Dot1a contains three nuclear localization signals and regulates the epithelial Na+ channel (ENaC) at multiple levels
Dot1a contains three nuclear localization signals and regulates the epithelial Na+ channel (ENaC) at multiple levels
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DOI：
10.1152/ajprenal.00105.2010
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发表时间：
2010-07-01
影响因子：
4.2
通讯作者：
Zhang, Wenzheng
中科院分区：
文献类型：
--
作者：
Reisenauer, Mary Rose;Wang, Steven W.;Zhang, Wenzheng
Dot1a包含三种核定位信号，并在多个水平上调控上皮Na+通道（ENaC）。[J] .中华医学杂志，2010,31(5):563 - 567。首次发表于2010年4月28日；doi: 10.1152 / ajprenal.00105.2010。-前期报道Dot1a位于细胞质和细胞核中（Reisenauer MR, Anderson M, Huang L, Zhang Z, Zhou Q, Kone BC, Morris AP, Lesage GD, Dryer SE, Zhang W. J生物化学284:35659-35669,2009），通过其组蛋白H3K79甲基转移酶活性检测在肾脏中广泛表达（张伟，Hayashizaki Y, Kone BC）。张伟，夏晓霞，陈志强，王志强，等。上皮细胞Na+通道亚单位α基因（α ENaC）的转录调控[J] .中国生物医学工程学报，2004,31(2):641-651。[J] .中国生物医学工程学报，2009,31(2):556 - 556。醛固酮通过降低Dot1a及其伴体AF9的表达来释放α - ENaC的抑制作用（Zhang W, Xia X, Reisenauer MR, Hemenway CS, Kone BC）。张伟，夏霞，Reisenauer MR ., Rieg T ., Lang F ., Kuhl D . Vallon V ., Kone BC。[J] .中国临床医学杂志，2009,31(2):387 - 398。这个网络似乎也调节其他几个醛固酮靶基因的转录。在这里，我们提供的证据表明Dot1a包含至少三个潜在的核定位信号（NLSs）。共聚焦显微镜显示，这些NLSs的缺失导致绿色荧光蛋白融合的Dot1a融合体几乎完全定位于293T细胞的细胞质中。NLSs的缺失消除了dot1a介导的M1细胞中α - enact启动子荧光素酶结构的抑制。AF9在小鼠肾脏中广泛表达。与α - ENaC类似，β - ENaC、γ - ENaC和Sgk1的mRNA水平也因Dot1a和AF9过表达而下调。小干扰rna介导的Dot1a和AF9的下调或醛固酮治疗可导致相反的效果。在IMCD3和M1细胞中使用单细胞荧光成像或等效短路电流，我们发现观察到的转录改变对应于ENaC和Sgk1蛋白水平的变化以及苯甲胺敏感的Na+转运。简而言之，Dot1a和AF9下调Na+转运，很可能是通过调节ENaC mRNA和随后的蛋白表达和ENaC活性。
Reisenauer MR, Wang SW, Xia Y, Zhang W. Dot1a contains three nuclear localization signals and regulates the epithelial Na+ channel (ENaC) at multiple levels. Am J Physiol Renal Physiol 299: F63-F76, 2010. First published April 28, 2010; doi: 10.1152/ajprenal.00105.2010.-We have previously reported that Dot1a is located in the cytoplasm and nucleus (Reisenauer MR, Anderson M, Huang L, Zhang Z, Zhou Q, Kone BC, Morris AP, Lesage GD, Dryer SE, Zhang W. J Biol Chem 284: 35659-35669, 2009), widely expressed in the kidney as detected by its histone H3K79 methyltransferase activity (Zhang W, Hayashizaki Y, Kone BC. Biochem J 377: 641-651, 2004), and involved in transcriptional control of the epithelial Na+ channel subunit-alpha gene (alpha ENaC) (Zhang W, Xia X, Jalal DI, Kuncewicz T, Xu W, Lesage GD, Kone BC. Am J Physiol Cell Physiol 290: C936-C946, 2006). Aldosterone releases repression of alpha ENaC by reducing expression of Dot1a and its partner AF9 (Zhang W, Xia X, Reisenauer MR, Hemenway CS, Kone BC. J Biol Chem 281: 18059-18068, 2006) and by impairing Dot1a-AF9 interaction via Sgk1-mediated AF9 phosphorylation (Zhang W, Xia X, Reisenauer MR, Rieg T, Lang F, Kuhl D, Vallon V, Kone BC. J Clin Invest 117: 773-783, 2007). This network also appears to regulate transcription of several other aldosterone target genes. Here, we provide evidence showing that Dot1a contains at least three potential nuclear localization signals (NLSs). Deletion of these NLSs causes green fluorescent protein-fused Dot1a fusions to localize almost exclusively in the cytoplasm of 293T cells as revealed by confocal microscopy. Deletion of NLSs abolished Dot1a-mediated repression of alpha ENaC-promoter luciferase construct in M1 cells. AF9 is widely expressed in mouse kidney. Similar to alpha ENaC, the mRNA levels of beta ENaC, gamma ENaC, and Sgk1 are also downregulated by Dot1a and AF9 overexpression. Small interference RNA-mediated knockdown of Dot1a and AF9 or aldosterone treatment leads to an opposite effect. Using single-cell fluorescence imaging or equivalent short-circuit current in IMCD3 and M1 cells, we show that observed transcriptional alterations correspond to changes in ENaC and Sgk1 protein levels as well as benzamil-sensitive Na+ transport. In brief, Dot1a and AF9 downregulate Na+ transport, most likely by regulating ENaC mRNA and subsequent protein expression and ENaC activity.