Edited by Alex TokerWe 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 druggable targets for regulating gene promoter activity. Figure 9. Four rotational views of the final DNA-C/EBP-Spi1-DNA composite model. Shown are the locations of the two bZIP chains and the Spi1 wHTH DBD, along with the likely long-range enhancer-promoter DNA interaction, for four 90°rotational views in association with DNA segments representing the long-range enhancer and promoter sites of the IL1B gene.
We previously reported that expression of the human IL1B gene coding for Interleukin 1β in LPS-activated macrophages requires long-range chromatin looping mediated by a mutual DNA binding domain (DBD) interaction between two transcription factors, Spi-1/PU.1 (Spi1) at the promoter and C/EBPβ at a far-upstream enhancer. We have now combined preexisting X-ray structures and advanced computational techniques with additional biochemical and cell-based studies in order to further investigate the structure of this critical interaction. In vitro binding was used to generate a surface interaction map for the two proteins that suggested an association similar to that of a C/EBPβ·Myb X-ray structure reported by others. The distinction is that, in addition to a C/EBPβ leucine zipper interaction similar to that with the Myb DBD, there is an interaction between a carboxyl-tail extension of one of the C/EBPβ leucine zipper monomers with Spi1. This is consistent with our previous report that the carboxyl-tail of C/EBPβ is critical for association with arginine 232 within a pocket at one end of the Spi1 DNA recognition helix. L-arginine, a known anti-inflammatory, was computationally identified to bind in this interaction pocket. In vivo studies of LPS-activated THP-1 macrophages treated with L-arginine decreased IL1B transcription in parallel with a reduction in C/EBPβ association with Spi1 on the IL1B promoter. No significant change in direct binding of Spi1 and C/EBPβ to DNA was observed. These results suggest that L-arginine inhibits IL1B gene expression by competitive inhibition of C/EBPβ binding to Spi1 in the absence of any effect on direct DNA binding for either factor, and support the predicted structure of the C/EBPβ·Spi1 interaction.
Interleukin 1β (IL‐1β) is a proinflammatory cytokine that is normally expressed and released by monocytes in response to infection and injury. Chronic expression of IL‐1β has been associated with numerous inflammatory diseases. We previously reported chromatin looping for the IL1B gene that is mediated by a mutual DNA‐binding domain (DBD) interaction between two transcription factors, Spi‐1/PU.1 (Spi‐1) at the promoter and C/EBPβ at a distal enhancer. The nature of this critical interaction between the Spi‐1 wHTH domain and the C/EBPβ leucine zipper was investigated by using the existing X‐ray structures of these two proteins bound to DNA as a guide in designing mutants for in vitro protein‐protein interaction studies. This generated surface interaction maps for the two proteins that in concert with computational techniques supported an association mode similar to, but distinct from, the C/EBPβ·c‐Myb X‐ray structure reported by others. The distinction was that, in addition to the C/EBPβ leucine zipper interaction with the c‐Myb wHTH domain, Spi‐1 utilizes an additional pseudo‐symmetrical bidentate interaction between the carboxyl‐tail extensions of each of the C/EBPβ leucine zipper monomers with the two ends of the Spi‐1 DNA recognition helix. This is consistent with our previous report that the carboxyl‐tail of C/EBPβ is critical for Spi‐1 association, suggesting that pocket‐like surfaces at the ends of the Spi‐1 DNA recognition helix might be targets for inhibition. Candidate small molecules were generated using a virtual screening approach. L‐arginine, a known anti‐inflammatory molecule, was identified from this approach as having potential to bind to each end of the Spi‐1 recognition helix, possibly inhibiting IL1B gene expression. Studies in 2.5 h LPS‐treated human THP‐1 macrophages in the presence of L‐arginine revealed a concentration‐dependent blunting of transcription in parallel with a decrease in C/EBPβ association with Spi‐1 on the IL1B gene promoter, while Spi‐1 binding to DNA was unaffected. In the same experiment, there was no significant change in direct binding of C/EBPβ to DNA at a well‐characterized site in the IL6 gene. In conclusion, these studies are consistent with an L‐arginine‐dependent decrease in IL1B gene expression that is due to its ability to inhibit the protein‐protein interaction between C/EBPβ and Spi‐1 in the absence of any effect on direct DNA binding for either factor. This is consistent with the predicted structure of the C/EBPβ·Spi‐1 interaction.
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