Combinatorial Regulation of Gene Expression by Basic Helix-Loop-Helix Proteins

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The Lopes laboratory has discovered that the yeast Ino4p basic helix-loop-helix (bHLH) protein dimerizes with five other bHLH proteins and regulates at least 159 genes. The primary goal of this project is to understand how different combinations of yeast bHLH transcription factors regulate gene expression. Yeast has seven well-studied bHLH proteins: Ino4p, Ino2p, Pho4p, Rg1p, Rtg3p, Sgc1p, and Cbf1p. This project will define the complete bHLH protein interaction map (PIM) by testing each possible pair-wise combination using the yeast two-hybrid system and co-purification of epitope-tagged recombinant proteins. This part of the study will also include two proteins recently assigned to the HLH class, Hms1p and YGR290Wp. The Lopes lab has used microarray hybridization to identify 159 genes that are either induced or repressed by Ino4p. The fact that Ino4p interacts with multiple bHLH proteins suggests that the target gene sets for each bHLH protein will overlap the Ino4p target gene set. Microarray hybridization will be used to identify target genes for the each bHLH protein. The binding sites for each heterodimer will be determined using epitope-tagged bHLH recombinant proteins coupled with an electrophoretic mobility shift assay (EMSA). The ability of bHLH optimal binding sites to direct transcription in vivo will be tested using reporter genes. The promoters of the bHLH target genes will be screened for the presence of the bHLH binding sites using the ScanACE program. This project will also investigate the mechanisms whereby bHLH proteins regulate target gene expression. The ability of bHLH dimers to regulate transcription may require direct binding to DNA or sequestration and prevention of bHLH proteins from binding to their target promoters. INO4 represses expression of two PHO genes and the MFalpha1 gene. A DNA-binding defective Ino4p mutant will be used to determine if this regulation is due to direct binding of Ino4p to the target genes or sequestration of a bHLH transcription factor. How cells turn on and turn off genes is a central question in biology. It is well established that proteins called transcription factors play an important role in controlling these events. This project will investigate how a group of transcription factors form multiple pairings to maximize the number of genes that they can turn on and turn off.