Altered S-AdenosylMethionine availability impacts dNTP pools in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has long been used as a model organism to study genome instability. The SAM1 and SAM2 genes encode AdoMet synthetases, which generate S-AdenosylMethionine (AdoMet) from Methionine (Met) and ATP. Previous work from our group has shown that deletions of the SAM1 and SAM2 genes cause changes to AdoMet levels and impact genome instability in opposite manners. AdoMet is a key product of methionine metabolism and the major methyl donor for methylation events of proteins, RNAs, small molecules, and lipids. The methyl cycle is interrelated to the folate cycle which is involved in de novo synthesis of purine and pyrimidine deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP). AdoMet also plays a role in polyamine production, essential for cell growth and used in detoxification of reactive oxygen species (ROS) and maintenance of the redox status in cells. This is also impacted by the methyl cycle's role in production of glutathione, another ROS scavenger and cellular protectant. We show here that sam2∆/sam2∆ cells, previously characterized with lower levels of AdoMet and higher genome instability, have a higher level of each dNTP (except dTTP), contributing to a higher overall dNTP pool level when compared to wildtype. Unchecked, these increased levels can lead to multiple types of DNA damage which could account for the genome instability increases in these cells.

Profiling the compendium of changes in Saccharomyces cerevisiae due to mutations that alter availability of the main methyl donor S-Adenosylmethionine.

The SAM1 and SAM2 genes encode for S-Adenosylmethionine (AdoMet) synthetase enzymes, with AdoMet serving as the main cellular methyl donor. We have previously shown that independent deletion of these genes alters chromosome stability and AdoMet concentrations in opposite ways in Saccharomyces cerevisiae. To characterize other changes occurring in these mutants, we grew wildtype, sam1Δ/sam1Δ, and sam2Δ/sam2Δ strains in 15 different Phenotypic Microarray plates with different components and measured growth variations. RNA-Sequencing was also carried out on these strains and differential gene expression determined for each mutant. We explored how the phenotypic growth differences are linked to the altered gene expression, and hypothesize mechanisms by which loss of the SAM genes and subsequent AdoMet level changes, impact pathways and processes. We present 6 stories, discussing changes in sensitivity or resistance to azoles, cisplatin, oxidative stress, arginine biosynthesis perturbations, DNA synthesis inhibitors, and tamoxifen, to demonstrate the power of this novel methodology to broadly profile changes due to gene mutations. The large number of conditions that result in altered growth, as well as the large number of differentially expressed genes with wide-ranging functionality, speaks to the broad array of impacts that altering methyl donor abundance can impart. Our findings demonstrate that some cellular changes are directly related to AdoMet-dependent methyltransferases and AdoMet availability, some are directly linked to the methyl cycle and its role in production of several important cellular components, and others reveal impacts of SAM gene mutations on previously unconnected pathways.

Profiling The Compendium Of Changes In Saccharomyces cerevisiae Due To Mutations That Alter Availability Of The Main Methyl Donor S-Adenosylmethionine.

The SAM1 and SAM2 genes encode for S-AdenosylMethionine (AdoMet) synthetase enzymes, with AdoMet serving as the main methyl donor. We have previously shown that independent deletion of these genes alters chromosome stability and AdoMet concentrations in opposite ways in S. cerevisiae. To characterize other changes occurring in these mutants, we grew wildtype, sam1∆/sam1∆, and sam2∆/sam2∆ strains in 15 different Phenotypic Microarray plates with different components, equal to 1440 wells, and measured for growth variations. RNA-Sequencing was also carried out on these strains and differential gene expression determined for each mutant. In this study, we explore how the phenotypic growth differences are linked to the altered gene expression, and thereby predict the mechanisms by which loss of the SAM genes and subsequent AdoMet level changes, impact S. cerevisiae pathways and processes. We present six stories, discussing changes in sensitivity or resistance to azoles, cisplatin, oxidative stress, arginine biosynthesis perturbations, DNA synthesis inhibitors, and tamoxifen, to demonstrate the power of this novel methodology to broadly profile changes due to gene mutations. The large number of conditions that result in altered growth, as well as the large number of differentially expressed genes with wide-ranging functionality, speaks to the broad array of impacts that altering methyl donor abundance can impart, even when the conditions tested were not specifically selected as targeting known methyl involving pathways. Our findings demonstrate that some cellular changes are directly related to AdoMet-dependent methyltransferases and AdoMet availability, some are directly linked to the methyl cycle and its role is production of several important cellular components, and others reveal impacts of SAM gene mutations on previously unconnected pathways.

Heterozygous Mutations in Aromatic Amino Acid Synthesis Genes Trigger TOR Pathway Activation in Saccharomyces cerevisiae.

The highly conserved complexes of Target of Rapamycin (TORC1 and TORC2) are central regulators to many vital cellular processes including growth and autophagy in response to nutrient availability. Previous research has extensively elucidated exogenous nutrient control on TORC1/TORC2; however, little is known about the potential alteration of nutrient pools from mutations in biosynthesis pathways and their impact on Tor pathway activity. Here, we analyze the impacts of heterozygous mutations in aromatic amino acid biosynthesis genes on TOR signaling via differential expression of genes downstream of TORC1 and autophagy induction for TORC1 and TORC2 activity.

