Autophagy involves a long process that starts with signaling, followed by LC3 protein lipidation and autophagosome formation, leading to fusion with lysosomes and ubiquitin mediated protein degradation. Each of the above processes is controlled by autophagy specific genes conserved from yeast to humans. Our initial microarray and ChIP-on chip analyses indicated that some of autophagy related genes are downregulated in IRF8-/- DCs relative to IRF8+/+ DC. The follow-up study confirmed that IRF8 is required for expression of a large number of autophagy specific genes in both macrophages and DC. Some of these genes are induced by IFN and IRF8 bound to the upstream promoter regions. Consistent with these findings, IRF8 -/- macrophages were defective in lipidation mediated conversion of LC3 and nutophagosome formation. These results raise the possibility that IRF8 utilizes autophagy to eliminate certain pathogens including TB. Because IRF8 and several other IRF members are modified by both ubiquitin and SUMO, we asked whether these modifications occur in other proteins during innate immune responses. Macrophages were stimulated with IFN and a toll like receptor (TLR) ligand, CpG which activates the TLR pathway and triggers transcription of many genes important for macrophage function. Some of these genes are responsible for resistance against pathogens and other genes are involved in inflammatory responses in the host. To detect global ubiquitin or SUMO modifications of nuclear proteins in stimulated macrophages, we performed immunoblot analysis using antibody for ubiquitin, SUMO1 or SUMO2/3. To this end we tested nuclear fractions obtained from stimulated RAW264.7 macrophage cells. While there is only one ubiquitin protein, there are three SUMO molecules. SUMO1 is distinct from SUMO2 and SUMO3 that are more than 90% identical in amino acid sequence, and currently there is no antibody that distinguishes SUMO2 and SUMO3. We found that ubiquitin linked proteins were dramatically increased following IFNCpG stimulation. IFN treatment alone resulted in significant increase in ubiquitin-liked proteins. However, additional stimulationby CpG further increased the amount of ubiquitin bound proteins. Not only CpG, but other TLR ligands tested including LPS and poly IC also caused increased ubiquitination, provided that macrophages were prestimulated with IFN Similarly, IFN/TLR stimulation caused a marked increase in proteins conjugated to SUMO1 or SUMO2/3. Our data demonstrate that ubiquitin and SUMO modifications are integral part of innate immune responses, likely coupled with large changes in transcription and other nuclear activities, which helps accommodate a rapid response to pathogen stimuli. Transcriptionally active genes are embedded in chromatin that is dynamically exchanged, whereas silenced genes are surrounded by more stable chromatin. Chromatin environment
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