Checkpoints of TNF Gene Regulation

 描述（由申请方提供）：肿瘤坏死因子（TNF）位于与光毒素（LT）α和β基因相关的基因密集位点。该基因在单核细胞/巨噬细胞中被脂多糖（LPS）高度诱导，并且其表达通过预暴露于干扰素（IFN）-γ或IFN-γ引发而进一步增强。紧密连锁的LT α和LTβ基因是 在活化的T淋巴细胞沿着TNF表达，但在单核细胞中不表达。与此一致，我们发现TNF/LT基因座在原代人单核细胞和T细胞中经历不同的染色质重塑事件，并且在两种细胞类型中检测到的染色质特征是不同的。然而，在这两种细胞类型中，TNF转录起始位点上游约8 kB的远端DNA酶超敏（DH）位点在LPS诱导的人TNF转录的IFN-γ引发和活化T细胞中的TNF表达期间均起增强子作用。在IFN-γ处理后的原代人巨噬细胞中，hHS-8变得更容易被DNA酶I消化，显示出增加的组蛋白H3 K27 me 3水平，并结合转录因子IRF 1。在IFN-γ引发的细胞的LPS刺激后，H3 K27 me 3特异性脱甲基酶JMJD 3和乙酰化酶p300被募集到hHS-8，乙酰化H3 K27水平显著增加，而H3 K27 me 3水平降低，并且eRNA被转录。使用与KRAB抑制结构域连接的催化失活的“死”Cas9在THP-1细胞的染色质环境中特异性靶向其hHS-8 IRF 1结合位点，消除了LPS诱导的TNF的IFN-γ增强，而基因的LPS诱导不受影响。相反，在活化的原代T细胞中，NFATp被募集到hHS-8，该位点用H3 K27 Ac修饰，并产生hHS-8 eRNA。eRNA转录被环孢菌素A处理阻断，进一步强调了NFATp在该位点的重要性。此外，TNF水平在HUT-78 hHS-8 NFATp靶向细胞中显著降低。在HUT-78 hHS-8 NFATp细胞中，LT α和LTβ基因的调控也明显减弱，提示hHS-8可能是T细胞中的一个位点控制元件，并与新鉴定的LT调控元件发生染色体内接触。基于这些发现，我们假设在活化的巨噬细胞中，顺序的H3 K27甲基化、去甲基化和乙酰化事件分别平衡然后触发增强子活性。我们将检验这一假设，我们也将研究是否IL-6和IL-12-B基因，也引发在活化的巨噬细胞，类似的调节。我们将测试IRF 1和NFATp作为“先锋因子”发挥作用，并与eRNA一起介导hHS-8启动子DNA环和触发增强子活性的假设。我们还将测试新鉴定的LT基因附近的元件在dCas 9/CRISPR编辑的人类细胞系和我们创建的CRSPR小鼠中与hHS-8相互作用的假设。我们希望获得有关细胞类型和刺激特异性的长程增强子功能的基本信息，并确定潜在的TNF基因表达调控的新靶点。

 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.