plasmid DNA can be easily transformed into yeast, as well as integrated into yeast chromosomes thanks to yeast’s highly efficient homologous recombination system. Additionally, yeast can be maintained in either haploid or diploid state. Furthermore, haploid yeast of opposite mating types (“a” or “⍺”) can be easily mated to introduce two different plasmids into the same cell.

While unicellular yeast cells may not seem like a relevant model to study the complex processes of highly specialized cells such as neurons, many neurodegenerative disease proteins have been successfully studied in yeast. Indeed, the first neurodegenerative disease protein to be modeled in yeast was ⍺-synuclein, which is encoded by the Parkinson’s- linked gene SNCA (Outeiro et al, 2003). Proteins from other neurodegenerative diseases soon followed, including Alzheimer’s and Huntigton’s disease (Outeiro et al, 2003,

Treusch et al, 2011). Soon after the discovery of FUS and TDP-43 involvement in subtypes of ALS, several groups established models of FUS and TDP-43 in

Saccharomyces cerevisiae (Ju et al, 2011; Sun et al, 2011; Johnson et al 2008). In these models, the overexpression of wild-type FUS and TDP-43 can recapitulate the cytotoxicity, cytosolic mislocalization, and inclusion formation (Figure 6) seen in ALS-affected neurons

(Ju et al, 2011; Sun et al, 2011; Johnson et al, 2008).

Figure 6. The overexpression of wild-type (WT) FUS and TDP-43 in yeast reduces yeast cell fitness and produces cytosolic inclusions. (A) the figure illustrates serial dilution growth assays. Each column of the assay shows a colony of yeast that was diluted from the previous column. Each row of the assay represents a unique strain of yeast. The legend at the left of the assay shows with what gene each strain is transformed (GFP = green fluorescent protein, as negative control). These genes are under the control of a Gal1 promoter, so growth on glucose media represses gene repression, while galactose media induces it. As shown, the expression of wild-type FUS and TDP-43 cause reduced yeast cell fitness, indicated by the less growth compared to GFP; (B) the figure shows fluorescent microscopy studies illustrating the cytosolic inclusions caused by the overexpression of FUS and TDP-43 in yeast. The legends at the top of the figure show the type of microscopy performed (GFP = emitted wavelength excites green fluorescent protein, DAPI = emitted wavelength excites fluorescent stain of DNA). As shown, compared to the diffuse cellular expression of GFP, FUS and TDP-43 overexpression result in the formation of cytosolic inclusions. Images adapted from Ju et al, 2011 and Johnson et al, 2008.

The conservation of essential cellular biology between yeast and higher eukaryotes allows us to make inferences about human cell biology from yeast-based studies.

Furthermore, yeast and humans share a large number of orthologous genes, representing 1/3 of the yeast genome (Laurent et al, 2016). Kachroo et al. (2015) performed a study in which they replaced 414 yeast genes with their human orthologs and found that roughly half could complement the loss of their yeast counterpart. Notably, rather than sequence similarity, replaceability was best predicted by gene module (if the genes functioned in similar pathways in their respective hosts). This study represents a growing arena of research interested in yeast “humanization”. Yeast humanization represents the altering of yeast to resemble human biology (Laurent et al, 2016). This can be as simple as the heterologous expression of a human gene in yeast (as in the case of FUS and TDP-43, which have no yeast orthologs), or as complex as the complete transformation of whole yeast cellular pathways

(Laurent et al, 2016). Indeed, this is a new and exciting arena of yeast research.

OVEREXPRESSING A HUMAN GENE LIBRARY IN YEAST

Given its ease of use and genetic tractability, yeast is also particularly useful for performing high-throughput genome-wide assays. Using this technique, massive genome- scale libraries can be rapidly and efficiently expressed in yeast to discover hundreds of novel genetic interactions (Botstein and Fink, 2011, Khurana and Lindquist, 2011). Previous research has demonstrated success in discovering novel genetic modifiers (suppressors and enhancers) of FUS and TDP-43 toxicity in yeast through the use of genome-wide association studies (Sun et al, 2011; Ju et al, 2011; Hayden et al, 2020). These early studies, however,

were limited to screening the yeast genome, which consists of roughly 6,000 genes. To form a more comprehensive picture of the possible interactions between FUS and TDP-43 with other genes, the Ju lab has undertaken screening of the largest known human ORF library

(representing over 13,500 human ORFs). Dr. Elliott Hayden in our lab utilized this library to perform a mating-based overexpression screen for genetic modifiers (including suppressors and enhancers) of FUS and TDP43-induced toxicity in yeast, from which he identified hundreds of potential interactors of FUS and TDP-43 (Hayden et al, 2020).

HYPOTHESIS

Yeast models of overexpressed WT FUS and TDP-43 mimic the cytotoxicity and cytosolic mislocalization of these proteins seen in ALS-affected neurons. High-throughput overexpression screening has been shown to be a useful method for identifying yeast genetic modifiers (suppressors and enhancers) of FUS and TDP-43 toxicity. We propose a human gene library can be overexpressed in yeast to identify previously unknown human genetic interactors for two ALS genes, FUS and TDP-43. These genetic interactions can then be isolated for targeted studies that aim to uncover the toxic mechanism of FUS and TDP43 in yeast.

SUMMARY

Saccharomyces cerevisiae is an exceptionally versatile model organism when it comes to molecular and cellular biological techniques, particularly for high-throughput genome-scale association studies. FUS and TDP-43 are two human genes that have causal links to a familial form of ALS, in which they lead to the aberrant mislocalization and

aggregation of toxic proteins in cytosolic inclusions in neurons. These pathological hallmarks can be recapitulated in yeast. While FUS and TDP-43 are known to have extensive roles in

RNA processing, as well as roles in DNA damage response, the pathophysiology of the proteins in ALS remains unclear. The use of overexpression screening of large gene libraries has proved successful at identifying human interactors that may reveal mechanisms of FUS and TDP-43 induced cytotoxicity.

