Functional Genomics Laboratory, Arizona Respiratory Center, University of Arizona, Tucson, Arizona 85719, USA.
J Biol Chem. 2010 Mar 12;285(11):8256-67. doi: 10.1074/jbc.M109.041004. Epub 2010 Jan 5.
Expression of the cytokine interleukin-13 (IL13) is critical for Th2 immune responses and Th2-mediated allergic diseases. Activation of human IL13 expression involves chromatin remodeling and formation of multiple DNase I-hypersensitive sites throughout the locus. Among these, HS4 is detected in the distal IL13 promoter in both naive and polarized CD4(+) T cells. We show herein that HS4 acts as a position-independent, orientation-dependent positive regulator of IL13 proximal promoter activity in transiently transfected, activated human CD4(+) Jurkat T cells and primary murine Th2 cells. The 3'-half of HS4 (HS4-3') was responsible for IL13 up-regulation and bound nuclear factor (NF) 90 and NF45, as demonstrated by DNA affinity chromatography coupled with tandem mass spectrometry, chromatin immunoprecipitation, and gel shift analysis. Notably, the CTGTT NF45/NF90-binding motif within HS4-3' was critical for HS4-dependent up-regulation of IL13 expression. Moreover, transfection of HS4-IL13 reporter vectors into primary, in vitro differentiated Th2 cells from wild-type, NF45(+/-), or NF90(+/-) mice showed that HS4 activity was exquisitely dependent on the levels of endogenous NF45 (and to a lesser degree NF90), because HS4-dependent IL13 expression was virtually abrogated in NF45(+/-) cells and reduced in NF90(+/-) cells. Collectively, our results identify NF45 and NF90 as novel regulators of HS4-dependent human IL13 transcription in response to T cell activation.
细胞因子白细胞介素-13（IL13）的表达对于 Th2 免疫反应和 Th2 介导的过敏性疾病至关重要。人 IL13 表达的激活涉及染色质重塑和整个基因座中多个 DNA 酶 I 超敏位点的形成。在这些超敏位点中，HS4 存在于幼稚和极化的 CD4(+)T 细胞中的远端 IL13 启动子中。我们在此表明，HS4 作为一个位置独立、方向依赖的正向调节剂，在瞬时转染的、激活的人 CD4(+)Jurkat T 细胞和原代小鼠 Th2 细胞中，发挥作用于 IL13 近端启动子活性。HS4 的 3'- 半部分（HS4-3'）负责 IL13 的上调，并与核因子（NF）90 和 NF45 结合，如 DNA 亲和层析与串联质谱、染色质免疫沉淀和凝胶迁移分析所示。值得注意的是，HS4-3' 内的 CTGTT NF45/NF90 结合基序对于 HS4 依赖性 IL13 表达上调至关重要。此外，将 HS4-IL13 报告载体转染到来自野生型、NF45(+/-)或 NF90(+/-)小鼠的体外分化的 Th2 细胞中，表明 HS4 活性非常依赖于内源性 NF45（以及较小程度上的 NF90）的水平，因为 HS4 依赖性 IL13 表达在 NF45(+/-)细胞中几乎被消除，在 NF90(+/-)细胞中减少。总的来说，我们的结果表明 NF45 和 NF90 是新型的调节因子，可调节 T 细胞激活后 HS4 依赖性人 IL13 转录。
