Tumor necrosis factor (TNF) is located in a gene dense locus with the lymphotoxin (LT) ? and ? genes. The gene is highly induced in monocytes/macrophages by lipopolysaccharide (LPS) and its expression is further enhanced by pre-exposure to interferon (IFN)-?, or IFN-? priming. The tightly linked LT ? and LT? genes are expressed in activated T lymphocytes along with TNF, but not in monocytic cells. Consistent with this, we have found that the TNF/LT locus undergoes distinct chromatin remodeling events in primary human monocytic cells and T cells, and the chromatin signatures detected in the two cell types are distinct. However, in both cell types a distal DNase Hypersensitivity (DH) site ~8 kB upstream of the TNF transcription start site functions as an enhancer during both IFN-? priming of LPS-induced human TNF transcription and in TNF expression in activated T cells. In primary human macrophages after IFN-? treatment, hHS-8 becomes more accessible to DNase I digestion, displays increased levels of histone H3K27me3, and binds the transcription factor IRF1. Upon LPS stimulation of IFN-?-primed cells, the H3K27me3-specific demethylase JMJD3 and the acetylase p300 are recruited to hHS-8 and acetylated H3K27 levels significantly increase while H3K27me3 levels decrease, and eRNA is transcribed. Specific targeting the hHS-8 IRF1 binding site in THP-1 cells in its chromatin context using a catalytically inactive `dead' Cas9 linked to the KRAB repressive domain, abolishes IFN-? augmentation of LPS-induced TNF while LPS induction of the gene is unaffected. By contrast in activated primary T cells, NFATp is recruited to hHS-8, the site is decorated with H3K27Ac, and hHS-8 eRNA is produced. eRNA transcription is blocked by cyclosporine A treatment, further underscoring the importance of NFATp at this site. Furthermore, TNF levels are significantly reduced in HUT-78 hHS-8 NFATp targeted cells. LT ? and LT? gene regulation was also significantly decreased in these HUT-78 hHS-8 NFATp cells indicating that hHS-8 may be a locus control element in T cells and make intrachromosomal contacst with the newly identified LT regulatory elements. Based on these findings, we hypothesize that in activated macrophages, sequential H3K27 methylation, demethylation and acetylation events, poise and then trigger enhancer activity, respectively. We will test this hypothesis, and we will also investigate whether the IL-6 and IL12-B genes that are also primed in activated macrophages, are similarly regulated. We will test the hypothesis that IRF1 and NFATp function as `pioneer factors' and together with eRNA mediate hHS-8- promoter DNA looping and triggering of enhancer activity. We will also test the hypothesis that newly identified elements near the LT genes interact with hHS-8 in both dCas9/CRISPR edited human cell lines and in CRSPR mice we created. We expect to gain fundamental information about cell type- and stimulus-specific long-range enhancer function in general and to identify novel targets for potential modulation of TNF gene expression.
Proper expression of the protein TNF has a profound impact on a number of autoimmune and infectious diseases. The protein is encoded by the TNF gene, the regulation of which is controlled by a number of DNA sequences that are highly conserved among different species. Depending on the type of immune cell in which TNF is expressed, distinct sets of these sequences combine to direct the expression of the TNF gene. We have recently discovered that sequences that located kilobases away from the TNF regulatory region play a major role in cell type specific TNF gene regulation. Understanding both the nature of and interactions between DNA sequences and host proteins that control TNF expression is critical for designing therapeutic strategies to counteract disease states that arise from improper regulation of TNF in specific cell types.
