Articles in PresS. Am J Physiol Renal Physiol (September 11, 2013). doi:10.1152/ajprenal.00202.2013
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Mineralocorticoid receptor antagonizes Dot1a-Af9 complex to increase αENaC transcription
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Xi Zhang1$, Qiaoling Zhou2,*, Lihe Chen3, Stefan Berger4, Hongyu Wu1, Zhou Xiao1,2, David Pearce5,
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Xiaodong Zhou1 and Wenzheng Zhang1,3,*
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Texas 77030
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410008, PR. China.
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Houston, Houston, Texas 77030
Department of Internal Medicine, The University of Texas Medical School at Houston, Houston,
Department of Internal Medicine, Xiangya Hospital, Central South University, Changsha, Hunan
Graduate School of Biomedical Sciences, The University of Texas Health Science Center at
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280, Heidelberg, Germany.
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94143, USA
German Cancer Research Center, Division Molecular Biology of the Cell I, Im Neuenheimer Feld
Division of Nephrology, Department of Medicine, University of California, San Francisco, California
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$: Current address: Department of Biology, College of Natural Sciences, University of Texas at
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Austin, Austin, TX 78712
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Running title: MR as a new Af9 binding partner and regulator
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*Address correspondence to: Wenzheng Zhang, Department of Internal Medicine, The University of
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Texas Medical School at Houston, 6431 Fannin, MSB 5.135, Houston, Texas 77030. Tel: 713-500-
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6862; Fax: 713-500-6882; E-Mail: [email protected] and Qiaoling Zhou, Department of
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Copyright © 2013 by the American Physiological Society.
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Internal Medicine, Xiangya Hospital, Central South University, Changsha, Hunan 410008, PR. China.
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Tel: 86-731-8423-7238; Fax: 86-731-8432-7348; E-mail: [email protected].
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Abstract
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Aldosterone is a major regulator of Na+ absorption and acts by activating the
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mineralocorticoid receptor to stimulate the epithelial Na+ channel (ENaC). MR-/- mice exhibited
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pseudohypoaldosteronism type 1 (hyponatremia, hyperkalemia, salt wasting, and high levels
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of aldosterone) and died around P10. However, if and how MR regulates ENaC transcription
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remain incompletely understood. Our earlier work demonstrated that aldosterone activates
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αENaC transcription by reducing expression of Dot1a and Af9 and by impairing Dot1a-Af9
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interaction. Most recently, we reported identification of a major Af9 binding site in the αENaC
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promoter and upregulation of αENaC mRNA expression in mouse kidneys lacking Dot1a.
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Despite these findings, the putative antagonism between the MR/aldosterone and Dot1a-Af9
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complexes has never been addressed. The molecular defects leading to PHA-1 in MR-/- mice
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remain elusive. Here, we report that MR competes with Dot1a to bind Af9. MR/aldosterone and
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Dot1a-Af9 complexes mutually counterbalance ENaC mRNA expression in IMCD3 cells. Real-
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time RT-qPCR revealed that 5-day-old MR-/- vs. MR+/+ mice had significantly lower αENaC
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mRNA levels. This change was associated with an increased Af9 binding and H3 K79
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hypermethylation in the αENaC promoter. Therefore, this study identified MR as a novel
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binding partner and regulator of Af9 and a novel mechanism coupling MR-mediated activation
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with relief of Dot1a-Af9-mediated repression via MR-Af9 interaction. Impaired ENaC
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expression due to failure to inhibit Dot1a-Af9 may play an important role in the early stages of
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PHA-1 (prior to P8) in MR-/- mice.
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Key words: aldosterone, mineralocorticoid receptor, Af9, Dot1a, pseudohypoaldosteronism type 1
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Introduction.
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The renin-angiotensin-aldosterone system (RAAS) plays a major role in the control of blood pressure,
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extracellular fluid volume, and electrolyte balance, largely through the regulation of urinary Na+
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excretion. Perturbations in the normal or adaptive mechanisms controlling this system, which are
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commonly encountered in clinical practice, can result in organ system dysfunction and even death. In
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the kidney, aldosterone-dependent regulation of Na+ reabsorption takes place in the aldosterone-
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sensitive distal nephron, which comprises the late distal convoluted tubule, connecting tube, and
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collecting duct. Transepithelial Na+ absorption occurs by apical Na+ entry via the epithelial Na+
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channel (ENaC) and basolateral Na+ exit via the Na+,K+-ATPase.