Mutations in the S-Adenosylmethionine Synthetase Genes SAM1 and SAM2 Differentially Affect Genome Stability in Saccharomyces cerevisiae.

Maintenance of genome integrity is a crucial cellular focus that involves a wide variety of proteins functioning in multiple processes. Defects in many different pathways can result in genome instability, a hallmark of cancer. Utilizing a diploid Saccharomyces cerevisiae model, we previously reported a collection of gene mutations that affect genome stability in a haploinsufficient state. In this work we explore the effect of gene dosage on genome instability for one of these genes and its paralog; SAM1 and SAM2 These genes encode S-Adenosylmethionine (AdoMet) synthetases, responsible for the creation of AdoMet from methionine and ATP. AdoMet is the universal methyl donor for methylation reactions and is essential for cell viability. It is the second most used cellular enzyme substrate and is exceptionally well-conserved through evolution. Mammalian cells express three genes, MAT1A, MAT2A, and MAT2B, with distinct expression profiles and functions. Alterations to these AdoMet synthetase genes, and AdoMet levels, are found in many cancers, making them a popular target for therapeutic intervention. However, significant variance in these alterations are found in different tumor types, with the cellular consequences of the variation still unknown. By studying this pathway in the yeast system, we demonstrate that losses of SAM1 and SAM2 have different effects on genome stability through distinctive effects on gene expression and AdoMet levels, and ultimately separate effects on the methyl cycle. Thus, this study provides insight into the mechanisms by which differential expression of the SAM genes have cellular consequences that affect genome instability.

Single-Gene Deletions Contributing to Loss of Heterozygosity in Saccharomyces cerevisiae: Genome-Wide Screens and Reproducibility.

Loss of heterozygosity (LOH) is a phenomenon commonly observed in cancers; the loss of chromosomal regions can be both causal and indicative of underlying genome instability. Yeast has long been used as a model organism to study genetic mechanisms difficult to study in mammalian cells. Studying gene deletions leading to increased LOH in yeast aids our understanding of the processes involved, and guides exploration into the etiology of LOH in cancers. Yet, before in-depth mechanistic studies can occur, candidate genes of interest must be identified. Utilizing the heterozygous Saccharomyces cerevisiae deletion collection (≈ 6500 strains), 217 genes whose disruption leads to increased LOH events at the endogenously heterozygous mating type locus were identified. Our investigation to refine this list of genes to candidates with the most definite impact on LOH includes: secondary testing for LOH impact at an additional locus, gene ontology analysis to determine common gene characteristics, and positional gene enrichment studies to identify chromosomal regions important in LOH events. Further, we conducted extensive comparisons of our data to screens with similar, but distinct methodologies, to further distinguish genes that are more likely to be true contributors to instability due to their reproducibility, and not just identified due to the stochastic nature of LOH. Finally, we selected nine candidate genes and quantitatively measured their impact on LOH as a benchmark for the impact of genes identified in our study. Our data add to the existing body of work and strengthen the evidence of single-gene knockdowns contributing to genome instability.

Implementation and assessment of a yeast orphan gene research project: involving undergraduates in authentic research experiences and progressing our understanding of uncharacterized open reading frames.

Saccharomyces cerevisiae was the first eukaryotic organism to be sequenced; however, little progress has been made in recent years in furthering our understanding of all open reading frames (ORFs). From October 2012 to May 2015 the number of verified ORFs had only risen from 75.31% to 78%, while the number of uncharacterized ORFs had decreased from 12.8% to 11% (representing > 700 genes still left in this category; http://www.yeastgenome.org/genomesnapshot). Course-based research has been shown to increase student learning while providing experience with real scientific investigation; however, implementation in large, multi-section courses presents many challenges. This study sought to test the feasibility and effectiveness of incorporating authentic research into a core genetics course, with multiple instructors, to increase student learning and progress our understanding of uncharacterized ORFs. We generated a module-based annotation toolkit and utilized easily accessible bioinformatics tools to predict gene function for uncharacterized ORFs within the Saccharomyces Genome Database (SGD). Students were each assigned an uncharacterized ORF, which they annotated using contemporary comparative genomics methodologies, including multiple sequence alignment, conserved domain identification, signal peptide prediction and cellular localization algorithms. Student learning outcomes were measured by quizzes, project reports and presentations, as well as a post-project questionnaire. Our results indicate that the authentic research experience had positive impacts on students' perception of their learning and their confidence to conduct future research. Furthermore, we believe that creation of an online repository and adoption and/or adaptation of this project across multiple researchers and institutions could speed the process of gene function prediction.

Sequences associated with centromere competency in the human genome.

Centromeres, the sites of spindle attachment during mitosis and meiosis, are located in specific positions in the human genome, normally coincident with diverse subsets of alpha satellite DNA. While there is strong evidence supporting the association of some subfamilies of alpha satellite with centromere function, the basis for establishing whether a given alpha satellite sequence is or is not designated a functional centromere is unknown, and attempts to understand the role of particular sequence features in establishing centromere identity have been limited by the near identity and repetitive nature of satellite sequences. Utilizing a broadly applicable experimental approach to test sequence competency for centromere specification, we have carried out a genomic and epigenetic functional analysis of endogenous human centromere sequences available in the current human genome assembly. The data support a model in which functionally competent sequences confer an opportunity for centromere specification, integrating genomic and epigenetic signals and promoting the concept of context-dependent centromere inheritance.

