Transcriptional regulation by a novel deafness gene in the DFNA27 locus

DFNA27 基因座中新型耳聋基因的转录调控

• 批准号: 9513317
• 负责人: Botond Banfi
• 金额: $ 32.54万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9513317
• 关键词: ACTL6B gene; Affect; Alleles; Alternative Splicing; Audiology; Cell Survival; Cells; Chromatin Remodeling Factor; Clinics and Hospitals; Complex; Cultured Cells; DNA Binding; DNA-Binding Proteins; Data; Defect; Development; Diagnostic; Epigenetic Process; Equilibrium; Exons; Frequencies; Functional disorder; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genotype; Goals; Hair Cells; Hearing; Histone Deacetylase; Histone Deacetylase Inhibitor; Human; Impairment; Inner Hair Cells; Iowa; Knowledge; Labyrinth; Lead; Messenger RNA; Methods; Microarray Analysis; Molecular; Molecular Diagnosis; Mus; Mutant Strains Mice; Mutation; Neuroglia; Neurons; Organ; Organ of Corti; Outer Hair Cells; Pathologic; Patient Care; Patients; Pharmaceutical Preparations; Pharmacology; Pharmacotherapy; Phenotype; Preventive treatment; Public Health; RNA Splicing; Regulation; Repression; Research; Rest; Sensory Hair; Site; Terminator Codon; Testing; Transcriptional Regulation; Transgenic Mice; Universities; base; clinical care; deafness; diagnostic screening; drug efficacy; efficacy testing; equilibration disorder; gene function; gene repression; hearing impairment; improved; insight; mouse model; neuron development; novel; novel therapeutics; prevent; programs; promoter; recruit; relating to nervous system; screening program; transcription factor; transcriptome

Summary The identification and characterization of novel “deafness genes” have been a fruitful approach to gain insights into the molecular mechanisms underlying the development and function of hair cells (HCs). The identification of deafness-causing mutations is also a key step towards developing new therapies. However, the pathological mutations are typically found in genes whose products cannot be manipulated favorably with existing drugs. Our preliminary data suggest that a previously unidentified “deafness gene” in the DFNA27 locus is an exception. DFNA27 is associated with autosomal-dominant, progressive, non-syndromic hearing loss. One of the genes in this locus encodes REST, a DNA binding protein that represses the transcription of its target genes by recruiting histone deacetylases (HDACs). In non-neuronal cells, REST represses the expression of hundreds of neuronal genes. In postmitotic neurons, REST is itself repressed by at least two mechanisms: a reduction of transcription, and incorporation of an alternative exon that contains a STOP codon (exon-S) into the REST mRNA. The objective of the proposed research is to establish how REST function is affected by DFNA27, how REST is regulated in HCs, which genes it represses, and how responsive it is to drug treatment. Our central hypothesis is that alternative splicing of Rest is critical for the de-repression of a cascade of gene-expression changes in HCs, and that HDAC inhibitors can compensate for the DFNA27-caused defect in the alternative splicing-dependent inactivation of Rest in mouse models. This hypothesis is based on our preliminary studies showing that: 1) in mice heterozygous for the global deletion of exon-S (Rest+/dS), HCs degenerate; 2) in mice heterozygous for the HC-specific deletion of exon-S, HCs also degenerate; 3) Rest+/dS mice do not have any obvious defects other than deafness and impaired balance; 4) DFNA27 subjects harbor a mutation in REST; 5) in cultured cells, the DFNA27 mutation inhibits the alternative splicing-dependent inactivation of REST; 6) HDAC inhibitors prevent the loss of HCs in organ of Corti cultures of Rest+/dS mice; 7) impaired inactivation of Rest leads to reduced expression of the epigenetic regulator Baf53b; and 8) the lack of Baf53b causes hearing loss in mice. We propose 3 specific aims: 1) determine the contributions of alternative splicing and transcriptional regulation to the repression of Rest in HCs versus neurons; 2) determine the effects of HDAC inhibitors on the phenotype of Rest-mutant mice and evaluate the frequency of REST mutations in a diagnostic program for the hearing impaired; and 3) establish the effect of exon-S splicing on the HC transcriptome and establish the function of the Rest-repressed epigenetic regulator Baf53b in HCs. These aims will be achieved using a variety of methods ranging from transcriptome analysis to drug efficacy tests in mice. The proposed studies are significant because defining the effect of DFNA27 on the regulation and function of REST will reveal key features of the transcriptional program of differentiating HCs, and has the potential to lead to the development of a preventative treatment for DFNA27-associated hearing loss.

概括 新颖的“耳聋基因”的识别和表征是一种获得见解的富有成果的方法 进入毛细胞发育和功能（HCS）的分子机制。标识 引起死亡的突变也是发展新疗法的关键一步。但是，病理 突变通常是在无法用现有药物经常操作的基因中发现的。 我们的初步数据表明，DFNA27基因座中先前未识别的“耳聋基因”是一个 例外。 DFNA27与常染色体主导，进行性，非综合性听力损失有关。之一 该基因座中的基因编码REST，这是一种反映其靶标转录的DNA结合蛋白 通过募集组蛋白脱乙酰基酶（HDACS）的基因。在非神经元细胞中，休息抑制了 数百个神经元基因。在有丝分裂后神经元中，休息本身至少由两种机制反映：a 减少转录并掺入包含终止密码子（外显子S）的替代外显子 其余的mRNA。拟议研究的目的是确定REST功能如何受到 DFNA27，如何在HC中调节REST，其基因反映的基因以及对药物治疗的反应程度。 我们的核心假设是，休息的替代剪接对于去抑制至关重要 HCS的基因表达变化，HDAC抑制剂可以补偿DFNA27引起的缺陷 小鼠模型中休息的替代剪接依赖性灭活。这个假设是基于我们的 初步研究表明：1）在外显子S（REST+/DS）全球缺失的杂合中，HCS 退化; 2）在外显子S的HC特异性缺失的杂合子中，HC也退化； 3）休息+/ds 除了死亡和平衡受损之外，小鼠没有明显的缺陷。 4）DFNA27主题港口 休息中的突变； 5）在培养的细胞中，DFNA27突变抑制替代剪接依赖性的 休息的失活； 6）HDAC抑制剂可防止静脉+/ds小鼠的皮质培养物中HC的损失； 7） 休息失活受损会导致表观遗传调节剂BAF53B的表达降低； 8）缺乏 BAF53B导致小鼠听力损失。我们提出3个具体目的：1）确定替代方案的贡献 对HCS与神经元中休息表达的剪接和转录调节； 2）确定效果 HDAC抑制剂在静止小鼠的表型上的抑制剂，并评估A中休息突变的频率 听力障碍的诊断计划； 3）建立外显子剪接对HC的影响 转录组并建立HCS中剩余抑制的表观遗传调节剂BAF53B的功能。这些目标 将使用从转录组分析到小鼠药物效率测试的多种方法来实现。 提出的研究很重要，因为定义了DFNA27对调节和功能的影响 休息将揭示区分HCS的转录程序的关键特征，并有可能领导 开发针对DFNA27相关的听力损失的预防治疗。