FMR1 protein and chromatin insulators mediate chromatin function and genome integrity

FMR1 蛋白和染色质绝缘体介导染色质功能和基因组完整性

• 批准号: 9019715
• 负责人: Mariano Labrador-San Jose
• 金额: $ 22.65万
• 依托单位: UNIVERSITY OF TENNESSEE KNOXVILLE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9019715
• 关键词: Address; Adult; Affect; Alleles; Attention; Behavior; Binding; Biochemical; Biological Models; Cell Nucleus; Cells; Chromatin; Chromatin Loop; Chromatin Structure; Chromosomes; Circadian Rhythms; Co-Immunoprecipitations; Complex; Coupled; Courtship; DNA; DNA Damage; DNA Repair; DNA biosynthesis; Data; Development; Drosophila genus; Dysmorphology; Elements; Embryonic Development; Enhancers; Eukaryota; Exhibits; FMR1; Face; Foundations; Fragile X Syndrome; Future; Genes; Genetic; Genetic Transcription; Genetic Translation; Genome; Gypsies; Health; Histones; Hyperactive behavior; Immunofluorescence Microscopy; Intellectual functioning disability; Investigation; Laboratories; Link; Locomotion; Mammals; Mediating; Memory; Messenger RNA; Modeling; Modification; Molecular; Molecular Biology; Molecular Genetics; Mutation; Neurons; Nuclear; Organism; Orthologous Gene; Output; Pathway interactions; Patients; Phenotype; Physiology; Play; Point Mutation; Property; Proteins; Publishing; RNA; RNA Binding; RNA Recognition Motif; RNA Sequences; RNA immunoprecipitation sequencing; RNA-Binding Proteins; Regulation; Resolution; Retrotransposon; Role; Single-Gene Defect; Sleep Disorders; Stem cells; Stress; Study models; Suppressor Genes; Synapses; Techniques; Testing; Therapeutic; Tissue Differentiation; Transcriptional Regulation; Translations; Wing; Work; autistic behaviour; base; cognitive ability; dFMR1 gene; fly; genetic manipulation; genome integrity; in vivo; insight; loss of function; loss of function mutation; male; mutant; novel; promoter; protein complex; protein function; relating to nervous system; research study; response; stem; success

 DESCRIPTION (provided by applicant): Fragile X syndrome (FXS) occurs through mutation of the FMR1 gene and is a common form of heritable intellectual disability. The FMR1 protein (FMRP) modulates neuronal function via binding and regulation of select RNA molecules. Recent studies show that FMRP function is more complex and includes roles in mediating a replication stress-induced DNA damage response (DDR) via direct binding to chromatin. The interaction of FMRP with chromatin comes from its ability to bind a subset of methylated histones via the function of its tandem Agenet domain, but more detailed mechanisms by which FMRP interacts with chromatin to modulate the DDR, whether there are additional functions for chromatin-associated FMRP, and the impact these properties have on behavior phenotypes of FXS are very much unknown. To conduct the proposed studies, we use the fruit fly D. melanogaster as a model, exploiting the ability to visualize chromatin at high resolution in the form of polytene chromosomes, the amenability of the organism to molecular genetic manipulations, and the fact that fruit flies have an ortholog of FMR1 (dfmr1; dFMRP) which shares all known functional domains with its mammalian counterparts. Moreover, flies mutant for dfmr1 exhibit behavior phenotypes with significant parallels to those observed in FXS patients. In the first Aim of this proposal, we will explore a connection between chromatin insulators and dFMRP. Recent work from my laboratory has expanded insulator function to include modulating changes in chromatin structure necessary for DNA replication and repair, and in response to exogenous stresses. These results suggest a functional link between chromatin insulators and dFMRP that may further explain how FMRP exerts an effect on chromatin. We have performed experiments to test whether dfmr1 and the insulator protein-encoding gene suppressor of Hairy wing [su(Hw)] function in common pathways, and find that a loss-of-function mutation of dfmr1 can suppress the wing margin development phenotype of ct6, a mutation mediated by the binding of Su(Hw) in the insulator sequences of a gypsy transposon inserted between the promoter and wing margin enhancer of the cut (ct) gene. This result is supported by finding that dFMRP and Su(Hw) protein co-localize on polytene chromosomes. We will characterize this interaction to discern the mechanism(s) by which dFMRP suppresses the ct6 phenotype, using existing alleles of dfmr1 that selectively disrupt chromatin or RNA binding domains of dFMRP. We expect that these studies will illuminate novel mechanisms by which dFMRP regulates chromatin function. In the second Aim, we address a connection between the chromatin-binding Agenet domain of dFMRP and behavior phenotypes of FXS, using techniques for controlling temporal and spatial expression of dfmr1, and the robust memory and circadian locomotion phenotypes of dfmr1 mutants that have parallels with the behavior deficits of FXS patients. The studies in this proposal may give insights into mechanisms of FMRP nuclear function that are necessary to develop additional therapeutic avenues for FXS.

 描述(由申请人提供)：脆性X综合征(FXS)通过FMR1基因突变而发生，是一种常见的遗传性智力残疾。FMR1蛋白(FMRP)通过结合和调节特定的RNA分子来调节神经元的功能。最近的研究表明，FMRP的功能更加复杂，包括通过直接与染色质结合来介导复制应激诱导的DNA损伤反应(DDR)。FMRP与染色质的相互作用来自于它通过其串联Agenet结构域与甲基化的组蛋白子集结合的能力，但FMRP与染色质相互作用调节DDR的更详细机制，是否有额外的功能与染色质相关的FMRP，以及这些特性对FXS行为表型的影响是非常未知的。为了进行拟议的研究，我们以黑腹果蝇为模型，利用其以多线染色体的形式以高分辨率显示染色质的能力，有机体对分子遗传操作的适应性，以及果蝇具有FMR1(dfmr1；dFMRP)的同源基因这一事实，该同源基因与哺乳动物的同行共享所有已知的功能域。此外，dfmr1突变的果蝇表现出与FXS患者显著相似的行为表型。在这项提议的第一个目标中，我们将探索染色质绝缘体和dFMRP之间的联系。我的实验室最近的工作扩展了绝缘体的功能，包括调节DNA复制和修复所需的染色质结构的变化，以及对外部压力的反应。这些结果表明染色质绝缘体和dFMRP之间存在功能联系，这可能进一步解释FMRP如何对染色质产生影响。我们已经进行了实验，以测试dfmr1和毛翼绝缘蛋白编码基因抑制子[su(Hw)]是否在共同的途径中发挥作用，并发现dfmr1的功能缺失突变可以抑制CT6的翼缘发育表型，这是一种突变，由插入在Cut(Ct)基因的启动子和翼缘增强子之间的吉普赛转座子的绝缘子序列中的SU(HW)结合介导。这一结果得到了dFMRP和SU(HW)蛋白在多线染色体上共定位的发现的支持。我们将利用dfmr1的现有等位基因选择性地破坏dFMRP的染色质或RNA结合域，来表征这种相互作用，以了解dFMRP抑制CT6表型的机制(S)。我们期望这些研究将阐明dFMRP调节染色质功能的新机制。在第二个目标中，我们利用控制dfmr1的时间和空间表达的技术，以及与FXS患者行为缺陷相似的dfmr1突变体的健壮记忆和昼夜运动表型，解决了dFMRP的染色质结合Agenet结构域与FXS行为表型之间的联系。这项提案中的研究可能会对FMRP核功能的机制提供见解，这些机制对于开发FXS的其他治疗途径是必要的。