The long term objective of our research is to understand the molecular mechanisms by which growth promoting signals at the cell surface lead to alterations in gene expression in the nucleus. We are focusing on the transcriptional regulatory sequences located in the long terminal repeat (LTR) of the avian retrovirus, Rous sarcoma virus (RSV). These sequences contain a strong enhancer and promotor that are constitutively active in many cell types; however, the transcriptional activity of the RSV LTR is rapidly induced 3 to 5-fold when quiescent cells re exposed to serum or individual growth factors such as EGF. We wish to establish the molecular basis for the rapid responsiveness of the RSV LTR to mitogenic stimulation. Two CArG boxes are present within the 200 bp LTR enhancer and promotor. This sequence motif has been shown to be a pivotal cis acting element mediating the rapid transcriptional induction of a family of """"""""early response"""""""" genes, including cfos, Egr1, Egr2 and cytoskeletal actin. Multiple nuclear proteins have been shown to interact with the CArG box upstream of the cfos promotor, including the well characterized SRF. However, the molecular details by which the CArG box responds to mitogenic stimulation are not known. We have found that the promotor-proximal CArG box (called EFIIIa) in the RSV LTR does not respond rapidly to EGF or other mitogens, despite the fact that it binds the SRF with high affinity. We have identified a new nuclear protein, EFIV, which binds with high affinity to cfos SRE but not EFIIIa DNA. A high affinity binding site for EFIV is also present adjacent to the second CArG box (EFIIb) in the LTR enhancer. We plan to determine if the second LTR CArG box, in conjunction with the EFIV binding site, mediates the rapid transcriptional response of the LTR to mitogenic stimulation. The involvement of other LTR sequences will also be ascertained. We plan to examine the possible role of EFIV in the rapid activation of both the LTR and cfos SRE. This will be in conjunction with our efforts to characterize the molecular mechanisms responsible for the rapid induction by EGF and other mitogens of specific LTR cis elements and their cognate transcription factors. Once the transcription factor(s) which are nuclear target(s) for mitogenic signaling pathways have been identified, we will analyze the immediate post receptor events required to elicit the activation of these specific transcription factor(s) upon exposure of cells to polypeptide mitogens such as EGF. Transcription of early response genes is one of the earliest known nuclear responses to growth factor treatment, and it is thought that many of the products of early response genes regulate subsequent transcriptional events which are necessary to commit cells to traverse the cell cycle and divide. Consequently, elucidating the mechanisms responsible for the transcriptional induction of early response genes is critical to understanding the pathways regulating proliferation in normal cells and the defects in this regulatory network which result in neoplastic transformation.
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