Dynamic Protein Associations Define Two Phases of IL-1β Transcriptional Activation

IL-1beta is a key proinflammatory cytokine with roles in multiple diseases. Monocytes package the IL-1beta promoter into a "poised architecture" characterized by a histone-free transcription start site and constitutive transcription factor associations. Upon LPS stimulation, multiple proteins inducibly associate with the IL-1beta gene. To understand how the complex combination of constitutive and inducible transcription factors activate the IL-1beta gene from a poised structure, we measured temporal changes in NF-kappaB and IFN regulatory factor (IRF) association with IL-1beta regulatory elements. Association of the p65 subunit of NF-kappaB peaks 30-60 min post-monocyte stimulation, and it shortly precedes IRF-4 recruitment to the IL-1beta enhancer and maximal mRNA production. In contrast, IRF-8/enhancer association decreases poststimulation. To test the importance of delayed IRF-4/enhancer association, we introduced a mutated PU.1 protein shown to prevent PU.1-mediated IRF-4 recruitment to the enhancer sequence. Mutated PU.1 initially increased IL-1beta mRNA followed by decreased mRNA levels 2-3 h poststimulation. Taken together, these data support a dynamic model of IL-1beta transcriptional activation in which a combination of IRF-8 and p65 drives the initial phase of IL-1beta transcription, while PU.1-mediated IRF-4 recruitment to the enhancer is important for the second phase. We further demonstrate that activation of both NF-kappaB and IRF-4 depends on CK2 kinase activity. Because IRF-4/enhancer association requires CK2 but not p65 activation, we conclude that CK2 triggers the IRF-4 and p65 pathways independently to serve as a master regulator of IL-1beta transcription.