Establishing coordinate gene regulation during Drosophila dosage compensation

在果蝇剂量补偿过程中建立协调基因调控

• 批准号: 8158948
• 负责人: Erica Nicole Larschan
• 金额: $ 29.34万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8158948
• 关键词: Address; Affinity; Affinity Chromatography; Autistic Disorder; Beryllium; Binding; Biological Assay; Biological Models; Cell Line; Chromatin; Chromatin Structure; Chromosomes; Chromosomes, Human, Pair 5; Chromosomes, Human, Pair 6; Chromosomes, Human, Pair 7; Cis-Acting Sequence; Complex; Copy Number Polymorphism; Coupling; DNA Sequence; Data; Development; Disease; Dosage Compensation (Genetics); Drosophila genus; Elements; Epitopes; Exhibits; Feedback; Female; Functional RNA; Gene Dosage; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Screening; Genetic Transcription; Genome; Genomics; Goals; In Vitro; Knowledge; Length; Link; Mammals; Mediating; Mission; Modeling; Molecular; Nuclear; Nucleosomes; Organism; Process; Protein Binding; Proteins; Public Health; RNA; Recruitment Activity; Regulation; Relative (related person); Research; Site; Specificity; Staging; System; Testing; Time; Transcript; Work; X Chromosome; Zinc Fingers; chromatin immunoprecipitation; disorder prevention; in vivo; innovation; insight; male; novel; prevent; protein function; protein protein interaction; reconstitution; tool

DESCRIPTION (provided by applicant): All organisms must regulate their genes precisely for normal development and to prevent disease states. However, significant gene copy number variation exists across genomes 1; therefore, coordinate regulation is required to equalize transcription levels 2 3. Our long-term goal is to describe the molecular mechanisms used to target genes for coordinate regulation, the essential initial step in their regulation. Dosage compensation is one of the best model systems for studying this process because all of the genes on a single chromosome are specifically identified and co-regulated. Drosophila, like mammals 4, increase the transcript levels of a large number of diversely regulated genes along the length of the single male X-chromosome precisely two-fold relative to each female X-chromosome 5. The objective of this application is to understand how dosage compensation in Drosophila is established, the critical first step in the regulatory process. The Drosophila Male Specific Lethal (MSL) complex is central to dosage compensation; it first identifies the X chromosome using a combination of cis-acting DNA sequences 6 and co-transcriptional recruitment by its roX (RNA on X) non-coding RNA components7 and then spreads into the bodies of active genes 8. However, we do not know how the MSL complex specifically identifies the MSL Recognition Element (MRE) sequences on the male X because known MSL components are insufficient for direct recognition of MREs in vitro 9. We used an innovative genetic screen for new regulators of dosage compensation that function in both males and females and thereby identified the essential CLAMP zinc-finger protein. Guided by strong preliminary data, we propose the following novel mechanism for identifying genes for coordinate regulation: CLAMP and the MSL complex associate inter-dependently, thereby generating a positive feedback amplification system that creates X- specificity from a two-fold X-enrichment of MRE sequences. The rationale for this work is that determining how the MSL complex specifically targets the X-chromosome will yield key insight into how genes are identified for coordinately regulation within sub-nuclear domains. We will test our novel mechanism using three specific aims: 1) We will define DNA sequence requirements for CLAMP binding in vivo and in vitro; 2) We will identify CLAMP interacting proteins that mediate its interaction with the MSL complex. At the same time, we will define new interaction partners of a previously unstudied essential transcriptional regulator; 3) We will establish the mechanism by which CLAMP and the MSL complex function inter-dependently at high affinity sites. Our proposed research is significant because we expect to describe for the first time the previously unknown mechanism that allows MSL complex to identify its high affinity sites, thereby defining the critical first step in establishing coordinate gene regulation. Defining the novel mechanism by which CLAMP and the MSL complex function together to generate a domain of enhanced transcription is likely to provide key insight into how genes are identified for coordinate regulation across species. PUBLIC HEALTH RELEVANCE: Our proposed research is relevant to public health because loss of precise regulation of genes underlies a large number of diseases including autism 10 and Chron's disease 11. Our research addresses the critical initial step in the coordinate regulation of genes, identifying target genes for subsequent regulation. Our findings are therefore relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will provide tools for mitigating diseases.

描述（由申请人提供）：所有生物体都必须精确调节其基因才能正常发育并预防疾病状态。然而，显着的基因拷贝数的变化存在于整个基因组1，因此，协调调节需要均衡转录水平2 - 3。我们的长期目标是描述用于靶向基因协调调控的分子机制，这是其调控的重要初始步骤。剂量补偿是研究这一过程的最佳模型系统之一，因为单个染色体上的所有基因都被特异性识别和共同调控。果蝇，像哺乳动物一样，沿着单个雄性X染色体的长度，相对于每个雌性X染色体5，增加了大量的双稳态调节基因的转录水平精确地两倍。本申请的目的是了解果蝇的剂量补偿是如何建立的，这是监管过程中关键的第一步。果蝇雄性特异性致死（MSL）复合物是剂量补偿的核心;它首先使用顺式作用DNA序列6和其roX（X上的RNA）非编码RNA组分7的共转录募集的组合来识别X染色体，然后扩散到活性基因的体内8。然而，我们不知道MSL复合物如何特异性识别雄性X上的MSL识别元件（MRE）序列，因为已知的MSL组分不足以在体外直接识别MRE 9。我们使用了一种创新的基因筛选新的调节剂的剂量补偿功能，在男性和女性，从而确定了必要的CLAMP锌指蛋白。在强有力的初步数据的指导下，我们提出了以下用于鉴定用于协调调节的基因的新机制：CLAMP和MSL复合物相互依赖地缔合，从而产生正反馈扩增系统，其从MRE序列的两倍X富集产生X特异性。这项工作的基本原理是，确定MSL复合物如何特异性靶向X染色体将产生关键的洞察力，以了解基因如何被识别为亚核结构域内的协调调节。我们将使用三个具体目标来测试我们的新机制：1）我们将定义CLAMP在体内和体外结合的DNA序列要求; 2）我们将鉴定介导其与MSL复合物相互作用的CLAMP相互作用蛋白。同时，我们将定义一个以前未研究的重要转录调节因子的新的相互作用伙伴; 3）我们将建立CLAMP和MSL复合物在高亲和力位点相互依赖的机制。我们提出的研究是有意义的，因为我们希望首次描述以前未知的机制，使MSL复合物，以确定其高亲和力位点，从而确定关键的第一步，在建立协调基因调控。确定CLAMP和MSL复合物共同作用产生增强转录的结构域的新机制可能为如何识别跨物种协调调控的基因提供关键见解。 公共卫生关系：我们提出的研究与公共卫生有关，因为基因精确调控的丧失是大量疾病的基础，包括自闭症10和克罗恩病11。我们的研究解决了基因协调调控的关键初始步骤，确定了后续调控的靶基因。因此，我们的发现与NIH的使命的一部分有关，即开发基础知识，为减轻疾病提供工具。