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

描述（由申请方提供）：人类和果蝇雄性必须进行剂量补偿，以使XX雌性和XY雄性之间的X染色体剂量相等。虽然这些物种使用不同的方法进行剂量补偿，但X染色体与常染色体特异性区分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如何特异性地鉴定X染色体，因为沿着X染色体沿着的所有活性基因都是靶向的，并且大量的遗传学、生物化学和基因组方法是可用的。在果蝇雄性中，X连锁基因的转录增加两倍，这是由MSL（雄性特异性致死）复合体介导的过程，MSL复合体特异性地识别并沿沿着雄性X染色体传播。然而，由蛋白质和RNA成分组成的MSL复合物不直接与DNA相互作用，并且其特异性靶向男性X染色体的机制仍然未知。最近发现的一种锌指蛋白CLAMP（Coupling Lethal Adapter for MSL Proteins）通过与称为MRE（MSL Recognition Elements）的复杂和短DNA序列结合将MSL复合物募集到X染色体上。因此，CLAMP的识别为理解X识别的初始阶段和随后的动态复杂扩展提供了关键机会。该项目的第一个目标是确定MSL复合物如何特异性识别果蝇雄性X染色体上的基因。我们的目标是捕捉动态的MSL复合物传播沿着X诱导MSL表达在雌性果蝇细胞，在那里它通常是不存在的，创造一个“假男性”系统。然后，我们将在不同的时间点测定沿着X染色体扩散的MSL复合物，这代表了一个太快而无法在雄性细胞中实时捕获的过程。通过这些实验，我们希望回答关键问题：CLAMP功能性地将MSL复合物连接到X染色体的机制是什么？我们的第二个目标是确定CLAMP蛋白的非性别特异性功能，该蛋白与组蛋白基因座体（HLB）的已知组分共定位。我们将在各种体内系统中使用显微镜以及基于细胞的报告，以确定这种特定的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。我们希望解决的关键问题：什么是非性别特异性的CLAMP定位在染色质上的功能？

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?