II: MATERIALS AND METHODS

YEAST STRAINS AND MEDIA

Yeast strains used for the experiments in this thesis are listed in Table 1. For all experiments, the yeast strain W303 was used. The genotype of this strain (MATa/MAT⍺ leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) contains mutations in genes that prevent growth on media lacking histidine, leucine, tryptophan, or uracil. After transforming this strain with plasmids containing auxotrophic markers (genes which encode for the aforementioned amino acids), we can isolate the transformed strain by growing colonies on selective media (media containing all amino acids except those selected for by the plasmid). All plasmids contained a GAL1 promoter to allow for gene induction in yeast grown on galactose-containing media, gene repression on glucose-containing media, and no effect on gene expression when grown on raffinose-containing media. Yeast strains transformed with plasmids integrated into a chromosome were pre-cultured in YPD (yeast-peptone-dextrose; rich media that contains all necessary nutrients in abundance). Yeast strains carrying non-integrated plasmids were pre- cultured in selective media (synthetic complete with a corresponding dropout) to prevent loss of the plasmid.

HUMAN GENE LIBRARY

The human ORF clones used to generate the yeast-expression plasmids are Gateway entry clones kindly provided by Dr. Marc Vidal (Dana-Farber Cancer Institute and Department of

Genetics, Harvard Medical School). This collection, including the clones from hORFeome version 8.1 (http://horfdb.dfci.harvard.edu/) and clones from the ORFeome collaboration

Table 1: List of yeast strains used in this study

Strain Genotype Reference W303a MATa leu2-3,112 trp1-1 can1-100 ura3-1 ATCC ade2-1 his3-11,15 W303α MATα leu2-3,112 trp1-1 can1-100 ura3-1 ATCC ade2-1 his3-11,15 W303a/2XTDP-43-GFP W303 MATa his3::pRS303-GAL1-TDP-43- Tardiff et al, GFP, leu2, trp1::pRS304-GAL1-TDP-43- 2012 GFP, ura3, pdr1::Kan, pdr3::Kan W303a/1XFUS W303 MATa leu2-3,112 trp1-1 can1-100 Ju et al, ura3-1 ade2-1 his3-11,15:: pRS303GAL1- 2011 FUS W303a/FUS-YFP W303 MATa leu2-3,112 trp1-1 can1-100 Ju et al, ura3-1 ade2-1 his3-11,15:: pRS413-GAL1- 2011 FUS-eYFP (HIS3) W303a/pRS416Gal1ccdB W303 MATa leu2-3,112 trp1-1 can1-100 This study ura3-1 ade2-1 his3-11,15:: pRS416-GAL1- ccdB (URA3) W303a/pRS413Gal1ccdB W303 MATa leu2-3,112 trp1-1 can1-100 This Study ura3-1 ade2-1 his3-11,15:: pRS413-GAL1- ccdB (HIS3) W303a/pRS416Gal1CKS1 W303 MATa leu2-3,112 trp1-1 can1-100 This study ura3-1 ade2-1 his3-11,15:: pRS416-GAL1- CKS1 (URA3) W303a/pRS416Gal1CDK2 W303 MATa leu2-3,112 trp1-1 can1-100 This study ura3-1 ade2-1 his3-11,15:: pRS416-GAL1- CDK2 (URA3)

(http://www.orfeomecollaboration.org), is the largest known human ORF library in which each gene has been individually cloned, and sequence verified. All the human genes on the entry vector were then cloned by the Ju-Zhong lab into the yeast destination vector pRS416Gal-ccdB (Figure 7) using Gateway cloning (Hayden et al, 2020). The resulting expression clones of human genes on pRS416Gal1ccdB were subsequently transformed into the haploid yeast strain w303⍺, and aliquots of each strain containing a unique human gene on pRS416Gal1ccdB were saved as long-term glycerol stock in wells of 96-well plates (total

154 plates) at -80°C (Hayden et al, 2020).

SCREENING FOR HUMAN GENES TOXIC TO YEAST

Upon each cycle of the screening, 20 plates of the library, representing yeast strains containing ~2000 human genes, were thawed from the glycerol stock, and were used to inoculate 20 new plates with synthetic media using 2% raffinose as a carbon source and containing all nutrients except uracil (the selectable marker for the library). Following 48hrs of growth at 30°C, each plate was briefly vortexed, and the cultures were spotted onto two plates of synthetic media using a Singer ROTOR robotic spotting machine. One plate contained 2% glucose to repress gene expression (as a control) and the other contained 2% galactose to induce gene expression. The robotic spotting machine was programed to spot in quadrants (from one well of the source plate to four spots on the destination agar plates) to increase the robustness of the screen. The spotted plates were then grown at 30°C over four days, and pictures were taken each day. Cellular fitness was assessed based on growth relative to background on the galactose plate. The candidate genes that were toxic to yeast were only selected when 4 spots in the same quadrant showed the same growth phenotype.

Figure 7. Plasmid map of pRS416Gal1-ccdB. The ~13,500 human genes were cloned into this yeast expression vector. This gateway compatible centromere plasmid (stable and usually

1 copy per cell) contains two Gateway cloning sites (attR1 and attR2), which can be used by

LR Clonase to insert the gene of interest from the Gateway entry vector. Immediately upstream of attR1 site is the Gal1 promoter, so expression of gene of interest can be regulated by switching carbon source (gene expression was turned off by glucose and highly induced by galactose). The plasmid also contains ampicillin resistance gene AmpR for amplification in bacteria and URA3 auxotrophic marker for yeast selection. The yeast strain

W303 contains a deletion in this gene; thus, only yeast containing this plasmid will be able to grow on media lacking uracil. Similarly, this study also uses pRS413Gal1-ccdB (not shown) which shared the same backbone as pRS416Gal1-ccdB, except that it contains a HIS3 auxotrophic marker and allows for yeast growth on media lacking histidine.

