Rapid evolutionary repair by secondary perturbation of a primary disrupted transcriptional network.

The discrete steps of transcriptional rewiring have been proposed to occur neutrally to ensure steady gene expression under stabilizing selection. A conflict-free switch of a regulon between regulators may require an immediate compensatory evolution to minimize deleterious effects. Here, we perform an evolutionary repair experiment on the Lachancea kluyveri yeast sef1Δ mutant using a suppressor development strategy. Complete loss of SEF1 forces cells to initiate a compensatory process for the pleiotropic defects arising from misexpression of TCA cycle genes. Using different selective conditions, we identify two adaptive loss-of-function mutations of IRA1 and AZF1. Subsequent analyses show that Azf1 is a weak transcriptional activator regulated by the Ras1-PKA pathway. Azf1 loss-of-function triggers extensive gene expression changes responsible for compensatory, beneficial, and trade-off phenotypes. The trade-offs can be alleviated by higher cell density. Our results not only indicate that secondary transcriptional perturbation provides rapid and adaptive mechanisms potentially stabilizing the initial stage of transcriptional rewiring but also suggest how genetic polymorphisms of pleiotropic mutations could be maintained in the population.

Multiple intermolecular interactions facilitate rapid evolution of essential genes.

Essential genes are commonly assumed to function in basic cellular processes and to change slowly. However, it remains unclear whether all essential genes are similarly conserved or if their evolutionary rates can be accelerated by specific factors. To address these questions, we replaced 86 essential genes of Saccharomyces cerevisiae with orthologues from four other species that diverged from S. cerevisiae about 50, 100, 270 and 420 Myr ago. We identify a group of fast-evolving genes that often encode subunits of large protein complexes, including anaphase-promoting complex/cyclosome (APC/C). Incompatibility of fast-evolving genes is rescued by simultaneously replacing interacting components, suggesting it is caused by protein co-evolution. Detailed investigation of APC/C further revealed that co-evolution involves not only primary interacting proteins but also secondary ones, suggesting the evolutionary impact of epistasis. Multiple intermolecular interactions in protein complexes may provide a microenvironment facilitating rapid evolution of their subunits.

Plastic Rewiring of Sef1 Transcriptional Networks and the Potential of Nonfunctional Transcription Factor Binding in Facilitating Adaptive Evolution.

Prior and extensive plastic rewiring of a transcriptional network, followed by a functional switch of the conserved transcriptional regulator, can shape the evolution of a new network with diverged functions. The presence of three distinct iron regulatory systems in fungi that use orthologous transcriptional regulators suggests that these systems evolved in that manner. Orthologs of the transcriptional activator Sef1 are believed to be central to how iron regulatory systems developed in fungi, involving gene gain, plastic network rewiring, and switches in regulatory function. We show that, in the protoploid yeast Lachancea kluyveri, plastic rewiring of the L. kluyveri Sef1 (Lk-Sef1) network, together with a functional switch, enabled Lk-Sef1 to regulate TCA cycle genes, unlike Candida albicans Sef1 that mainly regulates iron-uptake genes. Moreover, we observed pervasive nonfunctional binding of Sef1 to its target genes. Enhancing Lk-Sef1 activity resuscitated the corresponding transcriptional network, providing immediate adaptive benefits in changing environments. Our study not only sheds light on the evolution of Sef1-centered transcriptional networks but also shows the adaptive potential of nonfunctional transcription factor binding for evolving phenotypic novelty and diversity.

Genome plasticity in Paramecium bursaria revealed by population genomics.

