S. cerevisiae Cells Can Grow without the Pds5 Cohesin Subunit.

During DNA replication, the newly created sister chromatids are held together until their separation at anaphase. The cohesin complex is in charge of creating and maintaining sister chromatid cohesion (SCC) in all eukaryotes. In Saccharomyces cerevisiae cells, cohesin is composed of two elongated proteins, Smc1 and Smc3, bridged by the kleisin Mcd1/Scc1. The latter also acts as a scaffold for three additional proteins, Scc3/Irr1, Wpl1/Rad61, and Pds5. Although the HEAT-repeat protein Pds5 is essential for cohesion, its precise function is still debated. Deletion of the ELG1 gene, encoding a PCNA unloader, can partially suppress the temperature-sensitive pds5-1 allele, but not a complete deletion of PDS5. We carried out a genetic screen for high-copy-number suppressors and another for spontaneously arising mutants, allowing the survival of a pds5Δ elg1Δ strain. Our results show that cells remain viable in the absence of Pds5 provided that there is both an elevation in the level of Mcd1 (which can be due to mutations in the CLN2 gene, encoding a G1 cyclin), and an increase in the level of SUMO-modified PCNA on chromatin (caused by lack of PCNA unloading in elg1Δ mutants). The elevated SUMO-PCNA levels increase the recruitment of the Srs2 helicase, which evicts Rad51 molecules from the moving fork, creating single-stranded DNA (ssDNA) regions that serve as sites for increased cohesin loading and SCC establishment. Thus, our results delineate a double role for Pds5 in protecting the cohesin ring and interacting with the DNA replication machinery. IMPORTANCE Sister chromatid cohesion is vital for faithful chromosome segregation, chromosome folding into loops, and gene expression. A multisubunit protein complex known as cohesin holds the sister chromatids from S phase until the anaphase stage. In this study, we explore the function of the essential cohesin subunit Pds5 in the regulation of sister chromatid cohesion. We performed two independent genetic screens to bypass the function of the Pds5 protein. We observe that Pds5 protein is a cohesin stabilizer, and elevating the levels of Mcd1 protein along with SUMO-PCNA accumulation on chromatin can compensate for the loss of the PDS5 gene. In addition, Pds5 plays a role in coordinating the DNA replication and sister chromatid cohesion establishment. This work elucidates the function of cohesin subunit Pds5, the G1 cyclin Cln2, and replication factors PCNA, Elg1, and Srs2 in the proper regulation of sister chromatid cohesion.

Mutagenic mechanisms of cancer-associated DNA polymerase ϵ alleles.

A single amino acid residue change in the exonuclease domain of human DNA polymerase ϵ, P286R, is associated with the development of colorectal cancers, and has been shown to impart a mutator phenotype. The corresponding Pol ϵ allele in the yeast Saccharomyces cerevisiae (pol2-P301R), was found to drive greater mutagenesis than an entirely exonuclease-deficient Pol ϵ (pol2-4), an unexpected phenotype of ultra-mutagenesis. By studying the impact on mutation frequency, type, replication-strand bias, and sequence context, we show that ultra-mutagenesis is commonly observed in yeast cells carrying a range of cancer-associated Pol ϵ exonuclease domain alleles. Similarities between mutations generated by these alleles and those generated in pol2-4 cells indicate a shared mechanism of mutagenesis that yields a mutation pattern similar to cancer Signature 14. Comparison of POL2 ultra-mutator with pol2-M644G, a mutant in the polymerase domain decreasing Pol ϵ fidelity, revealed unexpected analogies in the sequence context and strand bias of mutations. Analysis of mutational patterns unique to exonuclease domain mutant cells suggests that backtracking of the polymerase, when the mismatched primer end cannot be accommodated in the proofreading domain, results in the observed insertions and T>A mutations in specific sequence contexts.

Telomeres and stress in yeast cells: When genes and environment interact.

Telomeres are structures composed of simple DNA repeats and specific proteins that protect the eukaryotic chromosomal ends from degradation, and facilitate the replication of the genome. They are central to the maintenance of the genome integrity, and play important roles in the development of cancer and in the process of aging in humans. The yeast Saccharomyces cerevisiae has greatly contributed to our understanding of basic telomere biology. Our laboratory has carried out systematic screen for mutants that affect telomere length, and identified ∼500 genes that, when mutated, affect telomere length. Remarkably, all ∼500 TLM (Telomere Length Maintenance) genes participate in a very tight homeostatic process, and it is enough to mutate one of them to change the steady-state telomere length. Despite this complex network of balances, it is also possible to change telomere length in yeast by applying several types of external stresses. We summarize our insights about the molecular mechanisms by which genes and environment interact to affect telomere length.

The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51.

Homologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. The Saccharomyces cerevisiae yeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2's helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a new SRS2 allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of the srs2(1-850) allele suggest a role for Srs2's helicase activity in sister chromatid cohesion. Our results also indicate that Srs2's function becomes more central in diploid cells.IMPORTANCE Homologous recombination (HR) is a key mechanism that repairs damaged DNA. However, this process has to be tightly regulated; failure to regulate it can lead to genome instability. The Srs2 helicase is considered a regulator of HR; it was shown to be able to evict the recombinase Rad51 from DNA. Cells lacking Srs2 exhibit sensitivity to DNA-damaging agents, and in some cases, they display defects in DNA replication. The relative roles of the helicase and Rad51 removal activities of Srs2 in genome stability remain unclear. To address this question, we created a new Srs2 mutant which has only the DNA helicase domain. Our study shows that only the DNA helicase domain is needed to deal with DNA damage and assist in DNA replication during vegetative growth and in meiosis. Thus, our findings shift the view on the role of Srs2 in the maintenance of genome integrity.

Clonal Heterogeneity Influences the Fate of New Adaptive Mutations.

The joint contribution of pre-existing and de novo genetic variation to clonal adaptation is poorly understood but essential to designing successful antimicrobial or cancer therapies. To address this, we evolve genetically diverse populations of budding yeast, S. cerevisiae, consisting of diploid cells with unique haplotype combinations. We study the asexual evolution of these populations under selective inhibition with chemotherapeutic drugs by time-resolved whole-genome sequencing and phenotyping. All populations undergo clonal expansions driven by de novo mutations but remain genetically and phenotypically diverse. The clones exhibit widespread genomic instability, rendering recessive de novo mutations homozygous and refining pre-existing variation. Finally, we decompose the fitness contributions of pre-existing and de novo mutations by creating a large recombinant library of adaptive mutations in an ensemble of genetic backgrounds. Both pre-existing and de novo mutations substantially contribute to fitness, and the relative fitness of pre-existing variants sets a selective threshold for new adaptive mutations.

Disentangling genetic and epigenetic determinants of ultrafast adaptation.

A major rationale for the advocacy of epigenetically mediated adaptive responses is that they facilitate faster adaptation to environmental challenges. This motivated us to develop a theoretical-experimental framework for disclosing the presence of such adaptation-speeding mechanisms in an experimental evolution setting circumventing the need for pursuing costly mutation-accumulation experiments. To this end, we exposed clonal populations of budding yeast to a whole range of stressors. By growth phenotyping, we found that almost complete adaptation to arsenic emerged after a few mitotic cell divisions without involving any phenotypic plasticity. Causative mutations were identified by deep sequencing of the arsenic-adapted populations and reconstructed for validation. Mutation effects on growth phenotypes, and the associated mutational target sizes were quantified and embedded in data-driven individual-based evolutionary population models. We found that the experimentally observed homogeneity of adaptation speed and heterogeneity of molecular solutions could only be accounted for if the mutation rate had been near estimates of the basal mutation rate. The ultrafast adaptation could be fully explained by extensive positive pleiotropy such that all beneficial mutations dramatically enhanced multiple fitness components in concert. As our approach can be exploited across a range of model organisms exposed to a variety of environmental challenges, it may be used for determining the importance of epigenetic adaptation-speeding mechanisms in general.

Lack of replication of higher genetic risk load in men than in women with systemic lupus erythematosus.

INTRODUCTION: We aimed to replicate a recent study which showed higher genetic risk load at 15 loci in men than in women with systemic lupus erythematosus (SLE). This difference was very significant, and it was interpreted as indicating that men require more genetic susceptibility than women to develop SLE. METHODS: Nineteen SLE-associated loci (thirteen of which are shared with the previous study) were analyzed in 1,457 SLE patients and 1,728 healthy controls of European ancestry. Genetic risk load was calculated as sex-specific sum genetic risk scores (GRS(s)). RESULTS: Our results did not replicate those of the previous study at either the level of individual loci or the global level of GRS(s). GRS(s) were larger in women than in men (4.20 ± 1.07 in women vs. 3.27 ± 0.98 in men). This very significant difference (P < 10(-16)) was more dependent on the six new loci not included in the previous study (59% of the difference) than on the thirteen loci that are shared (the remaining 41%). However, the 13 shared loci also showed a higher genetic risk load in women than in men in our study (P = 6.6 × 10(-7)), suggesting that heterogeneity of participants, in addition to different loci, contributed to the opposite results. CONCLUSION: Our results show the lack of a clear trend toward higher genetic risk in one of the sexes for the analyzed SLE loci. They also highlight several limitations of assessments of genetic risk load, including the possibility of ascertainment bias with loci discovered in studies that have included mainly women.

Influence of Atg5 mutation in SLE depends on functional IL-10 genotype.

