Abstract
Endotoxin tolerance occurs to protect the organism from hyperactivation of innate immune responses, primarily mediated by macrophages. Regulation of endotoxin tolerance occurs at multiple levels of cell responses and requires significant changes in gene expression. In the process of macrophage activation, induced expression of microRNA (miR)-155 and miR-146a contributes to the regulation of the inflammatory response and endotoxin tolerance. In this article, we demonstrate that expression of both miRNAs is coordinately regulated during endotoxin tolerance by a complex mechanism that involves monoallelic interchromosomal association, alterations in histone methyl marks, and transcription factor binding. Upon activation of naive macrophages, Histone3 was trimethylated at lysine4 and NFκBp65 was bound on both miR-155 and miR-146a gene loci. However, at the stage of endotoxin tolerance, both miR gene loci were occupied by C/EBPβ, NFκBp50, and the repressive Histone3 marks trimethylation of K9 of H3. DNA fluorescence in situ hybridization experiments revealed monoallelic interchromosomal colocalization of miR-155 and miR-146a gene loci at the stage of endotoxin tolerance, whereas RNA-DNA-fluorescence in situ hybridization experiments showed that the colocalized alleles were silenced, suggesting a common repression mechanism. Genetic ablation of Akt1, which is known to abrogate endotoxin tolerance, abolished induction of loci colocalization and C/EBPβ binding, further supporting that this mechanism occurs specifically in endotoxin tolerance. Overall, this study demonstrates that two miRNAs are coordinately regulated via gene colocalization at the three-dimensional chromatin space, same transcriptional machinery, and similar Histone3 methylation profile, contributing to the development of endotoxin tolerance.