Gene by Environment Interactions reveal new regulatory aspects of signaling network plasticity.

Phenotypes can change during exposure to different environments through the regulation of signaling pathways that operate in integrated networks. How signaling networks produce different phenotypes in different settings is not fully understood. Here, Gene by Environment Interactions (GEIs) were used to explore the regulatory network that controls filamentous/invasive growth in the yeast Saccharomyces cerevisiae. GEI analysis revealed that the regulation of invasive growth is decentralized and varies extensively across environments. Different regulatory pathways were critical or dispensable depending on the environment, microenvironment, or time point tested, and the pathway that made the strongest contribution changed depending on the environment. Some regulators even showed conditional role reversals. Ranking pathways' roles across environments revealed an under-appreciated pathway (OPI1) as the single strongest regulator among the major pathways tested (RAS, RIM101, and MAPK). One mechanism that may explain the high degree of regulatory plasticity observed was conditional pathway interactions, such as conditional redundancy and conditional cross-pathway regulation. Another mechanism was that different pathways conditionally and differentially regulated gene expression, such as target genes that control separate cell adhesion mechanisms (FLO11 and SFG1). An exception to decentralized regulation of invasive growth was that morphogenetic changes (cell elongation and budding pattern) were primarily regulated by one pathway (MAPK). GEI analysis also uncovered a round-cell invasion phenotype. Our work suggests that GEI analysis is a simple and powerful approach to define the regulatory basis of complex phenotypes and may be applicable to many systems.

Turnover and bypass of p21-activated kinase during Cdc42-dependent MAPK signaling in yeast.

Mitogen-activated protein kinase (MAPK) pathways regulate multiple cellular behaviors, including the response to stress and cell differentiation, and are highly conserved across eukaryotes. MAPK pathways can be activated by the interaction between the small GTPase Cdc42p and the p21-activated kinase (Ste20p in yeast). By studying MAPK pathway regulation in yeast, we recently found that the active conformation of Cdc42p is regulated by turnover, which impacts the activity of the pathway that regulates filamentous growth (fMAPK). Here, we show that Ste20p is regulated in a similar manner and is turned over by the 26S proteasome. This turnover did not occur when Ste20p was bound to Cdc42p, which presumably stabilized the protein to sustain MAPK pathway signaling. Although Ste20p is a major component of the fMAPK pathway, genetic approaches here identified a Ste20p-independent branch of signaling. Ste20p-independent signaling partially required the fMAPK pathway scaffold and Cdc42p-interacting protein, Bem4p, while Ste20p-dependent signaling required the 14-3-3 proteins, Bmh1p and Bmh2p. Interestingly, Ste20p-independent signaling was inhibited by one of the GTPase-activating proteins for Cdc42p, Rga1p, which unexpectedly dampened basal but not active fMAPK pathway activity. These new regulatory features of the Rho GTPase and p21-activated kinase module may extend to related pathways in other systems.

Conserved signaling modules regulate filamentous growth in fungi: a model for eukaryotic cell differentiation.

Eukaryotic organisms are composed of different cell types with defined shapes and functions. Specific cell types are produced by the process of cell differentiation, which is regulated by signal transduction pathways. Signaling pathways regulate cell differentiation by sensing cues and controlling the expression of target genes whose products generate cell types with specific attributes. In studying how cells differentiate, fungi have proved valuable models because of their ease of genetic manipulation and striking cell morphologies. Many fungal species undergo filamentous growth-a specialized growth pattern where cells produce elongated tube-like projections. Filamentous growth promotes expansion into new environments, including invasion into plant and animal hosts by fungal pathogens. The same signaling pathways that regulate filamentous growth in fungi also control cell differentiation throughout eukaryotes and include highly conserved mitogen-activated protein kinase (MAPK) pathways, which is the focus of this review. In many fungal species, mucin-type sensors regulate MAPK pathways to control filamentous growth in response to diverse stimuli. Once activated, MAPK pathways reorganize cell polarity, induce changes in cell adhesion, and promote the secretion of degradative enzymes that mediate access to new environments. However, MAPK pathway regulation is complicated because related pathways can share components with each other yet induce unique responses (i.e. signal specificity). In addition, MAPK pathways function in highly integrated networks with other regulatory pathways (i.e. signal integration). Here, we discuss signal specificity and integration in several yeast models (mainly Saccharomyces cerevisiae and Candida albicans) by focusing on the filamentation MAPK pathway. Because of the strong evolutionary ties between species, a deeper understanding of the regulation of filamentous growth in established models and increasingly diverse fungal species can reveal fundamentally new mechanisms underlying eukaryotic cell differentiation.

