The human immunodeficiency virus (HIV‐1) long terminal repeat (LTR) contains two binding sites for NF‐kappa B in close proximity to three binding sites for the constitutive transcription factor, Sp1. Previously, stimulation of the HIV enhancer in response to mitogens has been attributed to the binding of NF‐kappa B to the viral enhancer. In this report, we show that the binding of NF‐kappa B is not by itself sufficient to induce HIV gene expression. Instead, a protein‐protein interaction must occur between NF‐kappa B and Sp1 bound to an adjacent site. Cooperativity both in DNA binding and in transcriptional activation of NF‐kappa B and Sp1 was confirmed by electrophoretic mobility shift gel analysis, DNase footprinting, chemical cross‐linking and transfection studies in vivo. With a heterologous promoter, we find that the interaction of NF‐kappa B with Sp1 is dependent on orientation and position, and is not observed with other elements, including GATA, CCAAT or octamer. An increase in the spacing between the kappa B and Sp1 elements virtually abolishes this functional interaction, which is not restored when these sites are brought back into the same helical position. Several other promoters regulated by NF‐kappa B also contain kappa B in proximity to Sp1 binding sites. These findings suggest that an interaction between NF‐kappa B and Sp1 is required for inducible HIV‐1 gene expression and may serve as a regulatory mechanism to activate specific viral and cellular genes.

Induction of human immunodeficiency virus type 1 (HIV-1) gene expression in stimulated T cells has been attributed to the activation of the transcription factor NF-KB. The twice-repeated KB sites within the HIV-1 long terminal repeat are in close proximity to three binding sites for Spl. We have previously shown that a cooperative interaction of NF-KB with Spl is required for the efficient stimulation of HIV-1 transcription. In this report, we define the domains of each protein responsible for this effect. Although the transactivation domains seemed likely to mediate this interaction, we find, surprisingly, that this interaction occurs through the putative DNA-binding domains of both proteins. Spl specifically interacted with the amino-terminal region of ReIA(p65). Similarly, RelA bound directly to the zinc finger region of Spl. This interaction was specific and resulted in cooperative DNA binding to the icB and Spl sites in the HIV-1 long terminal repeat. Furthermore, the amino-terminal region of RelA did not associate with several other transcription factors, including MyoD, E12, or Koxl5, another zinc finger protein. These findings suggest that the juxtaposition of DNA-binding sites promotes a specific protein interaction between the DNA-binding regions of these transcription factors. This interaction is required for HIV transcriptional activation and may provide a mechanism to allow for selective activation of KB-regulated genes.

Vascular cell adhesion molecule 1 (VCAM-1) is expressed in both endothelial and epithelial cell types, where it contributes to lymphocyte migration to sites of inflammation. Its expression is regulated by cytokines, in part through two KB-like regulatory elements. Because NF-KB can be composed of multiple alternative subunits with differential effects on gene expression, the role of different specific NF-KB family members subunits in VCAM-1 regulation is unknown. In this report, we define the contribution of different NF-KB family members to VCAM-1 gene regulation. We show that both KB sites in the VCAM-1 enhancer are required to optimally stimulate gene expression, but the enhancer is differentially regulated by specific combinations of NF-cB subunits. At low concentrations, RelA(p65) acted in concert with the -50-kDa product of p105 NF-KB, NF-KBI(p5O), to stimulate transcription, and at high concentrations, RelA(p65) alone stimulated the VCAM-1 promoter. In contrast, NF-KB2 inhibited functional activation of the VCAM reporter by p65. Consistent with this finding, an additional binding complex was detected by using recombinant NF-KB2(p49)/RelA(p65) with radiolabeled VCAM KB site probes. Interestingly, the human immunodeficiency virus enhancer responded differently to stimulation by NF-KB subunits, with optimal response to p49(100)/p65. Analysis of NF-KB mRNA in human umbilical vein endothelial cells revealed that nflcbl, nflcb2, and reL4 NF-KB but not c-rel were induced by tumor necrosis factor alpha and lipopolysaccharide, which also induce VCAM-1. These data suggest that specific subunits of NF-KB regulate VCAM-1 and differentially activate other genes in these cells.