The major focus of research in my laboratory is transcriptional control of the pro-opiomelanocortin (POMC) gene. In particular we are interested in transcriptional factors that mediate the effects of stress and neuroimmune modulation on the POMC gene. We have previously described a transcriptional activator of POMC, designated PO-B, that interacts with the POMC promoter. PO-B represents the first defined transcriptional activator of POMC whose function has been verified by mutational analysis. Mutation of the PO-B binding site decreases basal transcription by 70% indicating a major role for this protein in POMC regulatory mechanisms. However, we do not know the physiological or pharmacological agents that regulate signal transduction pathways involving PO-B. Towards this goal we have recently purified PO-B to more than 90% homogeneity from HeLa cells. Interestingly deophosphorylation of the purified protein leads to a more 30-fold increase in DNA binding affinity. In this proposal we wish to rapidly extend these early observations by obtaining a full length cDNA clone of the PO-B gene. This is a defined project of limited duration that will enhance our knowledge of POMC transcriptional regulation and be a useful resource for other work ongoing in the laboratory. Since this laboratory was the first to identify and purify PO-B we would also like to obtain the full length clone in a timely fashion.
The specific aims are: 1) To obtain reliable N-terminal and internal amino acid sequence data from purified PO-B. """"""""Best-guess' oligonucleotide probes will be designed from these sequences. Specific PO-B cDNA sequences will be amplified with PCR from a human cDNA library using pairs of these oligonucleotides representing N-terminal and internal sequence. Amplified fragments will be recovered and sequenced to determine if they code for PO-B amino acid sequence. If this approach is unsuccessful, or only N-terminal sequence is obtained, we will use conventional screening of a HeLa lambda gt10 cDNA library identifying positive PO-B cDNA clones by hybridization with the 'best- guess' oligonucleotide probes. 2) PCR and conventional screening methods are not always successful approaches to identifying correct cDNA clones. Therefore, in parallel with the above experiments, we will also use expression screening. This method does not rely on obtaining any amino acid sequence from PO-B. For these studies we will screen a human fibroblast plasmid cDNA library that can be transiently expressed in mammalian cells. The identity of the full length clone will be confirmed by specific binding of the in vitro translated product to the PO-B cognate DNA binding element. The in vivo activity of the full length clone will be confirmed by the ability of transfected PO-B expression vectors to induce transcription from promoters harboring the PO-B binding site. If progress on PO-B cloning is rapid, we will examine its precise role in POMC transcription. We will perform mutational analysis of the protein to determine which domains are required for DNA binding, phosphorylation status and transcriptional activation. It is expected that these experiments will yield valuable information on the signal transduction pathways in which PO-B is involved.
| Lu, Y; Riegel, A T (1994) The human DNA-binding protein, PO-GA, is homologous to the large subunit of mouse replication factor C: regulation by alternate 3' processing of mRNA. Gene 145:261-5 |
| Lu, Y; Zeft, A S; Riegel, A T (1993) Cloning and expression of a novel human DNA binding protein, PO-GA. Biochem Biophys Res Commun 193:779-86 |