YEAST MATING TO BRING PLASMIDS TOGETHER

Yeast of opposite mating type and containing plasmids with different auxotrophic markers were streaked in a cross orientation on a YPD agar plate. Following one day of growth, yeast from the center of the cross was re-streaked onto synthetic media containing all amino acids except those selected for by each plasmid. Plates were incubated for 2-4 days at 30°C to allow growth of diploid colonies, which would include plasmids from each mating type.

SERIAL DILUTION GROWTH ASSAY

Yeast strains were pre-cultured in selective 2% raffinose media by growing at 30°C shaking

(200rpm) over 48 hours. This generates a culture of yeast cells in stationary phase (a community that is not actively growing). Pre-cultures were then diluted to an OD600 of 0.1 and grown under the same conditions to exponential phase over 14-16 hours. By starting cultures at the same OD, we are able to harvest all cultures in roughly exponential phase 14-

16 hours later. Harvesting cultures from the same growth phase prevents confounding factors due to altered genetic expression profile. Cultures were then diluted to OD600 of 1, and 5X serially diluted in a row of a 96-well plate. The dilutions were then spotted onto selective synthetic media containing 2% glucose or galactose. Plates were grown at 30°C for 4 days, with pictures taken each day.

FLUORESCENCE MICROSCOPY

Yeast pre-cultures were grown to stationary phase and then diluted to OD600 of 0.1. Cultures were grown for 14-16 hours, centrifuged, and then resuspended in selective 2% galactose to induce gene expression for 6 hours at 30℃ shaking. Following galactose induction, cultures

were centrifuged and resuspended in ddH20. Aliquots of 2ul were placed onto microscope slides with coverslips. Images were taken at 40X magnification using an Olympus IX83 inverted fluorescent microscope with an Olympus DP74 digital camera. Fluorescently tagged

TDP-43 and FUS protein (with GFP and YFP, respectively) was visualized using FITC and

EYFP filter cubes.

WESTERN BLOTTING

PAGE gel, run at 100V for 1 hour and then transferred to PVDF membrane (Millipore) at

50V for 2 hours. The membrane was rinsed with water and blocked with 1XTBST and 5% nonfat dry milk for 1 hour. The membrane was then probed overnight at 4°C with primary antibody at a 1:10,000 dilution in 1XTBST w/ 5% nonfat milk. The primary antibodies used include anti-FUS (Abcam), anti-GFP (Abcam), and anti-PGK1 (Invitrogen). The membrane was washed 3 X 10 min in 1XTBST and probed with secondary antibodies for 1 hour. Anti- mouse and anti-rabbit antibodies conjugated with alkaline phosphatase were used at a

1:10,000 dilution in 1XTBST w/ 5% nonfat milk. Membranes were washed 3 X 10 min in

1XTBST and then developed with NBT/BCIP solution.

GENE ONTOLOGY (GO) ENRICHMENT ANALYSIS

GO enrichment analysis was performed to indicate whether an identified gene set was overrepresented (or under-represented) relative to the whole library in the GO aspects of biological process, molecular function, and cellular components. The aspects enriched suggest the specific relationship between them and the phenotype being screening against.

The commonly used online tool PANTHER v15.0 was chosen for this analysis

(http://pantherdb.org/). Briefly, the query gene set (including toxic human genes, FUS enhancers, and TDP-43 enhancers) was uploaded to the system, and 13570 Entrez gene IDs corresponding to all the cloned human ORFs in our library was used as a reference list.

Overrepresentation testing was performed for each GO aspects: biological process, molecular function, and cellular component. Statistical analyses include Fisher's Exact Test followed by

FDR (False Detect Rate) correction. Only results statistically significant with FDR < 0.05 were displayed.

III. RESULTS

The JuZhong labs have available a ~13,500 human gene library representing over

60% of the human genome, the largest collection of human ORFs that we know of. This library includes genes from all major functional groups and does not contain biases to any particular group. Using this library, a previous member our lab, Dr. Elliott Hayden, performed a mating based screen for genetic modifiers (suppressors and enhancers) of TDP-

43 and FUS-induced toxicity in yeast (Figure 8). Thus, overexpression screening of the human gene library in haploid w303⍺ Saccharomyces cerevisiae serves two purposes: (1) to expand our knowledge about human gene expression in yeast, and (2) to refine a list of TDP-

43 and FUS enhancers.

AIM I: GENETIC SCREEN FOR TOXIC HUMAN GENES IN YEAST

Using our extensive human gene library, we first sought to determine the number of human genes which are toxic when overexpressed in Saccharomyces cerevisiae. In addition to providing information about the ability of yeast to be humanized, this screen will also allow us to refine and analyze previous and future genetic association screens for genetic modifiers of two ALS-linked genes, FUS and TDP-43 (discussed below).

The human library glycerol stock was revived and cultured in selective media and then spotted onto glucose and galactose agar plates using a Singer ROTOR robotic spotting machine. This robot was programmed to spot each colony from one well of the source plate to four spots on the agar destination plate. The colony growth on the galactose agar plates were then monitored for 4 days during which time some colonies could be observed to grow slower compared to the growth of the majority of colonies on the plates (Figure 9). This suggests that the overexpressed human genes within those select colonies is toxic to yeast.

Figure 8. Mating-based overexpression screening of a human gene library. Since yeast can be maintained in a haploid state, mating can be used to easily introduce two plasmids into one yeast cell. Here, we show the mating of yeast transformed with a human gene and yeast containing FUS (could alternatively be TDP-43). Because these genes are under the control of a GAL1 promoter, yeast growth on glucose represses the gene, while growth on galactose induces the gene. On galactose, human genetic suppressors (green colony) will show improved cellular fitness, while human genetic enhancers (red colony) will show reduced cellular fitness. Since human genes can potentially have an unrelated mechanism of toxicity related to FUS or TDP-43, this project aims to remove these false positive hits from a previously performed screen for human genetic enhancers.

