The TSC22D, WNK, and NRBP gene families exhibit functional buffering and evolved with Metazoa for cell volume regulation.

The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity.

Mutational robustness and the role of buffer genes in evolvability.

Organisms rely on mutations to fuel adaptive evolution. However, many mutations impose a negative effect on fitness. Cells may have therefore evolved mechanisms that affect the phenotypic effects of mutations, thus conferring mutational robustness. Specifically, so-called buffer genes are hypothesized to interact directly or indirectly with genetic variation and reduce its effect on fitness. Environmental or genetic perturbations can change the interaction between buffer genes and genetic variation, thereby unmasking the genetic variation's phenotypic effects and thus providing a source of variation for natural selection to act on. This review provides an overview of our understanding of mutational robustness and buffer genes, with the chaperone gene HSP90 as a key example. It discusses whether buffer genes merely affect standing variation or also interact with de novo mutations, how mutational robustness could influence evolution, and whether mutational robustness might be an evolved trait or rather a mere side-effect of complex genetic interactions.

A Simple Evolutionary Model of Genetic Robustness After Gene Duplication.

When a dispensable gene is duplicated (referred to the ancestral dispensability denoted by O+), genetic buffering and duplicate compensation together maintain the duplicate redundancy, whereas duplicate compensation is the only mechanism when an essential gene is duplicated (referred to the ancestral essentiality denoted by O-). To investigate these evolutionary scenarios of genetic robustness, I formulated a simple mixture model for analyzing duplicate pairs with one of the following states: double dispensable (DD), semi-dispensable (one dispensable one essential, DE), or double essential (EE). This model was applied to the yeast duplicate pairs from a whole-genome duplication (WGD) occurred about 100 million years ago (mya), and the mouse duplicate pairs from a WGD occurred about more than 500 mya. Both case studies revealed that the proportion of essentiality for those duplicates with ancestral essentiality [PE(O-)] was much higher than that for those with ancestral dispensability [PE(O+)]. While it was negligible in the yeast duplicate pairs, PE(O+) (about 20%) was shown statistically significant in the mouse duplicate pairs. These findings, together, support the hypothesis that both sub-functionalization and neo-functionalization may play some roles after gene duplication, though the former may be much faster than the later.

Feedback to the central dogma: cytoplasmic mRNA decay and transcription are interdependent processes.

Transcription and RNA decay are key determinants of gene expression; these processes are typically considered as the uncoupled beginning and end of the messenger RNA (mRNA) lifecycle. Here we describe the growing number of studies demonstrating interplay between these spatially disparate processes in eukaryotes. Specifically, cells can maintain mRNA levels by buffering against changes in mRNA stability or transcription, and can also respond to virally induced accelerated decay by reducing RNA polymerase II gene expression. In addition to these global responses, there is also evidence that mRNAs containing a premature stop codon can cause transcriptional upregulation of homologous genes in a targeted fashion. In each of these systems, RNA binding proteins (RBPs), particularly those involved in mRNA degradation, are critical for cytoplasmic to nuclear communication. Although their specific mechanistic contributions are yet to be fully elucidated, differential trafficking of RBPs between subcellular compartments are likely to play a central role in regulating this gene expression feedback pathway.

A yeast phenomic model for the influence of Warburg metabolism on genetic buffering of doxorubicin.

