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Comparing DNA binding specificities between orthologous Pho4 transcription factors using extended length probes that include flanking genomic sequences.

ABSTRACT: Combinatorial control by multiple transcription factors (TFs) is a hallmark of eukaryotic gene regulation. Despite its prevalence and crucial roles in enhancing specificity and integrating information, the mechanisms behind why eukaryotic TFs depend on one another, and whether such interdependence evolves, are not well understood. We exploit natural variation in co-TF dependence in the yeast phosphate starvation (PHO) response to address this question. In the model yeast Saccharomyces cerevisiae, the main TF, Pho4, relies on the co-TF Pho2 to regulate ~28 genes. In a related yeast pathogen, Candida glabrata, its Pho4 exhibits significantly reduced Pho2 dependence and has an expanded target set of ~70 genes. Chomatin IP based motifs for the two orthologous Pho4s are highly similar, with no detectable difference in their core E-box motif. However, past studies found that preference for sequences flanking the core motif can provide additional specificities to TFs in the same family. To determine if ScPho4 and CgPho4 differ in their flanking nucleotide preference, we conducted the genomic context Protein Binding Microarray (gcPBM) using 36 bp oligos centered on E-box motifs from ChIP-identified Pho4 binding locations in both S. cerevisiae and C. glabrata genomes. The results confirmed that the two Pho4 orthologs do not show divergence in their sequence preference even after including the genomic context.

ORGANISM(S): Nakaseomyces glabratus Saccharomyces cerevisiae

PROVIDER: GSE293214 | GEO | 2025/04/02

REPOSITORIES: GEO

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Project description:Binding of transcription factors to DNA is a key regulatory step in the control of gene expression. DNA sequences with high affinity for transcription factors occur more frequently in the genome than instances of genes bound or regulated by these factors. How specific gene regulation is achieved by transcription factors remains unclear. We used genome-wide approaches to study how trans factors shape the binding and regulatory landscape of Pho4, a budding yeast transcription factor that activates gene expression in response to phosphate limitation. In no phosphate (0mM phosphate) medium, Pho4 is transported into the nucleus and activates transcription of a set of genes (PHO) that are necessary for cell survival in phosphate limited environment. In high phosphate (10mM phosphate) medium, Pho4 is transported outside of the nucleus and the transcription program of PHO genes are turned off. Here we examined the transcription profiling of S. cerevisiae (W303) in various strains treated in media with 10mM or 0mM inorganic phosphate, to study the the transcription activation by Pho4 its cooperative binding factor Pho2, and its competitive binding factor Cbf1. (I) To infer how much Pho4 itself, Pho2 itself, and the interaction between Pho2 and Pho4 contribute to transcriptional activation in response to Pi starvation, we applied mutant cycle analysis to compare wild type, pho2Δ, pho4Δ and pho2Δ pho4Δ strains in both 10 mM and 0 mM Pi conditions. Mutant cycle analysis is a cyclic comparison among all various mutants. For example, by comparing pho2Δ and pho2Δ pho4Δ, we can directly infer the contribution from Pho4 alone to gene activation. And by comparing wild type and pho2Δ, we can infer the contribution from Pho2 alone together with that from the interaction between Pho2 and Pho4. (II) Compare two strains or one strain in two treatments in multiple replicates with dye-swap method. wt no vs high Pi, to identify genes that are induced in response to Pi starvation. cbf1Δ vs wt in high Pi, to identify the influence of Cbf1 on gene expression in high Pi condition. cbf1Δpho4Δ vs wt in high Pi, to identify the influence of Cbf1 on gene expression that is mediated through Pho4, in high Pi condition. rtg3Δtye7Δ vs wt in high Pi, to identify the influence of Rtg3 and Tye7 on gene expression in high Pi condition. pho80Δ vs pho80Δpho4Δ, to identify the genes that are activated Pho4 when Pho4 is fully nuclear localized. pho80Δcbf1Δ vs pho80Δcbf1Δpho4Δ, to identify the influence of Cbf1 on gene expression that is mediated through Pho4, when Pho4 is fully nuclear localized.

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Comparing DNA binding specificities between orthologous Pho4 transcription factors using extended length probes that include flanking genomic sequences.

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