Figure 9. The representative glucose (top) and galactose agar plates (bottom) from the human gene library screen for toxic human genes. The two plates were spotted from the same 96-well source plate. A Singer ROTOR robotic spotting machine was programed to spot from one well of a 96-well source plate to four spots on a glucose plate (Gene “Off”) and galactose agar plate (Gene “On”). Thus, a quadrant of colonies represents a single yeast species. The agar plates were incubated and monitored over 4 days for yeast colonies that displayed slow growth (see the highlighted square).

Ultimately, this screen produced a set of 474 human ORFs that are toxic when overexpressed in haploid w303⍺ yeast grown on galactose; furthermore, the strength of toxicity could be roughly categorized as strong, intermediate, or weak (Figure 10). This represents approximately 3% of the human gene library, demonstrating that there is no non- specific toxic phenotype due to overexpression alone.

ENRICHMENT ANALYSIS OF TOXIC HUMAN GENES

To determine if any cellular functions or pathways may be overrepresented among human genes which are toxic to yeast, we used PANTHER online classification system to perform enrichment analysis of the 474 human genes achieved from our screen. PANTHER operates by linking genes to functionally relevant categorical terms. These terms are provided by the Gene Ontology (GO) Consortium and are organized into three categories: (1) molecular function, (2) biological process, and (3) cellular component.

We used PANTHER to parse our list of 474 toxic human genes for any overrepresented GO terms from each of the three categories. We discovered the toxic human gene set to be enriched for genes involved in the regulation of organelle assembly, RNA metabolic processes, gene expression, and RNA polymerase II-mediated transcription (Table

2). These pathways being particularly susceptible to disruption by human genes suggest they are conserved in human cells and could be a potential avenue of interest for disease mechanisms in human cells.

Figure 10. ~3% human genes are toxic to yeast. An overexpression screen of a ~13,500 human gene library in haploid w303⍺ yeast yielded a set of 474 toxic human genes, representing about 3% of the entire library. The toxicity of these genes could be categorized as strong, medium, or weak. Of the 474 toxic human genes, 22 were shown to have a strong toxic phenotype (little to no growth on the galactose plate). The names of these genes were listed on the right.

Table 2. Go term analysis for toxic human genes. GO term enrichment analysis was performed on a set of 474 human genes that are toxic when overexpressed in yeast. The first column shows the enriched annotation category followed by the corresponding GO category in the second column. Note that many GO terms are embedded as children among more general parent terms. For example, the term “regulation of biosynthetic process” is a parent term to “regulation of RNA metabolic process”; moreover, all genes annotated to the child term are also annotated to the parent term. The third column shows the number of genes that map to that annotation from all human genes in the library, while the fourth column shows the number of genes that map from the toxic human gene set. The fifth column shows the number of genes that were expected to map to that annotation by random chance. The sixth column shows the fold enrichment of the actual toxic human genes over the expected by random chance. The seventh column shows the false discovery rate (FDR). The results are organized by GO category (molecular function, biological process, or cellular component), and then by decreasing fold enrichment. Only results with statistic significance are displayed

(p<0.05 and FDR<0.05).

# in # in # Fold Annotation Term GO Category FDR library toxic set expected Enrichment regulation of organelle Biological 95 17 4.53 3.75 1.30E-02 assembly (GO:1902115) Process positive regulation of nucleobase-containing Biological 1046 84 49.9 1.68 7.00E-03 compound metabolic Process process (GO:0045935) positive regulation of Biological RNA metabolic process 957 74 45.66 1.62 4.66E-02 Process (GO:0051254) positive regulation of Biological gene expression 1125 85 53.67 1.58 2.93E-02 Process (GO:0010628) regulation of transcription by RNA Biological 1165 87 55.58 1.57 3.28E-02 polymerase II Process (GO:0006357) regulation of nucleobase-containing Biological 1926 139 91.88 1.51 2.17E-03 compound metabolic Process process (GO:0019219) regulation of RNA Biological metabolic process 1801 128 85.92 1.49 5.72E-03 Process (GO:0051252) regulation of cellular macromolecule Biological 1872 129 89.31 1.44 1.27E-02 biosynthetic process Process (GO:2000112) regulation of macromolecule Biological 1944 133 92.74 1.43 1.07E-02 biosynthetic process Process (GO:0010556) regulation of gene Biological expression 2180 149 104 1.43 5.44E-03 Process (GO:0010468) regulation of nucleic acid-templated Biological 1684 115 80.34 1.43 4.88E-02 transcription Process (GO:1903506) regulation of RNA Biological biosynthetic process 1688 115 80.53 1.43 4.70E-02 Process (GO:2001141)

regulation of cellular Biological biosynthetic process 2024 134 96.56 1.39 3.50E-02 Process (GO:0031326) regulation of nitrogen Biological compound metabolic 2879 185 137.35 1.35 4.48E-03 Process process (GO:0051171) regulation of primary Biological metabolic process 2970 188 141.69 1.33 7.68E-03 Process (GO:0080090) regulation of metabolic Biological 3318 208 158.29 1.31 5.58E-03 process (GO:0019222) Process regulation of macromolecule Biological 3057 191 145.84 1.31 1.10E-02 metabolic process Process (GO:0060255) regulation of cellular Biological metabolic process 3047 189 145.36 1.3 1.34E-02 Process (GO:0031323) mRNA 3'-UTR binding Molecular 44 11 2.1 5.24 1.92E-02 (GO:0003730) Function mRNA binding Molecular 132 22 6.3 3.49 3.13E-03 (GO:0003729) Function RNA polymerase II cis- regulatory region Molecular 353 35 16.84 2.08 4.35E-02 sequence-specific DNA Function binding (GO:0000978) cis-regulatory region Molecular sequence-specific DNA 366 36 17.46 2.06 4.66E-02 Function binding (GO:0000987) cis-regulatory region Molecular 371 36 17.7 2.03 4.73E-02 binding (GO:0035326) Function DNA-binding transcription factor Molecular activity, RNA 543 47 25.91 1.81 4.71E-02 Function polymerase II-specific (GO:0000981) RNA binding Molecular 800 67 38.17 1.76 1.36E-02 (GO:0003723) Function DNA binding Molecular 1129 86 53.86 1.6 1.53E-02 (GO:0003677) Function nucleic acid binding Molecular 1816 136 86.64 1.57 1.10E-04 (GO:0003676) Function organic cyclic Molecular 2867 180 136.78 1.32 1.34E-02

