Abstract
We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1β, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer-binding protein (C/EBPβ) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBPβ leucine zipper is critical for its association with Spi1 via an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining in vitro interaction studies with computational docking and molecular dynamics of existing X-ray structures for the Spi1 and C/EBPβ DBDs, along with the C/EBPβ C-terminal tail sequence, we found that the tail sequence is intimately associated with Arg-232 of Spi1. The Arg-232 pocket was computationally screened for small-molecule binding aimed at IL1B transcription inhibition, yielding l-arginine, a known anti-inflammatory amino acid, revealing a potential for disrupting the C/EBPβ-Spi1 interaction. As evaluated by ChIP, cultured lipopolysaccharide (LPS)-activated THP-1 cells incubated with l-arginine had significantly decreased IL1B transcription and reduced C/EBPβ's association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBPβ to cognate DNA and in transcription of the C/EBPβ-dependent IL6 gene in the same cells. These results support the notion that disordered sequences extending from a leucine zipper can mediate protein-protein interactions and can serve as druggable targets for regulating gene promoter activity.
Highlights
We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer– binding protein (C/EBP) at a far-upstream enhancer
We reported that the DNA-binding domain of constitutively promoter-bound Spi1 directly interacts by long-range chromatin looping [2] with the DBD of the C/EBP transcription factor, which in turn binds to a far upstream superenhancer following lipopolysaccharide (LPS)/ Toll-like receptor 4 (TLR4) signaling
One hint was that the two proteins are capable of a direct interaction that depends upon the C terminus of C/EBP and the integrity of a single amino acid in the Spi1 DBD [5]
Summary
A combined computational and experimental approach reveals the structure of a C/EBP–Spi interaction required for IL1B gene transcription. Auron‡§§2 From the Departments of ‡Biological Sciences and §Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania 15282, the ¶Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, the ʈDepartment of Medicine, UPMC Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, the **Department of Computational and Systems Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, ‡‡exeResearch, East Lansing, Michigan 48823, and the §§Department of Microbiology & Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15219
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