The primary aim of our research is to define the molecular mechanisms by which the Rous sarcoma virus (RSV) long terminal repeat (LTR) enhancer activates transcription in a wide variety of eukaryotic cells. We have chosen to study the RSV LTR because this retrovirus appears to have been extremely adept at coopting cellular transcriptional machinery to promote its own expression. The enhancer and promotor elements located in the U3 region of the RSV LTR are among the strongest transactivating sequences identified in eukaryotic cells. Their potency and widespread function in many different cell types suggests that the transcription factors mediating LTR enhancer/promotor function will be essential proteins that normally play critical roles in regulating cellular gene expression. Consequently, understanding LTR enhancer function may provide valuable insights into the complex transcriptional regulatory network governing cell growth and/or differentiation that when altered, leads to neoplastic transformation. The molecular picture of the RSV LTR enhancer that has emerged from our studies over the past four years certainly supports this prediction. The major players mediating LTR enhancer function are a ubiquitously expressed, multi-component CCAAT-binding complex (CBC), the serum response factor (SRF), one or more members of the C/EBP family of transcription factors, and Myc, most likely with its partner Max. We propose that the LTR enhancer can be viewed as a bipartite structure. Transactivation is mediated by the distal one/third of the enhancer which binds C/EBP-related factors or Myc/Max, depending upon the relative balance of these transcription factors in the host cell. The CCAAT- binding complex and SRF, binding to the proximal two/thirds of the enhancer, create a specific protein-DNA architecture which conducive to high level enhancer function because it brings the more distally bound transactivators and basal transcription initiation machinery into close proximity. Research designed to test the validity of this view will be focused on (1) defining the protein composition of the multi-component CBC through reconstitution with recombinant proteins, (2) characterizing the RNA and DNA binding properties of a putative member of this complex, EFI/A, as well as pursuing state-of-the-art biophysical techniques to analyze the protein-DNA structure created when two CBCs and SRF bind to the proximal segment of the LTR enhancer in vitro, (3) positive identification of the C/EBP-related EFII factors via microprotein sequencing, including confirmation of preliminary data suggesting EFIIa is LIP and (4) further analysis of Myc and Max binding to the EFII cis element in vitro and potentially, in vivo. Information gained in these areas will provide a better understanding of how these various factors work together to achieve very high levels of transcriptional activation mediated by the LTR enhancer. At the same time, valuable information on the function of several critical regulators of cellular gene expression can be expected.
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