Identification of novel plant cysteine oxidase inhibitors from a yeast chemical genetic screen.

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an in vivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity in vitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress.

RAD52 influences the effect of BRCA1/2 missense variants on homologous recombination and gene reversion in Saccharomyces cerevisiae.

The breast and ovarian cancer susceptibility genes, BRCA1 and BRCA2, are key players in the homologous recombination (HR) repair pathway and act as tumor suppressors by maintaining genome stability. The yeast Saccharomyces cerevisiae has no BRCA1/2 homolog; however, a number of HR genes are evolutionary conserved between human and yeast. Among them, RAD52 is involved in DNA double strand break (DSB) repair by HR, and promotes genome stability. We previously reported that the heterologous expression of cancer-associated BRCA1/2 missense variants in growing yeast cultures affects both spontaneous HR and gene reversion (GR) suggesting that yeast could be a reliable system to assess the functional impact of variants. Because inhibition of Rad52p is lethal in BRCA1/2 mutated tumors, and Rad52p is conserved between humans and yeast, we asked if the effect of BRCA1/2 variants on HR and GR could be affected by loss of RAD52. We found that the rad52∆ mutation predominantly suppressed the stimulation of HR in yeast by pathogenic BRCA1 variants but also facilitated increased GR by pathogenic variants. Conversely, the rad52∆ mutation stimulated HR by a pathogenic BRCA2 variant in yeast but had no effect on GR. These results demonstrate a functional interplay between the pathogenic BRCA1/2 variants and Rad52p in budding yeast, supporting the use of budding yeast as a suitable system for evaluating potential chemotherapeutic strategies.

Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants.

Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in Saccharomyces cerevisiae increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants.

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions.

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5'UTR and 3'UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband's group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3'UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.

Yeast-based assays for the functional characterization of cancer-associated variants of human DNA repair genes.

Technological advances are continuously revealing new genetic variants that are often difficult to interpret. As one of the most genetically tractable model organisms, yeast can have a central role in determining the consequences of human genetic variation. DNA repair gene mutations are associated with many types of cancers, therefore the evaluation of the functional impact of these mutations is crucial for risk assessment and for determining therapeutic strategies. Owing to the evolutionary conservation of DNA repair pathways between human cells and the yeast Saccharomyces cerevisiae, several functional assays have been developed. Here, we describe assays for variants of human genes belonging to the major DNA repair pathways divided in functional assays for human genes with yeast orthologues and human genes lacking a yeast orthologue. Human genes with orthologues can be studied by introducing the correspondent human mutations directly in the yeast gene or expressing the human gene carrying the mutations; while the only possible approach for human genes without a yeast orthologue is the heterologous expression. The common principle of these approaches is that the mutated gene determines a phenotypic alteration that can vary according to the gene studied and the domain of the protein. Here, we show how the versatility of yeast can help in classifying cancer-associated variants.

Effect of BRCA1 missense variants on gene reversion in DNA double-strand break repair mutants and cell cycle-arrested cells of Saccharomyces cerevisiae.

Evaluation of the functional impact of germline BRCA1 variants that are likely to be associated to breast and ovarian cancer could help to investigate the mechanism of BRCA1 tumorigenesis. Expression of pathogenic BRCA1 missense variants increased homologous recombination (HR) and gene reversion (GR) in yeast. We thought to exploit yeast genetics to shed light on BRCA1-induced genome instability and tumorigenesis. We determined the effect on GR of several neutral and pathogenic BRCA1 variants in the yeast strain RSY6wt and its isogenic DSB repair mutants, such as mre11∆, rad50∆ and rad51∆. In the RSY6wt, four out of five pathogenic and two out of six neutral variants significantly increased GR; rad51∆ strain, the pathogenic variants C61G and A1708E induced a weak but significant increase in GR. On the other hand, in rad50∆ mutant expressing the pathogenic variants localised at the BRCT domain, a further GR increase was seen. The neutral variant N132K and the VUS A1789T induced a weak GR increase in mre11∆ mutant. Thus, BRCA1 missense variants require specific genetic functions and presumably induced GR by different mechanisms. As DNA repair is regulated by cell cycle, we determined the effect on GR of BRCA1 variants in cell cycle-arrested RSYwt cells. GR is highly BRCA1-inducible in S-phase-arrested cells as compared to G1 or G2. Sequence analysis of genomic DNA from ILV1 revertant clones showed that BRCA1-induced ilv1-92 reversion by base substitution when GR is at least 6-fold over the control. Our study demonstrated that BRCA1 may interfere with yeast DNA repair functions that are active in S-phase causing high level of GR. In addition, we confirmed here that yeast could be a reliable model to investigate the mechanism and genetic requirements of BRCA1-induced genome instability. Finally, developing yeast-based assays to characterise BRCA1 missense variants could be useful to design more precise therapies.