Gain- and loss-of-function alleles within signaling pathways lead to phenotypic diversity among individuals.

Understanding how phenotypic diversity is generated is an important question in biology. We explored phenotypic diversity among wild yeast isolates (Saccharomyces cerevisiae) and found variation in the activity of MAPK signaling pathways as a contributing mechanism. To uncover the genetic basis of this mechanism, we identified 1957 SNPs in 62 candidate genes encoding signaling proteins from a MAPK signaling module within a large collection of yeast (>1500 individuals). Follow-up testing identified functionally relevant variants in key signaling proteins. Loss-of-function (LOF) alleles in a PAK kinase impacted protein stability and pathway specificity decreasing filamentous growth and mating phenotypes. In contrast, gain-of-function (GOF) alleles in G-proteins that were hyperactivating induced filamentous growth. Similar amino acid substitutions in G-proteins were identified in metazoans that in some cases were fixed in multicellular lineages including humans, suggesting hyperactivating GOF alleles may play roles in generating phenotypic diversity across eukaryotes. A mucin signaler that regulates MAPK activity was also found to contain a prevalance of presumed GOF alleles amoung individuals based on changes in mucin repeat numbers. Thus, genetic variation in signaling pathways may act as a reservoir for generating phenotypic diversity across eukaryotes.

Ecological inducers of the yeast filamentous growth pathway reveal environment-dependent roles for pathway components.

Signaling modules, such as mitogen-activated protein kinase (MAPK) pathways, are evolutionarily conserved drivers of cell differentiation and stress responses. In many fungal species including pathogens, MAPK pathways control filamentous growth, where cells differentiate into an elongated cell type. The convenient model budding yeast Saccharomyces cerevisiae undergoes filamentous growth by the filamentous growth (fMAPK) pathway; however, the inducers of the pathway remain unclear, perhaps because pathway activity has been mainly studied in laboratory conditions. To address this knowledge gap, an ecological framework was used, which uncovered new fMAPK pathway inducers, including pectin, a material found in plants, and the metabolic byproduct ethanol. We also show that induction by a known inducer of the pathway, the non-preferred carbon source galactose, required galactose metabolism and induced the pathway differently than glucose limitation or other non-preferred carbon sources. By exploring fMAPK pathway function in fruit, we found that induction of the pathway led to visible digestion of fruit rind through a known target, PGU1, which encodes a pectolytic enzyme. Combinations of inducers (galactose and ethanol) stimulated the pathway to near-maximal levels, which showed dispensability of several fMAPK pathway components (e.g., mucin sensor, p21-activated kinase), but not others (e.g., adaptor, MAPKKK) and required the Ras2-protein kinase A pathway. This included a difference between the transcription factor binding partners for the pathway, as Tec1p, but not Ste12p, was partly dispensable for fMAPK pathway activity. Thus, by exploring ecologically relevant stimuli, new modes of MAPK pathway signaling were uncovered, perhaps revealing how a pathway can respond differently to specific environments. IMPORTANCE Filamentous growth is a cell differentiation response and important aspect of fungal biology. In plant and animal fungal pathogens, filamentous growth contributes to virulence. One signaling pathway that regulates filamentous growth is an evolutionarily conserved MAPK pathway. The yeast Saccharomyces cerevisiae is a convenient model to study MAPK-dependent regulation of filamentous growth, although the inducers of the pathway are not clear. Here, we exposed yeast cells to ecologically relevant compounds (e.g., plant compounds), which identified new inducers of the MAPK pathway. In combination, the inducers activated the pathway to near-maximal levels but did not cause detrimental phenotypes associated with previously identified hyperactive alleles. This context allowed us to identify conditional bypass for multiple pathway components. Thus, near-maximal induction of a MAPK pathway by ecologically relevant inducers provides a powerful tool to assess cellular signaling during a fungal differentiation response.

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2Δ cells that appears to result directly from impaired decapping rather than elevated transcription. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Edc3, or Scd6; whereas most of the remaining transcripts utilize nonsense-mediated mRNA decay factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2Δ confers widespread changes in relative translational efficiencies (TEs) that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2Δ, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are upregulated, and both mitochondrial function and cell filamentation are elevated in dcp2Δ cells, suggesting that decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

The Caenorhabditis elegans innexin INX-20 regulates nociceptive behavioral sensitivity.

