reproduced verbatim): The N-methyl-D-aspartate subunit 1 (NR1) is a pivotal component of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. NR1 expression in developing neurons ensures function of the NMDA receptor in brain development. The functionality of NR1 gene expression has been examined by the investigator via isolation of a 5.4-kb promoter and analysis of several putative cis-acting elements within the proximal 919-bp sequence. The long term goal of this study is to elucidate the genetic mechanism of NR1 expression and to genetically modulate NMDA receptor strength in order to improve physiological function of the receptor and reduce its pathological effects. The immediate goal is to search for crucial cis-elements in the 5.4-kb promoter that interact with trans-factors and govern transcription activation of the NR1 gene in neuronal differentiation. The present application aims 1) to assess the contribution of promoter activation to induced NR1 gene expression in cells undergoing neuronal differentiation and 2) to define cis-elements regulating activation of the NR1 promoter in neuronal differentiation. Our strategy combines technologies in molecular biology, cell biology, and biochemistry to investigate transcriptional activation of the NR1 gene and define crucial cis-elements in the promoter. Our approach for aim 1 is to apply nuclear run-on assays to directly evaluate the role of transcription rate in neurogenic induction and differentiation-mediated increase in NR1 gene expression;
for aim 2 to define functional cis-elements using a reporter gene and DNA-protein interaction studies in cells undergoing neuronal differentiation. In general, active cis-elements will be screened out by their ability to drive the reporter transgene and then pinpointed by DNA footprinting. Functionality of the cis-elements will be further confirmed by modifications of the pinpointed sequences. Identification of critical cis-elements for NR1 gene transcription in neuronal differentiation will significantly help us to understand the genetic mechanism of NR1 gene expression and ultimately provide us targets for pharmacological intervention.
