Characterization of Hox transcription factor complexes using proteomics

使用蛋白质组学表征 Hox 转录因子复合物

基本信息

批准号：
8325227
负责人：
Namiko Abe
金额：
$ 5.22万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8325227
关键词：
Acute Myelocytic Leukemia Adverse effects Amino Acid Motifs Animals Architecture Binding Binding Proteins Biochemical Body Patterning Cells Chromatin Comb animal structure Complex DNA DNA Binding Development Drosophila genus Embryo Embryonic Development Family Gene Activation Gene Expression Gene Targeting Genes Genetic Genetic Enhancer Element Genetic Transcription Goals Hematopoiesis Homeostasis Individual Laboratories Link Malignant Neoplasms Maps Mass Spectrum Analysis Methods Molecular Organogenesis Process Proteins Proteomics Recruitment Activity Regulation Regulatory Element Research Design Salivary Glands Specificity Techniques Testing Tissues Transcription Coactivator Transcription Repressor/Corepressor Transcriptional Activation Transcriptional Regulation Vertebrates Western Blotting Work base cancer therapy cell type chromatin immunoprecipitation cofactor embryo cell gene function gene repression in vivo insight liquid chromatography mass spectrometry malignant breast neoplasm mutant novel protein complex protein function research study sex transcription factor

项目摘要

DESCRIPTION (provided by applicant): Hox genes comprise an evolutionarily conserved family of transcription factors first described for their function in segmental body patterning during development. These genes have since been demonstrated to be important regulators of hematopoiesis and tissue homeostasis, and their misregulation is strongly linked to cancer. Like many transcription factors, Hox proteins can act as transcriptional activators or repressors depending on the target gene and cellular context, but how the sign of transcriptional regulation is determined remains unclear. A detailed characterization of the factors that work with Hox proteins to control gene expression has been lacking due to limitations in conventional genetic and biochemical methods. The Mann laboratory recently developed a novel technique called cell- and gene-specific chromatin immunoprecipitation (cgChIP) to isolate specific fragments of chromatin from specific subsets of cells in Drosophila. This technique will be extended to isolate Hox protein complexes associated with a single regulatory element in a single cell type. Proteomic profiling of single cis-regulatory elements targeted by individual Hox proteins is essential for understanding how Hox activity is defined in a context-dependent manner. Specific aims: The goal of this proposal is to test the hypothesis that 1) novel co-activators or co-repressors interact with Hox proteins to determine the sign of transcriptional activity, and that 2) the recruitment of these components is dependent on the Hox molecular architecture required for DNA target selection. To test this hypothesis, initial studies will focus on the mechanism by which the Drosophila Hox protein sex combs reduced (Scr) activates fkh transcription by the enhancer element fkh250 through the following specific aims: (1) Optimize the biochemical isolation of fkh250-activating Scr complexes from embryos by cell- and gene-specific chromatin immunoprecipitation (cgChIP). (2) Identify novel components of the fkh250-activating Scr complex and test Scr motifs required for binding. (3) Determine whether Scr-bound proteins are necessary for fkh250 activation in vivo. Study design: The cgChIP method will be used to purify the Scr complex specifically bound to fkh250 from Drosophila embryos. To identify components of this Scr complex, bound proteins will be analyzed by mass-spectrometry. To gain insight into how these proteins are recruited specifically to this locus, motifs in Scr that are required for their interaction will be mapped. Additional experiments will test whether these binding proteins function as transcriptional co- activators by analyzing mutant embryos for their ability to activate fkh250.

描述（由申请人提供）：Hox基因包含进化上保守的转录因子家族，最初描述其在发育过程中节段身体模式中的功能。这些基因已被证明是造血和组织稳态的重要调节因子，它们的失调与癌症密切相关。与许多转录因子一样，Hox 蛋白可以根据靶基因和细胞环境充当转录激活子或阻遏子，但如何确定转录调控的信号仍不清楚。由于传统遗传和生化方法的限制，一直缺乏与 Hox 蛋白一起控制基因表达的因素的详细表征。曼恩实验室最近开发了一种称为细胞和基因特异性染色质免疫沉淀 (cgChIP) 的新技术，用于从果蝇特定细胞亚群中分离特定染色质片段。该技术将扩展到分离与单一细胞类型中的单一调节元件相关的 Hox 蛋白复合物。单个 Hox 蛋白靶向的单个顺式调控元件的蛋白质组学分析对于理解如何以上下文依赖的方式定义 Hox 活性至关重要。具体目标：本提案的目标是检验以下假设：1) 新型共激活因子或共抑制因子与 Hox 蛋白相互作用以确定转录活性的迹象，2) 这些成分的招募取决于 Hox DNA 靶标选择所需的分子结构。为了验证这一假设，初步研究将重点关注果蝇Hox蛋白性别梳减少（Scr）通过增强子元件fkh250激活fkh转录的机制，具体目标如下：（1）优化fkh250激活Scr的生化分离通过细胞和基因特异性染色质免疫沉淀 (cgChIP) 从胚胎中提取复合物。 (2) 鉴定 fkh250 激活 Scr 复合物的新成分并测试结合所需的 Scr 基序。 (3)确定Scr结合蛋白是否是体内fkh250激活所必需的。研究设计：cgChIP 方法将用于从果蝇胚胎中纯化与 fkh250 特异性结合的 Scr 复合物。为了鉴定该 Scr 复合物的成分，将通过质谱分析结合的蛋白质。为了深入了解这些蛋白质如何被特异性招募到该位点，将绘制它们相互作用所需的 Scr 中的基序。其他实验将通过分析突变胚胎激活 fkh250 的能力来测试这些结合蛋白是否作为转录共激活剂发挥作用。