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ENaC is composed of three subunits α, β, and γ. Defects in ENaC subunits are responsible for two
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human
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pseudohypoaldosteronism type 1 (PHA-1). The former is manifested by early onset of hypertension
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coupled with normal or low plasma aldosterone levels, and is induced by an inappropriately high rate
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of Na+ absorption by the collecting duct due to gain-of-function mutations in βENaC or γENaC. In
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contrast, PHA-1 is a salt wasting syndrome with hypotension and high plasma aldosterone levels,
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resulting from loss-of-function mutations in any of the three subunits. Inactivation of each of the three
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genes encoding the ENaC subunits leads to perinatal-lethal phenotype, characterized by lung fluid-
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clearance failure, and/or by an acute PHA-1 with severe hyperkalemia and metabolic acidosis
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(reviewed in (35)).
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Aldosterone, like other steroid hormones, modulates gene transcription by interaction with two distinct
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but similar types of nuclear receptors: mineralo- and glucocorticoid receptors (MR and GR), which
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function as ligand-dependent transcription factors. These receptors regulate transcription by
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recognizing palindromic gluococorticoid response element (GRE), normally presented in the 5’
genetic
diseases,
Liddle’s
syndrome
and
the
autosomal
recessive
form
of
4
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flanking region of target genes, upon homo- or heterodimerization of the ligand-receptor complex (1),
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and produce long-lasting physiological effects of aldosterone stimulation. In contrast to direct trans-
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activation and repression, steroid receptors may also control gene expression by protein-protein
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interactions between the receptor and other trans-acting factors. Unique receptor-containing
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complexes may target distinct cis-acting elements, where the receptor does not contribute to DNA
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binding (43). The ligand-dependent modulation of transcription by the ligand-receptor complex has
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been termed “genomic” and is sensitive to inhibitors of transcription and translation.
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The biological significance of MR in maintaining Na+ homeostasis is demonstrated by the phenotype
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of mice deficient in MR (MR-/-) (2). MR-/- mice die in the second week after birth, showing at day 8
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PHA-1 phenotype with hyponatremia, hyperkalemia, high renal salt wasting, and a strongly activated
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RAAS (2). The MR-/- mice can be rescued by matched NaCl substitutions starting at day 5 (5).
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Although these observations confirm the essential role of MR in controlling Na+ balance, whether and
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how MR regulates ENaC transcription remain incompletely defined.
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In addition, how the ligand-receptor complex gains the accessibility to the DNA, which is packed into
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chromatin, remains obscure. Many genes have been identified to be up- or down-regulated by
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aldosterone in different systems including the renal collecting duct (18, 32, 41), IMCD3 (14), and M1
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cells (24). However, whether any of these genes play a role in histone modifications or chromatin
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remodeling is virtually unknown, until our identification of Dot1a as the first histone modifier in
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aldosterone-mediated transcriptional control.
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Disruption of telomeric silencing 1 (Dot1) was originally cloned as a gene affecting telomeric silencing
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in Saccharomyces cerevisiae. It is highly conserved from yeast to human (13, 37). Members of the
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Dot1 family including mouse Dot1l specifically methylate histone H3 K79 (45, 54). Dot1l plays an
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important role in multiple biological processes (21, 26, 33, 37, 55). Mouse Dot1l encodes five
5
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alternative splicing variants (Dot1a-e), with Dot1a being highly expressed in kidney and other tissues
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(54).
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Our earlier work linked histone methylation to aldosterone-mediated regulation of αENaC
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transcription. Under basal conditions, Dot1a and ALL1-fused gene from chromosome 9 protein (Af9)
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form a repression complex, which directly or indirectly binds to specific sites of the αENaC promoter,
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leading to targeted histone H3 K79 hypermethylation and repression of αENaC. Af9 possesses a
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YEATS (YNL107w, ENL, Af-9, and TFIIF small subunit) domain and a nuclear targeting sequence,
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consistent with a role as a transcription factor. Aldosterone attenuates the Dot1a-Af9 complex by
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reducing expression of Dot1a (55) and Af9 (56) and by inducing expression of Sgk1, which impairs
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Dot1a-Af9 interaction through phosphorylating Af9 (57), leading to histone H3 K79 hypomethylation of
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the αENaC promoter and release of αENaC repression. While H3 K79 hypomethylation occurs at
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multiple subregions, it takes place primarily at the R3 subregion of the αENaC promoter (57). Most
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recently, we reported that Af9 directly binds a cis-element in the R3 subregion and that specific
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inactivation of Dot1l in mouse kidney results in upregulation of αENaC (58), demonstrating the
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relevance and significance of Dot1a-Af9-mediated repression of αENaC in vivo in mouse kidney.
