Increasing Clinician-Scientist Workforce Diversity through the National Institute of General Medical Sciences' Medical Scientist Training Program.

The National Institute of General Medical Sciences Medical Scientist Training Program (MSTP) has been successful in producing clinician-scientists, with a majority of graduates pursuing research-related careers. However, there are a number of areas of continuing concern for the program. In particular, women and individuals from certain racial and ethnic backgrounds remain persistently underrepresented in MSTPs relative to the average college-aged U.S. population and to students receiving life sciences bachelor's degrees. The authors, who include leaders of NIGMS, identify a number of challenges and opportunities for enhancing diversity, equity and inclusion in the MSTPs and suggest strategies for addressing them.

The funders' perspective: Lessons learned from the National Institutes of Health Diversity Program Consortium evaluation.

Advancing diversity in the biomedical research workforce is critical to the ability of the National Institutes of Health (NIH) to achieve its mission. The NIH Diversity Program Consortium is a unique, 10-year program that builds upon longstanding training and research capacity-building activities to promote workforce diversity. It was designed to rigorously evaluate approaches to enhancing diversity in the biomedical research workforce at the student, faculty, and institutional level. In this chapter we describe (a) the program's origins, (b) the consortium-wide evaluation, including plans, measures, challenges, and solutions, and (c) how lessons learned from this program are being leveraged to strengthen NIH research-training and capacity-building activities and evaluation efforts.

MutSα mismatch repair protein stability is governed by subunit interaction, acetylation, and ubiquitination.

In eukaryotes, DNA mismatch recognition is accomplished by the highly conserved MutSα (Msh2/Msh6) and MutSß (Msh2/Msh3) complexes. Previously, in the yeast Saccharomyces cerevisiae, we determined that deleting MSH6 caused wild-type Msh2 levels to drop by ∼50%. In this work, we determined that Msh6 steady-state levels are coupled to increasing or decreasing levels of Msh2. Although Msh6 and Msh2 are reciprocally regulated, Msh3 and Msh2 are not. Msh2 missense variants that are able to interact with Msh6 were destabilized when Msh6 was deleted; in contrast, variants that fail to dimerize were not further destabilized in cells lacking Msh6. In the absence of Msh6, Msh2 is turned over at a faster rate and degradation is mediated by the ubiquitin-proteasome pathway. Mutagenesis of certain conserved lysines near the dimer interface restored the levels of Msh2 in the absence of Msh6, further supporting a dimer stabilization mechanism. We identified two alternative forms of regulation both with the potential to act via lysine residues, including acetylation by Gcn5 and ubiquitination by the Not4 ligase. In the absence of Gcn5, Msh2 levels were significantly decreased; in contrast, deleting Not4 stabilized Msh2 and Msh2 missense variants with partial function. The stabilizing effect on Msh2 by either the presence of Msh6 or the absence of Not4 are dependent on Gcn5. Taken together, the results suggest that the wild-type MutSα mismatch repair protein stability is governed by subunit interaction, acetylation, and ubiquitination.

Developing a culture of safety in biomedical research training.

The National Institute of General Medical Sciences (NIGMS) at the U.S. National Institutes of Health (NIH) is committed to supporting the safety of the nation's biomedical research and training environments. Institutional training grants affect many trainees and can have a broad influence across their parent institutions, making them good starting points for our initial efforts to promote the development and maintenance of robust cultures of safety at U.S. academic institutions. In this Perspective, we focus on laboratory safety, although many of the strategies we describe for improving laboratory safety are also applicable to other forms of safety including the prevention of harassment, intimidation, and discrimination. We frame the problem of laboratory safety using a number of recent examples of tragic accidents, highlight some of the lessons that have been learned from these and other events, discuss what NIGMS is doing to address problems related to laboratory safety, and outline steps that institutions can take to improve their safety cultures.

