A combined computational and experimental approach reveals the structure of a C/EBP–Spi1 interaction required for IL1B gene transcription

Sree H. Pulugulla, Riley Workman, Nathan W. Rutter, Zhiyong Yang, Juraj Adamik, Brian Lupish, David A. Macar, Samir el Abdouni, Emilio Xavier Esposito, Deborah L. Galson, Carlos J. Camacho, Jeffry D. Madura, Philip E. Auron

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Abstract

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 have also reported that the C-terminal tail sequence beyond the C/EBP 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/EBP DBDs, along with the C/EBP 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/EBP–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/EBP’s association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBP to cognate DNA and in transcription of the C/EBP-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 drug-gable targets for regulating gene promoter activity.

Original languageEnglish (US)
Pages (from-to)19942-19956
Number of pages15
JournalJournal of Biological Chemistry
Volume293
Issue number52
DOIs
StatePublished - Jan 1 2018
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Pulugulla, S. H., Workman, R., Rutter, N. W., Yang, Z., Adamik, J., Lupish, B., Macar, D. A., Abdouni, S. E., Esposito, E. X., Galson, D. L., Camacho, C. J., Madura, J. D., & Auron, P. E. (2018). A combined computational and experimental approach reveals the structure of a C/EBP–Spi1 interaction required for IL1B gene transcription. Journal of Biological Chemistry, 293(52), 19942-19956. https://doi.org/10.1074/jbc.RA118.005627