No abstract

Antigenic stimulation through cross-linking the IgE receptor and epithelial cell-derived cytokine IL-33 are potent stimuli of mast cell (MC) activation. However, target genes induced by the combined antigen and IL-33 stimulation have not been investigated in human skin mast cells in a genome-wide manner. Furthermore, epigenetic mechanisms by which IL-33 acts synergically with antigenic stimulation to induce gene transcription have not been investigated. We identified the target genes that responded to the combined antigen and IL-33 stimulation using RNA-seq. These genes can be divided into three groups. Genes in the first group are target genes where antigenic stimulation and IL-33 stimulation achieve high levels of synergy in promoting these gene transcription (high synergy target genes). Genes in the second group are target genes for low levels of synergy mediated by the combined antigen and IL-33 stimulation (low synergy target genes). And genes in the third group are not targeted for the antigen and IL-33 synergy (non-synergy target genes). We found that pro-inflammatory cytokine and chemokine genes were enriched among the high synergy target genes. We demonstrate that the combined antigen and IL-33 stimulation-induced or -increased chromatin accessible areas in the regulatory regions of the high synergy target genes but not of the low synergy target genes. Transcription factor binding motif analysis revealed more binding sites for NF-κB, AP-1, GABPA, and RAP1 in the induced or increased chromatin accessible regions of the high synergy target genes. Our study shines a light on an epigenetic mechanism by which how epithelial cell-derived cytokine IL-33 might cause exacerbation of skin MC-mediated allergic inflammation.

Macrophages play a critical role in inflammatory responses during infections. Activated macrophages by infections through stimulation of TLRs expressed their cell surface produce pro-inflammatory cytokines, including TNF. However, distal enhancers that regulate TNF gene transcription in human macrophages have not been investigated. In this study, we identified the five putative TNF enhancers using H3K27ac ChIP-seq and ATAC-seq. We showed proximal enhancer (PE), E-16.0, and E-6.5 possessed enhancer activity in a reporter gene assay. Deletion of the distal TNF E-16.0 enhancer resulted in 73% reduction in TNF gene transcription in human macrophage cell line THP-1 in response to ploy(I:C) stimulation. Our study identifies a novel distal enhancer that regulates TNF gene transcription in human macrophages.

Histamine is a critical mediator of anaphylaxis, a neurotransmitter, and a regulator of gastric acid secretion. Histidine decarboxylase is a rate-limiting enzyme for histamine synthesis. However, in vivo regulation of Hdc, the gene that encodes histidine decarboxylase is poorly understood. We identified potential enhancers in the Hdc gene using H3K27a histone modification and chromatin accessibility; we assessed the enhancer activity of the potential enhancers in a reporter gene assay; and we deleted noncoding sequences that possessed enhancer activity using CRISPR sgRNA guides. Deletion of the GC box reduced Hdc gene transcription and histamine synthesis in the CFTL-15 mouse mast cell line and the LAD2 human mast cell line. Mast cells, basophils, brain cells, and stomach cells from GC box-deficient mice transcribed the Hdc gene much less than similar cells from wild-type mice and Hdc GC box-deficient mice failed to develop anaphylaxis. Thus, a GC box within the proximal enhancer in the mouse Hdc gene is essential for Hdc gene transcription, histamine synthesis, and histamine-mediated anaphylaxis in vivo. Our study also reveals a critical role of the GC box in human HDC gene transcription and histamine synthesis.