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In addition to aldosterone, Af17 can also relieve Dot1a-Af9-mediated repression. Af17 and Af9
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competitively bound the same domain of Dot1a in multiple assays and had antagonistic effects on
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expression of ENaC in 293T cells (30). Af17 facilitated Dot1a nuclear export, decreased its nuclear
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expression, and relieved Dot1a-Af9-mediated repression (30). More importantly, changes in ENaC
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transcription corresponded to alternation in benzamil-sensitive Na+ currents, as measured by whole-
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cell patch clamping (30). Similar findings were made using single-cell fluorescence imaging and
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equivalent short-circuit current to measure ENaC activity in more physiologically relevant mouse
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collecting duct IMCD3 and M1 cells (31, 49). In mice, deletion of Af17 led to increased dimethylation
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of histone H3 K79, reduced ENaC function, increased Na+ excretion and decreased blood pressure
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(8). In contrast, inducing high levels of plasma aldosterone by a variety of methods completely
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compensated for Af17 deficiency with respect to sodium handling and blood pressure (BP) (8). The
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clinical relevance of the Dot1a-Af9 pathway in regulating blood pressure is also supported by a recent
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clinical study. In this study, a polymorphism in DOT1L (rs2269879) could be associated with blood
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pressure response to hydrochlorothiazide in Caucasians and a polymorphism in AF9 (rs12350051)
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may be associated with untreated blood pressure in African-Americans (10).
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Nevertheless, two major issues remain unclear. 1) Does the MR stimulate αENaC mRNA
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expression? 2) Do the MR and Dot1a-Af9 complex control αENaC transcription by interacting with
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each other? If so, do they mutually antagonize their opponents’ effect on the αENaC promoter? Here,
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we report that 1) MR competes with Dot1a to bind the same domain of Af9; 2) MR and Dot1a impair
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their opponents’ function in controlling αENaC mRNA abundance in IMCD3 cells; 3) MR-/- mice at the
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age of 5 days have impaired αENaC mRNA expression, possibly due to increased association of Af9
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and H3 K79 hypermethylation with the R3 and other subregions of the αENaC promoter. Hence, this
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study uncovered a novel mechanism coupling MR-mediated activation with relief of Dot1a-Af9-
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mediated repression via competitive interactions between MR-Af9 and Dot1a-Af9.
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Experimental Procedures
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Reagents. LIPOFECTAMINETM 2000 reagent (Invitrogen), and antibodies against Af9 (Bethyl) and
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MR (Santa Cruz) were obtained and used according to the manufacturer’s instructions. Constructs
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encoding GFP-Dot1a, GFP-Dot1a 479-659, FLAG-Af9, RFP-Af9, GST-Af9 397-557, GAL4-AD-Af9
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and GFP-MR have been described previously (27, 54, 56). The Af9 insert in the GAL4-AD-Af9
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construct was released as an NdeI-XhoI fragment and cloned into pGBKT7 at the same enzyme sites
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to produce the plasmid for expressing GAL4-BD-Af9. A fragment encoding Af9 2-406 was amplified
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and cloned into pGBKT7 at EcoRI-XhoI for expressing GAL4-BD-Af9 2-406. Human MR coding
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region was amplified and cloned into pGADT7 at NdeI-XhoI to generate constructs expressing GAL4-
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AD-MR. Restriction enzyme digestion and DNA sequencing were performed to verify the presence of
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the inserts and their sequences in the constructs.
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Cell culture and transient transfections. IMCD3 cells were maintained with DMEM/F12 plus 10%
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FBS. For transcriptional analyses using transiently transfected IMCD3 cells, cells were seeded and
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cultured with DMEM/F12 plus 10% FBS for 24 hours, then switched to DMEM/F12 plus 10%
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charcoal-stripped FBS for 16 hours before transfection with LIPOFECTAMINETM 2000 reagent mixed
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with the plasmids indicated in the figure legend. Twenty-four hours later, cells were treated with
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aldosterone (100 nM) for 16 hours, followed by real-time RT-qPCR.
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immunoblotting,
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immunoprecipitation. These assays were conducted according to our published protocols (7, 30,
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48, 54, 56, 57) and briefly described in the figure legends.
