Transcription profiling by array of yeast single and double deletion mutants of gene-specific transcription factors
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ABSTRACT: Contains gene expression profiles of yeast single and double deletion mutants of gene-specific transcription factors. Genetic interactions were studied by comparing gene expression changes of double mutants with gene expression changes in the respective single mutants. Pairs of gene-specific transcription factors were chosen based on previous evidence for epistasis, including synthetic genetic interactions as well as common DNA binding.
Project description:Contains gene expression profiles of yeast single and double deletion mutants of gene-specific transcription factors. Genetic interactions were studied by comparing gene expression changes of double mutants with gene expression changes in the respective single mutants. Pairs of gene-specific transcription factors were chosen based on previous evidence for epistasis, including synthetic genetic interactions as well as common DNA binding.
Project description:Contains gene expression profiles of yeast single and double deletion mutants of gene-specific transcription factors. Genetic interactions were studied by comparing gene expression changes of double mutants with gene expression changes in the respective single mutants. Pairs of gene-specific transcription factors were chosen based on previous evidence for epistasis, including synthetic genetic interactions as well as common DNA binding. Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Using the Erlenmeyer growth protocol up to five deletion strains were grown on a single day. In the tecan platereader, up to eleven deletion strains could be grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.
Project description:To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene-sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multi-process control.
Project description:To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene-sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multi-process control. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used for each separate hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Up to five deletion strains were grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.
Project description:RNA-seq is commonly used to identify genetic modules that respond to perturbations. In single cells, transcriptomes have been used as phenotypes, but this concept has not been applied to whole-organism RNA-seq. Linear models can quantify expression effects of individual mutants and identify epistatic effects in double mutants. However, interpreting these high-dimensional measurements is unintuitive. We developed a single coefficient to quantify transcriptome-wide epistasis which accurately reflects the underlying interactions. To demonstrate the power of our approach, we sequenced four single and two double Caenorhabditis elegans mutants. From these mutants, we successfully reconstructed the known hypoxia pathway. Using this approach, we uncovered a class of 31 genes that have opposing changes in expression in egl-9(lf) and vhl-1(lf) but for which the egl-9(lf);vhl-1(lf) mutant phenocopies egl-9(lf). These changes violate the classical model of HIF-1 regulation, but can be explained by postulating a role of hydroxylated HIF-1 in transcriptional control.
Project description:The study of climacteric fruit ripening in tomato has been facilitated by the spontaneous ripening mutants Colorless non-ripening (Cnr), non-ripening (nor), and ripening inhibitor (rin). These mutants effect the genes encoding ripening transcription factors (TFs) SPL-CNR, NAC-NOR, and MADS-RIN causing pleiotropic defects to the ripening program. Here, we demonstrate that some ripening processes occur in the mutant fruit but at later stages of development compared to the wild type. The rin and nor mutant fruit exhibit similar quality traits to wildtype at later stages of ripening and senescence and delayed expression of ripening-associated genes. In addition, we propose that the Cnr mutant has a broader range of effects to fruit development than just fruit ripening. Cnr fruit show distinct differences from wild type in ripening phenotypic traits and gene expression profiles prior to the initiation of ripening. We provide new evidence that some mutants can produce more ethylene than basal levels and demonstrate ABA accumulation is also affected by the mutations. Studies have examined the relationship between the CNR, RIN, and NOR TFs based on protein-protein interactions and transcriptional regulation during fruit ripening. We describe the genetic interactions affecting specific fruit traits by using homozygous double mutants. Cnr predominantly influences the phenotype of the Cnr/nor and Cnr/rin double mutants but additional defects beyond either single mutation is evident in the transcriptome of the Cnr/nor double mutant. Our reevaluation of the Cnr, nor, and rin mutants provides new insights the utilization of the mutants in breeding and studying fruit development.
Project description:Lung adenocarcinoma, the most common subtype of lung cancer, is genomically complex, with tumors containing tens to hundreds of non-synonymous mutations. However, little is understood about how genes interact with each other to enable the evolution of cancer in vivo, largely due to a lack of methods for investigating genetic interactions in a high-throughput and quantitative manner. Here, we employed a novel platform to generate tumors with all pairwise inactivation of ten diverse tumor suppressor genes within an autochthonous mouse model of oncogenic KRAS-driven lung cancer. By quantifying the fitness of tumors with every single and double mutant genotype, we show that most tumor suppressor genetic interactions exhibited negative epistasis, with diminishing returns on tumor fitness. In contrast, Apc inactivation showed positive epistasis with the inactivation of several other genes, including synergistic effects on tumor fitness in combination with Lkb1 or Nf1 inactivation. Sign epistasis was extremely rare, suggesting a surprisingly accessible fitness landscape during lung tumorigenesis. These findings greatly expand our understanding the evolutionary interactions that drive tumorigenesis in vivo.
Project description:To improve both drought stress tolerance and growth of plants, we generated transgenic Arabidopsis plants that overexpress two transcription factors: DREB1A and PIF4. It was reported that DREB1A improves drought stress tolerance in various crops but causes dwarfism, and PIF4 enhances cell elongation through activation of cell wall synthesis. We performed microarray experiments using a single overexpressor of PIF4 and the double overexpressor of DREB1A and PIF4 to study the potential interactions of the gene networks regulated by the two transcription factors in the double overexpressor.
Project description:To improve both drought stress tolerance and growth of plants, we generated transgenic Arabidopsis plants that overexpress two transcription factors: DREB1A and OsPIL1. It was reported that DREB1A improves drought stress tolerance in various crops but causes dwarfism, and OsPIL1 enhances cell elongation through activation of cell wall synthesis in rice. We performed microarray experiments using a single overexpressor of OsPIL1 and the double overexpressor of DREB1A and OsPIL1 to study the potential interactions of the gene networks regulated by the two transcription factors in the double overexpressor.
Project description:To improve both drought stress tolerance and growth of plants, we generated transgenic Arabidopsis plants that overexpress DREB1A and GA5. It was reported that DREB1A improves drought stress tolerance in various crops but causes dwarfism, and GA5 encodes a GA synthetic enzyme which enhances plant growth. We performed microarray experiments using a single overexpressor of GA5 and the double overexpressor of DREB1A and GA5 to study the potential interactions of the gene networks regulated by the two factors in the double overexpressor.