Whole-Genome Sequence and Variant Analysis of W303, a Widely-Used Strain of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has emerged as a superior model organism. Selection of distinct laboratory strains of S. cerevisiae with unique phenotypic properties, such as superior mating or sporulation efficiencies, has facilitated advancements in research. W303 is one such laboratory strain that is closely related to the first completely sequenced yeast strain, S288C. In this work, we provide a high-quality, annotated genome sequence for W303 for utilization in comparative analyses and genome-wide studies. Approximately 9500 variations exist between S288C and W303, affecting the protein sequences of ∼700 genes. A listing of the polymorphisms and divergent genes is provided for researchers interested in identifying the genetic basis for phenotypic differences between W303 and S288C. Several divergent functional gene families were identified, including flocculation and sporulation genes, likely representing selection for desirable laboratory phenotypes. Interestingly, remnants of ancestor wine strains were found on several chromosomes. Finally, as a test of the utility of the high-quality reference genome, variant mapping revealed more accurate identification of accumulated mutations in passaged mismatch repair-defective strains.

From the NIH: A Systems Approach to Increasing the Diversity of the Biomedical Research Workforce.

The National Institutes of Health (NIH) is committed to attracting, developing, and supporting the best scientists from all groups as an integral part of excellence in training. Biomedical research workforce diversity, capitalizing on the full spectrum of skills, talents, and viewpoints, is essential for solving complex human health challenges. Over the past few decades, the biomedical research workforce has benefited from NIH programs aimed at enhancing diversity. However, there is considerable room for improvement, particularly at the level of independent scientists and within scientific leadership. We provide a rationale and specific opportunities to develop and sustain a diverse biomedical research workforce through interventions that promote the successful transitions to different stages on the path toward completion of training and entry into the biomedical workforce.

The Eukaryotic Mismatch Recognition Complexes Track with the Replisome during DNA Synthesis.

During replication, mismatch repair proteins recognize and repair mispaired bases that escape the proofreading activity of DNA polymerase. In this work, we tested the model that the eukaryotic mismatch recognition complex tracks with the advancing replisome. Using yeast, we examined the dynamics during replication of the leading strand polymerase Polε using Pol2 and the eukaryotic mismatch recognition complex using Msh2, the invariant protein involved in mismatch recognition. Specifically, we synchronized cells and processed samples using chromatin immunoprecipitation combined with custom DNA tiling arrays (ChIP-chip). The Polε signal was not detectable in G1, but was observed at active origins and replicating DNA throughout S-phase. The Polε signal provided the resolution to track origin firing timing and efficiencies as well as replisome progression rates. By detecting Polε and Msh2 dynamics within the same strain, we established that the mismatch recognition complex binds origins and spreads to adjacent regions with the replisome. In mismatch repair defective PCNA mutants, we observed that Msh2 binds to regions of replicating DNA, but the distribution and dynamics are altered, suggesting that PCNA is not the sole determinant for the mismatch recognition complex association with replicating regions, but may influence the dynamics of movement. Using biochemical and genomic methods, we provide evidence that both MutS complexes are in the vicinity of the replisome to efficiently repair the entire spectrum of mutations during replication. Our data supports the model that the proximity of MutSα/ß to the replisome for the efficient repair of the newly synthesized strand before chromatin reassembles.

Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants.

Resistance to cancer therapy is a major obstacle in the long-term treatment of cancer. A greater understanding of drug resistance mechanisms will ultimately lead to the development of effective therapeutic strategies to prevent resistance from occurring. Here, we exploit the mutator phenotype of mismatch repair defective yeast cells combined with whole genome sequencing to identify drug resistance mutations in key pathways involved in the development of chemoresistance. The utility of this approach was demonstrated via the identification of the known CAN1 and TOP1 resistance targets for two compounds, canavanine and camptothecin, respectively. We have also experimentally validated the plasma membrane transporter HNM1 as the primary drug resistance target of mechlorethamine. Furthermore, the sequencing of mitoxantrone-resistant strains identified inactivating mutations within IPT1, a gene encoding inositolphosphotransferase, an enzyme involved in sphingolipid biosynthesis. In the case of bactobolin, a promising anticancer drug, the endocytosis pathway was identified as the drug resistance target responsible for conferring resistance. Finally, we show that that rapamycin, an mTOR inhibitor previously shown to alter the fitness of the ipt1 mutant, can effectively prevent the formation of mitoxantrone resistance. The rapid and robust nature of these techniques, using Saccharomyces cerevisiae as a model organism, should accelerate the identification of drug resistance targets and guide the development of novel therapeutic combination strategies to prevent the development of chemoresistance in various cancers.

Mutation rates, spectra, and genome-wide distribution of spontaneous mutations in mismatch repair deficient yeast.

