Philip Hieter

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
DNA:RNA hybrid genome-wide profiling and links to genome instability (2014)

No abstract available.

Defects in messenger RNA processing and biogenesis of RNA polymerases contribute to eukaryotic genome instability (2013)

Genome instability has been observed in mutants involved in various aspects of transcription and RNA processing. The prevalence of this mechanism among essential chromosome instability (CIN) genes remains unclear. In this thesis, it is shown that RNA biogenesis mutants exhibit elevated sensitivity to DNA damaging agents. A secondary screen for increased Rad52 foci in CIN mutants, representing ~25% of essential genes, identified seven essential subunits of the mRNA cleavage and polyadenylation (mCP) machinery. Genome-wide analysis of fragile sites by ChIP-chip of phosphorylated-H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of RNA:DNA hybrids known as R-loops, which were subsequently observed in mCP mutants. Among the CIN mutants with elevated Rad52 foci levels were the GPN proteins, a poorly-characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2 and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here, it is shown that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1, and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition and defects in RNA polymerase II nuclear localization. Importantly, previously unrecognized RNA polymerase III localization defects were observed in GPN2, GPN3 and IWR1 mutant backgrounds but no localization defects of unrelated nuclear proteins or of RNA polymerase I were found. In this study, it was shown that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1 suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common function in RNA polymerase assembly and subsequent transport. These findings demonstrate how mRNA cleavage and polyadenylation and proper RNA polymerase assembly contribute to maintenance of genome integrity and may be relevant to certain human cancers.

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Evolutionarily conserved synthetic lethal interaction networks reveal targets for anticancer therapeutic development (2012)

Cancer is a multigenic disease. The genetic distinctness of cancer cells offers aweakness that can be exploited: for example, nearly all cancers carry mutations in processesrelating to the maintenance of genomic stability. As this is an essential process, this presentsa weakness that can be leveraged towards inviability – a concept known as syntheticlethality. The ideal cancer therapeutic would have a broad spectrum, but genetic techniquesin human cells are not sufficiently developed to identify the spectrum of synthetic lethalinteractions of sets of genome stability genes easily. The use of model organisms canfacilitate the identification of second-site targets for the development of anticancertherapeutics, and allows the construction of synthetic lethal interaction networks. This has thepotential to identify “hub” genes having synthetic lethal interactions with many cancermutatedorthologs. If these synthetic lethal interactions are found to be conserved in humancells, these highly connected hub genes are potential targets for therapeutic development.Assembly of a synthetic lethal interaction network of yeast orthologs of 10 genes mutated incolorectal cancer, based on data in Saccharomyces cerevisiae, previously identified five suchsynthetic lethal hub genes in yeast. In this thesis, the evolutionary conservation of thisnetwork is interrogated in mammalian cells. The interactions between orthologs of colorectalcancer CIN genes in yeast were found to be highly conserved in human cells. A highthroughputassay to screen for small-molecule inhibitors of the protein encoded by one suchgene, FEN1, was developed and used to identify 13 compounds that inhibited FEN1 in vitrowith IC50 values in the low-micromolar range or less. These compounds were applied to cellsbearing mutation in the tumor suppressor CDC4, and two compounds were found to yieldselective killing of CDC4-deficient cells. Finally, yeast genetic techniques were used to characterize CTF4, a second highly connected hub gene within the colon cancer CIN genenetwork, and to expand the therapeutic range of cancers that could be selectively killed byinhibitors of Ctf4/WDHD1 or Rad27/FEN1. Taken together, these data demonstrate theconsiderable power of applying model organisms genetics to the discovery of new anticancertherapeutic targets.

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A cross species approach to identify potential therapeutic targets through synthetic lethal interactions (2011)

Chromosome instability (CIN) is characterized by the loss or gain of large portions of DNA and is characteristic of ~85% of solid tumours. Sequencing of the human orthologues of ~200 genes that cause CIN in yeast identified mutations in approximately 25% of tumours tested. Mutations in cohesin genes and CDC4 represented the two major mutational categories identified. Large scale genetic interaction networks in model organisms can provide insight into the biology underlying tumour mutations and can identify potential therapeutic targets. This approach is based on the concept of synthetic lethality (SL); cell death resulting from the combination of two sub-lethal mutations. Therapies that take advantage of SL distinguish a cancer cell from a normal cell based on their genetic background. This thesis investigated genetic interactions of three cohesin genes, SMC1, SCC1, and SCC2, using high throughput synthetic genetic array (SGA) methods in S. cerevisiae. The overlay of these three genome wide SGA screens and validation using growth curve analysis found that sub-optimal cohesin requires the presence of proteins that mediate replication fork progression. The protruding vulva assay was developed to identify genetic interactions in the somatic cells of C. elegans. It was used to test whether the cohesin interactions were conserved in a multicellular animal. 80% of the validated interactions identified with cohesin in yeast are conserved in C. elegans. Additional fork mediators, namely the pme/PARP family of genes was found to interact with him-1/SMC1 in both C. elegans and human cells. Human cells depleted of SMC1 by siRNA are selectively sensitive to the PARP inhibitor olaparib, currently being evaluated in phase II clinical trials. Additional genetic interaction testing found that CDC4 has a distinct genetic interaction profile from that of cohesin, suggesting different mutational consequences. Work in C. elegans with the lin-23 mutant suggested LIN-23 is involved in controlling CYE-1/Cyclin E levels. lin-23 mutants and human CDC4-/- cells are unable to properly respond to alkylating DNA damage, suggesting CDC4 is important for the DNA damage response. Keywords: colon tumours, chromosome instability, cohesin, CDC4, genetic interactions, SGA, replication fork, PARP.

