The Small Ubiquitin-like Modifier-Deconjugating Enzyme Sentrin-Specific Peptidase 1 Switches IFN Regulatory Factor 8 from a Repressor to an Activator during Macrophage Activation

Abstract

Macrophages, when activated by IFN-γ and TLR signaling, elicit innate immune responses. IFN regulatory factor 8 (IRF8) is a transcription factor that facilitates macrophage activation and innate immunity. We show that, in resting macrophages, some IRF8 is conjugated to small ubiquitin-like modifiers (SUMO) 2/3 through the lysine residue 310. SUMO3-conjugated IRF8 failed to induce IL12p40 and other IRF8 target genes, consistent with SUMO-mediated transcriptional repression reported for other transcription factors. SUMO3-conjugated IRF8 showed reduced mobility in live nuclei and bound poorly to the IL12p40 gene. However, macrophage activation caused a sharp reduction in the amount of SUMOylated IRF8. This reduction coincided with the induction of a deSUMOylating enzyme, sentrin-specific peptidase 1 (SENP1), in activated macrophages. In transfection analysis, SENP1 removed SUMO3 from IRF8 and enhanced expression of IL12p40 and other target genes. Conversely, SENP1 knockdown repressed IRF8 target gene expression. In parallel with IRF8 deSUMOylation, macrophage activation led to the induction of proteins active in the SUMO pathway and caused a global shift in nuclear protein SUMOylation patterns. Together, the IRF8 SUMO conjugation/deconjugation switch is part of a larger transition in SUMO modifications that takes place upon macrophage activation, serving as a mechanism to trigger innate immune responses.

Functional characterization of the human dendritic cell immunodeficiency associated with the IRF8K108E mutation

“…41 In addition, SUMOylation via SUMO2 and SUMO3 keeps IRF8 in a repressed functional state and can also target IRF8 for proteasomal degradation. 33 Following activation of macrophages, IRF8 is deSUMOylated by SENP1, which releases IRF8 for transcriptional activity. 33 We investigated the effect of K108E on regulatory posttranslational modification of IRF8 ( Figure 6D-E).…”

“…The IL-12p40-pGL3, IRF8-hemagglutinin (HA)-pcDNA3, and IRF1-pcDNA3 vectors were generated as previously described. 32 The V5-small ubiquitinlike modifier (SUMO)3 and HA-sentrin specific peptidase (SENP)1 plasmids were as previously described 33 and were kindly provided by Keiko Ozato (National Institutes of Health).…”

“…It is also possible that the K108R variant has a more subtle effect on IRF8 structure that may affect access to regulatory enzymes such as Cbl/DUBs (for ubiquitination) and E2I/SENP1 (for SUMOylation), thereby displacing the regulatory equilibrium between active IRF8 and inactive SUMOylated/ubiquitinated protein. 33,41 Finally, studies in cells expressing both the K108E mutant and the WT protein demonstrate that K108E does not behave as a dominant negative and does not alter targeting of the WT protein to the nucleus. This is in agreement with the recessive mode of inheritance of the immunodeficiency and granulocytic hyperplasia phenotypes associated with the K108E mutation in this family.…”

“…33 We investigated the effect of K108E on regulatory posttranslational modification of IRF8 ( Figure 6D-E). In transfected HEK 293T cells, we observed for both WT and K108E mutant, a high-molecular-mass smear typical of ubiquitinated proteins, but this smear was more intense for E108, suggesting increased ubiquitination of the mutant compared with WT ( Figure 6D).…”

Interferon Regulatory Factor 8 (IRF8) Impairs Induction of Interferon Induced with Tetratricopeptide Repeat Motif (IFIT) Gene Family Members

“…More recently, post-translational modification has emerged as a method of regulating the ability of IRF8 to activate or repress gene transcription. Sumoylation of IRF8 has been shown to remove its ability to activate gene transcription, switching it from an activator to a repressor (25). As such, IRF8 may be capable of repressing Ifit1 gene induction in cells such as pDCs and B cells, where it is present at high endogenous levels.…”