Project description:Effects of genetic modification of trans-acting transcription factors and a chromatin remodeler on noncoding RNA transcription and trans-acting factor binding at loci in yeast.
Project description:Both cis- and trans-regulatory mutations contribute to gene expression divergence within and between species. To estimate their relative contributions, we examined two yeast strains, BY4741 (a laboratory strain) and RM11-1a (a wild strain), for their gene expression divergence first by microarray. By incorporating the obtained array data with ChIP-chip data, we generated a set of single-regulator-regulated genes that showed expression divergence between BY4741 and RM11-1a. We randomly selected 50 of these genes for further study. We found substantial variations in the DNA sequences of the promoter regions of these genes and in their regulators between the two strains, which are potential sources of expression divergence. We then developed a step-by-step approach to assess the relative contributions of cis- and trans-variations to expression divergence by using pyrosequencing to quantify the mRNA levels of the two alleles of BY4741 and RM11-1a in co-cultures and in hybrid diploids. Among the 50 genes studied, 4 genes showed no significant expression difference when the two strains were grown in separate cultures or in the same culture (co-culture) and 6 genes showed significant expression difference in separate cultures but not in co-culture. The remaining 40 genes showed expression divergence between the two strains in co-culture. Among these 40 genes, pyrosequencing of diploids showed that 18 cases of expression divergence (45%=18/40) can be attributed to differences in trans-acting factors alone, 7 cases (17.5%) mainly to trans-variations, 8 cases (20%) to both cis- and trans-acting factors, 3 cases (7.5%) mainly to cis-variations, and 4 cases (10%) to cis-acting factors alone. In addition, we replaced the BY promoter by the RM promoter in each of 10 BY genes that were found from our microarray data to have expression divergence between BY and RM, and in each case our quantitative PCR analysis revealed the cis effect of the promoter replacement on gene expression. In summary, our study suggests that trans-acting factors play the major role in expression evolution between yeast strains, but the role of cis variation is also important. Keywords: comparative gene expression between strains
Project description:Transcriptional networks have been shown to evolve very rapidly, prompting questions as to how such changes arise and are tolerated. Recent comparisons of transcriptional networks across species have implicated variations in the cis-acting DNA sequences near genes as the main cause of divergence. What is less clear is how these changes interact with trans-acting changes occurring elsewhere in the genetic circuit. Here, we report the discovery of a system of compensatory trans and cis mutations in the yeast AP-1 transcriptional network that allows for conserved transcriptional regulation despite continued genetic change. We pinpoint a single species, the fungal pathogen Candida glabrata, in which a trans mutation has occurred very recently in a single AP-1 family member distinguishing it from its Saccharomyces ortholog. Comparison of chromatin immunoprecipitation profiles between Candida and Saccharomyces shows that, despite their different DNA binding domains, the AP-1 orthologs regulate a conserved block of genes. This conservation is enabled by concomitant changes in the cis-regulatory motifs upstream of each gene. Thus, both trans and cis mutations have perturbed the yeast AP-1 regulatory system in such a way as to compensate for one another. This demonstrates an example of “co-evolution” between a DNA-binding transcription factor and its cis-regulatory site, reminiscent of the co-evolution of protein binding partners. 3 Experiments were performed. Three replicates CgAp1-TAP was ChIPped under MMS treatment (GSM397447..GSM397449), two replicates each of ScYap1R79K and ScYap4K252R (GSM594724..GSM594727), were ChIPped under MMS treatment.
Project description:Transcriptional networks have been shown to evolve very rapidly, prompting questions as to how such changes arise and are tolerated. Recent comparisons of transcriptional networks across species have implicated variations in the cis-acting DNA sequences near genes as the main cause of divergence. What is less clear is how these changes interact with trans-acting changes occurring elsewhere in the genetic circuit. Here, we report the discovery of a system of compensatory trans and cis mutations in the yeast AP-1 transcriptional network that allows for conserved transcriptional regulation despite continued genetic change. We pinpoint a single species, the fungal pathogen Candida glabrata, in which a trans mutation has occurred very recently in a single AP-1 family member distinguishing it from its Saccharomyces ortholog. Comparison of chromatin immunoprecipitation profiles between Candida and Saccharomyces shows that, despite their different DNA binding domains, the AP-1 orthologs regulate a conserved block of genes. This conservation is enabled by concomitant changes in the cis-regulatory motifs upstream of each gene. Thus, both trans and cis mutations have perturbed the yeast AP-1 regulatory system in such a way as to compensate for one another. This demonstrates an example of “co-evolution” between a DNA-binding transcription factor and its cis-regulatory site, reminiscent of the co-evolution of protein binding partners.
Project description:Natural genetic variation can cause significant differences in gene expression, but little is known about the polymorphisms that affect gene regulation. We analyzed regulatory variation in a cross between laboratory and wild strains of Saccharomyces cerevisiae. Clustering and linkage analysis defined groups of coregulated genes and the loci involved in their regulation. Most expression differences mapped to trans-acting loci. Positional cloning and functional assays showed that polymorphisms in GPA1 and AMN1 affect expression of genes involved in pheromone response and daughter cell separation, respectively. We also asked whether particular classes of genes were more likely to contain trans-regulatory polymorphisms. Notably, transcription factors showed no enrichment, and trans-regulatory variation seems to be broadly dispersed across classes of genes with different molecular functions
Project description:Natural genetic variation can cause significant differences in gene expression, but little is known about the polymorphisms that affect gene regulation. We analyzed regulatory variation in a cross between laboratory and wild strains of Saccharomyces cerevisiae. Clustering and linkage analysis defined groups of coregulated genes and the loci involved in their regulation. Most expression differences mapped to trans-acting loci. Positional cloning and functional assays showed that polymorphisms in GPA1 and AMN1 affect expression of genes involved in pheromone response and daughter cell separation, respectively. We also asked whether particular classes of genes were more likely to contain trans-regulatory polymorphisms. Notably, transcription factors showed no enrichment, and trans-regulatory variation seems to be broadly dispersed across classes of genes with different molecular functions Keywords: other
Project description:Yeast cells can be affected during their growth to several stress conditions. One of the most known and characterised is the osmotic stress and most of the studies about osmotic sterss response in yeast have been focused on salt or sorbitol stress. However, during yeast growth in industrially relevant processes (for instance throughout alcoholic fermentation on the must to produce alcoholic beverages) the osmotic stress is mainly due to the high sugar(in particular glucose) concentration (200-250 g/L).
Project description:Gene regulation occurs through trans-acting factors (e.g. transcription factors) acting on cis-regulatory elements (e.g. enhancers). Massively parallel reporter assays (MPRAs) functionally survey large numbers of cis-regulatory elements for regulatory potential, but do not identify the trans-acting factors that mediate any observed effects. Here we include preliminary data from a pilot transMPRA experiment — a reporter assay that efficiently combines multiplex CRISPR-mediated perturbation and MPRAs to identify trans-acting factors that modulate the regulatory activity of specific enhancers.
Project description:In a previous study, we identified extensive natural variation in the response to acute ethanol treatment in three yeast strains: a lab strain derived from the commonly used S288c (DBY8268), vineyard isolate M22, and oak soil strain YPS163. Many expression differences persisted across several modules of co-regulated genes, implicating trans-acting systemic differences in ethanol sensing and/or response. To understand the genetic basis for these expression differences, we performed eQTL mapping analysis of the response to acute ethanol stress in ~100 F2 strains from two crosses: DBY8268x M22 and DBY8268 x YPS163.