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

• 批准号: 8196414
• 负责人: Namiko Abe
• 金额: $ 4.84万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 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/8196414
• 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

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. PUBLIC HEALTH RELEVANCE: Misregulation of Hox transcription factors is linked to a number of cancers, including acute myeloid leukemia (AML) and breast cancer. However, targeting Hox genes for cancer therapy is complicated by tissue-specific differences in their activities and downstream genes, potentially causing unwanted side effects. Thus, the identification and characterization of Hox cofactors that regulate Hox activity in a cell- and gene-specific manner is critical to developing specific anti-cancer therapies.

描述（由申请人提供）：HOX基因构成了一个进化保守的转录因子家族，首先描述了其在发育过程中其在节段体模式中的功能。此后，这些基因已被证明是造血和组织稳态的重要调节剂，它们的正调与癌症密切相关。像许多转录因子一样，HOX蛋白可以根据靶基因和细胞环境充当转录激活剂或阻遏物，但是如何确定转录调节的迹象尚不清楚。由于常规遗传和生化方法的局限性，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新型技术，称为细胞和基因特异性染色质免疫沉淀（CGCHIP），以分离果蝇中特定细胞的特定细胞子集的特定片段。该技术将扩展到与单个细胞类型中的单个调节元件相关的HOX蛋白复合物。单个HOX蛋白针对的单个顺式调节元件的蛋白质组学分析对于理解如何以上下文依赖性方式定义HOX活性至关重要。具体目的：该提案的目的是检验以下假设：1）新型的共激活因子或共抑制剂与HOX蛋白相互作用以确定转录活性的迹象，并且2）2）这些成分的募集取决于DNA靶标选择所需的HOX分子结构。 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）确定结合所需的FKH250激活SCR复合物和测试SCR基序的新成分。 （3）确定体内FKH250激活是否需要SCR结合蛋白。研究设计：CGCHIP方法将用于纯化果蝇胚胎特异性与FKH250的SCR复合物。为了识别该SCR复合物的组成部分，将通过质谱法分析结合的蛋白质。为了深入了解如何专门募集这些蛋白质，将绘制其相互作用所需的SCR中的基序。其他实验将通过分析突变胚激活FKH250的能力来测试这些结合蛋白是否通过转录共激活剂作用。 公共卫生相关性：HOX转录因子的不正当与许多癌症有关，包括急性髓性白血病（AML）和乳腺癌。然而，将HOX基因用于癌症治疗，其活性和下游基因的组织特异性差异变得复杂，可能会导致不良的副作用。因此，以细胞和基因特异性调节HOX活性的HOX辅助因子的鉴定和表征对于开发特定的抗癌疗法至关重要。