Unknown,Transcriptomics,Genomics,Proteomics

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Transcription Factor Regulation and Chromosome Dynamics in Pseudohyphal Growth

ABSTRACT: Pseudohyphal growth is a developmental pathway seen in some strains of yeast, in which cells elongate and form multicellular filaments in response to environmental stresses. Regulation of this process is complex and involves multiple signaling pathways and a large number of transcription factors (TFs). We used multiplexed transposon M-bM-^@M-^\Calling CardsM-bM-^@M-^] to simultaneously record the genome-wide binding patterns of 28 TFs in nitrogen-starved yeast. We were able to identify TF targets relevant for pseudohyphal growth, producing a detailed map of the regulatory network that governs this process. Using tools from graph theory, we identified 8 transcription factors that lie at the center of this network, including Flo8, Mss11, and Mfg1, which bind as a complex. Surprisingly, the DNA binding preferences for these key transcription factors were not known. Using Calling Card data, we predicted the in vivo DNA binding motif for the Flo8-Mss11-Mfg1 complex and validated it using a reporter assay. We found that this complex binds several important targets, including FLO11, at both their promoter and termination sequences. We demonstrated that this binding pattern is the result of DNA-looping, which regulates the transcription of these targets and is stabilized by an interaction with the nuclear pore complex. This looping provides yeast cells with a transcriptional memory, enabling them to more rapidly execute the filamentous growth program when nitrogen-starved if they have been previously exposed to this condition. These data contain mapped insertion sites from Ty5 transposon "Calling Cards" in M-NM-#1278b yeast sequenced using an Illumina Hiseq. Other sequencing data present are RNA-seq reads.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: David Mayhew

PROVIDER: E-GEOD-54831 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Morphogenetic transitions are prevalent in the fungal kingdom. For a leading human fungal pathogen, Candida albicans, the capacity to transition between yeast and filaments is key for virulence. For the model yeast Saccharomyces cerevisiae, filamentation enables nutrient acquisition. A recent functional genomic screen in S. cerevisiae identified Mfg1 as a regulator of morphogenesis that acts in complex with Flo8 and Mss11 to enable transcriptional responses crucial for filamentation. In C. albicans, Mfg1 also interacts physically with Flo8 and Mss11 and is critical for filamentation in response to diverse cues, but the mechanisms through which it regulates morphogenesis remained elusive. Here, we explored the consequences of perturbation of Mfg1, Flo8, and Mss11 on C. albicans morphogenesis, and identified functional divergence of complex members. We observed that C. albicans Mss11 was dispensable for filamentation, and that overexpression of FLO8 caused constitutive filamentation even in the absence of Mfg1. Epistasis experiments suggested that Mfg1 acts downstream of protein kinase A, as does Flo8. Harnessing transcriptional profiling and chromatin immunoprecipitation coupled to microarray analysis, we identified divergence between transcriptional targets of Mfg1 and Flo8 in C. albicans. A key direct transcriptional target of Mfg1 is TEC1, for which overexpression was sufficient to restore filamentation in the absence of Mfg1. To identify additional Mfg1 downstream effectors, we employed a novel strategy to select for mutations that restore filamentation in the absence of Mfg1. Whole genome sequencing of filamentation-competent mutants revealed chromosome 6 amplification as a conserved adaptive mechanism. A key determinant of the amplification on chromosome 6 is FLO8, as deletion of one allele blocked morphogenesis, and chromosome 6 was not amplified in evolved lineages for which FLO8 was re-located to a different chromosome. Thus, this work highlights rewiring of key morphogenetic regulators over evolutionary time and aneuploidy as an adaptive mechanism driving fungal morphogenesis.

