AF17 Competes with AF9 for Binding to Dot1a to Up-regulate Transcription of Epithelial Na+ Channel α
AF17 Competes with AF9 for Binding to Dot1a to Up-regulate Transcription of Epithelial Na+ Channel α
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DOI：
10.1074/jbc.m109.038448
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发表时间：
2009-12-18
影响因子：
4.8
通讯作者：
Zhang, Wenzheng
中科院分区：
文献类型：
--
作者：
Reisenauer, Mary Rose;Anderson, Marc;Zhang, Wenzheng
我们以前报道过Dot 1a中心点AF 9复合物抑制小鼠内髓集合管mIMCD 3细胞和小鼠肾脏上皮Na+通道α亚基（α-ENaC）基因的转录。醛固酮通过各种机制下调复合物来缓解这种抑制。这些机制是否在人类细胞中是足够的和保守的，或者可以应用于其他醛固酮调控的基因，在很大程度上仍然是未知的。在这里，我们证明，人胚肾293 T细胞表达的三个ENaC亚基和所有的ENaC转录调节检查。这些细胞对醛固酮有反应，并显示苯甲脒敏感的Na+电流，如通过全细胞膜片钳所测量的。我们还表明，AF 17和AF 9竞争性地结合到同一域的Dot 1a在多个测定和具有拮抗作用的α-ENaC启动子荧光素酶构建体的表达。Dot 1a或AF 9的过表达降低了ENaC亚基及其转录调节因子的mRNA表达，并降低了苯甲脒敏感性Na+电流。AF 17过表达引起相反的效应，伴随着Dot 1a从细胞核重定向到细胞质和组蛋白H3 K79甲基化的减少。核输出抑制剂来普霉素B阻断了AF 17过表达对H3 K79低甲基化的影响。RNAi介导的AF 17敲低导致Dot 1a和组蛋白H3 K79超甲基化的核富集。与AF 9一样，AF 17显示与Sgk 1的细胞核和细胞质共定位。因此，AF 17与AF 9竞争结合Dot 1a，通过可能促进其核输出来降低Dot 1a核表达，并减轻Dot 1a中心点AF 9介导的对α-ENaC和其他靶基因的抑制。
We previously reported that Dot1a center dot AF9 complex represses transcription of the epithelial Na+ channel subunit alpha (alpha-ENaC) gene in mouse inner medullary collecting duct mIMCD3 cells and mouse kidney. Aldosterone relieves this repression by down-regulating the complex through various mechanisms. Whether these mechanisms are sufficient and conserved in human cells or can be applied to other aldosterone-regulated genes remains largely unknown. Here we demonstrate that human embryonic kidney 293T cells express the three ENaC subunits and all of the ENaC transcriptional regulators examined. These cells respond to aldosterone and display benzamil-sensitive Na+ currents, as measured by whole-cell patch clamping. We also show that AF17 and AF9 competitively bind to the same domain of Dot1a in multiple assays and have antagonistic effects on expression of an alpha-ENaC promoter-luciferase construct. Overexpression of Dot1a or AF9 decreased mRNA expression of the ENaC subunits and their transcriptional regulators and reduced benzamil-sensitive Na+ currents. AF17 overexpression caused the opposite effects, accompanied by redirection of Dot1a from the nucleus to the cytoplasm and reduction in histone H3 K79 methylation. The nuclear export inhibitor leptomycin B blocked the effect of AF17 overexpression on H3 K79 hypomethylation. RNAi-mediated knockdown of AF17 yielded nuclear enrichment of Dot1a and histone H3 K79 hypermethylation. As with AF9, AF17 displays nuclear and cytoplasmic co-localization with Sgk1. Therefore, AF17 competes with AF9 to bind Dot1a, decreases Dot1a nuclear expression by possibly facilitating its nuclear export, and relieves Dot1a center dot AF9-mediated repression of alpha-ENaC and other target genes.