Regulation of normal cell proliferation in higher eucaryotes is strongly linked to expression of the c-myc proto-oncogene. Abnormal expression of the c-myc gene, in certain cases, appear to be associated with the process of tumorigenesis. Therefore, understanding how transcription of the c-myc gene is regulated will lead to a better understanding of the mechanisms controlling both normal and abnormal cell proliferation. Transcription of the murine c-myc gene is regulated in part by sequences that flank the major promoter (P2). These include enhancer and dehancer/repressor-like elements located at a distance from the c-myc transcription start site. Upstream promoter elements that are close to the start sit play an essential role in regulating transcription in most promoters. However, a detailed analysis of these upstream promoter elements in the P2 promoter of the c-myc gene has not been forthcoming. Here, an in vitro transcription system was used as a starting point in an analysis of the upstream promoter elements in the P2 promoter of the murine c-myc gene. It was found that sequences between -113 to -24 relative to P2 contain the information necessary for maximal transcription in vitro. Within this region are multiple protein binding sites i.e. ME1a, UBP and putative SP1). Optimal transcription in vitro appears to be dependent on the binding of at least some of these factors. The ME1a element alone can increase transcription initiation from P2 and appears to be a novel promoter element. Further, the binding of a protein(s) to the ME1a element is significantly increased in extracts from serum stimulated fribroblasts. This grant proposes to continue this analysis of the upstream promoter elements in the P2 promoter of the murine c-myc gene. The long term goal is to define and characterize the elements and factors that are essential for transcription in the specific region 113 bases 5' of P2. Towards this end two broad objectives are proposed. The first objective is to further define the cis-acting elements adjacent to P2 that are necessary for transcription in vitro and in vivo. The goal is to determine how transcription is dependent on the position, orientation and arrangement of the UBP, SP1 and ME1a elements. The second objective is to isolate, purify and characterize the ME1a binding protein, isolate its cognate cDNA and determine whether this protein acts as a transcription factor.
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