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

描述（由申请人提供）：基因间转录因子（TF）结合基序的变体通常被认为是功能等同（简并）的，但其中一些可能实际上影响基因表达;此类变体称为“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，我们知之甚少。我们的目标是了解TF结合位点变异对基因组中基因之间或不同物种中基因之间表达变异的贡献程度。我们的目标是：A。使用计算方法表征酵母中的功能性TF结合位点变体，（a）开发稳健的方法以解释TF的多个结合位点、功能变体内的简并性、表达噪声以及基因和结合位点变体的全部比较的非独立性，（B）阐明变体特异性表达谱的保守模式，在严格意义上的酵母菌中，这些酵母菌与C.白色念珠菌中，以确定TF的靶基因的功能或基序的序列组成是否与功能变体的保守性相关。（c）表征靶基因启动子中可能引起物种B之间变体特异性表达谱转换的核苷酸取代。实验验证和表征功能变体。（a）使用定点诱变改变变体结合位点位置处的核苷酸以测试预测的表达变化。在S.将选择酿酒酵母进行实验验证。此外，变异的影响之间的S。cerevisiae和S.将通过杂交菌株中的等位基因特异性表达分析来验证paradoxus。（B）变体结合位点功能机制的测试模型。拟议的研究将增加我们对生理学和病理学基础的复杂顺式调控密码的理解。由于启动子中的突变与心脏病、HIV传播风险、α-地中海贫血、阿尔茨海默病和其他疾病相关，因此了解结合位点的全部功能对于评估与疾病病因相关的突变可能至关重要。

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.