Transcriptional regulation by a novel deafness gene in the DFNA27 locus

摘要 鉴定和表征新的“耳聋基因”是获得洞察力的卓有成效的方法。 研究毛细胞(HC)发育和功能的分子机制。身份识别 导致耳聋的突变也是开发新疗法的关键一步。然而，病态的 突变通常在基因中发现，其产物不能与现有药物进行有利的操作。 我们的初步数据表明，DFNA27基因座上一个以前未被识别的“耳聋基因”是一种 例外情况。DFNA27与常染色体显性遗传性、进行性、非综合征性听力损失有关。其中之一 该基因座的基因编码REST，这是一种DNA结合蛋白，抑制其靶标的转录 通过招募组蛋白脱乙酰酶(HDAC)来表达基因。在非神经细胞中，REST抑制了 成百上千个神经基因。在有丝分裂后神经元中，REST本身至少受到两种机制的抑制：a 转录减少，并将含有终止密码子的替代外显子(外显子-S)加入到 其余的mrna。拟议研究的目标是确定以下因素对静息功能的影响 DFNA27，REST在HCS中是如何调控的，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的另一种拼接对于抑制一系列 Hcs基因表达的变化，以及HDAC抑制剂可以弥补DFNA27引起的Hcs缺陷 小鼠模型中REST的选择性剪接依赖失活。这一假设是基于我们的 初步研究表明：1)在外显子-S(REST+/DS)杂合性缺失的小鼠中， 退化；2)外显子-S HC特异性缺失杂合子小鼠，Hcs也退化；3)REST+/DS 除耳聋和平衡受损外，小鼠没有任何明显的缺陷；4)DFNA27受试者 5)在培养细胞中，DFNA27突变抑制依赖选择性剪接的 REST失活；6)HDAC抑制剂防止REST+/DS小鼠Corti培养器官中HCS的丢失；7) REST的受损失活导致表观遗传调节因子Baf53b表达减少；以及8)缺乏 Baf53b会导致小鼠听力损失。我们提出了三个具体目标：1)确定备选方案的贡献 剪接和转录调控对Hcs对神经元REST的抑制；2)决定其影响 HDAC抑制剂对REST突变小鼠表型的影响，并评估REST突变的频率 3)建立外显子-S剪接对HC的影响 转录组，并建立休止抑制的表观遗传调控因子Baf53b在HCS中的功能。这些目标 将使用从转录组分析到小鼠药效测试的各种方法来实现。 拟议的研究具有重要意义，因为定义了DFNA27对调节和功能的影响 REST将揭示区分HCS的转录程序的关键特征，并有可能引领 对DFNA27相关性听力损失的预防性治疗的开发。

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.