BACKGROUND: The influence of the Warburg phenomenon on chemotherapy response is unknown. Saccharomyces cerevisiae mimics the Warburg effect, repressing respiration in the presence of adequate glucose. Yeast phenomic experiments were conducted to assess potential influences of Warburg metabolism on gene-drug interaction underlying the cellular response to doxorubicin. Homologous genes from yeast phenomic and cancer pharmacogenomics data were analyzed to infer evolutionary conservation of gene-drug interaction and predict therapeutic relevance. METHODS: Cell proliferation phenotypes (CPPs) of the yeast gene knockout/knockdown library were measured by quantitative high-throughput cell array phenotyping (Q-HTCP), treating with escalating doxorubicin concentrations under conditions of respiratory or glycolytic metabolism. Doxorubicin-gene interaction was quantified by departure of CPPs observed for the doxorubicin-treated mutant strain from that expected based on an interaction model. Recursive expectation-maximization clustering (REMc) and Gene Ontology (GO)-based analyses of interactions identified functional biological modules that differentially buffer or promote doxorubicin cytotoxicity with respect to Warburg metabolism. Yeast phenomic and cancer pharmacogenomics data were integrated to predict differential gene expression causally influencing doxorubicin anti-tumor efficacy. RESULTS: Yeast compromised for genes functioning in chromatin organization, and several other cellular processes are more resistant to doxorubicin under glycolytic conditions. Thus, the Warburg transition appears to alleviate requirements for cellular functions that buffer doxorubicin cytotoxicity in a respiratory context. We analyzed human homologs of yeast genes exhibiting gene-doxorubicin interaction in cancer pharmacogenomics data to predict causality for differential gene expression associated with doxorubicin cytotoxicity in cancer cells. This analysis suggested conserved cellular responses to doxorubicin due to influences of homologous recombination, sphingolipid homeostasis, telomere tethering at nuclear periphery, actin cortical patch localization, and other gene functions. CONCLUSIONS: Warburg status alters the genetic network required for yeast to buffer doxorubicin toxicity. Integration of yeast phenomic and cancer pharmacogenomics data suggests evolutionary conservation of gene-drug interaction networks and provides a new experimental approach to model their influence on chemotherapy response. Thus, yeast phenomic models could aid the development of precision oncology algorithms to predict efficacious cytotoxic drugs for cancer, based on genetic and metabolic profiles of individual tumors.

A Humanized Yeast Phenomic Model of Deoxycytidine Kinase to Predict Genetic Buffering of Nucleoside Analog Cytotoxicity.

Knowledge about synthetic lethality can be applied to enhance the efficacy of anticancer therapies in individual patients harboring genetic alterations in their cancer that specifically render it vulnerable. We investigated the potential for high-resolution phenomic analysis in yeast to predict such genetic vulnerabilities by systematic, comprehensive, and quantitative assessment of drug-gene interaction for gemcitabine and cytarabine, substrates of deoxycytidine kinase that have similar molecular structures yet distinct antitumor efficacy. Human deoxycytidine kinase (dCK) was conditionally expressed in the Saccharomycescerevisiae genomic library of knockout and knockdown (YKO/KD) strains, to globally and quantitatively characterize differential drug-gene interaction for gemcitabine and cytarabine. Pathway enrichment analysis revealed that autophagy, histone modification, chromatin remodeling, and apoptosis-related processes influence gemcitabine specifically, while drug-gene interaction specific to cytarabine was less enriched in gene ontology. Processes having influence over both drugs were DNA repair and integrity checkpoints and vesicle transport and fusion. Non-gene ontology (GO)-enriched genes were also informative. Yeast phenomic and cancer cell line pharmacogenomics data were integrated to identify yeast-human homologs with correlated differential gene expression and drug efficacy, thus providing a unique resource to predict whether differential gene expression observed in cancer genetic profiles are causal in tumor-specific responses to cytotoxic agents.

Using genetic buffering relationships identified in fission yeast to reveal susceptibilities in cells lacking hamartin or tuberin function.

