The role of CLAMP, a novel zinc-finger protein, in Drosophila dosage compensation

说明（由申请人提供）：人类和果蝇雄性都必须进行剂量补偿，以平衡XX雌性和XY雄性之间的x染色体剂量。尽管这些物种使用不同的方法进行剂量补偿，但对X染色体与常染色体进行后续调节的共同第一步仍然知之甚少。果蝇提供了一个理想的系统来研究X染色体是如何被特异性识别的，因为沿X染色体的所有活性基因都是目标，并且有大量的遗传、生化和基因组方法可用。在雄性果蝇中，x连锁基因的转录增加了两倍，这一过程由MSL（雄性特异性致死）复合体介导，该复合体特异性地识别并沿着雄性x染色体传播。然而，由蛋白质和RNA组成的MSL复合体并不直接与DNA相互作用，其特异性靶向男性x染色体的机制尚不清楚。最近发现的锌指蛋白CLAMP （MSL蛋白偶联致死适配器）通过与MSL复合体和称为MREs （MSL识别元件）的短DNA序列结合，将MSL复合体招募到x染色体上。因此，CLAMP的鉴定为了解X鉴定的初始阶段和随后的动态复杂传播提供了关键机会。该项目的第一个目标是确定MSL复合体如何特异性识别果蝇雄性x染色体上的基因。我们的目标是通过诱导MSL在雌性果蝇细胞中的表达（通常不存在MSL）来捕捉MSL复合物沿着X轴传播的动态，从而创建一个“伪雄性”系统。然后，我们将分析MSL复合体在不同时间点沿x染色体扩散，这代表了一个太快的过程，无法在男性细胞中实时捕获。通过这些实验，我们希望回答一个关键问题：CLAMP在功能上将MSL复合体与x染色体连接起来的机制是什么？我们的第二个目标是确定CLAMP蛋白的非性别特异性功能，它与组蛋白位点体（HLB）的已知成分共定位。我们将在多种体内系统中使用显微镜以及基于细胞的报告器来确定这种特定的CLAMP染色质定位在男性和女性中的功能。然后，我们将使用候选RNAi筛选来确定参与CLAMP招募的其他因素。我们希望解决关键问题：非性别特异性的CLAMP定位在染色质上的功能是什么？

DESCRIPTION (provided by applicant): Both human and Drosophila males must dosage compensate to equalize X-chromosome dosage between XX females and XY males. Although these species use diverse approaches for dosage compensation, the common first step by which the X chromosome is specifically distinguished from the autosomes for subsequent regulation remains poorly understood. Drosophila provides an ideal system in which to study how the X- chromosome is specifically identified because all of the active genes along the X-chromosome are targeted and a large number of genetic, biochemical, and genomic approaches are available. In Drosophila males, transcription of X-linked genes is increased two-fold, a process mediated by the MSL (Male Specific Lethal) complex, which specifically identifies and spreads along the male X-chromosome. However, the MSL complex, comprised of protein and RNA components, does not directly interact with DNA, and the mechanism by which it is specifically targeted to the male X-chromosome remains unknown. A recently identified zinc-finger protein, CLAMP (Coupling Lethal Adapter for MSL Proteins), recruits MSL complex to the X-chromosome by associating with both the complex and short DNA sequences called MREs (MSL Recognition Elements). Therefore, the identification of CLAMP provides a key opportunity to understand the initial stages of X identification and subsequent dynamic complex spreading. The first goal of this project is to determine how MSL complex specifically recognizes genes on the Drosophila male X-chromosome. We aim to capture the dynamics of MSL complex spreading along the X by inducing MSL expression in female Drosophila cells, where it is normally not present, creating a "pseudo-male" system. We will then assay MSL complex spreading along the X-chromosome at various time points, representing a process too rapid to capture in male cells in real-time. Via these experiments we hope to answer the key question: What is the mechanism by which CLAMP functionally links the MSL complex to the X-chromosome? Our second goal is to determine the non sex-specific function of the CLAMP protein, which colocalizes with known components of the Histone Locus Body (HLB). We will use microscopy in a variety of in vivo systems as well as a cell-based reporter to determine the function of this specific CLAMP chromatin localization in both males and females. We will then use a candidate RNAi screen to identify other factors involved in CLAMP recruitment. We hope to address the key question: What is the function of non sex-specific CLAMP localization on the chromatin?