Synthesis and biological evaluation of unnatural derivatives of narciclasine: 7-aza-nornarciclasine and its N-oxide.

Several unnatural derivatives of narciclasine were prepared in which the C-7 carbon was replaced with nitrogen. The 7-aza derivative and its N-oxide were prepared by the coupling of iodopicolinic acid with a conduramine unit derived chemoenzymatically from bromobenzene. Intramolecular Heck reaction was used to construct the isocarbostyryl ring system. The compounds were submitted to biological screening against cancer cell lines. Full experimental and spectra data are provided for all new compounds.

Af17 deficiency increases sodium excretion and decreases blood pressure.

The putative transcription factor AF17 upregulates the transcription of the epithelial sodium channel (ENaC) genes, but whether AF17 modulates sodium homeostasis and BP is unknown. Here, we generated Af17-deficient mice to determine whether deletion of Af17 leads to sodium wasting and low BP. Compared with wild-type mice, Af17-deficient mice had lower BP (11 mmHg), higher urine volume, and increased sodium excretion despite mildly increased plasma concentrations of aldosterone. Deletion of Af17 led to increased dimethylation of histone H3 K79 and reduced ENaC function. The attenuated function of ENaC resulted from decreased ENaC mRNA and protein expression, fewer active channels, lower open probability, and reduced effective activity. In contrast, inducing high levels of plasma aldosterone by a variety of methods completely compensated for Af17 deficiency with respect to sodium handling and BP. Taken together, these data identify Af17 as a potential locus for the maintenance of sodium and BP homeostasis and suggest that a particular histone modification is directly linked to these processes. Af17-mediated regulation of BP is largely, but not exclusively, the result of modulating ENaC, suggesting it has potential as a therapeutic target for the control of BP.

Histone H3 lysine 79 methyltransferase Dot1 is required for immortalization by MLL oncogenes.

Chimeric oncoproteins resulting from fusion of MLL to a wide variety of partnering proteins cause biologically distinctive and clinically aggressive acute leukemias. However, the mechanism of MLL-mediated leukemic transformation is not fully understood. Dot1, the only known histone H3 lysine 79 (H3K79) methyltransferase, has been shown to interact with multiple MLL fusion partners including AF9, ENL, AF10, and AF17. In this study, we utilize a conditional Dot1l deletion model to investigate the role of Dot1 in hematopoietic progenitor cell immortalization by MLL fusion proteins. Western blot and mass spectrometry show that Dot1-deficient cells are depleted of the global H3K79 methylation mark. We find that loss of Dot1 activity attenuates cell viability and colony formation potential of cells immortalized by MLL oncoproteins but not by the leukemic oncoprotein E2a-Pbx1. Although this effect is most pronounced for MLL-AF9, we find that Dot1 contributes to the viability of cells immortalized by other MLL oncoproteins that are not known to directly recruit Dot1. Cells immortalized by MLL fusions also show increased apoptosis, suggesting the involvement of Dot1 in survival pathways. In summary, our data point to a pivotal requirement for Dot1 in MLL fusion protein-mediated leukemogenesis and implicate Dot1 as a potential therapeutic target.

Widely expressed Af17 is likely not required for embryogenesis, hematopoiesis, and animal survival.

As a putative transcription factor, Af17 may play a role in multiple signaling pathways. However, the Af17 expression profile during development and in adult tissues remains largely uncharacterized. The importance of Af17 function in embryogenesis, hematopoiesis, and animal survival has never been addressed before. Here we report the generation of the first Af17 mutant mouse model and characterization of the Af17 temporal and spatial expression profile in various embryonic stages and adult tissues by X-gal staining, in situ hybridization, and RT-PCR. Af17 expression is detected in specific cell populations in all stages and in multiple tissues examined. In situ hybridization yielded a consistent Af17 expression pattern by X-gal staining. Homozygous mutant mice are viable, fertile, normal in size, and do not display any gross physical, behavioral, or hematopoietic abnormalities. Thus, our studies describe the generation of the first Af17 mutant mouse model, provide the first developmental profile of Af17 expression, and reveal that Af17 may be dispensable for normal embryogenesis, hematopoiesis, and animal survival.

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.

AF17 competes with AF9 for binding to Dot1a to up-regulate transcription of epithelial Na+ channel alpha.

We previously reported that Dot1a.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.AF9-mediated repression of alpha-ENaC and other target genes.

Aldosterone-induced Sgk1 relieves Dot1a-Af9-mediated transcriptional repression of epithelial Na+ channel alpha.

Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes--including that encoding the epithelial Na+ channel (ENaC), a key arbiter of Na+ transport in the kidney and other epithelia--have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCalpha by a complex containing the histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) and the putative transcription factor ALL1-fused gene from chromosome 9 (Af9) as well as the release of this repression by aldosterone treatment. Here we provide evidence from renal collecting duct cells and serum- and glucocorticoid-induced kinase-1 (Sgk1) WT and knockout mice that Sgk1 phosphorylated Af9, thereby impairing the Dot1a-Af9 interaction and leading to targeted histone H3 Lys79 hypomethylation at the ENaCalpha promoter and derepression of ENaCalpha transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCalpha and likely other aldosterone-induced genes.

Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha in an aldosterone-sensitive manner.

Aldosterone is a major regulator of epithelial Na(+) absorption and acts in large part through induction of the epithelial Na(+) channel (ENaC) gene in the renal collecting duct. We previously identified Dot1a as an aldosterone early repressed gene and a repressor of ENaCalpha transcription through mediating histone H3 Lys-79 methylation associated with the ENaCalpha promoter. Here, we report a novel aldosterone-signaling network involving AF9, Dot1a, and ENaCalpha. AF9 and Dot1a interact in vitro and in vivo as evidenced in multiple assays and colocalize in the nuclei of mIMCD3 renal collecting duct cells. Overexpression of AF9 results in hypermethylation of histone H3 Lys-79 at the endogenous ENaCalpha promoter at most, but not all subregions examined, repression of endogenous ENaCalpha mRNA expression and acts synergistically with Dot1a to inhibit ENaCalpha promoter-luciferase constructs. In contrast, RNA interference-mediated knockdown of AF9 causes the opposite effects. Chromatin immunoprecipitation assays reveal that overexpressed FLAG-AF9, endogenous AF9, and Dot1a are each associated with the ENaCalpha promoter. Aldosterone negatively regulates AF9 expression at both mRNA and protein levels. Thus, Dot1a-AF9 modulates histone H3 Lys-79 methylation at the ENaCalpha promoter and represses ENaCalpha transcription in an aldosterone-sensitive manner. This mechanism appears to be more broadly applicable to other aldosterone-regulated genes because overexpression of AF9 alone or in combination with Dot1a inhibited mRNA levels of three other known aldosterone-inducible genes in mIMCD3 cells.