EXCEED THE SPACE PROVIDED. The objective of the proposed research is to understand the mechanisms by which neuronal activity regulates gene; expression. To achieve this goal we have characterized the signal transduction pathways by which Ca^+ influx through L-type voltage sensitive Ca^+ channels stimulates the transcription of two genes in neurons, the o-fos proto-oncogene and the gene encoding brain derived neurotrophic factor (BDNF). BDNF gene transcription is activated by Ca^+ influx into neurons, and like c-fos transcription BDNF transcription is controlled by the transcription factor CREB. The 40 kB BDNF gene gives rise to eight distinct mRNAs whose synthesis is driven by four separate promoters. Our studies during the previous funding period revealed that BDNF promoter III is the most Ca~+ responsive of the BDNF promoters.. A CREB binding site locaied at -35 nucleotides relative to the site of initiation of BDNF promoter III transcription is critical for Ca2+ induction of BDNF transcription. In addition, a second -60 CaRE within the BDNF promoter cooperates with the CREB binding site to confer a Ca^+ response. The -60 CaRE binds an apparently novel transcription factor that may confer neuronal specificity to the BDNF Ca^+ response. Despite progress towards defining the mechanisms by which Ca^ regulates c-fos and BDNF transcription, a great deal still remains to be learned. There are as yet uncharacterized phosphorylation events that appear to be critical for Ca2+ induction of CREB-dependent gene transcription, and several not yet well defined transcription factors that in addition to CREB are capable of conferring a Ca~+ response in neurons. These transcription factors include members of the MEF2 family, and the transcription factor that binds to the -60 CaRE of the BDNF gene-. We propose three specific aims to address these unresolved issues: 1) To characterize phosphorylation modifications of the CREB/CBP complex, that in addition to CREB Ser-133 phosphorylation play a critical role in mediating Ca^+ responses, 2) To purify, clone, and characterize the transcription factor(s) that binds the -60 CaRE within BDNF promoter III, 3) To characterize the mechanism(s) by which Ca2+ regulates MEF2 activity, and to establish the importance of MEF2 proteins as mediators of calcium's effects on gene expression. The proposed research will provide insight into how Ca^+ regulates gene transcription, and promotes critical cellularresponses that underlieneuronal differentiation, survival, and plasticity. PERFOFIMANCE SITE(S) (organization, dry, state) Children's Hospital Boston, Massachusetts KEY PERSONNEL ========================================Section End===========================================
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