TRANSCRIPTIONAL MECHANISMS REGULATING ACTIVITY DEPENDENT GENE EXPRESSION

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The ability of the nervous system to be remodeled with experience, and of skeletal muscles to adapt to different environmental demands, results from the phenotypic changes of these cells in response to neural activity during maturation. These activity-dependent processes often require the coupling of synaptic signals to selective changes in gene expression. We have used the regulation of NMDA receptors in the cerebellum and of the contractile genes encoding muscle troponins, as model systems to identify the molecular mechanisms that mediate activity-transcription coupling in these tissues. The most obvious developmental change in NMDA receptor (NR) kinetics occurs in the cerebellum, which results from a switch in the heteromeric subunit composition of the receptor as granule cells are innervated by mossy fiber inputs. We found that neural activity down-regulates NR2B transcription through a 150 bp sequence flanking the basal promoter. In order to understand the cellular and molecular mechanisms that upregulate the NR2C subunit during the maturation of mossy fiber/granule cell synapses and to assess the function of this subunit, we engineered a knock-in mouse where the NR2C gene was replaced by the Lac Z reporter using homologous recombination. Using co-culture experiments of pontine neurons with cerebellar slices from heterozygote NR2C/beta-gal knock-in mice, we found that the mossy fiber inputs are necessary to activation expression of the NR2C gene. This is consistent with our previous data showing that the factor Nrg-1 plus the neurotransmitter glutamate, which are released from the presynaptic mossy fiber terminals and activate ErbB and NMDA receptors, are necessary to trigger NR2C expression. We have also shown that the co-signaling via these 2 receptors may occur at synapses because NMDA and ErbB receptors are enriched at postsynaptic densities (PSD) where they interact with the same proteins harboring PDZ protein- protein interaction domains. The PDZ-domain proteins are important because they scaffold receptors and channels to signaling molecules, thus coupling synaptic activity to signaling cascades in the postsynaptic neurons. The slow- and fast-twitch properties of muscles are largely determined by distinct patterns of depolarization, which selectively regulate the transcription of contractile genes. To identify the pathways that couple specific patterns of activity to selective changes in gene expression, using trangenic mice we have isolated the shortest enhancers known to direct fiber- type-specific transcription of troponin I genes in either slow- or fast-twitch muscles. We have identified 4 conserved cis-acting elements in both enhancers: 3 of these previously known motifs (E box, MEF-2 site and CACC) are necessary to direct muscle-specific transcription in all muscles, but the fourth motif, which is novel, is necessary to direct fiber- type-specificity. Experiments are in progress to identify the transcription factors binding this site. Considering the extraordinary evolutionary conservation of transcription factor pathways used to execute epigenetic and genetic regulatory programs, as exemplified by the basic/helix-loop- helix (b/HLH) factors in neural and muscle commitment, our long-term goal is to determine if the molecular mechanisms that couple activity to transcription are also conserved during maturation of these two plastic cell types.