Functional Dissection of IME1 Transcription Using Quantitative Promoter–Reporter Screening

Abstract

Transcriptional regulation is a key mechanism that controls the fate and response of cells to diverse signals. Therefore, the identification of the DNA-binding proteins, which mediate these signals, is a crucial step in elucidating how cell fate is regulated. In this report, we applied both bioinformatics and functional genomic approaches to scrutinize the unusually large promoter of the IME1 gene in budding yeast. Using a recently described fluorescent protein-based reporter screen, reporter-synthetic genetic array (R-SGA), we assessed the effect of viable deletion mutants on transcription of various IME1 promoter-reporter genes. We discovered potential transcription factors, many of which have no perfect consensus site within the IME1 promoter. Moreover, most of the cis-regulatory sequences with perfect homology to known transcription factor (TF) consensus were found to be nonfunctional in the R-SGA analysis. In addition, our results suggest that lack of conservation may not discriminate against a TF regulatory role at a specific promoter. We demonstrate that Sum1 and Sok2, which regulate IME1, bind to nonperfect consensuses within nonconserved regions in the sensu stricto Saccharomyces strains. Our analysis supports the view that although comparative analysis can provide a useful guide, functional assays are required for accurate identification of TF-binding site interactions in complex promoters.

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…The complex dynamics of gene expression during the cell cycle and after subjecting cells to stress have been assayed by ChIP analysis of the regulatory TFs and coactivators (McBride et al 1997;Bhoite et al 2001;Simon et al 2001;Horak et al 2002;Tan et al 2008;Ni et al 2009). Similarly, during sporulation (Kahana et al 2010) and glucose starvation (Young et al 2003;Tachibana et al 2005;Ratnakumar and Young 2010) multiple TFs bind upstream of the promoter of coregulated genes. In the sulfur metabolic network, the non-DNA-binding Met4 activator interacts with multiple cofactors that stabilize DNA binding (Lee et al 2010).…”

Regulated repression, and not activation, governs the cell fate promoter controlling yeast meiosis

“…Various transcription factors have been implicated in controlling in IME1 expression 12 . To further examine whether transcriptional activators are readily present at the IME1 promoter, we measured IME1 expression after depletion of Tup1 or Cyc8.…”

“…Notably, the transcription factor Sok2 controls IME1 promoter activity via the glucose responding element 11 . In addition, multiple other transcription factors contribute to regulation of IME1 transcription [12][13][14] . Over 50 transcription factors have a conserved consensus site in the IME1 promoter 12 .…”

“…In addition, multiple other transcription factors contribute to regulation of IME1 transcription [12][13][14] . Over 50 transcription factors have a conserved consensus site in the IME1 promoter 12 . A genome-wide reporter screen revealed that about 30 transcription factors may directly or indirectly control IME1 transcription 12 .…”

“…Over 50 transcription factors have a conserved consensus site in the IME1 promoter 12 . A genome-wide reporter screen revealed that about 30 transcription factors may directly or indirectly control IME1 transcription 12 . How different transcription factors and functional elements of the IME1 promoter interact to control IME1 expression and thus regulate the decision to enter meiosis is not well understood.…”