This proposal is focused on the molecular mechanisms underlying transcriptional silencing of the platelet-derived growth factor (PDGF) A-chain gene. Polypeptide growth factors (GFs) such as PDGF, EGF and TGFalpha, among many others, are important positive growth effectors in malignant cells, while others such as TGFbeta have been implicated as inhibitory regulators. However, very little is known about the mechanisms through which GF genes are repressed, nor about the mechanisms through which control of GF gene transcription is subverted in cancer. We have identified multiple silencer elements in the 5'-flanking region of the A-chain gene, one of which was localized to a 33 bp sequence and denoted the 5'-S1 nuclease-hypersensitive (5'SHS) silencer. Two 5'SHS-binding factors were identified as NM23-H1 and NM23-H2 (H1 and H2), proteins implicated previously in suppression of metastasis in breast cancer and melanoma. We have also shown that H1 and H2 expression is required for A-chain silencing, and that DNA-binding is required for their silencing and growth-suppressing activities. We have also observed binding of a number of other protein species (p97, p87, p70, p44/48) to the 5'SHS silencer element. These findings appear to place this project in an excellent position to study molecular mechanisms of transcriptional silencing in a GF gene model. The linkage between NM23s and silencer function also provides a foundation for study of mechanisms through which NM23s mediate transcriptional repression and thereby suppress tumor progression. The proposed studies will test the hypothesis that binding of NM23 and other 5'SHS-binding proteins is critical to function of the structurally related 5'SHS and intSHS silencers of the A-chain gene. In addition, we will determine the extent to which DNA-binding and structure-modifying activities of H1 and H2 are required for silencing and growth-suppression. DNA structure will be assessed by nuclease- and chemical-hypersensitivity assays, while DNA-protein interactions will be characterized by electrophoretic mobility shift assay (EMSA), Southwestern blot and DNA footprinting. Silencer function will be determined by transient transfection analysis in a panel of tumor cell lines that exhibit a range of A-chain silencer activities and expression levels of H1 and H2. Together, the proposed studies should provide important new insights into the mechanisms through which precise control of GF gene transcription is compromised in cancer.
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