Dot1a contains three nuclear localization signals and regulates the epithelial Na+ channel (ENaC) at multiple levels.
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 (alphaENaC) (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 alphaENaC 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 alphaENaC-promoter luciferase construct in M1 cells. AF9 is widely expressed in mouse kidney. Similar to alphaENaC, the mRNA levels of betaENaC, gammaENaC, 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.
Reisenauer
Mary Rose
MR
Department of Internal Medicine, University of Texas Medical School at Houston, Houston, TX 77030, USA.
Wang
Steven W
SW
Xia
Yang
Y
Zhang
Wenzheng
W
D021581:Active Transport, Cell Nucleus; D000450:Aldosterone / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D019175:DNA Methylation; D053503:Epithelial Sodium Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D011495:Histone-Lysine N-Methyltransferase; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic; D014162:Transfection; D000099184:Serum-Glucocorticoid Regulated Kinases
D000450:Aldosterone; D053503:Epithelial Sodium Channels; D011495:Histone-Lysine N-Methyltransferase; D017874:Immediate-Early Proteins; D008780:Methyltransferases; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012964:Sodium; D000099184:Serum-Glucocorticoid Regulated Kinases; C489902:Mllt3 protein, mouse; C507047:Scnn1a protein, mouse; C507049:Scnn1b protein, mouse; C507053:Scnn1g protein, mouse; C481527:Dot1l protein, mouse