NEURONAL TRANSFORMATION BY INDUCING NEURON-SPECIFIC TRANSCRIPTION FACTORS.

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We have investigated the molecular mechanism controlling specifity and variability of neurons in expect of its application for the therapy of neurodegenerative disorders in the future. At the first step, we focused on transcriptional regulation of the D1A dopamine receptor gene as a representative molecule which characterixes a subset of neurons and on the POU transcription factor gene family that are expressed widely but specifically in the central nervous system. The upstream genomic fragment of the D1A receptor gene was subcloned into a CAT reporter plasmid, deleted to various length by using PCR,and used for the analyzes on cis-element. We co-transfected Brn-4, a member of the POU gene family, and observed its transcriptional activation. Through this assay, a Brn-4 responsive element was found in the first intron of the D1A receptor gene which contained two consensus sequences for binding of POU factors. Gel mobility shift assays with recombinant GST-Brn-4 protein confirmed that th … More ese motifs were scrual binding sites. We then tested whether Brn-4 and D1A colocalizes in the striatal neurons by in situ hybridization, and found that these two molecules exist in a similar group of neurons. These results suggest that Brn-4 actually acts on its responsive element of the D1A dopamine receptor gene in vivo, and thereby influence on its transcriptional regulation. Furthermore, we found that POU family members differentially act on the same element. Oct2 and Brn-4 positively regulate transcription of the D1A gene, whereas Brn-2 Oct-3 and Oct-6 not. When Oct-6 was co-transfected with Brn-4, it copetitively inhibited transactivation by Brn-4. These results may suggest that some co-transcription factors which bind to specifiic POU members and modify their transactivation. We also found that, in a D1A-negative neuronal cell line, some repressing transcription factors act on the upstream of the D1A receptor gene. We are now trying to clone the unknown factors.These results obtained in this year showed a molecular relationship between POU transcription factors and D1A receptor, a neuronal gene which defines the specificity of neruons. In the next step, we wish to regulate the specific character of neurons by transfecting transcription factors or by other experimental approaches. Less