Functional epialleles at an endogenous human centromere.

Human centromeres are defined by megabases of homogenous alpha-satellite DNA arrays that are packaged into specialized chromatin marked by the centromeric histone variant, centromeric protein A (CENP-A). Although most human chromosomes have a single higher-order repeat (HOR) array of alpha satellites, several chromosomes have more than one HOR array. Homo sapiens chromosome 17 (HSA17) has two juxtaposed HOR arrays, D17Z1 and D17Z1-B. Only D17Z1 has been linked to CENP-A chromatin assembly. Here, we use human artificial chromosome assembly assays to show that both D17Z1 and D17Z1-B can support de novo centromere assembly independently. We extend these in vitro studies and demonstrate, using immunostaining and chromatin analyses, that in human cells the centromere can be assembled at D17Z1 or D17Z1-B. Intriguingly, some humans are functional heterozygotes, meaning that CENP-A is located at a different HOR array on the two HSA17 homologs. The site of CENP-A assembly on HSA17 is stable and is transmitted through meiosis, as evidenced by inheritance of CENP-A location through multigenerational families. Differences in histone modifications are not linked clearly with active and inactive D17Z1 and D17Z1-B arrays; however, we detect a correlation between the presence of variant repeat units of D17Z1 and CENP-A assembly at the opposite array, D17Z1-B. Our studies reveal the presence of centromeric epialleles on an endogenous human chromosome and suggest genomic complexities underlying the mechanisms that determine centromere identity in humans.

Utilizing Saccharomyces cerevisiae to identify aneuploidy and cancer susceptibility genes.

Cancers are commonly characterized as having an abnormal number of chromosomes, termed aneuploidy, which arise due to genomic instability. There is still debate over whether aneuploidy is a driving force of the disease or a resulting phenotype; however, the presence of aneuploidy can be used to grade the malignant potential of certain types of cancer. A simple hypothesis is that genome instability itself is tumorigenic in that it results in alterations in the number of chromosomes, which alters gene copy number and ultimately affects gene expression in cells.Many gene disruptions that result in a propensity for cells to become aneuploid were first identified through mutagenesis screens designed to generate null or missense mutations in haploid strains of Saccharomyces cerevisiae. In contrast, the susceptibility to develop cancer can be transmitted as an autosomal dominant trait with affected individuals being heterozygous carriers of null mutations. In this chapter, we will describe a technique that can be used to identify heterozygous mutations in dosage-sensitive genes that mediate genomic stability by performing genome-wide screens in yeast.

A robust estimator of mutation rates.

Fluctuation analysis is an established and widely used technique of estimating mutation rates in cultured cells. This paper presents a modified median estimator of mutation rates, which is novel because it allows for unequal population sizes N(t) of the parallel cultures, and helps to detect and reduce the estimation variability. Simulation results show a good accuracy and robustness of the modified median estimator compared with the median estimator and the maximum likelihood estimator. The proposed estimator, based on the Luria-Delbrück model, is applied to 20 yeast datasets collected during 3 different days for a study of chromosome loss and recombination in wild-type Saccharomyces cerevisiae strains. The estimates obtained display among-experiment variability, which is inflated with respect to the model predictions on simulated data. Further investigation in S. cerevisiae and Escherichia coli uncovers an empirical inverse relationship between the population sizes N(t) and the mutation rate estimates under certain experimental conditions. The impact of these effects on the practice of fluctuation analysis is discussed.

Heterozygous screen in Saccharomyces cerevisiae identifies dosage-sensitive genes that affect chromosome stability.

Current techniques for identifying mutations that convey a small increased cancer risk or those that modify cancer risk in carriers of highly penetrant mutations are limited by the statistical power of epidemiologic studies, which require screening of large populations and candidate genes. To identify dosage-sensitive genes that mediate genomic stability, we performed a genomewide screen in Saccharomyces cerevisiae for heterozygous mutations that increase chromosome instability in a checkpoint-deficient diploid strain. We used two genome stability assays sensitive enough to detect the impact of heterozygous mutations and identified 172 heterozygous gene disruptions that affected chromosome fragment (CF) loss, 45% of which also conferred modest but statistically significant instability of endogenous chromosomes. Analysis of heterozygous deletion of 65 of these genes demonstrated that the majority increased genomic instability in both checkpoint-deficient and wild-type backgrounds. Strains heterozygous for COMA kinetochore complex genes were particularly unstable. Over 50% of the genes identified in this screen have putative human homologs, including CHEK2, ERCC4, and TOPBP1, which are already associated with inherited cancer susceptibility. These findings encourage the incorporation of this orthologous gene list into cancer epidemiology studies and suggest further analysis of heterozygous phenotypes in yeast as models of human disease resulting from haplo-insufficiency.