FUNCTIONAL GENOMICS OF HEARING

在这项应用中，我们建议使用微阵列技术来研究毛细胞特异性转录因子BRN-3.1控制的基因。传统的研究转录因子靶标的方法既耗时又费力。它也缺乏在转录因子的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条理想的路线。此外，人类和其他基因组计划以及微阵列技术的结合应该能揭示由BRN-3.1和其他转录因子控制的功能通路。我们会进行三项主要研究。第一种是基于寡核苷酸阵列技术的三种互补方法，分离受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将提供强大的BRN-3.1生产控制，并使我们能够调查BRN-3.1靶标的最大数量的人类基因(35,000个)。对Corti细胞系器官的转染可能会识别可能需要调节BRN-3.1的辅助因子的靶点。比较BRN-3.1基因敲除小鼠卵圆囊和对照的表达谱，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，以确保分离候选基因。第二个目的是系统地表征BRN-3.1对候选基因调控区的激活和结合。将使用聚类分析来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BRN-3.1结合位点的检查，以便出现更多候选基因。此外，还将检查与BRN-3.1靶标位于同一簇中的基因的5‘调控区，以确定其他重要转录因子的共同结合基序。在赠款的第三个目标中，将研究目标基因在正常和BRN-3.1基因敲除小鼠的毛细胞中的表达；以便提供它们的表达与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。该项目的成功将提供有关BRN-3.1控制的基因及其功能途径的信息，并将为研究由Math1等其他转录因子控制的毛细胞发育建立一个模型。

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.