FUNCTIONAL GENOMICS OF HEARING

在这个应用中，我们建议使用微阵列技术来研究由毛细胞特异性转录因子Brn-3.1控制的基因。传统的转录因子靶点研究方法既耗时又费力。它也缺乏在转录因子控制下同时分离多个基因的方法。微阵列方法为这样的任务提供了一个理想的途径。此外，人类和其他基因组计划的结合以及微阵列技术将有助于揭示由Brn-3.1和其他转录因子控制的功能途径。将进行三项主要研究。第一种是利用三种互补的方法，基于寡核苷酸阵列技术，分离Brn- 3.1控制的候选基因。四环素在人骨肉瘤细胞系中诱导Brn-3.1将提供对Brn-3.1产生的强大控制，并使我们能够调查最多数量的人类基因（35,000个）Brn-3.1靶点。转染Corti细胞系器官可能会发现可能需要Brn-3.1调控的辅助因子的靶标。通过比较Brn-3.1敲除小鼠胞囊与对照组的表达谱，可以揭示Brn-3.1直接和间接调控的靶基因。这些方法的结合将建立足够的冗余，以确保候选基因的分离。第二个目的是系统表征Brn- 3.1对候选基因调控区域的激活和结合。聚类分析将用于确定候选基因在发育过程中的表达模式。将对簇中的其他基因进行检查，以寻找Brn-3.1的结合位点，从而出现更多的候选基因。此外，还将研究与Brn- 3.1靶点在同一簇内的基因的5'调控区域，以确定其他重要转录因子的共享结合基序。拨款的第三个目的是研究靶基因在正常小鼠和Brn- 3.1基因敲除小鼠毛细胞中的表达；以提供它们的表达与Brn-3.1发病之间的因果关系。抗体将被用来定位毛细胞中的蛋白质。该项目的成功将为研究Brn-3.1控制的基因及其功能通路提供信息，并为研究Math1等其他转录因子控制的毛细胞发育建立模型。

In this application we propose to use microarray technology to study genes controlled by Brn-3.1, a hair-cell- specific transcription factor. The traditional way of studying transcription factor targets has been time consuming and laborious. It also lacks a means of simultaneously isolating multiple genes under the control of, a transcription factor. The microarray approach provides an ideal route for such a task. In addition the combination of human and other genome projects along with microarray technology should shed light on functional pathways controlled by Brn-3.1 and other transcription factors. Three major studies will be carried out. The first is to use three complementary approaches, based on the oligonucleotide array technology, to isolate candidate genes controlled by Brn- 3.1. The tetracycline induction of Brn-3.1 in a human osteosarcoma cell line will provide robust control of production of Brn-3.1, and allow us to survey the greatest number of human genes (35,000) for Brn-3.1 targets. The transfection of the organ of Corti cell line will likely identify the targets which may require the co-factors for Brn-3.1 regulation. The comparison of expression profiles of Brn-3.1 knock-out mouse utricles with control may reveal both direct and in-direct target genes regulated by Brn-3.1. The combination of these approaches will build enough redundancies to ensure the isolation of the candidate genes. The second aim is to systematically characterize the activation and binding on the regulatory regions of candidate genes by Brn- 3.1. Cluster analysis will be used to identify the expression pattern of candidate genes during development. Other genes in the clusters will be examined for the binding site of Brn-3.1 such that more candidate genes will emerge. In addition the 5' regulatory regions of the genes within the same cluster as Brn- 3.1 targets will be examined to identify the shared binding motifs for other important transcription factors. In the third aim of the grant the target genes will be studied in relation to their expression in the hair cell of normal and Brn- 3.1 knock-out mice; in order to provide the casual relation between their expression and the onset of Brn-3.1. Antibodies will be used to localized the proteins in the hair cells. The success of the project will provide information regarding genes and their functional pathways controlled by Brn-3.1, and should establish a model for studying hair cell development controlled by other transcription factors such as Math1.