Organisms rely on chemical cues in their environment to indicate the presence or absence of food, reproductive partners, predators, or other harmful stimuli. In the nematode Caenorhabditis elegans, the bilaterally symmetric pair of ASH sensory neurons serves as the primary nociceptors. ASH activation by aversive stimuli leads to backward locomotion and stimulus avoidance. We previously reported a role for guanylyl cyclases in dampening nociceptive sensitivity that requires an innexin-based gap junction network to pass cGMP between neurons. Here, we report that animals lacking function of the gap junction component INX-20 are hypersensitive in their behavioral response to both soluble and volatile chemical stimuli that signal through G protein-coupled receptor pathways in ASH. We find that expressing inx-20 in the ADL and AFD sensory neurons is sufficient to dampen ASH sensitivity, which is supported by new expression analysis of endogenous INX-20 tagged with mCherry via the CRISPR-Cas9 system. Although ADL does not form gap junctions directly with ASH, it does so via gap junctions with the interneuron RMG and the sensory neuron ASK. Ablating either ADL or RMG and ASK also resulted in nociceptive hypersensitivity, suggesting an important role for RMG/ASK downstream of ADL in the ASH modulatory circuit. This work adds to our growing understanding of the repertoire of ways by which ASH activity is regulated via its connectivity to other neurons and identifies a previously unknown role for ADL and RMG in the modulation of aversive behavior.

Decapping factor Dcp2 controls mRNA abundance and translation to adjust metabolism and filamentation to nutrient availability.

Degradation of most yeast mRNAs involves decapping by Dcp1/Dcp2. DEAD-box protein Dhh1 has been implicated as an activator of decapping, in coupling codon non-optimality to enhanced degradation, and as a translational repressor, but its functions in cells are incompletely understood. RNA-Seq analyses coupled with CAGE sequencing of all capped mRNAs revealed increased abundance of hundreds of mRNAs in dcp2 Δ cells that appears to result directly from impaired decapping rather than elevated transcription, which was confirmed by ChIP-Seq analysis of RNA Polymerase II occupancies genome-wide. Interestingly, only a subset of mRNAs requires Dhh1 for targeting by Dcp2, and also generally requires the other decapping activators Pat1, Lsm2, Edc3 or Scd6; whereas most of the remaining transcripts utilize NMD factors for Dcp2-mediated turnover. Neither inefficient translation initiation nor stalled elongation appears to be a major driver of Dhh1-enhanced mRNA degradation. Surprisingly, ribosome profiling revealed that dcp2 Δ confers widespread changes in relative TEs that generally favor well-translated mRNAs. Because ribosome biogenesis is reduced while capped mRNA abundance is increased by dcp2 Δ, we propose that an increased ratio of mRNA to ribosomes increases competition among mRNAs for limiting ribosomes to favor efficiently translated mRNAs in dcp2 Δ cells. Interestingly, genes involved in respiration or utilization of alternative carbon or nitrogen sources are derepressed, and both mitochondrial function and cell filamentation (a strategy for nutrient foraging) are elevated by dcp2 Δ, suggesting that mRNA decapping sculpts gene expression post-transcriptionally to fine-tune metabolic pathways and morphological transitions according to nutrient availability.

New Aspects of Invasive Growth Regulation Identified by Functional Profiling of MAPK Pathway Targets in Saccharomyces cerevisiae.

MAPK pathways are drivers of morphogenesis and stress responses in eukaryotes. A major function of MAPK pathways is the transcriptional induction of target genes, which produce proteins that collectively generate a cellular response. One approach to comprehensively understand how MAPK pathways regulate cellular responses is to characterize the individual functions of their transcriptional targets. Here, by examining uncharacterized targets of the MAPK pathway that positively regulates filamentous growth in Saccharomyces cerevisiae (fMAPK pathway), we identified a new role for the pathway in negatively regulating invasive growth. Specifically, four targets were identified that had an inhibitory role in invasive growth: RPI1, RGD2, TIP1, and NFG1/YLR042cNFG1 was a highly induced unknown open reading frame that negatively regulated the filamentous growth MAPK pathway. We also identified SFG1, which encodes a transcription factor, as a target of the fMAPK pathway. Sfg1p promoted cell adhesion independently from the fMAPK pathway target and major cell adhesion flocculin Flo11p, by repressing genes encoding presumptive cell-wall-degrading enzymes. Sfg1p also contributed to FLO11 expression. Sfg1p and Flo11p regulated different aspects of cell adhesion, and their roles varied based on the environment. Sfg1p also induced an elongated cell morphology, presumably through a cell-cycle delay. Thus, the fMAPK pathway coordinates positive and negative regulatory proteins to fine-tune filamentous growth resulting in a nuanced response. Functional analysis of other pathways' targets may lead to a more comprehensive understanding of how signaling cascades generate biological responses.