Project description:Transcription factors direct gene expression, and so there is much interest in mapping their genome-wide binding locations. M-BM- Current methods do not allow for the multiplexed analysis of TF binding, and this limits their throughput. We describe a novel method for determining the genomic target genes of multiple transcription factors simultaneously. DNA-binding proteins are endowed with the ability to direct transposon insertions into the genome near to where they bind. The transposon becomes a M-bM-^@M-^\Calling CardM-bM-^@M-^] marking the visit of the DNA-binding protein to that location. A unique sequence M-bM-^@M-^\barcodeM-bM-^@M-^] in the transposon matches it to the DNA-binding protein that directed its insertion. The sequences of the DNA flanking the transposon (which reveal where in the genome the transposon landed) and the barcode within the transposon (which identifies the TF that put it there) are determined by massively-parallel DNA sequencing. To demonstrate the methodM-bM-^@M-^Ys feasibility, we determined the genomic targets of eight transcription factors in a single experiment. The Calling Card method promises to significantly reduce the cost and labor needed to determine the genomic targets of many transcription factors in different environmental conditions and genetic backgrounds. These data contain Ty5 insertion sites mapped by an Illumina GAII analyzer in the S. cerevisiae genome for the background strain without any Sir4 present (1 run), in strains expressing Sir4-tagged copies of three well-characterized TFs: Gal4, Leu3, and Gcn4 (1 run each), and a multiplex of eight Sir4-tagged TFs pooled in a single experiment (2 biological replicates), and insertions from the Thi2-Sir4 fusion expressed from its native locus in two conditions (1 run each). The format of each insertions file is [chromosome number] [position of genomic base] [direction of insertion] [number of reads at that position]. Raw sequencing data comes in two varieties. Paired-end data contains a 5 bp barcode at the beginning of read #2. Single-end data contains a 2 bp barcode on the beggining of read #1.

Project description:Effect of either FLO8 or MSS11 deletion and -overexpression on yeast transcript profiles compared to wild type in laboratory yeast strains Σ1278b and S288c - also the effect of FLO11 (MUC1) overexpression in the Σ1278b genetic background The aim of this study was to (1) perform a repeat analysis (to improve statistical analysis of these data sets) similar to data submitted previously (GSE17716) and also (2) study the effect of FLO11 over-expression on the transcriptome. Background: The outer cell wall of the yeast Saccharomyces cerevisiae serves as the interface with the surrounding environment and defines cell-cell and cell-surface interactions. Many of these interactions are facilitated by specific adhesins that belong to the Flo protein family. This family of mannoproteins has been implicated in phenotypes such as flocculation and substrate adhesion as well as pseudohyphal growth. Genetic data strongly suggest that individual Flo proteins are responsible for many specific cellular adhesion phenotypes. However, it remains unclear whether such phenotypes are determined solely by the nature of the expressed FLO genes or rather the result of a combination of FLO gene expression and other cell wall properties and cell wall proteins. Mss11p has been shown to be a central element of FLO1 and FLO11 gene regulation and acts together with the cAMP-PKA-dependent transcription factor Flo8p. We use genome wide transcript analysis to identify genes that are direct ly or indirectly regulated by Mss11p in the genetic backgrounds: Sigma1278b and S288c. Sigma 1278b is the strain historically used for the study of pseudohyphae (FLO11 expression) but we also included S288c as this strain is widely used in the research community and was used to determine the first full genome sequence (Thus correspond with SGD information). We also compare this data with transcriptome data from Sigma 1278b yeast over-expressing FLO8 to compare similarities/differences between these two signalling factors. Finally the effect of FLO11 over-expression in Sigma1278b on global transcription is studied so that we can differentiate between "direct" gene targets of Flo8p or Mss11p, and those regulated as a result by the "indirect" effect caused by modified cell wall Flo11p levels. We used two laboratory yeast strains that behave different with regard to adhesion phenotypes. By comparing yeast deleted in either FLO8 or MSS11 to wild type, or yeast overexpressing these genes, in both genetic backgrounds, we investigate the role of Flo8p and Mss11p on yeast transcription. In addition the effect of the over-expression of the adhesin gene FLO11 was studied in Sigma 1278b. By using similar growth conditions to what we use for adhesion phenotype determination we aim to correlate transcription profile changes to yeast behaviour (phenotypes).

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Transcription Factor Regulation and Chromosome Dynamics in Pseudohyphal Growth

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