A principal feature of keratinocytes governing their carcinogenic response to polycyclic aromatic hydrocarbons (PAHs) and related environmental agents, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is the expression of cytochrome P4501A1 (CYP1A1). This proposal explores the molecular basis for our finding that spontaneously immortalized rat epidermal cells silence the expression of CYP1A1 and 1B1 and thereby become insensitive to PAH toxicity. They have identified a novel negative regulatory DNA element (NeRD) present upstream of the rodent CYP1A1 gene which silences constitutive and inducible CYP1A1 gene expression. DNA binding analysis revealed a nuclear protein factor present in cells from rat, mouse, human and other species which specifically binds to the NeRD element. They hypothesize that silencing of the CYP1A1 gene in immortalized keratinocytes is mediated by postranslational modification of the NeRD binding protein and/or its interaction with another factor present in these cells. The presence of similar NeRD-binding proteins in all cells examined, including several of human origin, suggests that a similar silencing of gene expression could occur in other species and tissues as well. They propose to characterize the NeRD element and NeRD-binding proteins, focusing on those proteins from rat and human cells. Wild-type and mutant NeRD oligonucleotides will be used to examine the DNA sequence determinants necessary for DNA binding of the NeRD specific factor (using gel retardation analysis) as well as NeRD silencing activity (using transient transfection analysis). Competitive gel retardation analysis experiments will be used to determine the degree of similarity or difference between the NeRD element and previously identified silencer elements. The NeRD binding protein(s) will be characterized using a combination of DNA footprinting analysis, nucleotide modification techniques, UV and chemical cross-linking and southwestern blotting. The NeRD binding protein(s) will be purified from rat epidermal keratinocytes using a combination of conventional chromatographic and magnetic DNA recognition site affinity binding techniques and the purified proteins sequenced. Purified and partially purified NeRD-binding proteins will be used to develop monoclonal antibodies in mice. The results will provide a basis for understanding mechanism of silencer action and facilitate the future cloning and characterization of the NeRD-binding factor, examination of the mechanisms by which it negatively regulates gene expression, its tissue- and species-specific expression and determining the spectrum of genes it regulates.
