Harvard Catalyst Profiles

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Knowledge of gene regulation in the nervous system has implications for understanding normal nervous system function as well as neurological disease.1,2. The activation of transcription has been shown to be an important level for regulating the expression of specific genes. This project aims to elucidate some of the mechanisms that control gene transcription in neurons of the motor system by identifying cis- and trans-acting genomic regulatory elements using genes that are well- characterized and neuronally expressed. The calcitonin gene related peptide (alpha- and beta-CGRP) genes code for two, nearly identical neuropeptides that are expressed differentially in the nervous system and found in diencephalon, sensory ganglia, motor neurons, at the neuromuscular junction and in the autonomic nervous system. Because they appear to be independently regulated and have highly divergent promoter sequences, they offer an excellent model system in which to explore the determinants of tissue-specific gene expression in the mammalian nervous system.

The regulatory role of 5' upstream sequences of calcitonin gene related peptide genes (alpha- and beta-CGRP) will be investigated by analyzing the expression of gene constructs introduced into cells after transient or stable DNA transfection. Promoter regions will be fused to readily assayable reporter genes encoding chloramphenicol acetyl transferase (CAT) or luciferase. Specific sequences that confer neural tissue-specificity of CGRP gene expression will be determined using reporter gene expression in selected cell lines and in primary culture. Similarly, sequences that transduce hormonal signals (such as glucocorticoids and agents which increase cAMP) which effect CGRP transcriptional rates will be evaluated for their ability to effect reporter gene expression. Deletion mapping of the upstream promoter will define functional regions that mediate tissue specificity and hormonal effects. In addition DNAse footprinting and methylation interference assays will be used to demonstrate protein-DNA interactions at specific sites important for gene regulation.

Finally, in later phases of the project, a molecular isolation and characterization of transacting regulatory factors (DNA binding proteins) will be attempted using a yeast expression system. Mobility shift assays will demonstrate specific protein-DNA interaction in nuclear extracts. The existence of possible repressor factors will be studied using in vivo competition studies.

Funded by the NIH National Center for Advancing Translational Sciences through its Clinical and Translational Science Awards Program, grant number UL1TR002541.