Serotonin (5-hydroxytryptamine, 5-HT) and its receptors have been implicated in the etiology and therapeutic treatment of mental depression. An understanding of 5-HT receptor gene expression is fundamentally important for 5-HT mediated neural function and will provide insights into the molecular basis of mental depression. In the previous funding period a NF-AT like transcription factor complex, factor b, important for human 5-HT2A receptor (5-HT2AR) gene expression was identified. NF-AT is a key transcription factor for interleukin-2 production in activated T-cells. The objective of this project is to further characterize and clone the cDNA(s) encoding factor b.
Specific aims are as follows: 1. To further define the DNA binding sequence of factor b complex and its role in regulating transcription of the 5-HT2AR gene by gel retardation and promotor activity assays. 2. The interaction between factor b complex and transcription factor Sp1 will be studied in order to determine if factor b complex and Sp1 binding are independent, mutually exclusive, or synergistic. DNase 1 footprinting and promotor activity assays in human neuroblastoma (SHSY-5Y) cells will be performed. The interaction between transcription factors will also be studied by in vivo genomic footprinting. 3. UV cross-linking will be performed to determine whether factor b complex consists of a single or multiple polypeptide(s). Their immuno-reactive properties will be studied using NF45 and NF90 polyclonal antibodies. 4. To identify the cDNA(s) encoding polypeptide(s) with factor b activity. If factor b complex is composed of one polypeptide, its cDNA will be cloned by screening an expression library. If factor b complex is composed of two or more subunits, they will be purified by DNA affinity chromatography. Degenerate oligonucleotides will then be designed based on amino acid sequence(s) and used as probes. 5. To characterize and investigate the validity of the cDNA(s) encoding factor b. The validity of isolated factor b cDNA clone(s) will be studied by observing if the expressed polypeptides will (a) bind to the 0.35 kb fragment and (b) stimulate promotor activity of co-transfected 0.35 kb (or 0.70 kb) construct in NCI-H460 cells (lacking factor b activity and 5-HT2AR expression). The tissue distribution of factor b will be studied by Northern analysis. Factor b polypeptide(s) will also be expressed in resting Jurkat cells to see if it can stimulate IL-2 production.
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