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Dynamic Protein Associations Define Two Phases of IL-1β Transcriptional Activation¹

Yue Zhang^*, Simona Saccani, Hyunjin Shin^* and Barbara S. Nikolajczyk^2,*

^* Department of Microbiology, Boston University School of Medicine, Boston, MA 02118; and Department of Cellular and Molecular Immunology, Max-Planck Institute of Immunobiology, Freiburg, Germany

IL-1β is a key proinflammatory cytokine with roles in multiple diseases. Monocytes package the IL-1β 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-1β gene. To understand how the complex combination of constitutive and inducible transcription factors activate the IL-1β gene from a poised structure, we measured temporal changes in NF-B and IFN regulatory factor (IRF) association with IL-1β regulatory elements. Association of the p65 subunit of NF-B peaks 30–60 min post-monocyte stimulation, and it shortly precedes IRF-4 recruitment to the IL-1β 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-1β mRNA followed by decreased mRNA levels 2–3 h poststimulation. Taken together, these data support a dynamic model of IL-1β transcriptional activation in which a combination of IRF-8 and p65 drives the initial phase of IL-1β 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-B 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-1β transcription.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was funded by R01 AI54611 and an American Diabetes Association Research Grant to B.S.N.

² Address correspondence and reprint requests to Dr. Barbara S. Nikolajczk, Department of Microbiology, Boston University School of Medicine, 715 Albany Street L516, Boston, MA 02118. E-mail address: bnikol{at}bu.edu

³ Abbreviations used in this paper: C/EBPβ, CCAAT-enhancer binding protein β; CHART-PCR, chromatin accessibility by real-time PCR; ChIP, chromatin immunoprecipitation; IRF, IFN regulatory factor; MM6, Mono-Mac-6 human monocytes; MNase, micrococcal nuclease; mPU.1, PU.1 mutated serine to alanine at position 148; SSRP, structure-specific recognition protein; TBP, TATA-binding protein; TRAF6, TNFR-associated factor 6.