compound binding Function (GO:0097159) heterocyclic compound Molecular 2825 175 134.77 1.3 2.31E-02 binding (GO:1901363) Function chromosome Cellular 853 73 40.69 1.79 2.02E-03 (GO:0005694) Component nuclear chromosome Cellular 665 55 31.73 1.73 2.90E-02 (GO:0000228) Component nuclear lumen Cellular 2249 152 107.29 1.42 3.41E-03 (GO:0031981) Component nucleoplasm Cellular 1907 125 90.98 1.37 2.64E-02 (GO:0005654) Component organelle lumen Cellular 2866 182 136.73 1.33 2.82E-03 (GO:0043233) Component intracellular organelle Cellular 2866 182 136.73 1.33 2.11E-03 lumen (GO:0070013) Component membrane-enclosed Cellular 2866 182 136.73 1.33 1.69E-03 lumen (GO:0031974) Component Cellular nucleus (GO:0005634) 3310 201 157.91 1.27 5.23E-03 Component

AIM II: DISCOVERING ENRICHED PATHWAYS AMONG FUS AND TDP-43

ENHANCERS

It has been previously demonstrated that, when overexpressed in yeast, wild-type

FUS and TDP-43 cause yeast cell cytotoxicity and reduced cellular fitness (see Figure 6).

However, the mechanism by which FUS and TDP-43 cause toxicity in yeast remains a mystery. Our lab aims to uncover the cellular processes of FUS and TDP-43 in yeast by identifying genetic modifiers of FUS and TDP-43 toxicity; that is, genes whose expression either enhances or suppresses the toxicity induced by FUS and TDP-43 expression in yeast.

By studying the genetic modifiers, we can elucidate mechanisms on FUS and TDP-43 induced toxicity in yeast which may imply their pathological functions in human neurons.

REFINING A SET OF FUS AND TDP-43 ENHANCERS

A previous member of our lab performed an overexpression screen of the human gene library for FUS and TDP-43 genetic modifiers (Hayden et al, 2020). This was accomplished by mating the haploid w303⍺ human library with haploid yeast of the opposite mating type

(w303a) which contained integrated copies of FUS or TDP-43 (Figure 8). Since yeast can be maintained in a haploid state, mating represents a fast and efficient way to generate recombinant organisms without the need for excessive yeast transformation, particularly for high-throughput studies. Enhancers and suppressors of FUS and TDP43-induced toxicity were identified in this screen by visually inspecting growth on galactose plates for either reduced or improved cellular fitness compared to the background growth of the plate. This thesis will discuss the analysis of the enhancer hits produced by this screen. Analysis of the suppressor hits were performed separately (Hayden et al, 2020).

The first step in analyzing the FUS and TDP-43 enhancer hits aimed to refine the list by removing potential false positive hits. Since the overexpression of human genes may produce a toxic phenotype, it is possible that introducing FUS or TDP-43 would represent a compounding of two unrelated mechanisms of toxicity and not a synthetic lethal interaction.

To eliminate false positives, I juxtaposed the FUS and TDP-43 enhancer lists with the list of toxic human genes and removed the common values. This produced a refined list of 358 human genetic enhancers (out of 658 original hits) whose independent expression in yeast was either not toxic or very weakly toxic. Out of 358 human genes, 138 enhanced TDP-43 toxicity, 335 enhanced FUS toxicity, and 115 enhanced both. (Figure 11).

CONFIRMING FUS AND TDP-43 ENHANCERS

To further support the validity of the newly refined enhancers list, we used a

Hamilton Microlab STAR liquid handling robot to cherrypick the enhancers from the 13,500 human gene library and array them into 96-well plates. This library of haploid yeast was then revived and tested for enhancement of FUS and TDP-43 induced toxicity through mating- based screening according to the same method as the initial screen (Materials and Methods;

Figure 8). Following this test, all but 4 of the enhancers identified by the original screen were able to be replicated (NDST4, ODF3B, WEE2, CCDC7). Compared to the 335 FUS enhancers identified from the first screen, the screened screen identified 350 enhancers of

FUS, representing a loss of 3, and a gain of 18 enhancers from the initial screen.

Figure 11. Distribution profile of FUS and TDP-43 enhancers. An independently produced list of 687 enhancers was juxtaposed with a list of human genes toxic to yeast to eliminate the false positives (genes are toxic to yeast on their own). The resulting 358 enhancers were sorted into FUS only enhancers (220), TDP-43 only enhancers (23), and enhancers of both FUS and TDP-43 (115). Names of 23 genes that enhance TDP-43 only are listed on the bottom.

Compared to the 138 initial TDP-43 enhancers, the test identified 209 enhancers representing a loss of 18 and a gain of 89 enhancers from the initial screen. Moreover, each enhancer list was largely reproducible. Our next step was to survey the FUS and TDP-43 enhancer lists for any enriched cellular functions or pathways.

ENRICHMENT ANALYSIS OF FUS AND TDP-43 ENHANCERS

To determine if any cellular functions or pathways are enriched among FUS and

TDP-43 genetic enhancers (thereby, giving us a clue as to the possible toxic mechanisms of these proteins), we performed overrepresentation testing of the 358 enhancers using

PANTHER. First, all 358 enhancers refined from the initial screen were grouped into five gene sets: (1) those that enhance FUS (n=335), (2) those that enhance TDP-43 (n=138), (3) those that enhance FUS, but not TDP-43 (n=220), (4) those that enhance TDP-43, but not

FUS (n=23), (5) and those that enhance both FUS and TDP-43 (n=115). Each of these gene sets were uploaded to PANTHER for overrepresentation testing.

