Mechanisms of X chromosome dosage compensation in C. elegans

描述（由申请人提供）：染色体结构的调节对于正常细胞功能很重要，并且在疾病中经常被破坏。凝聚素是一种进化上保守的多亚基蛋白复合物，在细胞分裂过程中对染色体的凝聚和分离至关重要，并在转录调控和DNA修复中发挥关键作用。大多数生物体含有两种类型的凝聚素，它们执行不同的功能并定位于不同的染色体区域。C.线虫含有第三种类型的凝聚素，其特异性结合并抑制X染色体转录以在剂量补偿复合物（DCC）内实现剂量补偿。目前尚不清楚凝聚素如何靶向其结合位点，以及它们如何调节染色体结构和转录。我们的目标是通过确定DCC在C.优雅C.秀丽线虫DCC是研究凝聚素结合和功能的极好实验模型。DCC被特异性地募集到X染色体上，并抑制XX两性体中两条X染色体的转录，以使总体X连锁转录物水平与XO男性的水平相等。DCC首先靶向X特异性募集位点，然后扩散到物理连接的染色质上。铺展产生了沿着X的~90%的DCC结合位点。这些位点大多位于活性基因启动子处。DCC的结合和阻遏之间没有直接的关系，因此DCC可能在长距离上调节基因。我们对DCC机制的理解中的重要差距是DCC结合和转录在局部和全局水平上的关系，以及DCC扩散到染色体上的分子机制。本申请的目的是1）测试DCC结合是否足够并且是阻遏所需的，2）确定H4 K20 me 1在DCC结合中的作用，3）确定DCC如何影响X的染色体结构。我们将通过分析异位DCC结合、DCC结合位点缺失、X染色体重复的影响来实现这些目标，并分别使用ChIP-seq、RNA-seq和4C-seq在全基因组范围内测量DCC定位、基因表达和染色体结构。在我们的项目完成后，我们将更好地了解一个长期存在的问题：凝聚素如何与染色体结合并影响其结构和功能。这与健康有关，因为染色体结构和凝聚素功能对于正确的染色体分离、转录和DNA修复是必不可少的，这些过程在癌症和发育疾病中被破坏。

DESCRIPTION (provided by applicant): Regulation of chromosome structure is important for normal cell function and is often disrupted in disease. Condensins are evolutionarily conserved, multi-subunit protein complexes that are essential for chromosome condensation and segregation during cell division and play key roles in transcription regulation and DNA repair. Most organisms contain two types of condensins, which perform different functions and localize to different chromosomal regions. C. elegans contains a third type of condensin that specifically binds to and represses X chromosome transcription to accomplish dosage compensation within the dosage compensation complex (DCC). It is not known how condensins are targeted to their binding sites, and how they regulate chromosome structure and transcription. Our goal is to address this significant gap in our knowledge by determining the mechanism of DCC binding and function in C. elegans. C. elegans DCC serves as an excellent experimental model to study condensin binding and function. DCC is recruited specifically to the X chromosome and represses transcription of both X chromosomes in XX hermaphrodites to equalize overall X-linked transcript levels to that of XO males. DCC is first targeted to X- specific recruitment site, and then spreads onto physically connected chromatin. Spreading gives rise to ~90% of the DCC binding sites along the X. Majority of these sites are at active gene promoters. There is no direct relationship between DCC binding and repression, thus DCC may regulate genes at long-range. Important gaps in our understanding of DCC mechanism is the relation between DCC binding and transcription at the local and global level, and the molecular mechanism by which the DCC spreads onto the chromosome. The objective of this application is to 1) test if DCC binding is sufficient and required for repression, 2) determine the role of H4K20me1 in DCC binding 3) determine how the DCC affects chromosome structure of the X. We will accomplish these objectives by analyzing the effects of ectopic DCC binding, DCC binding site deletions, X chromosome duplications and measure DCC localization, gene expression and chromosome structure genome-wide using ChIP-seq, RNA-seq and 4C-seq, respectively. At the completion of our project we will have a better understanding of a persistent question: how do condensins bind to chromosomes and affect its structure and function. This is relevant to health, because chromosome structure and condensin function is essential for proper chromosome segregation, transcription, and DNA repair, processes that are disrupted in cancers and developmental diseases.