Increasing evidence supports the involvement of autophagy in the etiopathology of autoimmune diseases. Despite the identification of autophagy-related protein (Atg)-5 as one of the susceptibility loci in systemic Lupus erythematosus (SLE), the consequences of the carriage of these mutations for patients remain unclear. The present work analyzed the association of Atg5 rs573775 single nucleotide polymorphism (SNP) with SLE susceptibility, IFNα, TNFα and IL-10 serum levels, and clinical features, in 115 patients and 170 healthy individuals. Patients who where carriers of the rs573775 T* minor allele presented lower IFNα levels than those with the wild genotype, whereas the opposite result was detected for IL-10. Thus, since IL-10 production was regulated by rs1800896 polymorphisms, we evaluated the effect of this Atg5 mutation in genetically high and low IL-10 producers. Interestingly, we found that the rs573775 T* allele was a risk factor for SLE in carriers of the high IL-10 producer genotype, but not among genetically low producers. Moreover, IL-10 genotype influences SLE features in patients presenting the Atg5 mutated allele. Specifically, carriage of the rs573775 T* allele led to IL-10 upregulation, reduced IFNα and TNFα production and a low frequency of cytopenia in patients with the high IL-10 producer genotype, whereas patients with the same Atg5 allele that were low IL-10 producers presented reduced amounts of all these cytokines, had a lower prevalence of anti-dsDNA antibodies and the latest onset age. In conclusion, the Atg5 rs573775 T* allele seems to influence SLE susceptibility, cytokine production and disease features depending on other factors such as functional IL-10 genotype.

Identification of three new cis-regulatory IRF5 polymorphisms: in vitro studies.

BACKGROUND: Polymorphisms in the interferon regulatory factor 5 (IRF5) gene are associated with susceptibility to systemic lupus erythematosus, rheumatoid arthritis and other diseases through independent risk and protective haplotypes. Several functional polymorphisms are already known, but they do not account for the protective haplotypes that are tagged by the minor allele of rs729302. METHODS: Polymorphisms in linkage disequilibrium (LD) with rs729302 or particularly associated with IRF5 expression were selected for functional screening, which involved electrophoretic mobility shift assays (EMSAs) and reporter gene assays. RESULTS: A total of 54 single-nucleotide polymorphisms in the 5' region of IRF5 were genotyped. Twenty-four of them were selected for functional screening because of their high LD with rs729302 or protective haplotypes. In addition, two polymorphisms were selected for their prominent association with IRF5 expression. Seven of these twenty-six polymorphisms showed reproducible allele differences in EMSA. The seven were subsequently analyzed in gene reporter assays, and three of them showed significant differences between their two alleles: rs729302, rs13245639 and rs11269962. Haplotypes including the cis-regulatory polymorphisms correlated very well with IRF5 mRNA expression in an analysis based on previous data. CONCLUSION: We have found that three polymorphisms in LD with the protective haplotypes of IRF5 have differential allele effects in EMSA and in reporter gene assays. Identification of these cis-regulatory polymorphisms will allow more accurate analysis of transcriptional regulation of IRF5 expression, more powerful genetic association studies and deeper insight into the role of IRF5 in disease susceptibility.

Further evidence of subphenotype association with systemic lupus erythematosus susceptibility loci: a European cases only study.

INTRODUCTION: Systemic Lupus Erythematosus (SLE) shows a spectrum of clinical manifestations that complicate its diagnosis, treatment and research. This variability is likely related with environmental exposures and genetic factors among which known SLE susceptibility loci are prime candidates. The first published analyses seem to indicate that this is the case for some of them, but results are still inconclusive and we aimed to further explore this question. METHODS: European SLE patients, 1444, recruited at 17 centres from 10 countries were analyzed. Genotypes for 26 SLE associated SNPs were compared between patients with and without each of 11 clinical features: ten of the American College of Rheumatology (ACR) classification criteria (except ANAs) and age of disease onset. These analyses were adjusted for centre of recruitment, top ancestry informative markers, gender and time of follow-up. Overlap of samples with previous studies was excluded for assessing replication. RESULTS: THERE WERE THREE NEW ASSOCIATIONS: the SNPs in XKR6 and in FAM167A-BLK were associated with lupus nephritis (OR=0.76 and 1.30, P(corr) =0.007 and 0.03, respectively) and the SNP of MECP2, which is in chromosome X, with earlier age of disease onset in men. The previously reported association of STAT4 with early age of disease onset was replicated. Some other results were suggestive of the presence of additional associations. Together, the association signals provided support to some previous findings and to the characterization of lupus nephritis, autoantibodies and age of disease onset as the clinical features more associated with SLE loci. CONCLUSION: Some of the SLE loci shape the disease phenotype in addition to increase susceptibility to SLE. This influence is more prominent for some clinical features than for others. However, results are only partially consistent between studies and subphenotype specific GWAS are needed to unravel their genetic component.

