We have previously reported that Dot1a is located in the cytoplasm and nucleus, widely expressed in kidney as detected by its histone H3 K79 methyltransferase activity, and involved in transcriptional control of the epithelial Na+ channel subunit α gene (αENaC). Aldosterone releases repression of αENaC by reducing expression of Dot1a and its partner AF9 and by impairing Dot1a-AF9 interaction via Sgk1-mediated AF9 phosphorylation. 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 (NLS). Deletion of these NLSs causes GFP-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 αENaC-promoter luciferase construct in M1 cells. AF9 is widely expressed in mouse kidney. Similar to αENaC, the mRNA levels of βENaC, γENaC, and Sgk1 are also downregulated by Dot1a and AF9 overexpression. siRNA-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.
- nuclear localization signal
- benzofuran isophthalate acetoxymethyl ester (SBFI-AM)
- intracellular Na+ concentration
- Copyright © 2010, American Journal of Physiology - Renal Physiology