The only gene sets which yielded statistically significant results (p<0.05, FDR<0.05) were the set of all FUS enhancers and the set of all TDP-43 enhancers (sets 1 and 2 from above, respectively). GO biological process analysis of all TDP-43 enhancers revealed a significant number of genes involved in RNA metabolism and nucleic-acid templated transcription (Table 3). Similarly, GO cellular component analysis of all FUS enhancers revealed many genes that encode proteins which localize to the nuclear chromosome, the site of RNA transcription and processing. While GO biological process enrichment analysis of

TDP-43 enhancers revealed many overrepresented GO terms, the same biological process analysis for FUS enhancers did not reveal any significantly overrepresented terms.

Table 3. Enrichment analysis for GO biological process terms among the human genetic enhancers of TDP-43 induced toxicity in yeast. The terms are organized by decreasing fold enrichment. Note that many GO terms are child terms to more general parent terms. Thus, there is much overlap in the genes mapped to each GO term. Only results with a p<0.05 and

FDR<0.05 are displayed.

# of # of Fold Annotation Term # in library FDR enhancers expected Enrichment positive regulation of nitrogen compound 1772 38 19.27 1.97 2.48E-02 metabolic process (GO:0051173) regulation of nucleic acid-templated 1690 36 18.38 1.96 4.26E-02 transcription (GO:1903506) regulation of RNA biosynthetic process 1694 36 18.42 1.95 3.94E-02 (GO:2001141) regulation of RNA metabolic process 1806 38 19.64 1.93 2.70E-02 (GO:0051252) positive regulation of cellular metabolic 1856 39 20.19 1.93 4.95E-02 process (GO:0031325) positive regulation of macromolecule 1856 39 20.19 1.93 3.71E-02 metabolic process (GO:0010604) regulation of macromolecule 1947 39 21.18 1.84 4.48E-02 biosynthetic process (GO:0010556) positive regulation of metabolic process 2013 40 21.89 1.83 3.84E-02 (GO:0009893)

regulation of biosynthetic process 2082 41 22.64 1.81 4.19E-02 (GO:0009889) regulation of primary metabolic process 2967 55 32.27 1.7 1.63E-02 (GO:0080090) regulation of nitrogen compound metabolic 2876 53 31.28 1.69 2.21E-02 process (GO:0051171) regulation of cellular metabolic process 3045 54 33.12 1.63 3.22E-02 (GO:0031323) regulation of macromolecule 3051 54 33.18 1.63 2.72E-02 metabolic process (GO:0060255) regulation of metabolic 3311 56 36.01 1.56 4.14E-02 process (GO:0019222)

CELL CYCLE REGULATORS ENHANCE FUS OR TDP-43 TOXICITY IN YEAST

Our next step was to survey the set of FUS and TDP-43 enhancers for functionally related genes which mapped to the same enriched GO terms. By identifying smaller groups of genes, this gives us the opportunity to perform smaller scale, targeted experimental studies into the nature of these genetic interactions.

Functional classification of the genes involved in FUS and TDP-43 enriched pathways and cell parts revealed several genes which encode regulators of the cell cycle.

These include cyclin-dependent kinase 1 and 2 (CDK1,2), cyclin-dependent kinase’s regulatory subunit 1 (CKS1), and cyclin B (CCNB). All four of these genes were shown to have an enhancing effect on both FUS and TDP-43 toxicity in yeast. Interestingly, CKS2 did not show up as a hit in the genetic screen despite sharing over 80% protein sequence homology with CKS1.

To validate the cell cycle proteins as enhancers of FUS and TDP-43 toxicity, we performed a more sensitive serial dilution assay (Figure 12). Diploid yeast containing both the cell cycle gene (CDK2 or CKS1) and disease gene (FUS or TDP-43) were generated by yeast mating as done in the genetic screens. Liquid cultures of theses diploid strains were grown and serially diluted on glucose (genes are repressed) and galactose (genes are expressed) agar plates. Compared to the controls (diploid yeast expressing a disease gene and the empty, functionless vector, pRS416Gal1-ccdB), yeast expressing a disease gene plus

CDK2 or CKS1 grew noticeably worse. This suggests that CDK2 and CKS1 may be interacting with the same pathway as FUS and TDP-43 to enhance toxicity. Given that CDK2 and CKS1 both promote progression through the cell division cycle, this pathway may represent a toxic mechanism of FUS and TDP-43 in yeast.

Figure 12 Cell cycle regulators enhance FUS (A) and TDP43-induced toxicity (B) when overexpressed in yeast. The accompanying figure illustrates a serial dilution growth assay.

Each column of the assay shows a colony of yeast that was 5X diluted from the previous column. Each row of the assay represents a unique strain of yeast. The yeast strains in this assay were generated by mating haploid yeast strains of opposite mating types and containing different plasmids. The legend at the left of the assay shows with what gene each strain is transformed (EV = pRS416Gal1-ccdB or pRS413Gal1-ccdB). Galactose media induces gene expression, while glucose represses gene expression. Pictures, representative of 3 independent experiments, show growth at 30°C for 3 days.

HYDROXYUREA ENHANCES FUS AND TDP-43 INDUCED TOXICITY

The overexpression of cell cycle promoting genes CDK2 and CKS1 lead to reduced yeast cell fitness when co-expressed with FUS and TDP-43. This suggests that the toxic mechanism of FUS and TDP-43 may involve perturbation of the cell cycle. Furthermore, if cell cycle defects play a role in the toxic mechanism of FUS and TDP43-induce toxicity, then introducing yeast to a cell cycle arresting agent, hydroxyurea, may further increase toxicity.

Hydroxyurea prevents cells from exiting S-phase by inhibiting the enzymatic process which reduces ribonucleoside diphosphatase to deoxyribonucleoside diphosphatase (retrieved from

PubChem, https://pubchem.ncbi.nlm.nih.gov/compound/Hydroxyurea).