BACKGROUND: Ciliates are an ancient and diverse eukaryotic group found in various environments. A unique feature of ciliates is their nuclear dimorphism, by which two types of nuclei, the diploid germline micronucleus (MIC) and polyploidy somatic macronucleus (MAC), are present in the same cytoplasm and serve different functions. During each sexual cycle, ciliates develop a new macronucleus in which newly fused genomes are extensively rearranged to generate functional minichromosomes. Interestingly, each ciliate species seems to have its way of processing genomes, providing a diversity of resources for studying genome plasticity and its regulation. Here, we sequenced and analyzed the macronuclear genome of different strains of Paramecium bursaria, a highly divergent species of the genus Paramecium which can stably establish endosymbioses with green algae. RESULTS: We assembled a high-quality macronuclear genome of P. bursaria and further refined genome annotation by comparing population genomic data. We identified several species-specific expansions in protein families and gene lineages that are potentially associated with endosymbiosis. Moreover, we observed an intensive chromosome breakage pattern that occurred during or shortly after sexual reproduction and contributed to highly variable gene dosage throughout the genome. However, patterns of copy number variation were highly correlated among genetically divergent strains, suggesting that copy number is adjusted by some regulatory mechanisms or natural selection. Further analysis showed that genes with low copy number variation among populations tended to function in basic cellular pathways, whereas highly variable genes were enriched in environmental response pathways. CONCLUSIONS: We report programmed DNA rearrangements in the P. bursaria macronuclear genome that allow cells to adjust gene copy number globally according to individual gene functions. Our results suggest that large-scale gene copy number variation may represent an ancient mechanism for cells to adapt to different environments.

Experimental evolution reveals a general role for the methyltransferase Hmt1 in noise buffering.

Cell-to-cell heterogeneity within an isogenic population has been observed in prokaryotic and eukaryotic cells. Such heterogeneity often manifests at the level of individual protein abundance and may have evolutionary benefits, especially for organisms in fluctuating environments. Although general features and the origins of cellular noise have been revealed, details of the molecular pathways underlying noise regulation remain elusive. Here, we used experimental evolution of Saccharomyces cerevisiae to select for mutations that increase reporter protein noise. By combining bulk segregant analysis and CRISPR/Cas9-based reconstitution, we identified the methyltransferase Hmt1 as a general regulator of noise buffering. Hmt1 methylation activity is critical for the evolved phenotype, and we also show that two of the Hmt1 methylation targets can suppress noise. Hmt1 functions as an environmental sensor to adjust noise levels in response to environmental cues. Moreover, Hmt1-mediated noise buffering is conserved in an evolutionarily distant yeast species, suggesting broad significance of noise regulation.

Simultaneous, rapid, and sensitive quantification of 8-hydroxy-2'-deoxyguanosine and cotinine in human urine by on-line solid-phase extraction LC-MS/MS: correlation with tobacco exposure biomarkers NNAL.

Cigarette smoke can increase oxidative DNA damage. The main component in cigarette smoke is nicotine. Nicotine is metabolized to cotinine, which can be regarded as a biomarker for measuring exposure to tobacco smoke. A sensitive, simple, and robust method based on on-line solid-phase extraction liquid chromatography with tandem mass spectrometry (on-line SPE LC-MS/MS) has been developed and validated for the simultaneous determination of 8-OHdG and cotinine. The matrix effects of 8-OHdG and cotinine were measured at 97.1 and 91.7 %, with values for CV at 4.4 and 4.2 %, respectively. The limits of detection of 8-OHdG and cotinine were 10.0 and 5.5 pg mL(-1), and the limits of quantification were 40.0 and 20.0 pg mL(-1), respectively. The total run time was 12 min. We quantified 8-OHdG and cotinine in the urine of 80 male subjects. The results showed the levels of 8-OHdG and cotinine in smokers were significantly higher than that in non-smokers. Furthermore, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide conjugate (defined as total NNAL) are the nitrosation metabolites of nicotine. In this study, urinary levels of 8-OHdG and cotinine were well correlated with urinary levels of total NNAL. This is also the first study to focus on the future risk of oxidative stress from exposure to cigarette smoke based on the relationship between 8-OHdG levels, cotinine levels, and total NNAL concentrations in the urine of humans. Graphical Abstract On-line SPE LC-MS/MS for the simultaneous determination of 8-OHdG and cotinine in human urine.