Mechanisms of epigenetic gene regulation by the Drosophila COMPASS-like complex

果蝇COMPASS样复合体的表观遗传基因调控机制

• 批准号: 1413331
• 负责人: Andrew Dingwall
• 金额: $ 69万
• 依托单位: Loyola University Stritch School of Medicine
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1413331&HistoricalAwards=false
• 关键词: Mechanisms; epigenetic; gene; regulation; Drosophila

The genetic code of every living organism is contained within chromosomes, referred to collectively as the genome. In all eukaryotes, the genome is compacted into the nucleus through the formation of condensed molecules known as chromatin. The smallest individual units of compaction are known as nucleosomes that are comprised of 147 base pairs of DNA wrapped around an octamer of small basic histone proteins. In order for regulatory factors and enzymes to access the genetic code and properly regulate processes essential for normal development and cell survival, the nucleosomes need to be moved aside or directly modified on specific amino acid residues in a process termed chromatin remodeling. The enzymes and other proteins that carry out chromatin remodeling are ancient and remarkably conserved, with increasing complexity from single celled organisms up through vertebrates. The overall impact of this project will be a better understanding of how chromatin remodeling controls expression of the information that is encoded within the genome. In addition, high school, undergraduate and graduate students will participate in mentored research. Women and minorities will be highly represented. Multidisciplinary training and education are vital to prepare for diverse science careers and this project employs the unique tools currently available for Drosophila studies. Students will be trained in molecular and developmental genetics, biochemistry, molecular biology and bioinformatics, structural and developmental biology. The project will enhance interactions among faculty scientists within the institution and with students at multiple undergraduate institutions to explore the rapidly emerging field of bioinformatics. Undergraduate students will be tasked with learning new informatics tools and approaches using experimental data derived from this project. In order to define the histone recognition and binding properties of Cmi and related mammalian domains and elucidate the potential mechanism of COMPASS-like complex targeting to gene enhancers, collaborative structural studies that include X-ray crystallography and nuclear magnetic resonance of the histone recognition domains and targeted mutagenesis combined with in vitro measurements of binding affinities and in vivo chromatin association will be employed. The project also uses next-generation high throughput chromatin and RNA analyses (ChIP-Seq, RNA-Seq) and target gene studies. ChIP-Seq using chromatin from cmi mutant animals will enable correlation of epigenetic marks with Cmi function and align the RNA-Seq gene expression data with chromatin binding to address key developmental functions. Tissue-specific targeted removal of both cmi and trr and overexpression of cmi using unique genetic tools developed for these studies will allow for direct testing of COMPASS-like functions on target genes. Leading edge chromatin technologies that examine physical connections between distant gene regions will elucidate novel regulatory roles for the Drosophila COMPASS-like complex in facilitating enhancer-promoter communication necessary for proper gene control. These studies will help to reveal essential and foundational properties of chromatin remodeling and modifying complexes and provide critical insight into the mechanisms of developmental gene regulation.

每个活着的有机体的遗传密码都包含在染色体中，统称为基因组。在所有真核生物中，基因组通过形成被称为染色质的浓缩分子而紧凑到细胞核中。最小的个体紧凑单位被称为核小体，它由147个碱基对的DNA组成，包裹在一个八聚体的小碱性组蛋白周围。为了让调节因子和酶获得遗传密码，并适当地调节对正常发育和细胞生存至关重要的过程，需要将核小体移到一边或直接对特定的氨基酸残基进行修饰，这一过程称为染色质重塑。进行染色质重塑的酶和其他蛋白质是古老的，非常保守，从单细胞生物体到脊椎动物都越来越复杂。这个项目的总体影响将是更好地理解染色质重塑如何控制基因组中编码的信息的表达。此外，高中生、本科生和研究生将参与辅导研究。妇女和少数族裔将有很高的代表性。多学科培训和教育对于为多样化的科学职业做准备至关重要，该项目使用了目前可用于果蝇研究的独特工具。学生将接受分子和发育遗传学、生物化学、分子生物学和生物信息学、结构和发育生物学方面的培训。该项目将加强学院内部教师和科学家之间的互动，以及与多个本科院校的学生之间的互动，以探索迅速崛起的生物信息学领域。本科生的任务是学习新的信息学工具和方法，使用从这个项目获得的实验数据。为了明确CMI和相关哺乳动物结构域的组蛋白识别和结合特性，并阐明COMPASS类复合体靶向基因增强子的潜在机制，我们将采用协作结构研究，包括组蛋白识别结构域的X射线结晶学和核磁共振，以及定向突变，并结合体外结合亲和力和体内染色质结合的测量。该项目还使用下一代高通量染色质和RNA分析(CHIP-Seq，RNA-Seq)和目标基因研究。ChIP-Seq使用CMI突变动物的染色质，将使表观遗传标记与CMI功能相关，并将RNA-Seq基因表达数据与染色质结合对齐，以定位关键的发育功能。利用为这些研究开发的独特的遗传工具，对CMI和TRR进行组织特异性靶向移除，并过度表达CMI，将允许直接测试目标基因上的COMPASS类似功能。前沿染色质技术检查遥远基因区域之间的物理联系，将阐明果蝇指南针状复合体在促进适当基因控制所需的增强子-启动子通信方面的新调节作用。这些研究将有助于揭示染色质重塑和修饰复合体的基本和基本性质，并为发育基因调控的机制提供重要的见解。