Diploid yeast containing the disease protein and an empty plasmid vector were cultured and then serially diluted onto glucose and galactose containing hydroxyurea at a concentration of 10mM, 20mM, and 30mM. At concentration of 20mM and 30mM, hydroxyurea causes significant toxicity to yeast (data not shown). At a concentration of

10mM, hydroxyurea had no effect on yeast expressing the empty plasmid vector alone but had a noticeable enhancing effect on yeast expressing FUS and TDP-43 (Figure 13). This demonstrates that the overexpression of FUS and TDP-43 makes yeast cell vulnerable to cell cycle disruption. Moreover, the overexpression of CDK2 and CKS1 may also be exacerbating toxicity through disrupting the cell cycle. To explore how CDK2 and CKS1 increase FUS and TDP-43 toxicity, future experiments will be aimed at how these proteins might be interacting with each other.

Figure 13. 10mM Hydroxyurea enhances FUS (A) and TDP-43 (B) induced toxicity in yeast. The accompanying serial dilution growth assay shows diploid yeast transformed with integrated wild-type FUS and TDP-43 plasmids grown on glucose and galactose agar with and without 10mM of hydroxyurea. Compared to controls, yeast transformed with FUS and

TDP-43 show reduced cell fitness when exposed to hydroxyurea (indicated by the fewer number of colonies on the galactose plate with +10mM hydroxyurea). Pictures, representative of 3 independent experiments, show growth at 30°C for 4 days.

CELL CYCLE REGULATORS DO NOT CHANGE FUS OR TDP-43 AGGREGATION

NOR LOCALIZATION

The expression of cell cycle regulators CDK2 and CKS1 increase cellular toxicity of

FUS and TDP-43 in yeast. One simple explanation is that these genes enhance toxicity through their direct effect on FUS/TDP-43 aggregation, mislocalization, or increase in

FUS/TDP-43 protein expression level. To determine FUS/TDP-43 aggregation and localization, we utilized microscopy to visualize fluorescently tagged FUS and TDP-43 protein constructs in the presence or absence of cell cycle regulating genes CDK2 and CKS1.

The number of FUS and TDP-43 protein aggregates did not appear to be changed per cell when co-expressed with CDK2 and CKS1 compared to controls (Figure 14). When expressed with an empty plasmid vector (pRS416Gal1-ccdB) or cell cycle regulators (CDK2 or CKS1), FUS formed 4-5 aggregates per cell, while TDP43 formed 1-2 aggregates per cell.

Additionally, the localization of the aggregates within the cell also appeared to be unchanged. This result suggests that increased toxicity is likely not caused by increased (or decreased) FUS and TDP-43 aggregation or mislocalization. To further suggest this result, we performed a Western Blot to directly assess the amount of FUS and TDP-43 protein in the presence or absence of CDK2 and CKS1.

CELL CYCLE REGULATORS DO NOT AFFECT FUS or TDP-43 PROTEIN LEVEL

To check possible change on FUS or TDP43 protein level, Western Blot was performed to detect FUS and TDP-43 protein level with and without the co-overexpression of CDK2 and CKS1. Our results indicated no significant change in the amount of FUS or

TDP-43 protein level in the presence of CDK2 and CKS1 (Figure 15).

Figure 14. CDK2 or CKS1 does not alter FUS or TDP-43 aggregate quantity or localization. The accompanying figure shows 6 yeast strains visualized with an Olympus

IX83 fluorescent microscope. The legends at the top and left of the figure show with what gene each strain is transformed (EV = pRS416Gal1-ccdB; CKS1 = pRS416Gal1-CKS1;

CDK2 = pRS416Gal1-CDK2). The TDP-43 gene contains a c-terminal GFP tag which was visualized with the FITC channel of the microscope. The FUS gene contains a c-terminal

YFP tag which was visualized with the YFP channel of the microscope. Yeast were analyzed after 6 hours of galactose induction of stationary-phase yeast cultures. Pictures are taken from representative field from three independent experiments (n=~1000).

Figure 15. FUS and TDP-43 protein level does not change when co-expressed with

CDK2 and CKS1. Diploid yeast strains containing a cell cycle regulator gene (CDK2 or

CKS1) and a disease gene (FUS or TDP-43) were cultured and grown in galactose media for

6 hours to induce gene expression. Crude protein extracts and Weston Blot was performed as described in materials and methods. FUS (75 kDa) contained a c-terminal YFP tag (28 kDa), and TDP-43 (45 kDa) contained c-terminal GFP tag (26 kDa). The primary antibodies used were targeting YFP, GFP, and PGK1 (as loading control). The legend at the top of each figure shows the genetic identity of the yeast strain from which the protein was extracted.

The legend at the left is a representation of the protein ladder in kDa. The western blot experiment was performed three times (n=3).

IV. DISCUSSION

There are two primary findings from this thesis which are applicable over wide and diverse areas of interest. Firstly, we have shown that the majority of human genes can be overexpressed in Saccharomyces cerevisiae without producing non-specific toxicity.

Secondly, we have shown that RNA pathways continue to be a key player in the pathological activity of FUS and TDP-43 in yeast, but also with new evidence supporting that cell cycle disruption could be an integral part to these toxic pathways.

In Aim I, we used the largest known sequence-verified human ORF library to show that the majority of human genes in our library (roughly 97%) can be overexpressed in yeast without producing a non-specific toxicity. While some of these non-toxic human genes may also be non-functional in yeast, our findings still demonstrate the immense capacity of yeast to be humanized. As reviewed by Laurent et al. 2016, among the highest degrees of yeast humanization studies include the transformation of whole human cellular pathways to yeast.

This would not only allow us to study individual functions of genes, but also how genes function together as a network. These network-level studies would be untenable if each gene within the pathway caused a toxic phenotype. Thus, the findings from our research leave the door open to even more ambitious studies of genetic networks. As of yet, very few attempts have been made into this new and exciting arena of research.

