TRANSCRIPTIONAL MECHANISMS REGULATING ACTIVITY DEPENDENT GENE EXPRESSION

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Nerve-elicited electrical activity is important for the formation of neural circuits during development and maturation of synaptic connections. These activity-dependent processes require the coupling of synaptic signals to selective changes in gene expression. We have used cerebellar granule cells and skeletal muscle as model systems to identify factors that mediate activity-transcription coupling. The electrophysiological properties of NMDA receptors (NR) are modified by a subunit switch that occurs when granule cells are innervated by glutamatergic mossy fibers, suggesting that activity and/or neurally-derived factors regulate the repression of NR2B expression and activation of the NR2C subunit gene. Using transgenic mice, we found that repression of the NR2B gene is conferred by 1.8 kb of 5'-flanking sequence. Regulatory elements were delineated further in transfected cultures of dissociated granule neurons; these cells develop functional synapses from 4-10 days in culture and repress NR2B expression in response to activity. A minimal NR2B promoter construct (-135/+15) that confers neural-specificity and activity-dependent repression was identified. This element is being used to identify transcription factors mediating the activity-dependent repression of the NR2B gene. In contrast to the NR2B subunit gene, NR2C expression increases after granule cell innervation. We found that activation of the NR2C gene requires 2 converging signals: neuregulin (Nrg) and activity through NMDA receptors. Nrg (also known as ARIA) is a neural factor that accumulates at the glutamatergic mossy fiber/granule cell synapse. We found that Nrg stimulates NR2C expression by >100-fold in cerebellar slice cultures. Addition of the activity inhibitors TTX (sodium channel blocker) or AP-5 (NR blocker) to the slices abolished the Nrg-dependent stimulation of NR2C; DNQX (an AMPA receptor blocker) had no effect. Consistent with these findings, we found that neuregulin receptors (erbB receptor tyrosine kinases) are expressed by granule cells prior to the NR2B/NR2C subunit switch. These results demonstrate that similar mechanisms and factors (i.e.. Nrg) are used to regulate receptor composition in muscle and the CNS during synaptogenesis. To understand how specific activity patterns regulate gene expression, we have studied how distinct depolarization patterns elicited by motoneurons differentially regulate transcription of either slow-or fast-contractile protein genes in skeletal muscle. Our studies on the muscle troponin I slow (TnIs) and fast (TnIf) genes, which are differentially stimulated by distinct depolarization frequencies (10 vs. 100 Hz, respectively), have focused on the identification of cis- and trans-acting factors that mediate the specific responses to activity patterns. We have identified a 128 bp slow upstream regulatory element (SURE) and a 144 bp fast intronic regulatory element (FIRE) that direct either slow- or fast-muscle-specific transcription in transgenic mice. Interestingly, the TnI SURE and FIRE have 4 common cis-acting elements: an A/T-rich sequence (binds MEF2), an E box (binds MyoD-related factors), a CACC box, and a novel motif (GCAGGCA) that we denoted the CAGG box. Electrophoretic mobility shift assays with muscle nuclear extracts demonstrate specific binding to these motifs. Functional studies performed in cultured myocytes and transgenic mice demonstrate that interaction of multiple protein-DNA complexes are necessary for enhancer function. Experiments are in progress to identify which of these elements and corresponding transcription factors mediate the frequency-specific response of the TnI genes, and may also participate in frequency-dependent regulation of neural genes.