Establishing coordinate gene regulation during Drosophila dosage compensation

描述(申请人提供)：所有生物体都必须精确地调节它们的基因，才能正常发育和预防疾病状态。然而，基因组1之间存在显著的基因拷贝数差异；因此，需要协调调节来平衡转录水平2 3。我们的长期目标是描述用于靶向基因协调调节的分子机制，这是它们调节的基本初始步骤。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地识别并共同调节。果蝇和哺乳动物4一样，沿着单个雄性X染色体的长度，使大量不同调控基因的转录水平精确地增加两倍于每个雌性X染色体5。这一应用的目的是了解果蝇如何建立剂量补偿，这是调节过程的关键第一步。果蝇雄性特异性致死(MSL)复合体是剂量补偿的中心；它首先通过顺式作用的DNA序列6和其ROX(X上的RNA)非编码RNA组分7的共转录招募来识别X染色体，然后传播到活性基因8的体内。然而，我们不知道MSL复合体如何具体识别雄性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遗传筛选来寻找在雄性和雌性中都起作用的新的剂量补偿调节因子，从而鉴定了必不可少的锌指蛋白。在强大的初步数据的指导下，我们提出了以下用于识别协调调控基因的新机制：钳制和MSL复合体相互依赖，从而产生一个正反馈扩增系统，该系统从两倍的MRE序列的X浓缩中产生X特异性。这项工作的基本原理是，确定MSL复合体如何特定靶向X染色体将产生关键的洞察力，即如何识别基因，以便在亚核区域内进行协调调控。我们将通过三个具体目标来测试我们的新机制：1)我们将在体内和体外定义钳子结合的DNA序列要求；2)我们将寻找介导其与MSL复合体相互作用的钳子相互作用蛋白。同时，我们将定义一个以前从未研究过的必要转录调控因子的新相互作用伙伴；3)我们将建立CLAMP和MSL复合体在高亲和力位点相互依赖的作用机制。我们的研究具有重要意义，因为我们希望首次描述以前未知的机制，使MSL复合体能够识别其高亲和力位点，从而定义建立协调基因调控的关键第一步。确定夹子和MSL复合体共同作用以产生增强转录区域的新机制，可能会为如何识别基因以进行跨物种的协调调控提供关键的见解。 公共卫生相关性：我们建议的研究与公共健康相关，因为基因精确调控的缺失是包括自闭症10和慢性疾病11在内的大量疾病的基础。我们的研究解决了基因协调调控的关键初始步骤，确定了后续调控的目标基因。因此，我们的发现与美国国立卫生研究院的任务有关，即发展基础知识，为减轻疾病提供工具。

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.