Previous results from our laboratory and others provide evidence that a point-mutated, over-expressed Ha-ras gene is sufficient for the generation of benign papillomas in the mouse skin model. Our earlier observations of elevated Ha-ras message in papillomas led us to focus during the previous finding period on an investigation of the factors which regulate the expression of the mouse Ha-ras oncogene in epidermal cells at the transcriptional level. We have developed a successful transfection method for primary adult epidermal keratinocytes, which has allowed us to test the transcriptional machinery components in a cell relevant to our tumor model. We have used this transfection method along with deletion analysis to characterize a negative Cis-regulatory element in the mouse Ha-ras gene. DNase footprinting studies have delineated a GC-rich DNA fragment 1 kb upstream from the Ha-ras promoter, which is capable of shutting off expression from a heterologous promoter by nearly 100%. Results of gel retardation studies, DNase footprinting, and site-directed mutagenesis indicate the presence of one or more negative regulatory proteins (NRPs) in mouse epidermal cells which bind to the NRE. Supershift experiments with antiSp1 antibody have confirmed that Sp1 factor is one of the proteins binding to the NRE fragment but not the only one. Our studies have yielded evidence for the presence of positive regulatory elements as well, including a putative positive cis-element in Intron 0 of the Ha-ras gene which can upregulate Ha-ras expression 4-fold in transfected keratinocytes. In view of the fact that expression of the mouse Ha-ras gene is low in epidermis, while the cloned promoter region of the gene is as powerful as the most potent mammalian viral promoters studied thus far, we hypothesize that expression of the mouse Ha-ras gene is modulated by both negative and positive regulatory elements. We further hypothesize that expression of the Ha-ras gene in normal epidermis is predominantly under negative control, and that this control is lost or 'over-ridden' during tumorigenesis. Support for this hypothesis comes from recent gel mobility shift assays demonstrating that a specific DNA binding factor(s) present in nuclear extract from a normal skin cell line is missing from extracts of papilloma and carcinoma cell lines. In this revised application, we propose the following specific aims to test this hypothesis: l. To determine the specific DNA sequences in the NRE required for negative function, identify/isolate the NRPs and establish whether they are missing or defective in tumors using gel mobility shift experiments, transient gene expression assays and in vivo footprinting; 2. To clone the gene coding for the negative regulatory protein (NRP); 3. To extend our analysis of the positive regulatory element (PRE) in Intron 0 and other areas of the Ha-ras gene, by characterizing the DNA sequences responsible for enhancing Ha-ras expression, by identifying positive regulatory proteins (PRPs) in epidermis which bind to the PREs, and by establishing whether the PRPs in normal skin differ in tumor cells; 4. To investigate whether the interaction of positive and negative regulatory elements in the mouse Ha-ras gene serves to modulate Ha-ras expression in normal epidermal cells and in cells undergoing tumorigenesis.
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