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

碱性螺旋-环-螺旋蛋白对基因表达的组合调控

• 批准号: 0110408
• 负责人: John Lopes
• 金额: $ 31.79万
• 依托单位: Wayne State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-10-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0110408&HistoricalAwards=false
• 关键词: Combinatorial; Regulation; Gene; Expression; Basic

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.

Lopes实验室发现，酵母Ino 4p碱性螺旋-环-螺旋（bHLH）蛋白与其他五种bHLH蛋白二聚化，并调节至少159个基因。 该项目的主要目标是了解酵母bHLH转录因子的不同组合如何调节基因表达。 酵母有七种研究充分的bHLH蛋白：Ino 4p，Ino 2 p，Pho 4p，Rg 1 p，Rtg 3 p，Sgc 1 p和Cbf 1 p。 该项目将通过使用酵母双杂交系统和表位标记的重组蛋白的共纯化测试每个可能的成对组合来定义完整的bHLH蛋白相互作用图谱（PIM）。 这部分研究还将包括最近被分配到HLH类的两种蛋白质，Hms 1 p和YGR 290 Wp。 Lopes实验室已经使用微阵列杂交来鉴定159个被Ino 4p诱导或抑制的基因。 Ino 4p与多种bHLH蛋白相互作用的事实表明，每种bHLH蛋白的靶基因集将与Ino 4p靶基因集重叠。 微阵列杂交将用于鉴定每种bHLH蛋白的靶基因。 将使用表位标记的bHLH重组蛋白结合电泳迁移率变动试验（EMSA）测定每种异二聚体的结合位点。 将使用报告基因检测bHLH最佳结合位点在体内指导转录的能力。 将使用ScanACE程序筛选bHLH靶基因的启动子是否存在bHLH结合位点。 本项目还将研究bHLH蛋白调节靶基因表达的机制。 bHLH二聚体调节转录的能力可能需要与DNA直接结合或螯合并防止bHLH蛋白与其靶启动子结合。 INO 4抑制两个PHO基因和MFalpha 1基因的表达。 将使用DNA结合缺陷型Ino 4p突变体来确定该调节是否是由于Ino 4p与靶基因的直接结合或bHLH转录因子的螯合。细胞如何开启和关闭基因是生物学的核心问题。 众所周知，称为转录因子的蛋白质在控制这些事件中起着重要作用。 该项目将研究一组转录因子如何形成多个配对，以最大限度地增加它们可以打开和关闭的基因数量。