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Deconvolution microscopy. IMCD3 cells cultured with DMEM/F12 plus 10% FBS on coverslips
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were cotransfected with constructs encoding GFP-MR and RFP-Af9. 24 hours later, cells were
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washed briefly in phosphate buffered saline (PBS) and fixed with 1% fresh prepared
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paraformaldehyde for 30 minutes at room temperature. Staining of the nucleus was done with 300 nM
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4',6-diamidino-2-phenylindole (DAPI, Sigma) for 15 minutes at room temperature. Coverslips were
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mounted onto microscope slides with Vectashield mounting medium (Vector Labs). Deconvolution
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microscopy was conducted at the Multi-User Fluorescence Imaging and Microscopy Core Facility,
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Department of Pathology and Laboratory Medicine, University of Texas Medical School, Houston, TX.
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The protocols for image analysis were described in our previous publications (30, 31, 56).
immunoprecipitation,
real-time
RT-qPCR,
and
chromatin
8
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Animal experiments. MR+/- mice were previously described and maintained with free access to
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water and normal Na+ (0.4%) diet (2). MR-/- and their WT littermates were obtained by inbreeding of
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the MR+/- mice. PCR-based genotyping was conducted with tail genomic DNA as described (7, 50,
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59). All animal studies were performed in accordance with NIH Guides for the Care and Use of
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Laboratory Animals and were approved by the University of the University of Texas Health Science
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Center at Houston Animal Welfare Committee.
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Statistical analysis. Due to small sample sizes, expression of each ENaC subunit gene was
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compared using non-parametric Kruskal-Wallis test, followed by Dunn’s multiple comparison post-test
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to compare one group with another in Fig. 4. For all other comparisons, unpaired Student t-test was
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carried out to determine the significance. In all cases, P<0.05 was considered significant.
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Results.
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MR interacts with Af9 in yeast two-hybrid assay. MR has been previously reported to interact with
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GR (22, 34) and multiple coactivators and corepressors (reviewed in (51)). On the other hand, Af9
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binds Dot1a (56) and Sgk1 (57) as well as Af4 (11), Aff4 (4), CBX8 (17), and BCoR (42). Since MR
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activates and Af9 represses αENaC, we hypothesize that MR and Af9 may interact to mutually
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antagonize their opponent effect on αENaC transcription. As the first step to test this hypothesis, we
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set to determine if MR interacts with Af9 in yeast two-hybrid assays. MR and Af9 were expressed as
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GAL4-AD and GAL4-BD fusions, respectively. Cotransformation of Gal-AD-MR and GAL4-BD-Af9
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constructs into yeast strain AH109 resulted in activation of three GAL4-dependent reporters, as
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evidenced by the Ade+ His+ and Mel1+ phenotype. However, this phenotype was abolished by
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replacement of one or both of these fusion constructs with the corresponding empty vectors (Fig. 1).
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Further analyses revealed that the N-terminal part of Af9 (aa 2-406) apparently was unable to bind
9
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MR. In contrast, the very C-terminal part of Af9 (aa 397-557) appeared to be sufficient for mediating
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the interaction. Interestingly, this fragment has been shown to binds Dot1a (30, 56). These data
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suggest that MR interacts specifically with Af9 in yeast two-hybrid assays and MR may compete with
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Dot1a to bind Af9 aa 397-557.
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MR co-immunoprecipitates with Af9. To independently verify MR-Af9 interaction and demonstrate
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that the interaction occurs at the endogenous protein level, we performed co-immunoprecipitation
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assay. Whole kidney lysates from MR+/+ and MR-/- mice as negative control were immunoprecipitated
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with a rabbit antibody specific for Af9 or the same amount of normal rabbit IgG as negative control.
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Immuoprecipitated proteins were subject to immunoblotting analyses with the same Af9 antibody or a
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rabbit antibody against MR. As shown in Fig. 2A, Af9 was detected in the input and
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immunoprecipitates of the Af9 antibody from both genotypes, but not in the reactions of the normal
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rabbit IgG, confirming the specificity of the Af9 antibody. Similarly, MR appeared in the input and the
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Af9 immunoprecipitates from MR+/+ mice, but not in the input and Af9 immunoprecipitates from MR-/-
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mice. MR was also absent from the IgG controls. Accordingly, we conclude that the endogenous MR
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and Af9 specifically interact in vivo in mouse kidney.
