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 i s 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.