DNA mismatch repair is a highly conserved DNA repair pathway. In humans, germline mutations in hMSH2 or hMLH1, key components of mismatch repair, have been associated with Lynch syndrome, a leading cause of inherited cancer mortality. Current estimates of the mutation rate and the mutational spectra in mismatch repair defective cells are primarily limited to a small number of individual reporter loci. Here we use the yeast Saccharomyces cerevisiae to generate a genome-wide view of the rates, spectra, and distribution of mutation in the absence of mismatch repair. We performed mutation accumulation assays and next generation sequencing on 19 strains, including 16 msh2 missense variants implicated in Lynch cancer syndrome. The mutation rate for DNA mismatch repair null strains was approximately 1 mutation per genome per generation, 225-fold greater than the wild-type rate. The mutations were distributed randomly throughout the genome, independent of replication timing. The mutation spectra included insertions/deletions at homopolymeric runs (87.7%) and at larger microsatellites (5.9%), as well as transitions (4.5%) and transversions (1.9%). Additionally, repeat regions with proximal repeats are more likely to be mutated. A bias toward deletions at homopolymers and insertions at (AT)n microsatellites suggests a different mechanism for mismatch generation at these sites. Interestingly, 5% of the single base pair substitutions might represent double-slippage events that occurred at the junction of immediately adjacent repeats, resulting in a shift in the repeat boundary. These data suggest a closer scrutiny of tumor suppressors with homopolymeric runs with proximal repeats as the potential drivers of oncogenesis in mismatch repair defective cells.

Cell-cycle and DNA damage regulation of the DNA mismatch repair protein Msh2 occurs at the transcriptional and post-transcriptional level.

DNA mismatch repair during replication is a conserved process essential for maintaining genomic stability. Mismatch repair is also implicated in cell-cycle arrest and apoptosis after DNA damage. Because yeast and human mismatch repair systems are well conserved, we have employed the budding yeast Saccharomyces cerevisiae to understand the regulation and function of the mismatch repair gene MSH2. Using a luciferase-based transcriptional reporter, we defined a 218-bp region upstream of MSH2 that contains cell-cycle and DNA damage responsive elements. The 5' end of the MSH2 transcript was mapped by primer extension and was found to encode a small upstream open reading frame (uORF). Mutagenesis of the uORF start codon or of the uORF stop codon, which creates a continuous reading frame with MSH2, increased Msh2 steady-state protein levels ∼2-fold. Furthermore, we found that the cell-cycle transcription factors Swi6, Swi4, and Mbp1-along with SCB/MCB cell-cycle binding sites upstream of MSH2-are all required for full basal expression of MSH2. Mutagenesis of the cell-cycle boxes resulted in a minor reduction in basal Msh2 levels and a 3-fold defect in mismatch repair. Disruption of the cell-cycle boxes also affected growth in a DNA polymerase-defective strain background where mismatch repair is essential, particularly in the presence of the DNA damaging agent methyl methane sulfonate (MMS). Promoter replacements conferring constitutive expression of MSH2 revealed that the transcriptional induction in response to MMS is required to maintain induced levels of Msh2. Turnover experiments confirmed an elevated rate of degradation in the presence of MMS. Taken together, the data show that the DNA damage regulation of Msh2 occurs at the transcriptional and post-transcriptional levels. The transcriptional and translational control elements identified are conserved in mammalian cells, underscoring the use of yeast as a model system to examine the regulation of MSH2.

Proteasome inhibition rescues clinically significant unstable variants of the mismatch repair protein Msh2.

MSH2 is required for DNA mismatch repair recognition in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with a common hereditary cancer syndrome. Previously, we characterized clinically identified MSH2 missense mutations, using yeast as a model system, and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we show that increased protein turnover is responsible for the reduced cellular levels. Increasing gene dosage of more than half of the missense alleles fully restored function. A titration experiment revealed that raising the expression level of one variant to less than wild-type levels restored mismatch repair, suggesting that overexpression is not always required to regain function. We found that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the Msh2 variants and identified the primary ubiquitin ligase as San1. Deletion of San1 restored protein levels for all but one variant, but did not elevate wild-type Msh2 levels. The unstable variants interacted with San1, whereas wild-type Msh2 did not. Additionally, san1Δ suppressed the mismatch repair defect of unstable variants. Of medical significance, the clinically approved drug Bortezomib partially restored protein levels and mismatch repair function for low-level variants and reversed the resistance to cisplatin, a common chemotherapeutic. Our results provide the foundation for an innovative therapeutic regime for certain mismatch-repair-defective cancers that are refractory to conventional chemotherapies.

