Uncovering novel transcriptional regulatory pathways in the cell cycle

描述(申请人提供)：失调的细胞周期进程是癌症的一个定义特征，因此清楚地了解正常的细胞周期进程对于了解癌症的发生至关重要。最近在酵母和哺乳动物中的研究表明，细胞周期依赖的转录比以前所了解的更广泛。酵母中近800个基因(占基因组的14%)表现出依赖于细胞周期的转录模式，但这些基因中只有不到40%受到已知的细胞周期转录因子的直接调控。这表明我们缺少与细胞周期进程相关的几条调控通路。这一建议旨在解决细胞周期生物学中的两个基本问题：控制细胞周期特异性转录的DNA-蛋白质相互作用是什么，以及核心细胞周期机制如何调节这些相互作用？具体目标1：确定调节细胞周期的新的DNA-蛋白质相互作用。我将使用转录因子缺失酵母菌株的流式细胞术来寻找正常细胞周期进展所需的转录因子。我将使用全基因组表达谱和染色质免疫沉淀来确定这些转录因子调控的靶基因，并使用一系列计算和实验工具确定相关的顺式调控位点。这一目标的结果将揭示新的细胞周期转录因子、它们的靶标和相关的顺式调控位点。具体目标2：了解细胞周期蛋白依赖的蛋白激酶CDC28如何通过与转录因子的相互作用来调节细胞周期进程。利用流式细胞术和保守的磷酸化位点的突变，我将确定其CDC28磷酸化位点是正常细胞周期进程所必需的转录因子。我将通过体外和体内的激酶试验来验证这些转录因子是真正的CDC28底物。我将使用全基因组表达谱来识别当CDC28对其同源转录因子的调控被取消时错误表达的靶基因。这项工作的成功完成将使人们更好地了解组成细胞周期转录调控网络的DNA-蛋白质相互作用，以及更好地理解该网络如何与核心细胞周期机制相互作用。 相关性：许多人类疾病，特别是癌症，涉及细胞分裂失去控制。了解细胞分裂是如何调节的，对于我们治疗和预防此类疾病的能力至关重要。该项目旨在通过使用基因组工具来研究细胞分裂的控制，以检查基因在细胞分裂周期中是如何调控的。

DESCRIPTION (provided by applicant): Misregulated cell cycle progression is a defining characteristic of cancer, thus a clear understanding of normal cell cycle progression is critical to an informed picture of carcinogenesis. Recent studies in yeast and mammals suggest that cell cycle-dependent transcription is more extensive than was previously apreciated. Nearly 800 genes (14% of the genome) in yeast exhibit cell cycle-dependent patterns of transcription, yet fewer than 40% of these genes are directly regulated by known cell cycle transcription factors. This indicates that we are missing several regulatory pathways involved in cell cycle progression. This proposal aims to address two fundamental questions in cell cycle biology: What are the DNA-protein interactions that control cell cycle-specific transcription, and how are these interactions regulated by the core cell cycle machinery? Specifi Aim 1: Idenitfy novel DNA-protein interactions that regulate the cell cycle. I will use flow cytometry of transcription factor deletion yeast strains to find transcription factors required for normal cell cycle progression. I will identify the target genes regulated by these transcription factors using genome-wide expression profiling and chromatin immunoprecipitation, and determine the associated cis-regulatory sites using an array of computational and experimental tools. The results of this aim will reveal novel cell cycle transcription factors, their targets, and associated cis-regulatory sites. Specific Aim 2: Understand how the cyclin-dependent kinase Cdc28 regulates cell cycle progression through interactions with transcription factors. Using flow cytometry and mutations at conserved phosphorylation sites, I will identify transcription factors whose Cdc28 phosphorylation sites are required for normal cell cycle progression. I will verify that these transcription factors are true Cdc28 substrates through in vitro and in vivo kinase assays. I will use genome-wide expression profiling to identify target genes that are misexpressed when Cdc28 regulation of their cognate transcription factors is abolished. Successful completion of this work will lead to a greater knowledge of the DNA-protein interactions that make up the cell cycle transcriptional regulatory network, as well as lead to a better understanding of how this network interacts with the core cell cycle machinery. Relevance: Many human diseases, notably cancer, involve the loss of control of cell division. Understanding how cell division is regulated is essential to our ability to treat and prevent such diseases. This project aims to study the control of cell division by using genomic tools to examine how genes are regulated during the cell division cycle.