Chang Tzu-Pei, Kim Myra, Vancurova Ivana
Department of Biology, St. John's University, 8000 Utopia Parkway, Queens, NY, 11439, USA.
Methods Mol Biol. 2014;1172:329-41. doi: 10.1007/978-1-4939-0928-5_30.
The immunosuppressive cytokines transforming growth factor β1 (TGFβ1) and interleukin-10 (IL-10) regulate a variety of biological processes including differentiation, proliferation, tissue repair, tumorigenesis, inflammation, and host defense. Aberrant expression of TGFβ1 and IL-10 has been associated with many types of autoimmune and inflammatory disorders, as well as with many types of cancer and leukemia. Patients with cutaneous T cell lymphoma (CTCL) have high levels of malignant CD4+ T cells expressing IL-10 and TGFβ1 that suppress the immune system and diminish the antitumor responses. The transcriptional regulation of TGFβ1 and IL-10 expression is orchestrated by several transcription factors, including NFκB. However, while the transcriptional regulation of pro-inflammatory and anti-apoptotic genes by NFκB has been studied extensively, much less is known about the NFκB regulation of immunosuppressive genes. In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the transcriptional regulation of TGFβ1 and IL-10 by measuring recruitment of NFκB p65, p50, c-Rel, Rel-B, and p52 subunits to TGFβ1 and IL-10 promoters in human CTCL Hut-78 cells.
免疫抑制细胞因子转化生长因子β1（TGFβ1）和白细胞介素-10（IL-10）调节多种生物学过程，包括分化、增殖、组织修复、肿瘤发生、炎症和宿主防御。TGFβ1和IL-10的异常表达与多种自身免疫性和炎症性疾病以及多种癌症和白血病有关。皮肤T细胞淋巴瘤（CTCL）患者的恶性CD4 + T细胞高水平表达IL-10和TGFβ1，这些细胞会抑制免疫系统并削弱抗肿瘤反应。TGFβ1和IL-10表达的转录调控由包括NFκB在内的几种转录因子精心编排。然而，虽然NFκB对促炎和抗凋亡基因的转录调控已得到广泛研究，但对NFκB对免疫抑制基因的调控了解较少。在本章中，我们描述了一种使用染色质免疫沉淀（ChIP）的方案，通过测量NFκB p65、p50、c-Rel、Rel-B和p52亚基在人CTCL Hut-78细胞中向TGFβ1和IL-10启动子的募集来分析TGFβ1和IL-10的转录调控。
