Identification and characterization of putative enhancer regions that direct Il6 transcription in murine macrophages.

Interleukin-6 (IL-6) plays a crucial role in various cellular functions, including innate and adaptive immune responses. Dysregulated expression of IL-6 is associated with hyperinflammation and chronic inflammatory diseases. In this study, we aimed to identify the enhancer regions responsible for robust Il6 mRNA expression in murine macrophages. Through comprehensive genome-wide ChIP- and ATAC-seq analyses, we identified two distinct clusters, termed E1 and E2 regions, located at -144 to -163 kb relative to the Il6 transcription start site in lipopolysaccharide (LPS)-activated murine macrophages. These clusters exhibited an accumulation of histone modification marks (H3K27ac and H3K4me1), as well as open chromatin, and were found to contain binding sites for the transcription factors PU.1, NF-κB, C/EBPß, and JunB. Upregulation of non-coding RNA (ncRNA) transcripts from the E1 and E2 regions was observed upon LPS stimulation, and repression of these ncRNAs resulted in abrogation of Il6 expression. Additionally, deletion of either E1 or E2 region significantly impaired Il6 expression, while CRISPR/dCas9 activation-mediated recruitment of the co-activator p300 to the E1 and E2 regions facilitated Il6 expression. Collectively, our findings suggest that the E1 and E2 regions serve as putative enhancers for Il6 expression.

Pathophysiological Role of Nucleic Acid-Sensing Pattern Recognition Receptors in Inflammatory Diseases.

Pattern recognition receptors (PRRs) play critical roles in recognizing pathogen-derived nucleic acids and inducing innate immune responses, such as inflammation and type I interferon production. PRRs that recognize nucleic acids include members of endosomal Toll-like receptors, cytosolic retinoic acid inducible gene I-like receptors, cyclic GMP-AMP synthase, absent in melanoma 2-like receptors, and nucleotide binding oligomerization domain-like receptors. Aberrant recognition of self-derived nucleic acids by these PRRs or unexpected activation of downstream signaling pathways results in the constitutive production of type I interferons and inflammatory cytokines, which lead to the development of autoimmune or autoinflammatory diseases. In this review, we focus on the nucleic acid-sensing machinery and its pathophysiological roles in various inflammatory diseases.

Regulation of Il6 expression by single CpG methylation in downstream of Il6 transcription initiation site.

The innate immune system is an immediate defense against infectious pathogens by the production of inflammatory cytokines and other mediators. Deficiencies of epigenetic regulatory enzymes, such as Tet1 and Dnmt1, cause dysregulation of cytokine expression. However, it is unclear if DNA methylation at a single CpG dinucleotide in a specific gene locus can regulate gene expression. In this study, we demonstrated that CpG+286 and CpG+348 in exon 2 of the Il6 gene are similar in various primary mouse cells. In lipopolysaccharide-stimulated condition, hypomethylated CpG+286 promoted Il6 expression whereas deletion of CpG+348 led to a reduction in Il6 expression associated with enhanced CTCF binding to the Il6 locus. Moreover, hypomethylation at CpG+286 in alveolar macrophages from aged mice led to higher Il6 expression in response to LPS compared with young mice. Thus, DNA methylation at specific CpG dinucleotides plays an important regulatory role in Il6 expression.

Identification and Analysis of a Gene with Potential to Repress the IMD Pathway in Drosophila melanogaster.

The IMD pathway is one of the signal transduction pathways that regulates innate immune responses in Drosophila. To understand the regulation mechanism of the IMD pathway, we performed a mis-expression screening and identified a gene, Dyro, which potentially repress the expression of the downstream target genes of the IMD pathway. We confirmed that Dyro was expressed in the fat body where the IMD pathway is functional and that the overexpression of Dyro increases susceptibility. However, we detected neither an increased expression of target genes nor reduced susceptibility in mutants. During the analysis, we observed that the Dyro mutant exhibits a female sterile phenotype, and observed oogenesis defects. The results suggest that Dyro have potential to suppress immune response but its main role is regulation of oogenesis.