The manners in which p50 and Bcl-3 interact represent an important paradigm shift for the Re1/IkappaB system: Bcl-3 is a nuclear IkappaB- like protein that acts specifically upon p50 homodimers as an adaptor molecule in vivo and in vitro to synergistically promote transcription. Using biochemical and molecular approaches, in combination with a new retroviral system that can rapidly produce high titre helper-free infectious recombinant retroviral vectors for the introduction of effector transcription factor cDNAs and reporter constructs into cells, it is proposed to investigate the biochemical basis for the roles p50 and Bcl-3 play in HIV and bcl-3 gene regulation: A. Bcl-3 and p50 purified from mammalian cells will he used to study the biochemical basis for their interaction as co-activator molecules. Studies will be performed on how Bcl-3 changes the affinity of (p50)2 for different classes of NF-kappaB binding sites and will determine both in vivo and in vitro those sites that can bind (p50)2 in a transcriptionally -active conformation and those which support Bcl-3 --co-activation. Studies will be undertaken to localize the transcriptional activation region of Bcl-3 and understand the relationship of Bcl-3 co-activator activity to phosphorylation. B. Given the precedents for autoregulation of transcription factors we will investigate whether p50 and Bcl-3 participate in their own regulation. We will focus upon two NF-kappaB-like sites in the promoter of Bcl-3 to understand what part Bcl-3 and p50 have in basal and mitogen- induced expression of Bcl-3. C. We will develop mutants of p50 and Bcl-3 proteins--phenotypically selected within mammalian cells using retroviral libraries of randomly mutated cDNAs--that are deficient in key biochemical properties. Selected trans-dominant mutants will he retrovirally introduced to cells in studies of p50 and Bcl-3 activity in endogenous gene regulation, HIV expression, and on reporter constructs.
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