Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the α-ENaC gene in a renal collecting duct cell line.
Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the α-ENaC gene in a renal collecting duct cell line.
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DOI：
10.1042/bsr20130086
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发表时间：
2013-10-25
影响因子：
4
通讯作者：
Kone BC
中科院分区：
文献类型：
--
作者：
Yu ZY;Kong Q;Kone BC
Rnf2 （RING finger protein 2）是PRC （Polycomb郁结复合体）1的核心成分，而Af9（来自9号染色体的all1融合基因）是醛固酮敏感的转录因子，在调节基础和醛固酮刺激的α-ENaC（上皮Na+通道α-亚基）基因的转录过程中，我们探索了Rnf2 （RING finger protein 2）和Af9 （all1融合基因）在mIMCD3 CD（集束管）细胞中的物理和功能相互作用。由于Rnf2缺乏dna特异性结合活性，必须有其他因子介导其位点特异性染色质募集。Rnf2和Af9在细胞核内共定位，共免疫沉淀。GST（谷胱甘肽转移酶）-Af9羧基末端融合蛋白在GST下拉实验中直接与体外翻译的Rnf2相互作用。Rnf2敲低可增强基础和醛固酮刺激的α-ENaC mRNA水平和α-ENaC启动子活性。ChIP/QPCR（染色质免疫沉淀/定量PCR）检测显示，在基础条件下，Rnf2、H2AK119（单泛素化组蛋白H2A赖氨酸119）和H3K27me3（组蛋白H3赖氨酸27三甲基化）（PRC2染色质标记）在与已知Af9富集区域对应的多个α-ENaC启动子亚区中富集。序贯ChIP证实Rnf2-Af9共占用α-ENaC启动子。醛固酮引起与α-ENaC启动子亚区相关的Rnf2、H2AK119泛素化和H3K27三甲基化的早期和持续耗损。因此，Af9介导了Rnf2在α-ENaC启动子上的位点选择性物理和功能募集，从而抑制了收集管细胞中α-ENaC的基础转录，而醛固酮逆转了这一过程。
The physical and functional interaction of Rnf2 (RING finger protein 2), a central component of the PRC (Polycomb repressive complex) 1 and Af9 (ALL1-fused gene from chromosome 9 protein), an aldosterone-sensitive transcription factor, in regulating basal and aldosterone-stimulated transcription of the α-ENaC (epithelial Na+ channel α-subunit) gene was explored in mIMCD3 CD (collecting duct) cells. Since Rnf2 lacks DNA-specific binding activity, other factors must mediate its site-specific chromatin recruitment. Rnf2 and Af9 co-localized in the nucleus and co-immunoprecipitated. A GST (glutathione transferase)–Af9 carboxy-terminal fusion protein directly interacted with in vitro translated Rnf2 in GST pull-down assays. Rnf2 knock down enhanced basal and aldosterone-stimulated α-ENaC mRNA levels and α-ENaC promoter activity. ChIP/QPCR (chromatin immunoprecipitation/quantitative PCR) assays demonstrated enrichment of Rnf2, H2AK119 (mono-ubiquitinated histone H2A lysine 119), and H3K27me3 (histone H3 lysine 27 trimethylated), a PRC2 chromatin mark, at multiple α-ENaC promoter subregions corresponding to regions of known Af9 enrichment, under basal conditions. Sequential ChIP confirmed Rnf2–Af9 co-occupancy of the α-ENaC promoter. Aldosterone provoked early and sustained depletion of Rnf2, ubiquitinated H2AK119, and trimethylated H3K27 associated with the subregions of the α-ENaC promoter. Thus, Af9 mediates site-selective physical and functional recruitment of Rnf2 to the α-ENaC promoter to constrain basal α-ENaC transcription in collecting duct cells, and aldosterone reverses this process.