Detecting Histone Modification Turnover in Living Cells

检测活细胞中的组蛋白修饰更新

• 批准号: 7927086
• 负责人: Karmella Ann Haynes
• 金额: $ 5.38万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7927086
• 关键词: Acetylation; Amino Acid Motifs; Antineoplastic Agents; Behavior; Binding; Biomedical Engineering; Cell Cycle; Cell Differentiation process; Cell division; Cells; Chemicals; Chromatin; DNA Repair; Development; Disease; Drosophila genus; Drug Delivery Systems; EZH2 gene; Engineering; Enzymes; Epigenetic Process; Feedback; Fellowship; Fluorescence; Gene Activation; Genes; Genetic; Genome; Histone Code; Histone H3; Histones; Human; In Vitro; Individual; Inherited; Instruction; Lead; Length; Life; Lysine; Mammalian Cell; Maps; Measures; Mediating; Memory; Messenger RNA; Methylation; Mitosis; Modification; Mus; Names; Nuclear; Output; Pattern; Peptides; Phosphorylation; Polycomb; Program Development; Protein Binding; Proteins; Reading; Recombinants; Reporter; Reporting; Repression; Research; Research Proposals; Signal Transduction; Silver; Specificity; Stimulus; Synthetic Genes; System; Tail; Technology; Testing; Time; Transcriptional Activation; Transgenic Organisms; Translating; Work; Zebrafish; base; biological systems; chromatin immunoprecipitation; chromatin modification; design; fly; gene repression; histone methyltransferase; histone modification; in vivo; mammalian genome; protein structure; research study; synthetic biology

DESCRIPTION (provided by applicant): In the post-genome era, one of the current challenges is to understand how epigenetic mechanisms impact cellular programming, development and disease. Histones, the proteins around which DMA is wrapped, carry covalent modifications that lead to gene activation or repression. Effector proteins read and translate this "histone code" through histone binding motifs. Recent studies have generated a wealth of information on patterns of histone modifications. Current technologies to assess histone modifications rely upon chromatin immunoprecipitation (ChIP). While powerful, ChIP has limited ability to probe the dynamics of epigenetic modification in living cells. I propose to use a modular histone tail binding motif from the polycomb (Pc) group of effector proteins to measure changes in histone methylation at a single locus in living cells. Synthetic biology is a new experimental paradigm that offers creative opportunities to investigate the mechanisms of epigenetic inheritance. The design of artificial biological systems is a means for testing our understanding of natural protein "modules." To this end, I will identify a peptide from PC that acts as a module that specifically recognizes methylated histone tails in mammalian cells. I will integrate this new modular part into a reporter system designed to track changes in histone methylation in dividing cells. Peptides that read specific histone modifications could serve as a new class of modular parts that interface bioengineered gene circuits with epigenetic signals that mark key steps in cell development. Heritable changes in the proteins bound to DMA (epigenetics) underlie mechanisms of cell development and disease. Research of the enzymes that mediate epigenetic changes has lead to the discovery of new cancer drug targets. Here, we use a bioengineering approach to probe the epigenetic phenomena. PHS 416-1 (Rev.10/05) Page 2 Number pages consecutively at the bottom throughout Form Page 2 the application. Do not use suffixes such as 2a, 2b. NAME OFAPPLICANT (Last, first, middle initial) Kirschstein-NRSA Individual Fellowship Application Haynes, Karmella A. (To becompletedbyapplicant - follow PHS 416-1 instructions)

描述（由申请人提供）： 在后基因组时代，当前的挑战之一是了解表观遗传机制如何影响细胞编程，发育和疾病。组蛋白，DMA包裹的蛋白质，携带导致基因激活或抑制的共价修饰。效应蛋白通过组蛋白结合基序读取并翻译这种“组蛋白密码”。最近的研究已经产生了丰富的信息组蛋白修饰的模式。目前评估组蛋白修饰的技术依赖于染色质免疫沉淀（ChIP）。虽然功能强大，但ChIP探测活细胞中表观遗传修饰动力学的能力有限。我建议使用一个模块化的组蛋白尾部结合基序从polycomb（Pc）组的效应蛋白来测量组蛋白甲基化在活细胞中的一个单一位点的变化。合成生物学是一种新的实验范式，为研究表观遗传机制提供了创造性的机会。人工生物系统的设计是测试我们对天然蛋白质“模块”理解的一种手段。“为此，我将从PC中鉴定出一种肽，它作为一种模块，特异性地识别哺乳动物细胞中的甲基化组蛋白尾部。我将把这个新的模块化部分整合到一个报告系统中，该系统旨在跟踪分裂细胞中组蛋白甲基化的变化。阅读特定组蛋白修饰的肽可以作为一类新的模块化部分，将生物工程基因电路与标记细胞发育关键步骤的表观遗传信号连接起来。与DMA（表观遗传学）结合的蛋白质的遗传变化是细胞发育和疾病机制的基础。对介导表观遗传变化的酶的研究导致了新的癌症药物靶点的发现。在这里，我们使用生物工程的方法来探测表观遗传现象。PHS 416-1（Rev.10/05）第2页在整个表格第2页的底部连续编号。不要使用后缀，如2a，2b。 申请人姓名（姓、名、中间名首字母）Kirschstein-NRSA个人奖学金申请Haynes，Karmella A。 (To由申请人填写-遵循PHS 416-1说明）