The enzyme GTP cyclohydrolase I (GTPCH) catalyzes the first and limiting step in the synthesis of tetrahydrobiopterin (BH4), the required cofactor for monoamine (MA) neurotransmitter production. BH4 availability is a control point for MA synthesis and is regulated by changes in GTPCH gene expression. GTPCH gene expression is controlled by signal transduction pathways that converge on the GTPCH promoter. Some of these signaling pathways utilize the second messenger cAMP and protein kinase A (PKA). In order to better understand how cAMP and PKA control GTPCH gene transcription we have cloned, sequenced and begun to characterize 5.8 kb of the rat GTPCH promoter. We have focused on the cis-acting CRE and adjacent CCAAT-box elements within the GTPCH core promoter because we have already shown them to be critical for basal and cAMP-dependent transcription. The overall goal of this application is to firmly establish that the trans-acting factors ATF-4 and NF-Y are bound by these DNA response elements and, through the actions of PKA, serve to mediate the cAMP-dependent enhancement of GTPCH transcription that we have observed in PC12 cells.
Specific Aim I will use recombinant ATF-4 and NF-Y, EMSA, supershift, and in vitro and in vivo DNA footprinting techniques to analyze the protein complexes recruited by the CRE and CCAAT-box elements.
This Aim will also investigate the spatial relationship between the CRE and CCAAT-box as well as the role of the non-canonical TATA-box in transcription. Specific i Aim 2 will use TPCH promoter reporter constructs combined with foreed expression of wild type and dominant negative forms of ATF-4 and NF-Y to test the hypothesis that these proteins and the eo-activator CBP play an essential role in basal and cAMP-dependent transcription. Specifie Aim 3 will use GTPCH promoter reporter eonstruets along with foreed expression of wild type and dominant negative subunits of PKA, a peptide inhibitor of PKA and a PKA-defieient eell line to establish the role of PKA in the control of transeription.
This Aim will also investigate whether ATF-4 and NF-Y are phosphorylated in vitro and in vivo by PKA. This researeh is important because dopamine (DA) synthesis by human nigrostriatal neurons is selectively vulnerable to genetic mutations in GTPCH that produce hereditary progressive dystonia (HPD), a disorder of movement that primarily effects young girls. Knowledge of how eAMP-eoupled signal transduetion pathways eontrol GTPCH gene transeription within DA neurons is therefore critical to our understanding of the underlying causes of the HPD phenotype and possibly other illnesses that involve nigrostriatal DA neurons, such as Parkinson's Disease.
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