Background: The nuclear receptor Nur77 (NR4A1) has a crucial function in macrophages’ resolution phase of inflammation. Nur77 can trans-repress the activity of transcription factor NFĸB (p65) thereby reducing downstream pro-inflammatory pathways. Previous ChIP-seq data revealed that Nur77 and p65 bind the DNA in similar areas around promoters of pro-inflammatory cytokines. However, only NFĸB response elements were found and Nur77-elements seem absent, which may indicate that just the protein-protein interaction between Nur77 and p65 is sufficient to inhibit p65 transcriptional activity. At present, the underlying molecular mechanism of this trans-repression remains largely unknown.

Results: In the current study we show, to our knowledge for the first time, that DNA binding of Nur77 is essential to inhibit the transcriptional activity of p65. Detailed analysis of mutants of Nur77 lacking (partial) domains revealed that some of these mutants no longer trans-repress p65, but still show protein-protein interaction with p65. Therefore, we propose that inhibition of p65 by Nur77 involves both DNA binding of Nur77 and modulation of the co-regulatory p65 complex. To identify the molecular composition of this complex, we will perform Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (RIME) in mouse macrophage RAW264.7 cell lines with inducible overexpression of Nur77. In these same cells, the gene sets regulated by Nur77-mediated trans-repression of p65 will be determined by RNA-sequencing after inflammatory stimulation.

In conclusion, the Nur77 DNA-binding domain (DBD) is essential to trans-repress p65, similar to Glucocorticoid Receptor (GR)-mediated trans-repression of NFĸB.