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.

每个生物体的遗传密码都包含在染色体内，统称为基因组。在所有真核生物中，基因组通过形成称为染色质的浓缩分子而被压缩到细胞核中。最小的压缩个体单位称为核小体，由包裹在小型碱性组蛋白八聚体周围的 147 个碱基对的 DNA 组成。为了使调节因子和酶能够访问遗传密码并正确调节正常发育和细胞存活所必需的过程，需要在称为染色质重塑的过程中将核小体移到一边或直接对特定氨基酸残基进行修饰。进行染色质重塑的酶和其他蛋白质是古老的，并且非常保守，从单细胞生物到脊椎动物，其复杂性不断增加。该项目的总体影响将是更好地了解染色质重塑如何控制基因组内编码信息的表达。此外，高中生、本科生和研究生将参与指导研究。妇女和少数族裔的比例将很高。多学科培训和教育对于为多样化的科学职业做好准备至关重要，该项目采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。该项目将加强机构内教师科学家之间以及与多个本科机构学生的互动，以探索快速新兴的生物信息学领域。本科生的任务是使用从该项目中获得的实验数据来学习新的信息学工具和方法。为了定义 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 样复合物靶向基因增强子的潜在机制，合作结构研究包括组蛋白识别结构域的 X 射线晶体学和核磁共振，以及将采用靶向诱变与结合亲和力和体内染色质关联的体外测量相结合。该项目还使用下一代高通量染色质和 RNA 分析（ChIP-Seq、RNA-Seq）和靶基因研究。使用 cmi 突变动物的染色质进行 ChIP-Seq 将能够将表观遗传标记与 Cmi 功能相关联，并将 RNA-Seq 基因表达数据与染色质结合进行比对，以解决关键的发育功能。使用为这些研究开发的独特遗传工具对 cmi 和 trr 进行组织特异性靶向去除以及 cmi 的过度表达将允许直接测试目标基因上的 COMPASS 样功能。检查遥远基因区域之间的物理联系的前沿染色质技术将阐明果蝇 COMPASS 样复合物在促进适当基因控制所需的增强子-启动子通讯方面的新调控作用。这些研究将有助于揭示染色质重塑和修饰复合物的本质和基础特性，并为发育基因调控机制提供重要的见解。