Bias in effect size of systemic lupus erythematosus susceptibility loci across Europe: a case-control study.

INTRODUCTION: We aimed to investigate whether the effect size of the systemic lupus erythematosus (SLE) risk alleles varies across European subpopulations. METHODS: European SLE patients (n = 1,742) and ethnically matched healthy controls (n = 2,101) were recruited at 17 centres from 10 different countries. Only individuals with self-reported ancestry from the country of origin were included. In addition, participants were genotyped for top ancestry informative markers and for 25 SLE associated SNPs. The results were used to compare effect sizes between the Central Eureopan and Southern European subgroups. RESULTS: Twenty of the 25 SNPs showed independent association with SLE, These SNPs showed a significant bias to larger effect sizes in the Southern subgroup, with 15/20 showing this trend (P = 0.019) and a larger mean odds ratio of the 20 SNPs (1.46 vs. 1.34, P = 0.02) as well as a larger difference in the number of risk alleles (2.06 vs. 1.63, P = 0.027) between SLE patients and controls than for Central Europeans. This bias was reflected in a very significant difference in the cumulative genetic risk score (4.31 vs. 3.48, P = 1.8 × 10-32). Effect size bias was accompanied by a lower number of SLE risk alleles in the Southern subjects, both patients and controls, the difference being more marked between the controls (P = 1.1 × 10-8) than between the Southern and Central European patients (P = 0.016). Seven of these SNPs showed significant allele frequency clines. CONCLUSION: Our findings showed a bias to larger effect sizes of SLE loci in the Southern Europeans relative to the Central Europeans together with clines of SLE risk allele frequencies. These results indicate the need to study risk allele clines and the implications of the polygenic model of inheritance in SLE.

Association of systemic lupus erythematosus clinical features with European population genetic substructure.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with a very varied spectrum of clinical manifestations that could be partly determined by genetic factors. We aimed to determine the relationship between prevalence of 11 clinical features and age of disease onset with European population genetic substructure. Data from 1413 patients of European ancestry recruited in nine countries was tested for association with genotypes of top ancestry informative markers. This analysis was done with logistic regression between phenotypes and genotypes or principal components extracted from them. We used a genetic additive model and adjusted for gender and disease duration. Three clinical features showed association with ancestry informative markers: autoantibody production defined as immunologic disorder (P = 6.8×10(-4)), oral ulcers (P = 6.9×10(-4)) and photosensitivity (P = 0.002). Immunologic disorder was associated with genotypes more common in Southern European ancestries, whereas the opposite trend was observed for photosensitivity. Oral ulcers were specifically more common in patients of Spanish and Portuguese self-reported ancestry. These results should be taken into account in future research and suggest new hypotheses and possible underlying mechanisms to be investigated. A first hypothesis linking photosensitivity with variation in skin pigmentation is suggested.

Cis-regulation of IRF5 expression is unable to fully account for systemic lupus erythematosus association: analysis of multiple experiments with lymphoblastoid cell lines.

INTRODUCTION: Interferon regulatory factor 5 gene (IRF5) polymorphisms are strongly associated with several diseases, including systemic lupus erythematosus (SLE). The association includes risk and protective components. They could be due to combinations of functional polymorphisms and related to cis-regulation of IRF5 expression, but their mechanisms are still uncertain. We hypothesised that thorough testing of the relationships between IRF5 polymorphisms, expression data from multiple experiments and SLE-associated haplotypes might provide useful new information. METHODS: Expression data from four published microarray hybridisation experiments with lymphoblastoid cell lines (57 to 181 cell lines) were retrieved. Genotypes of 109 IRF5 polymorphisms, including four known functional polymorphisms, were considered. The best linear regression models accounting for the IRF5 expression data were selected by using a forward entry procedure. SLE-associated IRF5 haplotypes were correlated with the expression data and with the best cis-regulatory models. RESULTS: A large fraction of variability in IRF5 expression was accounted for by linear regression models with IRF5 polymorphisms, but at a different level in each expression data set. Also, the best models from each expression data set were different, although there was overlap between them. The SNP introducing an early polyadenylation signal, rs10954213, was included in the best models for two of the expression data sets and in good models for the other two data sets. The SLE risk haplotype was associated with high IRF5 expression in the four expression data sets. However, there was also a trend towards high IRF5 expression with some protective and neutral haplotypes, and the protective haplotypes were not associated with IRF5 expression. As a consequence, correlation between the cis-regulatory best models and SLE-associated haplotypes, regarding either the risk or protective component, was poor. CONCLUSIONS: Our analysis indicates that although the SLE risk haplotype of IRF5 is associated with high expression of the gene, cis-regulation of IRF5 expression is not enough to fully account for IRF5 association with SLE susceptibility, which indicates the need to identify additional functional changes in this gene.