CCAAT/enhancer-binding protein beta
transcription cis-regulatory region binding
A combined computational and experimental approach reveals the structure of a C/EBPβ-Spi1 interaction required for <i>IL1B</i> gene transcription.
We previously reported that transcription of the human <i>IL1B</i> 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 <i>via</i> an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining <i>in vitro</i> 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 <i>IL1B</i> 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 <i>IL1B</i> transcription and reduced C/EBPβ's association with Spi1 on the <i>IL1B</i> 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 <i>IL6</i> 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.