Characterization of functional molecular domains of MeCP2

项目总结/摘要 Rett综合征，一种主要由基因突变引起的X连锁神经发育障碍 编码染色质调节剂甲基CpG结合蛋白2（MECP 2），是导致残疾的主要遗传原因 在世界各地的女孩。受影响的个体通常发展6-18个月的时间，此时疾病 导致发育倒退，失去有目的的手部动作，失去语言能力， 功能.目前的治疗选择仅限于对症治疗， 新的治疗方法至关重要。10%的致病MECP 2突变发生在其C- 末端域然而，尽管该区域的突变发生率很高，但很少有研究表明， 探讨其病理生理机制。此外，建议的RTT治疗策略 主要集中在补救功能丧失（LOF）突变发生在其他领域的MeCP 2- 甲基结合和NCoR相互作用结构域。以前的工作表明，C-末端可能会改变局部 染色质结合和构象，但不同类型的C-末端突变的彻底功能研究 缺乏。初步数据显示，一种最常见的C-末端突变，消除了 整个结构域，R294 X，在小鼠中产生截短的蛋白产物，随着年龄的增长而积累，并结合 与野生型MeCP 2相比，R294 X突变在染色质中的作用更紧密，这表明R294 X突变可能不是通过简单的 LOF。由此产生的假设是，一些C末端突变通过非LOF引起Rett综合征。 该建议旨在1）确定MeCP 2 C-末端对蛋白质功能的贡献 作为转录调节因子，2）阐明R294 X突变如何导致转录失调， 三个不同的大脑区域，和3）建立转基因互补的行为后果， R294 X等位基因。拟议的研究将涉及开发一种体外框架来探测C- 在染色质结合和共阻遏物相互作用的功能测定中， 使用分子和行为双重方法来剖析流行的致病机制， R294 X突变。这项研究将为复杂的神经发育障碍提供机制上的见解 Rett综合征，以及通过确定拟议治疗的可行性来协助临床决策， C-末端MECP 2突变的个体。拟议的研究将通过一个仔细的 精心设计的奖学金培训计划，包括发展申请人技术专长的机会， 批判性思维能力和科学沟通能力。此外，研究环境提供了轮流 由MSTP，范德比尔特脑研究所和儿科神经病学系是理想的建议， 研究和培训，拥有卓越的科学声誉、学院社区和强大的 注重学员发展。

PROJECT SUMMARY/ABSTRACT Rett syndrome, an X-linked neurodevelopmental disorder caused predominantly by mutations in the gene encoding chromatin modulator Methyl-CpG-binding protein 2 (MECP2), is a leading genetic cause of disability in girls worldwide. Affected individuals develop typically for a period of 6-18 months, at which time the disease causes developmental regression with loss of purposeful hand movements, loss of speech, and autistic features. Treatment options are currently limited to symptomatic management, making the development of novel therapeutic approaches critically important. 10% of disease-causing MECP2 mutations arise in its C- terminal domain. However, despite the high prevalence of mutations in this region, few studies have investigated their pathophysiological mechanism(s). Furthermore, proposed therapeutic strategies for RTT focus largely on remedying loss-of-function (LOF) mutations that occur in other domains of MeCP2 - the methyl-binding and NCoR-interaction domains. Previous work suggests that the C-terminus may alter local chromatin binding and conformation, but thorough functional studies of different types of C-terminal mutations are lacking. Preliminary data shows that one of the most common C-terminal mutations that eliminates the entire domain, R294X, yields a truncated protein product in mice that accumulates with age and binds chromatin more tightly than wild-type MeCP2, suggesting that the R294X mutation may not act through simple LOF. With the resulting hypothesis that some C-terminal mutations cause Rett syndrome via a non-LOF mechanism, this proposal aims to 1) determine the contributions of the MeCP2 C-terminus to protein function as a transcriptional modulator, 2) elucidate how the R294X mutation elicits transcriptional dysregulation in three different brain regions, and 3) establish the behavioral consequences of transgenic complementation of the R294X allele. The proposed studies will involve the development of an in vitro framework to probe C- terminal Mecp2 mutations in functional assays for chromatin binding and co-repressor interaction, as well as the use of dual molecular and behavioral approaches to dissect the pathogenic mechanism of the prevalent R294X mutation. This research will provide mechanistic insight into the complex neurodevelopmental disorder Rett syndrome, as well as assist clinical decision-making by identifying the viability of proposed therapies for individuals with C-terminal MECP2 mutations. The proposed research will be accomplished through a carefully crafted fellowship training plan that involves opportunities to develop the applicant's technical expertise, critical-thinking skills, and scientific communication skills. Further, the research environment provided in turns by the MSTP, Vanderbilt Brain Institute, and Department of Pediatric Neurology is ideal for the proposed research and training, harboring a reputation for scientific excellence, a collegial community, and a strong focus on trainee development.