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To demonstrate the biological relevance of the MR-Af9 interaction, we coexpressed green
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fluorescence protein-tagged (GFP)-MR with red fluorescence protein (RFP)-Af9 fusion in IMCD3 cells
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and examined their cellular distribution by deconvolution microscopy. As expected, GFP-MR partially
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colocalized with RFP-Af9 primarily, if not exclusively, in the nucleus (Fig. 2B).
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MR competes with Dot1a to bind Af9. We previously demonstrated that Af9 397-557 specifically
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interacts with Dot1a in multiple assays including GST pulldown. In this assay, GST-Af9 397-557
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expressed and purified from E. coli was shown to retain a green fluorescence protein (GFP) fusion
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harboring Dot1a 479-659. We verified the paucity of interaction between the two tags (GST and GFP)
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under the same conditions (56, 57). The finding that the Dot1a-interacting domain of Af9 can also
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bind MR in yeast two hybrid assay suggests that MR may compete with Dot1a to bind Af9, as
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illustrated in Fig. 3A. To validate this hypothesis, we performed GST pulldown assays to investigate
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Dot1a-Af9 interaction with MR as a competitor. As shown in Fig. 3B, increasing the amount of lysate
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containing overexpressed MR from 0 to 1000 μl gradually reduced the amount of the GFP-Dot1a
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fusion retained by a fixed amount of the GST-Af9 fusion. The relative Dot1a-Af9 binding efficiency in
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the reaction with 1000 μl of MR-overexpressing lysate was ~30% of the control in which the
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competitor was omitted (compare lane 4 with lane 2, Fig. 3B), indicating that MR inhibited Dot1a-Af9
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interaction.
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MR antagonizes Dot1a-Af9-mediated repression of αENaC mRNA expression. Aldosterone
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regulates αENaC transcription through an imperfect GRE in the 5’ flanking region (20, 23, 36). The
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GRE is also responsible for Ras-mediated repression of αENaC in salivary epithelial cells (53),
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indicating that the αENaC transcription control is achieved by the mutual antagonistic effect between
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MR/GR-dependent activation and Ras-dependent repression. In IMCD3 cells, use of GR- or MR-
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specific inhibitors suggested that both receptors contribute to the aldosterone-mediated effects on
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gene expression (14). Our earlier work suggests that aldosterone activates αENaC transcription by
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preventing the association of the Dot1a-Af9 complex with its binding site via Sgk1-mediated
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phosphorylation of Af9 and down-regulation of Dot1a and Af9 expression. We recently identified and
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characterized a major Af9 binding site in the 5’ flanking region of αENaC (58), which is also the first
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Af9 binding site identified so far.
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To test the hypothesis that MR activates ENaC transcription by antagonizing Dot1a-Af9-mediated
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repression, we continued to use IMCD3 cells as the model systems. Previous studies using primary
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cells derived from the inner medullary collecting duct (IMCD) indicated that IMCD is a target of
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aldosterone action and constitutes an important terminal site of Na+ reabsorption and acid secretion
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in the collecting duct (38, 39, 46). IMCD3 cells share many of the phenotypic properties of the IMCD
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in vivo (29). They have been shown to respond to aldosterone by us (55-57) and others (6, 14, 15,
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44), and express all known components of Dot1a-Af9 signaling pathways (Sgk1 (14), Dot1a, Af9, and
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ENaC genes (55, 56)). More importantly, we have reported that transcriptional changes of ENaC and
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Sgk1 are translated into changes in their protein levels and benzamil-sensitive Na+ transport in both
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IMCD3 and mouse cortical collecting duct cells M1 (31). Therefore, it is very likely that mechanisms
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defined in IMCD3 cells could be applicable to cortical collecting duct cells.
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Accordingly, IMCD3 were cultured with charcoal-stripped serum and transiently transfected with
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plasmid DNA mixture containing different combinations of pEGFP-Dot1a, pFLAG-Af9, and pEGFP-
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MR. The amount of pEGFP-Dot1a and pFLAG-Af9 were added equally across the transfections (50
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ng/plasmid) except the control in which only an empty vector (pCMV500) was included. Various
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amounts of pEGFP-MR were added. The empty vector pCMV500 was supplemented accordingly to
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keep the total amount of DNA the same among transfections. Twenty-four hours after transfection,
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cells were treated with aldosterone (100 nM) for 16 hour before harvest. Cells were analyzed by real-
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time RT-qPCR for expression of ENaC genes, with β-actin as internal control.