Two replication regions in the pJM1 virulence plasmid of the marine pathogen Vibrio anguillarum.

Vibrio anguillarum is a fish pathogen that causes vibriosis, a serious hemorrhagic septicemia, in wild and cultured fish. Many serotype O1 strains of this bacterium harbor the 65kb plasmid pJM1 carrying the majority of genes encoding the siderophore anguibactin iron transport system that is one of the most important virulence factors of this bacterium. We previously identified a replication region of the pJM1 plasmid named ori1. In this work we determined that ori1 can replicate in Escherichia coli and that the chromosome-encoded proteins DnaB, DnaC and DnaG are essential for its replication whereas PolI, IHF and DnaA are not required. The copy number of the pJM1 plasmid is 1-2, albeit cloned smaller fragments of the ori1 region replicate with higher copy numbers in V. anguillarum while in E. coli we did not observe an obvious difference of the copy numbers of these constructs which were all high. Furthermore, we were able to delete the ori1 region from the pJM1 plasmid and identified a second replication region in pJM1 that we named ori2. This second replication region is located on ORF25 that is within the trans-acting factor (TAFr) region, and showed that it can only replicate in V. anguillarum.

Reciprocal regulation of nuclear import of the yeast MutSalpha DNA mismatch repair proteins Msh2 and Msh6.

DNA mismatch recognition is performed in eukaryotes by two heterodimers known as MutSalpha (Msh2/Msh6) and MutSbeta (Msh2/Msh3) that must reside in the nucleus to function. Two putative Msh2 nuclear localization sequences (NLS) were characterized by fusion to green fluorescent protein (GFP) and site-directed mutagenesis in the context of Msh2. One NLS functioned in GFP targeting assays and both acted redundantly within Msh2. We examined nuclear localization of each of the MutS monomers in the presence and absence of their partners. Msh2 translocated to the nucleus in cells lacking Msh3 and Msh6; however, cells lacking Msh6 showed significantly decreased levels of nuclear Msh2. Furthermore, the overall protein levels of Msh2 were significantly diminished in the absence of Msh6, particularly if Msh2 lacked a functional NLS. Msh3 localized in the absence of Msh2, but Msh6 localization depended on Msh2 expressing functional NLSs. Overall, the nuclear levels of Msh2 and Msh6 decline when the other partner is absent. The data suggest a stabilization mechanism to prevent free monomer accumulation in the cytoplasm.

Ultrastructural analysis of cell fusion in yeast.

The process of creating a single cell from two progenitor cells requires molecular precision to coordinate the events leading to cytoplasmic continuity while preventing lethal cell lysis. Cell fusion characteristically involves the mobilization of fundamental processes, including signaling, polarization, adhesion, and membrane fusion. The yeast Saccharomyces cerevisiae is an ideal model system for examining the events of this critical and well-conserved process. Researchers employ yeast cells because they are rapidly growing, easy to manipulate, amenable to long-term storage, genetically tractable, readily transformed, and nonhazardous. The genetic and morphological characterizations of cell fusion in wild-type and fusion mutants have helped define the mechanism and temporal regulation required for efficient cell fusion. Ultrastructural studies, in particular, have contributed to the characterization of and revealed striking similarities within cell fusion events in higher organisms. This chapter details two yeast cell fusion ultrastructural methods. The first utilizes an ambient temperature chemical fixation, and the second employs a combination of high-pressure freezing and freeze substitution.

Functional characterization of pathogenic human MSH2 missense mutations in Saccharomyces cerevisiae.

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with defects in DNA mismatch repair. Mutations in either hMSH2 or hMLH1 underlie the majority of HNPCC cases. Approximately 25% of annotated hMSH2 disease alleles are missense mutations, resulting in a single change out of 934 amino acids. We engineered 54 missense mutations in the cognate positions in yeast MSH2 and tested for function. Of the human alleles, 55% conferred strong defects, 8% displayed intermediate defects, and 38% showed no defects in mismatch repair assays. Fifty percent of the defective alleles resulted in decreased steady-state levels of the variant Msh2 protein, and 49% of the Msh2 variants lost crucial protein-protein interactions. Finally, nine positions are predicted to influence the mismatch recognition complex ATPase activity. In summary, the missense mutations leading to loss of mismatch repair defined important structure-function relationships and the molecular analysis revealed the nature of the deficiency for Msh2 variants expressed in the tumors. Of medical relevance are 15 human alleles annotated as pathogenic in public databases that conferred no obvious defects in mismatch repair assays. This analysis underscores the importance of functional characterization of missense alleles to ensure that they are the causative factor for disease.

