Checkpoints of TNF Gene Regulation

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 DESCRIPTION (provided by applicant): Tumor necrosis factor (TNF) is located in a gene dense locus with the lymphotoxin (LT) α and β genes. The gene is highly induced in monocytes/macrophages by lipopolysaccharide (LPS) and its expression is further enhanced by pre-exposure to interferon (IFN)-γ, or IFN-γ priming. The tightly linked LT α and LTβ 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-γ priming of LPS-induced human TNF transcription and in TNF expression in activated T cells. In primary human macrophages after IFN-γ 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-γ-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-γ 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 α and LTβ 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.