Tuberous sclerosis complex is an autosomal dominant disorder characterized by benign tumors arising from the abnormal activation of mTOR signaling in cells lacking TSC1 (hamartin) or TSC2 (tuberin) activity. To expand the genetic framework surrounding this group of growth regulators, we utilized the model eukaryote Schizosaccharomyces pombe to uncover and characterize genes that buffer the phenotypic effects of mutations in the orthologous tsc1 or tsc2 loci. Our study identified two genes: fft3 (encoding a DNA helicase) and ypa1 (encoding a peptidyle-prolyl cis/trans isomerase). While the deletion of fft3 or ypa1 has little effect in wild-type fission yeast cells, their loss in tsc1Δ or tsc2Δ backgrounds results in severe growth inhibition. These data suggest that the inhibition of Ypa1p or Fft3p might represent an 'Achilles' heel' of cells defective in hamartin/tuberin function. Furthermore, we demonstrate that the interaction between tsc1/tsc2 and ypa1 can be rescued through treatment with the mTOR inhibitor, torin-1, and that ypa1Δ cells are resistant to the glycolytic inhibitor, 2-deoxyglucose. This identifies ypa1 as a novel upstream regulator of mTOR and suggests that the effects of ypa1 loss, together with mTOR activation, combine to result in a cellular maladaptation in energy metabolism that is profoundly inhibitory to growth.

The Hsp90-dependent proteome is conserved and enriched for hub proteins with high levels of protein-protein connectivity.

Hsp90 is one of the most abundant and conserved proteins in the cell. Reduced levels or activity of Hsp90 causes defects in many cellular processes and also reveals genetic and nongenetic variation within a population. Despite information about Hsp90 protein-protein interactions, a global view of the Hsp90-regulated proteome in yeast is unavailable. To investigate the degree of dependency of individual yeast proteins on Hsp90, we used the "stable isotope labeling by amino acids in cell culture" method coupled with mass spectrometry to quantify around 4,000 proteins in low-Hsp90 cells. We observed that 904 proteins changed in their abundance by more than 1.5-fold. When compared with the transcriptome of the same population of cells, two-thirds of the misregulated proteins were observed to be affected posttranscriptionally, of which the majority were downregulated. Further analyses indicated that the downregulated proteins are highly conserved and assume central roles in cellular networks with a high number of protein interacting partners, suggesting that Hsp90 buffers genetic and nongenetic variation through regulating protein network hubs. The downregulated proteins were enriched for essential proteins previously not known to be Hsp90-dependent. Finally, we observed that downregulation of transcription factors and mating pathway components by attenuating Hsp90 function led to decreased target gene expression and pheromone response, respectively, providing a direct link between observed proteome regulation and cellular phenotypes.

POPULATION GENETIC STRUCTURE OF CECROPIA OBTUSIFOLIA, A TROPICAL PIONEER TREE SPECIES.

Theoretical analyses of the genetic organization of pioneer species have postulated two very different scenarios. Some models have predicted that such species would show strong population substructuring, whereas other models have suggested that extinction and recolonization can augment gene flow and reduce interpopulation differentiation. We tested these alternative scenarios by analyzing the genetic structure of eight loci from populations of the pioneer dioecious tree, Cecropia obtusifolia, in the tropical rain forest region of Los Tuxtlas, México. The populations studied exhibit low overall FST values, no clear pattern of isolation by distance, and high estimates of gene flow. These results suggest either that the species is not at a genetic equilibrium under present levels of gene flow with populations derived from each other in the recent past, or that pollen and seed dispersal in this species occur over long distances (up to more than 100 km). Mating among relatives appears higher than expected by chance based on significantly positive fixation indices (F) and FIS values at some loci. However, no direct evidence for biparental inbreeding was found. The multilocus and single-locus outcrossing rates for C. obtusifolia were estimated at tm = 0.974 (SE = 0.024) and ts = 0.980 (SE = 0.035), respectively. These are not significantly different from 1, and the difference, tm - ts = - 0.006 (SE = 0.018), is not significantly different from 0. These estimates, however, could be biased because in all enzymes, except PGM-1, we found statistically significant departures from the mixed-mating model used to estimate them. Two rare alleles were found only in seeds collected from the soil, and the greatest number of different alleles were found also in soil seeds. It is hypothesized that the seed bank may play an important role in the genetic buffering of C. obtusifolia. Significantly positive or negative fixation indices in adults at some loci and significantly different heterozygosities among different life stages (from seeds to adults) suggest the action of selection at some loci.