Role of transcription factor Kar4 in regulating downstream events in the Saccharomyces cerevisiae pheromone response pathway.

Yeast Kar4 is a putative transcription factor required for karyogamy (the fusion of haploid nuclei during mating) and possibly other functions. Previously known to be required only for the transcriptional induction of KAR3 and CIK1, microarray experiments identified many genes regulated by Kar4 in both mating and mitosis. Several gene clusters are positively or negatively regulated by mating pheromone in a Kar4-dependent manner. Chromatin immunoprecipitation and gel shift assays confirmed that Kar4 binds to regulatory DNA sequences upstream of KAR3. Together with one-hybrid experiments, these data support a model in which both Kar4 and Ste12 bind jointly to the KAR3 promoter. Analysis of the upstream regions of Kar4-induced genes identified a DNA sequence motif that may be a binding site for Kar4. Mutation within the motif upstream of KAR3 eliminated pheromone induction. Genes regulated by Kar4, on average, are delayed in their temporal expression and exhibit a more stringent dose response to pheromone. Furthermore, the induction of Kar4 by pheromone is necessary for the delayed temporal induction of KAR3 and PRM2, genes required for efficient nuclear fusion during mating. Accordingly, we propose that Kar4 plays a critical role in the choreography of the mating response.

Lrg1p Is a Rho1 GTPase-activating protein required for efficient cell fusion in yeast.

To identify additional cell fusion genes in Saccharomyces cerevisiae, we performed a high-copy suppressor screen of fus2Delta. Higher dosage of three genes, BEM1, LRG1, and FUS1, partially suppressed the fus2Delta cell fusion defect. BEM1 and FUS1 were high-copy suppressors of many cell-fusion-defective mutations, whereas LRG1 suppressed only fus2Delta and rvs161Delta. Lrg1p contains a Rho-GAP homologous region. Complete deletion of LRG1, as well as deletion of the Rho-GAP coding region, caused decreased rates of cell fusion and diploid formation comparable to that of fus2Delta. Furthermore, lrg1Delta caused a more severe mating defect in combination with other cell fusion mutations. Consistent with an involvement in cell fusion, Lrg1p localized to the tip of the mating projection. Lrg1p-GAP domain strongly and specifically stimulated the GTPase activity of Rho1p, a regulator of beta(1-3)-glucan synthase in vitro. beta(1-3)-glucan deposition was increased in lrg1Delta strains and mislocalized to the tip of the mating projection in fus2Delta strains. High-copy LRG1 suppressed the mislocalization of beta(1-3) glucan in fus2Delta strains. We conclude that Lrg1p is a Rho1p-GAP involved in cell fusion and speculate that it acts to locally inhibit cell wall synthesis to aid in the close apposition of the plasma membranes of mating cells.

Characterization of pathogenic human MSH2 missense mutations using yeast as a model system: a laboratory course in molecular biology.

This work describes the project for an advanced undergraduate laboratory course in cell and molecular biology. One objective of the course is to teach students a variety of cellular and molecular techniques while conducting original research. A second objective is to provide instruction in science writing and data presentation by requiring comprehensive laboratory reports modeled on the primary literature. The project for the course focuses on a gene, MSH2, implicated in the most common form of inherited colorectal cancer. Msh2 is important for maintaining the fidelity of genetic material where it functions as an important component of the DNA mismatch repair machinery. The goal of the project has two parts. The first part is to create mapped missense mutation listed in the human databases in the cognate yeast MSH2 gene and to assay for defects in DNA mismatch repair. The second part of the course is directed towards understanding in what way are the variant proteins defective for mismatch repair. Protein levels are analyzed to determine if the missense alleles display decreased expression. Furthermore, the students establish whether the Msh2p variants are properly localized to the nucleus using indirect immunofluorescence and whether the altered proteins have lost their ability to interact with other subunits of the MMR complex by creating recombinant DNA molecules and employing the yeast 2-hybrid assay.