Exploiting a gain-of-function SKN-1 mutant to discover novel mechanisms of transcription factor regulation

利用功能获得性 SKN-1 突变体发现转录因子调控的新机制

• 批准号: 10371198
• 负责人: Christian David Turner
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-03 至 2023-04-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371198
• 关键词: Affinity; Alleles; Alzheimer' s Disease; Animals; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Process; CRISPR/Cas technology; Caenorhabditis elegans; Cell Nucleus; Cell physiology; Cells; ChIP-seq; Chromatin; Clinical; Collaborations; Consensus; Consensus Sequence; Development; Disease; Disease Progression; Dissociation; Drug Metabolic Detoxication; EMSA; Elements; Ensure; Enzymes; Escherichia coli; Exclusion; Expression Profiling; Fractionation; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Genetic study; Genome; Genomics; Human; In Vitro; Infrastructure; Intestines; Kinetics; Knowledge; Left; Libraries; Life; Link; Longevity; Maintenance; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Measures; Metabolism; Modification; Molecular; Mutagenesis; Mutate; Mutation; Natural Immunity; Nuclear; Nucleotides; Oxidative Stress; Pathology; Phase; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Proteins; RNA Interference; Reactive Oxygen Species; Regulation; Regulatory Pathway; Reporter; Resistance; Stress; Surface Plasmon Resonance; System; Techniques; Testing; Transcriptional Activation; Transgenic Organisms; Variant; Xenobiotics; base; biological adaptation to stress; chromatin immunoprecipitation; environmental stressor; experimental study; gain of function; genome-wide; in vivo; interest; knock-down; lipid metabolism; mutant; novel; overexpression; promoter; screening; stoichiometry; transcription factor; transcriptome; transcriptome sequencing; tumorigenesis

Project Summary: Homeostatic control mechanisms are vital components of biological maintenance within the cell. Reactive oxygen species (ROS) are kept under homeostatic control, in part, by “Cap’n’Collar”(CnC) transcription factors, like SKN-1 in C. elegans. A mutagenesis screen uncovered SKN-1 gain-of-function(gf) alleles that display increased resistance to oxidative stress, but surprisingly, deregulated metabolism and shortened lifespans. These early findings suggest a link between uncontrolled regulation of cytoprotective transcription factors and disease pathology. Regulation of SKN-1 has been documented primarily through genetic studies. This has left many mechanistic details of SKN-1 regulation unstudied. This proposal leverages the opportunity to examine a constitutively active transcription factor to characterize aberrant regulation normally applied in the wild type condition. This proposal will CRISPR/cas9- generated GFP tagged variants of SKN-1 and SKN-1gf at the endogenous locus, which avoids problems with multi- copy transgenic arrays used in previous studies. In Aim 1, I will utilize ChIP-seq to assess target selection as well as a Biacore surface plasmon resonance (SPR) studies to define binding affinity and dissociation constants, which based on our RNAseq studies are predicted to be different across target genes. Preliminarily ChIP-qPCR experiments have been performed for AIM1 and have revealed that SKN-1gf occupies target gene promoters more than wildtype. We will expand this approach to a genome-wide assessment and begin with these established targets for our binding studies. In Aim 2, I will examine the interplay of phosphorylation and OGlcNAcylation post-translational modifications on SKN-1 activity and target selection. Phosphorylation on SKN-1 is critical for its regulation as well as OGlcNAcylation. A preliminary assessment of phosphorylation stoichiometry has been performed via PhosTag SDS PAGE, where an increase in phosphorylation on SKN-1gf compared to wildtype was observed. It is unclear what kinases are causing this shift, but my combined genetic and biochemical approach will define these important regulators. Taken together these biochemical assessments will fill a critical knowledge gap for SKN-1 activity and target gene selection, which is a topic of great interest to the study of transcription factor regulation.

项目概要： 稳态控制机制是细胞内生物维持的重要组成部分。活性氧 物种（ROS）保持在稳态控制下，部分是由“帽环”（CnC）转录因子，如SKN-1 in C.优雅的突变筛选发现了SKN-1功能获得性（gf）等位基因， 氧化应激，但令人惊讶的是，失调的新陈代谢和缩短寿命。这些早期发现表明 细胞保护性转录因子的不受控制的调节与疾病病理学之间的联系。调控 SKN-1主要通过基因研究记录。这留下了SKN-1的许多机械细节 未研究的规则该提案利用了检查组成型活性转录因子的机会， 表征通常在野生型条件下应用的异常调节。CRISPR/cas9- 在内源基因座产生了SKN-1和SKN-1gf的GFP标记的变体，这避免了多基因突变的问题。 复制先前研究中使用的转基因阵列。在目标1中，我将利用ChIP-seq来评估靶标选择以及 a Biacore表面等离子体共振（SPR）研究以定义结合亲和力和解离常数，其基于 在我们的RNAseq研究中，预测在靶基因之间是不同的。ChIP-qPCR实验具有以下特征： 对AIM 1进行了研究，结果表明SKN-1gf比野生型更能占据靶基因启动子。我们 我将把这种方法扩展到全基因组评估，并开始与这些既定的目标，我们的结合， 问题研究在目标2中，我将研究磷酸化和OGlcNAc化翻译后修饰的相互作用， SKN-1的活性和目标选择SKN-1上的磷酸化对于其调节以及 OGlcNAc酰化。通过PhosTag SDS对磷酸化化学计量进行了初步评估 PAGE，其中观察到与野生型相比SKN-1gf上的磷酸化增加。目前还不清楚是什么 激酶引起了这种转变，但我结合遗传学和生物化学的方法将定义这些重要的 监管部门总之，这些生化评估将填补SKN-1活性的关键知识空白， 目的基因选择是转录因子调控研究的热点。