Since it is unlikely that there is a non-specific overexpression toxicity, then this means that the human genes found to be toxic to yeast may also be functional, either act as conserved yeast homologs, or through conserved interactions with yeast proteins. However, when we conclude which human genes are toxic to yeast, we have to be careful that by no means, the genes identified in our study is a complete list, not only because our library covers

about two thirds of human genome, but also because toxicity of human genes in yeast may be dependent on the growth conditions or their expression levels. It will be very interesting to test other conditions, such as growing yeast in fermentable carbon source (which is dependent on functional mitochondria). It will also be interesting to check how much our list of toxic genes would change if human genes are expressed at higher level.

Enrichment analysis of the human genes which caused a toxic phenotype in yeast revealed a large number of genes involved in RNA metabolic pathways, primarily RNA transcription. This evidence indicates that yeast cell fitness may be particularly vulnerable to disruption in RNA transcription, and other RNA-related pathways. Given the conservation of

RNA pathways between yeast and higher eukaryotes, this suggests that human cells could also be sensitive to RNA dysregulation. Indeed, RNA dysregulation is a rapidly developing area of interest in many human diseases, including neurodegenerative diseases, such as ALS.

The practicality of our results from Aim I are clearly demonstrated through their application in Aim II. Knowing which human genes cause toxicity in yeast allowed us to refine a previously generated list of human genetic enhancers for two ALS-linked genes,

FUS and TDP-43. These two genes do not have yeast orthologs and are toxic when overexpressed in yeast. By studying human genetic enhancers and their interactions with these two disease genes, we may be able to discover how they cause toxicity in yeast, which may imply how their mutated forms may cause neurodegeneration in humans.

Interestingly, we identified a larger number of FUS enhancers (n=335) compared to

TDP-43 enhancers (n=138). This could mean that FUS has more prolific protein interactions than TDP-43. However, it is important to note that, compared to TDP-43, yeast integrated with FUS produces a much stronger toxic phenotype. This leaves a much smaller window to

screen for FUS enhancers. Thus, the list of FUS enhancers may be exaggerated compared to

TDP-43. Likewise, the screen for FUS and TDP-43 suppressors (genes that rescue toxicity) ultimately produced similar numbers of hits.

Our investigations into the human genetic enhancers of FUS and TDP-43 toxicity revealed a group of cell cycle regulator genes, including CDK1, CDK2, CKS1, and CCNB.

Notably, CKS1 has a homolog known as CKS2. The two proteins share 80% amino acid similarity, functioning together in regulating the cyclin dependent complexes and regulating transcription, and have been shown to be overexpressed in many cancers (Khattar et al, 2013, del Rincon et al, 2015). Interestingly, CKS2 was not identified in our genetic screen to be an enhancer like CKS1. Notably, CKS1 has been observed as a monomer, while CKS2 primarily forms dimers, in vitro (Arvai et al, 1995). The functional significance of these architectural differences is not yet fully understood, but likely suggests that CKS1 could be involved in a distinct pathway from CKS2 that enhances FUS and TDP-43 toxicity.

FUTURE EXPERIMENTS

While the combination of our fluorescence microscopy (Figure 14) and western blot studies (Figure 15B) indicated that FUS protein level is not significantly altered with the co- overexpression of CDK2 and CKS1. The signal from the western blot on FUS protein level was weak. This is likely due to the aged primary antibody. Using fresh antibody very likely will address the issue.

Interestingly, many cell cycle regulator proteins have now been implicated in FUS and TDP-43 toxicity. In addition to CDK1, CDK2, and CKS1 identified by our screens, other cell cycle regulators including CDK4, CDK6, and CCND have also been implicated. These

proteins function to promote progression through the cell cycle during different phases. For example, CDK4 and CDK6 regulate entry into G1 phase, CDK2 regulates progression through S phase, and CDK1 regulates progression through G2 phase (Figure 5). Thus, it would be interesting to investigate if FUS and TDP-43 toxicity involves generalized cell cycle disruption or disruption at specific stages.

If FUS or TDP-43 favors arrest at a particular cell cycle stage, then the proportion of cells arrested in this stage will be greater. Cell cycle arrest could be determined by assessing the morphology of these yeast cells due to their distinct sizes and shapes throughout the cell cycle (Figure 16). Furthermore, since CDK2 and CKS1 enhance FUS and TDP-43 toxicity in yeast, it would also be expected that the overexpression of the genes would increase the observed number of yeast cells arrested at a particular stage.

Similarly, Western Blot can be performed to identify yeast cell cycle arrest using the antibodies targeting cyclins. Eukaryotes are well known to conditionally express different types of cyclins at specific points of the cell cycle. For example, the cyclin Cln3 is only expressed during G1. The conditional expression of endogenous yeast cell cyclins could be exploited to determine if overexpression of FUS and TDP-43 arrest cells at particular stages.

It will also be very interesting to check whether cell cycle regulator genes co-localize with FUS or TDP43. A direct interaction could strongly support that they may be involved in the same pathways.

Figure 16. Yeast cellular and nuclear morphology changes during progression through the stages of the cell cycle. The emergence and growth of a bud is indicative of progression through S-phase. Nuclear migration begins after entrance into G2-phase. Nuclear division is indicative of entrance into M phase. Adapted from Calvert et al, 2008.

CONCLUSION

The majority of human ORFs (around 97%) can be overexpressed in yeast without producing a non-specific toxicity. This means that any toxic phenotype observed is likely due to the inherent functional properties of the encoded protein. The toxic mechanisms of FUS and TDP-43 in yeast likely involves dysregulation of RNA pathways, particularly RNA transcription. We discovered that a significant number of genetic interactors of FUS and

TDP-43 are involved in RNA metabolism, including several cell cycle regulators. Among these, CDK1, CDK2, and CKS1 were previously unknown to interact with FUS and TDP-43.

This suggests that the toxic functions of FUS and TDP-43 in yeast potentially rely upon an intersection between RNA metabolism and the cell cycle.

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