Cis-regulatory motif variants and evolution of gene regulation in yeasts

描述(由申请人提供)：转录因子(TF)结合基序在基因之间的变体通常被认为在功能上是等价的(退化)，但其中一些实际上可能影响基因的表达；这样的变体被称为“功能”变体。关于这些功能变体如何或何时起作用，以及如何检测它们，人们知之甚少。我们的目标是了解TF结合位点变异在多大程度上促进了基因组中基因之间或不同物种基因之间的表达差异。我们的目标是：a.使用计算方法表征酵母中的功能性转铁蛋白结合位点变异，(A)开发稳健的方法来解释转铁蛋白的多个结合位点、功能变异体内的简并、表达噪声以及基因和结合部位变异体的全部比较的非独立性，(B)阐明变异体特异性表达谱的保守模式，观察严格意义上的酵母以及这些物种与白色念珠菌之间的差异，以确定转铁蛋白的靶基因的功能或基序的序列组成是否与功能变异体的保守有关，(C)鉴定目标基因启动子中可能导致物种B之间变异体特异性表达谱切换的核苷酸替换。通过实验验证和表征功能变异体，(A)使用定点突变改变不同结合部位的核苷酸，以测试预测的表达变化。将选择酿酒酵母中的一些变异位点进行实验验证。此外，酿酒酵母和矛盾链霉菌之间的变异切换的影响将通过在杂交菌株中的等位基因特异性表达分析来验证，(B)变异结合位点功能机制的测试模型。这项拟议的研究将增加我们对构成生理学和病理学基础的复杂顺式调控代码的理解。由于启动子的突变与心脏病、艾滋病毒传播风险、阿尔法地中海贫血、阿尔茨海默氏症和其他疾病有关，了解结合位点的全部功能对于评估与疾病病因学相关的突变至关重要。

DESCRIPTION (provided by applicant): Variants of a transcription factor (TF) binding motif among genes are commonly thought to be functionally equivalent (degenerate), but some of them may in fact influence gene expression; such a variant is called a "functional" variant. Little is known about how or when these functional variants act, and how to detect them. Our goal is to understand the extent that TF binding site variants contribute to expression variation among genes in a genome or between genes in different species. Our aims are to: A. Characterize functional TF binding site variants in yeast using computational approaches, (a) Develop robust methods to account for multiple binding sites of the TF, degeneracy within functional variants, expression noise, and non-independence of all-by-all comparisons of genes and binding site variants, (b) Elucidate the patterns of conservation of variant-specific expression profiles, looking across Saccharomyces sensu stricto species and between these species and C. albicans to determine whether the function of target genes of TFs or the sequence composition of the motif is associated with the conservation of functional variants, (c) Characterize nucleotide substitutions in target gene promoters that may have caused a switch in variant-specific expression profiles between species B. Experimentally validate and characterize functional variants, (a) Alter nucleotides at variant binding site positions using site-directed mutagenesis to test for predicted expression changes. A number of variant sites in S. cerevisiae will be selected for experimental validation. Further, the effect of variant switches between S. cerevisiae and S. paradoxus will be validated by allele-specific expression analysis in a hybrid strain, (b) Test models for the mechanism of variant binding site function. The proposed research will increase our understanding of the complex cis-regulatory code that underlies physiology and pathology. As mutations in promoters have been associated with heart disease, HIV transmission risk, alpha-thalassemia, Alzheimer's and other diseases, understanding the full range of function of a binding site could be critical for assessing mutations that are associated with disease etiology.