Establishing coordinate gene regulation during Drosophila dosage compensation

描述（由申请人提供）：所有生物必须精确调节其基因以正常发育和预防疾病状态。然而，基因组间存在显著的基因拷贝数变异1；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识别和共同调节。果蝇，像哺乳动物一样，在单个雄性x染色体的长度上增加了大量不同调控基因的转录水平，相对于每个雌性x染色体精确地增加了两倍。本应用的目的是了解果蝇的剂量补偿是如何建立的，这是调控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；它首先通过顺式作用的DNA序列和X非编码RNA成分的共转录招募来识别X染色体，然后扩散到活性基因的体内。然而，我们不知道MSL复合物是如何特异性识别男性X染色体上的MSL识别元件（MRE）序列的，因为已知的MSL成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方法来寻找在男性和女性中都起作用的剂量补偿的新调节因子，从而确定了必需的CLAMP锌指蛋白。在强有力的初步数据的指导下，我们提出了以下鉴定协调调控基因的新机制：CLAMP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MRE序列的两倍X富集中产生X特异性。这项工作的基本原理是，确定MSL复合体如何特异性靶向x染色体，将对亚核结构域内如何识别协调调节的基因产生关键见解。我们将通过三个具体目标来测试我们的新机制：1)我们将定义体内和体外CLAMP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基本转录调控因子的新的相互作用伙伴；3)我们将建立CLAMP和MSL复合体在高亲和位点相互依赖的作用机制。我们提出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，从而确定建立协调基因调控的关键第一步。定义CLAMP和MSL复合物共同作用产生增强转录域的新机制可能为了解基因如何被识别为跨物种协调调节提供关键见解。

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.