DNA element downstream of the κB site in the Lcn2 promoter is required for transcriptional activation by IκBζ and NF-κB p50

The nuclear protein I kappa B zeta activates transcription of a subset of NF-kappa B-dependent innate immune genes such as Lcn2 encoding the antibacterial protein lipocalin-2. I kappa B zeta functions as a coactivator via its interaction with NF-kappa B p50, which contains a DNA-binding Rel-homology domain but lacks a transcriptional activation domain. However cis-regulatory elements involved in I kappa B zeta function have remained unknown. Here, we show that, although I kappa B zeta by itself is unable to associate with the Lcn2 promoter, I kappa B zeta interacts with the promoter via p50 binding to the NF-kappa B-binding site (kappa B site) and the interaction also requires the pyrimidine-rich site (CCCCTC) that localizes seven bases downstream of the kappa B site. The pyrimidine-rich site is also essential for I kappa B zeta-mediated activation of the Lcn2 gene. Introduction of both sites into an I kappa B zeta-independent gene culminates in I kappa B zeta-p50-DNA complex formation and transcriptional activation. Furthermore, spacing between the two sites is crucial for both I kappa B zeta-DNA interaction and I kappa B zeta-mediated gene activation. Thus, the pyrimidine-rich I kappa B zeta-responsive site plays an essential role in productive interaction of I kappa B zeta with the p50-DNA complex.