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Systems Biology of Cellular Signalling -- Quantitative Experimentation and Systems Genetics Approaches (2009)

No abstract available.

Master's Student Supervision (2010 - 2018)
Identification of cancer relevant synthetic genetic interactions with cohesin mutations in Saccharomyces cerevisiae (2017)

Cancer therapy is changing. Whole genome sequencing technologies are advancing at an unprecedented pace, opening new opportunities for the genotype-driven personalized treatment of cancer. Synthetic Lethality (SL) based therapeutics have emerged as promising approaches to target cancer-specific somatic mutations, by targeting a second gene that is required for viability in the presence of a tumor-specific mutation. The targetable set of SL partner genes can be expanded by screening for a conditional SL interaction, in which loss of function of two genes results in sensitivity to low doses of a DNA-damaging agent, a concept we have called Synthetic Cytotoxicity (SC). SC also has the potential to expand the number of genotypes that can be treated with existing chemotherapeutics and to improve the efficacy of these therapeutics. In contrast to SL and SC negative genetic interactions, Phenotypic Suppression (PS) describes a genetic interaction in which the double mutant cell is more fit than anticipated based on the fitness of each single mutant. The model organism, Saccharomyces cerevisiae was used to screen for SC interactions with cohesin-mutated genes, with the aim of identifying cross-species candidate genes that could be followed up in subsequent studies as SL-based cancer-drug targets. The cohesin complex is frequently mutated across a wide range of tumors and is conserved from yeast to man. We used Synthetic Genetic Array (SGA) technology, a high-throughput genetic method available in yeast, to screen cohesin-mutated strains for synthetic lethal genetic interactions against an array of 310 deletions affecting mainly DNA damage response genes. The screens were done in the presence and absence of four clinically-relevant genotoxic agents. We screened and analyzed 4,650 potential genetic interactions, identifying hundreds of negative and positive interactions, belonging to conserved biological pathways, and potentially relevant to cancer. Using ScanLag, a new validation method, we re-tested and validated several genetic interactions that represent potential therapeutic candidates. These strong SL, SC and PS interactions can be further analyzed in mammalian cells to potentially inform and improve individual cancer therapies as personalized medicine treatments, and lead to the discovery of new pathways or candidates for anti-cancer treatments.

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Ground penetrating radar applied : a model for quantifying interpretation of human burials in historical contexts (2015)

This thesis explores the applied use of Ground Penetrating Radar (GPR) technology in conducting comprehensive burial survey work in historic period, post-contact cemeteries. These results are based on research conducted from 2008 to 2011 within several First Nations post-contact cemeteries along with work done in other less-defined burial sites in Southwestern British Columbia. My research has been informed by other types of GPR surveys conducted during that period and through 2015, that have added experience in GPR project management, data collection, trace signal analysis, interpretation and reporting. I have developed a recursive, interpretive model that creates a simple, direct and more objective process for evaluating any single or group of potential burial locations situated in a variety of physical contexts. The basic analysis is done by linking GPR signal results obtained from over 300 cases with either prior historical or field-work based knowledge of related ground surface, physical, ethnographic and documentary evidence. The model developed here quantifies the interpretative analysis of these data and develops what I refer to as a Burial Confidence Index (BCI) from a set of parameters or variables that reflects the full extent of our knowledge of any specific location. This allows for testing and statistical comparison of known versus previously unknown locations where GPR evidence is recovered. Other important aspects of GPR-related work in the community are also addressed in brief to provide more complete coverage of the many contexts involved, including professional, academic and social considerations.

