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.

总结 新的“耳聋基因”的鉴定和表征是获得洞察力的富有成效的方法 毛细胞（HCs）发育和功能的分子机制。识别 基因突变也是开发新疗法的关键一步。然而，病理 突变通常存在于基因中，其产物无法用现有药物进行有效操纵。 我们的初步数据表明，DFNA 27位点上一个先前未被识别的“耳聋基因”是一个与耳聋相关的基因。 例外. DFNA 27与常染色体显性、进行性、非综合征性听力损失相关。之一 该基因座中的基因编码REST，REST是一种抑制其靶基因转录的DNA结合蛋白 组蛋白去乙酰化酶（HDAC）。在非神经元细胞中，REST抑制 数百个神经元基因在有丝分裂后的神经元中，REST本身受到至少两种机制的抑制： 减少转录，并将含有终止密码子的选择性外显子（外显子-S）掺入 REST mRNA拟议研究的目标是确定REST功能如何受到以下因素的影响 DFNA 27，REST如何在HC中调节，它抑制哪些基因，以及它对药物治疗的反应如何。 我们的中心假设是，Rest的选择性剪接对于一个级联反应的去抑制是至关重要的。 基因表达的变化，HDAC抑制剂可以弥补DFNA 27引起的缺陷， 小鼠模型中Rest的选择性剪接依赖性失活。这个假设是基于我们的 初步研究表明：1）在外显子-S整体缺失（Rest+/dS）杂合子小鼠中，HC 简并; 2）在HC特异性缺失外显子S的杂合小鼠中，HC也简并; 3）Rest+/dS 小鼠除了耳聋和平衡受损外没有任何明显的缺陷; 4）DFNA 27受试者具有 REST突变; 5）在培养的细胞中，DFNA 27突变抑制了选择性剪接依赖性的 REST的失活; 6）HDAC抑制剂防止Rest+/dS小鼠的Corti器官培养物中HC的损失; 7） Rest失活受损导致表观遗传调节因子Baf 53 b表达减少;和8）缺乏 Baf 53 b导致小鼠听力损失。我们提出了3个具体目标：1）确定备选方案的贡献 剪接和转录调控对HCs和神经元中Rest抑制的影响; 2）确定HCs和神经元中Rest抑制的影响 HDAC抑制剂对REST突变小鼠表型的影响，并评估在一个受试者中REST突变的频率。 3）确定外显子S剪接对HC的影响 转录组，并建立休息阻遏表观遗传调节Baf 53 b在HC中的功能。这些目标 将使用从转录组分析到小鼠药物功效测试的各种方法来实现。 所提出的研究是重要的，因为确定DFNA 27对调节和功能的影响， REST将揭示分化HC的转录程序的关键特征，并有可能导致 开发DFNA 27相关听力损失的预防性治疗。