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Requiem protein links RelB/p52 and the Brm-type SWI/SNF complex in a noncanonical NF-kappaB pathway.

Requiem 蛋白以非经典 NF-kappaB 途径连接 RelB/p52 和 Brm 型 SWI/SNF 复合物。

基本信息

批准号：
21590507
负责人：
MIZUTANI Taketoshi
金额：
$ 2.91万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21590507/
关键词：
SWI/SNF複合体 HIV-1 NF-κB レトロウイルス SWI/SNF NF-kappaB SWI SNF

项目摘要

In this study, we identify the human requiem protein(REQ/DPF2) as an adaptor molecule that links the NF-kappaB and SWI/SNF chromatin remodeling factor.Using sensitive 293FT reporter cell clones that had integrated a SWI/SNF-dependent NF-κB reporter gene, we find in this study that the overexpression of DPF1, DPF2, DPF3a, DPF3b, and PHF10 significantly potentiates the transactivating activity of typical NF-κB dimers. Knockdown analysis using 293FT reporter cells that endogenously express these five proteins at low levels clearly showed that DPF3a and DPF3b, which are produced from the DPF3 gene by alternative splicing, are the most critical for the RelA/p50 NF-κB heterodimer transactivation induced by TNF-αstimulation. Our data further show that this transactivation requires the SWI/SNF complex. DPF3a and DPF3b are additionally shown to interact directly with RelA, p50, and several subunits of the SWI/SNF complex in vitro and to be co-immunoprecipitated with RelA/p50 and the SWI/SNF complex from the nuclear fractions of cells treated with TNF-α. In ChIP experiments, we further found that endogenous DPF3a/b and the SWI/SNF complex are continuously present on HIV-1 LTR, whereas the kinetics of RelA/p50 recruitment after TNF-αtreatment correlate well with the viral transcriptional activation levels.

在本研究中，我们将人类安魂蛋白（REQ/DPF2）确定为连接NF-kappaB和SWI/SNF染色质重塑因子的接头分子。使用整合了SWI/SNF依赖性NF-κB报告基因的敏感293FT报告细胞克隆，我们在本研究中发现DPF1、DPF2的过度表达， DPF3a、DPF3b 和 PHF10 显着增强典型 NF-κB 二聚体的反式激活活性。使用低水平内源表达这五种蛋白质的 293FT 报告细胞进行的敲低分析清楚地表明，DPF3a 和 DPF3b（通过选择性剪接从 DPF3 基因产生）对于 TNF-α 刺激诱导的 RelA/p50 NF-κB 异二聚体反式激活最为关键。我们的数据进一步表明这种反式激活需要 SWI/SNF 复合物。另外，DPF3a 和 DPF3b 在体外被证明可直接与 RelA、p50 和 SWI/SNF 复合物的几个亚基相互作用，并与来自 TNF-α 处理的细胞核部分的 RelA/p50 和 SWI/SNF 复合物进行免疫共沉淀。在 ChIP 实验中，我们进一步发现内源性 DPF3a/b 和 SWI/SNF 复合物持续存在于 HIV-1 LTR 上，而 TNF-α 处理后 RelA/p50 招募的动力学与病毒转录激活水平密切相关。