Defining a mechanism for targeting the X-chromosome during dosage compensation

描述（由申请人提供）：在异配子物种中，雄性和雌性之间的性连锁基因数量不平衡。因此，性别之间以及性染色体和常染色体之间的转录平衡至关重要，这一过程称为剂量补偿。虽然不同的剂量补偿机制已经进化，但它们都有一个共同的初始步骤，即区分X染色体和常染色体。模式生物黑腹果蝇（Drosophila melanogaster）已经进化出一种单步剂量补偿机制，为研究单个染色体如何与基因组的其他部分区分开来提供了一个简单的模型。在果蝇中，单个雄性X染色体的选择性两倍上调使两性之间的转录平衡。X染色体的选择性鉴定涉及核糖核蛋白复合物的募集，即仅在雄性中表达的雄性特异性致死（MSL）。MSL复合物最初定位于X染色体上的“种子位点”内的富含GA的序列，称为MSL识别元件（MRE）。然而，MSL复合物不能直接与MRE结合，这些序列分布在整个基因组中。Larschan实验室最近发现了一种以前未研究过的锌指蛋白，MSL蛋白的染色质连接接头（CLAMP），它将MSL复合物与MRE序列连接起来。此外，CLAMP在X染色体上高度富集，独立于MSL复合体，因此可能参与X鉴定的最早步骤。因此，CLAMP的鉴定提供了第一次机会来定义X染色体特征，从而促进其用于剂量补偿的鉴定。基于强有力的初步数据，我假设X染色体的鉴定需要初级基因组序列和由CLAMP蛋白介导的高阶核组织的贡献。我将通过定义线性和三维基因组组织在X染色体上富集CLAMP中的作用来验证我的假设。首先，使用染色质免疫沉淀- qPCR（ChIP-qPCR），我将确定是否富集的CLAMP的X染色体上发生协同或相加的串联MRE序列的数量增加。其次，我将确定是否假定的初始种子网站集群在三维核空间中使用染色质相互作用分析配对末端标签测序（ChIA-pet）方法。拟议的实验将使用最先进的方法，以提供关键的洞察如何实现X染色体的初始识别。

DESCRIPTION (provided by applicant): In heterogametic species there is an imbalance in the number of sex-linked genes between males and females. Therefore, it is essential that transcription is equalized between the sexes and between the sex chromosomes and autosomes, a process called dosage compensation. Though different dosage compensation mechanisms have evolved, they all share a common initial step of distinguishing the X-chromosome from the autosomes. The model organism Drosophila melanogaster has evolved a single-step mechanism for dosage compensation, providing a simple model for studying how a single chromosome is discriminated from the rest of the genome. In Drosophila, selective two-fold upregulation of the single male X-chromosome equalizes transcription between the sexes. The selective identification of the X-chromosome involves the recruitment of a ribonucleoprotein complex, Male-Specific Lethal (MSL) that is expressed only in males. The MSL complex initially localizes to GA-rich sequences within "seed sites" on the X-chromosome, termed MSL Recognition Elements (MREs). However, MSL complex cannot bind directly to MREs and these sequences are distributed throughout the genome. The Larschan laboratory has recently identified a previously unstudied zinc finger protein, Chromatin-Linked Adapter for MSL Proteins (CLAMP), that links the MSL complex to MRE sequences. Furthermore, CLAMP is highly enriched on the X-chromosome independent of MSL complex and therefore is likely to be involved in the earliest step of X-identification. Therefore, the identification of CLAMP provides the first opportunity to define the X-chromosome features that promote its identification for dosage compensation. Based on strong preliminary data, I hypothesize that identification of the X-chromosome requires contributions from primary genome sequence and higher-order nuclear organization mediated by the CLAMP protein. I will test my hypothesis by defining the role of linear and three-dimensional genomic organization in enrichment of CLAMP on the X-chromosome. First, using Chromatin Immunoprecipitation- qPCR (ChIP-qPCR), I will determine whether enrichment of CLAMP on the X-chromosome occurs cooperatively or additively as the number of tandem MRE sequences increases. Second, I will determine if putative initial seed sites cluster together in three-dimensional nuclear space using the Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-pet) method. The proposed experiments will use state-of-the- art approaches to provide key insight into how initial identification of the X-chromosome is achieved.