The main research interest of this laboratory is the neuroimmune modulation of proopiomelanocortin (POMC) gene transcription in pituitary, lymphocytes and macrophages. In this proposal we will focus on a single transcriptional regulator (PO-B) that we have recently discovered that contributes to 70% of POMC basal transcription, suggesting a major role for this protein in the control of POMC gene expression. The PO-B DNA binding element is critically positioned between the POMC TATA box and CAP site which we believe is necessary for its efficient effects on transcriptional induction. Furthermore, the PO-B DNA binding site is highly homologous to elements required for viral and interferon induction of transcription of other cellular genes, suggesting a role for PO-B in immune signal transduction pathways. Our experiments will address the hypothesis that PO-B is involved in neuroimmune signal transduction pathways influencing POMC transcription by virtue of its critical position in the POMC promoter. 1) We will biochemically characterize and purify PO-B and determine its relationship to other transcription factors. PO-B antibodies will be raised for use as probes for in vitro and tissue distribution studies. 2) Immune regulation of POMC transcription in pituitary, lymphocytes and macrophages will be examined. The role of PO-B in these responses will be determined by transient transfection assays. 3) We will determine if PO-B binds specifically to the elements required for viral and interferon induction of gene transcription. 4) We will use oligonucleotide substitution mutational analysis to determine if the PO-B element effects on transcription are position- and orientation-dependent. 5) We will determine if PO-B DNA interactions in in vitro represent the interactions of PO-B with the integrated POMC promoter in vivo. We will determine if immune-induced changes in POMC transcription are paralleled by changes in vivo PO-B DNA interactions. 6) We will extend our previous in vitro transcription studies to design optimal assay conditions such that the effects of purified PO-B on transcription can be observed. The effects of immune regulators on the transcriptional activity of cellular extracts will be determined and the role of PO-B in these changes will be assessed.
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| Dobrenski, A F; Zeft, A S; Wellstein, A et al. (1993) DNA binding of the transcription factor PO-B is regulated during differentiation of HL-60 cells. Cell Growth Differ 4:647-56 |
| Wellstein, A; Dobrenski, A F; Radonovich, M N et al. (1991) Purification of PO-B, a protein that has increased affinity for the pro-opiomelanocortin gene promoter after dephosphorylation. J Biol Chem 266:12234-41 |
| Riegel, A T; Lu, Y; Remenick, J et al. (1991) Proopiomelanocortin gene promoter elements required for constitutive and glucocorticoid-repressed transcription. Mol Endocrinol 5:1973-82 |