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Divergence in alternative splicing patterns between duplicated gene pairs in polyploid Brassica napus (2010)

Polyploidy is the process of genome doubling that gives rise to organisms with multiple sets of chromosomes. Expression patterns and levels of genes duplicated by polyploidy, termed homeologs, can change and gene silencing can occur after polyploidy. Alternative splicing (AS) creates multiple mature mRNAs from a single type of precursor mRNA. AS can change the level of gene expression by degradation of transcripts with premature stop codons, as well as create new protein isoforms. Little is known about how AS changes after a polyploidization event, either within a few generations after polyploidy or over evolutionary time, and what effects AS changes have on gene expression in polyploids. In this project, the evolution of alternative splicing patterns after genome duplication in allotetraploid Brassica napus and a synthetic allotetraploid B. napus was examined by RT-PCR assays of a set of 31 duplicated genes. Since genes can show different patterns of AS in different organ types and under different abiotic stresses, two different organ types (leaf and cotyledon), and two different abiotic stresses (heat and cold) were used. Comparing the AS patterns between the two homeologs in B. napus revealed that 18% of the gene pairs show AS in only one homeolog. In contrast 33% of the gene pairs in the synthetic allotetraploid showed AS in only one homeolog. Gene silencing was observed for 6% and 9% of genes in B. napus and synthetic B. napus, respectively. These results indicate that there are many changes in AS in both the synthetic B. napus and natural B. napus after polyploidy, but more AS changes occurred in the synthetic polyploid. The PASTICCINO gene showed partitioning of two AS events between the homeologs in the synthetic allopolyploid, suggesting subfunctionalization of AS forms. Results from this project indicate that AS patterns can change rapidly after polyploidy and suggest that changes in AS patterns are a major phenomenon in allopolyploids.

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Functionalization of cancer-associated mutant alleles (2013)

No abstract available.

Synthetic lethality and synthetic cytotoxicity strategies for selective killing of ATM deficient cells (2012)

Chromosome instability (CIN) is a hallmark of cancer cells and could, in theory, be exploited in the design of cancer therapeutics. Tumor cells harboring CIN mutations may be dependent on certain DNA repair pathways for viability. Thus, inhibition of specific DNA repair enzymes may enhance the CIN phenotype to an intolerable level, or may sensitize cells to DNA damage stress. To test this hypothesis, I focused on the CIN gene ATM, which is often mutated in human tumors. I hypothesized that knockdown of certain second site DNA repair genes would selectively kill ATM-deficient cells resulting in synthetic lethality (SL), or sensitize ATM-deficient cells to a sub-lethal dose of DNA damaging agent resulting in synthetic cytotoxicity (SC). The goal of this research is to use budding yeast as a model system to identify candidate SL or SC interaction partner genes for ATM with/without sub-lethal doses of DNA damaging agents, using mutations in the yeast ATM homologues, TEL1 and MEC1. I tested for interactions with TEL1 and MEC1 in a small matrix of three DNA repair genes (RAD27, TDP1 and TPP1) and four DNA damaging agents (hydroxyurea, 5-fluorouracil, bleomycin, and camptothecin). I also performed a genome-wide screen for interactions between TEL1 and ~5000 non-essential genes, both in the presence and absence of low doses of camptothecin. I discovered one SL interaction with MEC1 and fourteen SC interactions with TEL1. Most of the SC interaction partner genes are involved in DNA repair and show sensitivity specifically to camptothecin. These data provide a rationale for testing specific combination therapies for selective killing of cancer cells bearing ATM mutations. Specifically, the Shu complex, Ku complex, Rrm3, Rad27 and CK2β subunits can be further tested as potential combination therapeutic targets, together with a sub-lethal dose of camptothecin, to kill ATM-deficient cancer cells.

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Divergence in alternative splicing patterns between duplicated gene pairs in polyploid Brassica napus (2010)

Polyploidy is the process of genome doubling that gives rise to organisms with multiple sets of chromosomes. Expression patterns and levels of genes duplicated by polyploidy, termed homeologs, can change and gene silencing can occur after polyploidy. Alternative splicing (AS) creates multiple mature mRNAs from a single type of precursor mRNA. AS can change the level of gene expression by degradation of transcripts with premature stop codons, as well as create new protein isoforms. Little is known about how AS changes after a polyploidization event, either within a few generations after polyploidy or over evolutionary time, and what effects AS changes have on gene expression in polyploids. In this project, the evolution of alternative splicing patterns after genome duplication in allotetraploid Brassica napus and a synthetic allotetraploid B. napus was examined by RT-PCR assays of a set of 31 duplicated genes. Since genes can show different patterns of AS in different organ types and under different abiotic stresses, two different organ types (leaf and cotyledon), and two different abiotic stresses (heat and cold) were used. Comparing the AS patterns between the two homeologs in B. napus revealed that 18% of the gene pairs show AS in only one homeolog. In contrast 33% of the gene pairs in the synthetic allotetraploid showed AS in only one homeolog. Gene silencing was observed for 6% and 9% of genes in B. napus and synthetic B. napus, respectively. These results indicate that there are many changes in AS in both the synthetic B. napus and natural B. napus after polyploidy, but more AS changes occurred in the synthetic polyploid. The PASTICCINO gene showed partitioning of two AS events between the homeologs in the synthetic allopolyploid, suggesting subfunctionalization of AS forms. Results from this project indicate that AS patterns can change rapidly after polyploidy and suggest that changes in AS patterns are a major phenomenon in allopolyploids.

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