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正常细胞功能很重要，在疾病中经常被破坏。凝聚素是进化上保守的多亚基蛋白质复合体，在细胞分裂过程中对染色体凝聚和分离是必不可少的，并在转录调节和DNA修复中发挥关键作用。大多数生物含有两种类型的凝集素，它们执行不同的功能，并定位于不同的染色体区域。线虫含有第三种凝集素，它特异性地与X染色体转录结合并抑制X染色体转录，以在剂量补偿复合体(DCC)内完成剂量补偿。目前尚不清楚凝聚素是如何针对其结合部位的，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结合的机制和线虫的功能来解决我们知识中的这一重大差距。线虫DCC是研究凝集素结合和功能的一个很好的实验模型。DCC被特异性地招募到X染色体上，并抑制XX两性体中两条X染色体的转录，以使X-连锁转录本的总体水平与XO雄性的转录水平持平。DCC首先针对X特定的招募站点，然后扩散到物理连接的染色质上。DCC结合位点中约90%位于X染色体上，大部分位于活性基因启动子上。DCC结合与抑制没有直接关系，因此DCC可能对基因进行远程调控。在我们对DCC机制的理解中，重要的空白是DCC结合和转录在局部和全球水平上的关系，以及DCC扩散到染色体的分子机制。本应用程序的目的是1)测试DCC结合是否充分和抑制所需，2)确定H4K20me1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结合位点缺失和X染色体复制的影响来实现这些目标，并分别使用CHIP-SEQ、RNA-SEQ和4C-SEQ来测量DCC在全基因组的定位、基因表达和染色体结构。在我们的项目完成后，我们将对一个长期存在的问题有更好的理解：凝集素是如何与染色体结合并影响其结构和功能的。这与健康有关，因为染色体结构和凝集素功能对于适当的染色体分离、转录和DNA修复至关重要，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.