The Nuclear Matrix Protein, NRP/B, Acts as a Transcriptional Repressor of E2F-mediated Transcriptional Activity.
The Nuclear Matrix Protein, NRP/B, Acts as a Transcriptional Repressor of E2F-mediated Transcriptional Activity.
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核基质蛋白 NRP/B 作为 E2F 介导的转录活性的转录抑制因子。
DOI：
10.15430/jcp.2014.19.3.187
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发表时间：
2014-09
影响因子：
2.5
通讯作者：
Kim TA
中科院分区：
文献类型：
--
作者：
Choi J;Yang ES;Cha K;Whang J;Choi WJ;Avraham S;Kim TA
NRP/B是BTB/Kelch重复蛋白家族成员，与神经元和癌症的发展以及乳腺癌和脑癌中氧化应激反应的调节有关。我们以前的研究表明，NRP/B-BTB/POZ结构域参与了NRP/B的二聚化，并与肿瘤抑制因子视网膜母细胞瘤蛋白形成复合物。尽管有许多证据支持NRP/B作为肿瘤抑制因子的潜在作用，但NRP/B对E2 F转录因子作用的分子机制尚未阐明。使用NRP/B的三维建模来产生BTB/Kelch结构域中的点突变。建立了Tet-on诱导的NRP/B表达。使用慢病毒shNRP/B产生NRP/B缺陷型乳腺癌细胞系MDA-MB-231，以评价NRP/B对细胞增殖、侵袭和迁移的影响。免疫沉淀法检测NRP/B与E2 F和组蛋白去乙酰化酶（HDAC-1）的相互作用，Western blot分析NRP/B蛋白的表达水平。流式细胞仪检测细胞周期的变化。氯霉素乙酰转移酶（CAT）活性测定E2 F转录因子的转录活性。NRP/B的异位过表达表明NRP/B-BTB/POZ结构域在E2 F介导的转录活性中起关键作用。BTB/POZ结构域内的点突变恢复了NRP/B抑制的E2启动子活性。NRP/B缺失促进乳腺癌细胞的增殖和迁移。内源性NRP/B与E2 F和HDAC 1相互作用。用HDAC抑制剂阿司他丁A（TSA）消除NRP/B介导的E2启动子活性抑制。HeLa细胞中NRP/B的获得或丧失证实了NRP/B对E2 F靶基因、细胞周期蛋白E和HsORC（智人起源识别复合物）的转录抑制能力。本研究表明NRP/B通过与辅助抑制因子HDAC 1相互作用而发挥转录抑制因子的作用，为NRP/B抑制肿瘤的分子机制提供了新的见解。
NRP/B, a family member of the BTB/Kelch repeat proteins, is implicated in neuronal and cancer development, as well as the regulation of oxidative stress responses in breast and brain cancer. Our previous studies indicate that the NRP/B-BTB/POZ domain is involved in the dimerization of NRP/B and in a complex formation with the tumor suppressor, retinoblastoma protein. Although much evidence supports the potential role of NRP/B as a tumor suppressor, the molecular mechanisms of NRP/B action on E2F transcription factors have not been elucidated. Three-dimensional modeling of NRP/B was used to generate point mutations in the BTB/Kelch domains. Tet-on inducible NRP/B expression was established. The NRP/B deficient breast cancer cell line, MDA-MB-231, was generated using lentiviral shNRP/B to evaluate the effect of NRP/B on cell proliferation, invasion and migration. Immunoprecipitation was performed to verify the interaction of NRP/B with E2F and histone deacetylase (HDAC-1), and the expression level of NRP/B protein was analyzed by Western blot analysis. Changes in cell cycle were determined by flow cytometry. Transcriptional activities of E2F transcription factors were measured by chloramphenicol acetyltransferase (CAT) activity. Ectopic overexpression of NRP/B demonstrated that the NRP/B-BTB/POZ domain plays a critical role in E2F-mediated transcriptional activity. Point mutations within the BTB/POZ domain restored E2-promoter activity inhibited by NRP/B. Loss of NRP/B enhanced the proliferation and migration of breast cancer cells. Endogenous NRP/B interacted with E2F and HDAC1. Treatement with an HDAC inhibitor, trichostatin A (TSA), abolished the NRP/B-mediated suppression of E2-promoter activity. Gain or loss of NRP/B in HeLa cells confirmed the transcriptional repressive capability of NRP/B on the E2F target genes, Cyclin E and HsORC (Homo sapiens Origin Recognition Complex). The present study shows that NRP/B acts as a transcriptional repressor by interacting with the co-repressors, HDAC1, providing new insight into the molecular mechanisms of NRP/B on tumor suppression.