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While the aldosterone concentration in the medium was higher than the physiological concentration of
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the hormone, the actual effective intracellular concentration was probably much lower (14). This is
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because steroid hormones may not diffuse across cell membranes freely (14, 28). A wide range of
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aldosterone concentrations including 10 (9), 30 (12), 1000 (14, 30, 31, 40, 49, 55-58), and even 1500
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nM (16) have been used in IMCD3 cells, mouse cortical collecting duct M1 cells, and HEK 293T cells
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by others and us. In particular, earlier studies showed that aldosterone elicited the most dramatic
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effect in IMCD3 cells at 1000 nM, which can be completely blocked by MR and GR inhibitors, used
12
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alone or in combination (14). Therefore, we believe that the effect of MR overexpression in the
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presence of 100 nM aldosterone in the medium should be detectable.
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Indeed, as shown in Fig. 4A-C, cells transfected with Dot1a and Af9 constructs without addition of the
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MR plasmid reduced α-, β-, and γENaC mRNA levels by about 50%, 40% and 70%, respectively,
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compared with control cells. The Dot1a-Af9-mediated repression was abolished by addition of 50 ng
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of pEGFP-MR. This is evidenced by indistinguishable mRNA levels of the ENaC genes in the
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transfected cells vs. control. Increasing the amount of the MR construct to 100 ng further enhanced
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the transcript abundance of these genes, reaching 50%, 100% and 40% higher α-, β-, and γENaC
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mRNA levels in the corresponding transfected cells than in the vector-transfected control cells. These
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results suggest that MR is capable of antagonizing Dot1a-Af9-mediated repression of ENaC mRNA
265
expression in IMCD3 cells.
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Dot1a-Af9 blunts MR-mediated activation of ENaC mRNA expression. In reciprocal experiments
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aimed to test the hypothesis that overexpression of Dot1a-Af9 suppresses MR-mediated
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transcriptional activation of ENaC genes, IMCD3 cells were transiently transfected with a fixed
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amount of pEGFP-MR (100 ng) and increasing amounts of pEGFP-Dot1a and pFLAG-Af9 ranging
270
from 0 to 50 ng/plasmid. As shown in Fig. 4D-F, transfection with the MR construct alone resulted in
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α-, β-, and γENaC mRNA levels being 210%, 322% and 250% of the vector-transfected control,
272
respectively. Increasing amounts of the Dot1a and Af9 constructs added to the transfection mixture
273
progressively impaired the MR-mediated activation. Taken together, our data support the notion that
274
Dot1a-Af9 and MR mutually impair their opponent’s effect on ENaC transcription under the conditions
275
tested.
276
Deletion of MR decreases mRNA expression of ENaC genes in 5-day old neonate kidneys. If
277
MR antagonizes Dot1a-Af9-mediated repression in vivo in mouse kidney as in IMCD3, disruption of
13
278
MR should lead to decreased ENaC mRNA expression. Accordingly, we used MR knockout mice
279
(MR-/-) as the model system to address this question. We focused on 5-day old MR-/- neonates and
280
their WT littermates as control since around this day the activity of the RAAS is progressively
281
increasing.
282
MR-/- and their WT littermates were produced by inbreeding MR+/- mice and genotyped by PCR as we
283
reported before. We isolated the total kidney RNA from 5-day-old MR-/- mice and their WT littermates
284
and performed real-time RT-qPCR. We found that MR-/- mice reduced α, β, and γENaC mRNA levels
285
to 45%, 57%, and 65% of the WT littermates (Fig. 5, A-C). Hence, it appears that at day 5 the MR-/-
286
mice have impaired mRNA expression of ENaC genes.
287
MR deletion leads to an increase in Af9 binding and H3 K79 methylation at the αENaC
288
promoter in 5-day old neonate kidneys. If MR relieves Dot1a-Af9-mediated repression by
289
decreasing Af9 binding at the αENaC promoter, disruption of MR should be associated with an
290
increase in Af9 binding, which in turn recruits more Dot1a to enhance H3 K79 methylation. Indeed, in
291
the MR mutant animals, chromatin immunoprecipitation (ChIP) revealed that MR-/- vs. WT had 70%,
292
40% and 78% higher Af9 binding at R0, R1 and R3 subregions of αENaC promoter, respectively.
293
There were little or no Af9 binding detected in Ra and R2 subregions. Consistently, deletion of MR did
294
not induce a significant change in Af9 association with Ra and R2 subregions (Fig. 6, A-B). To
295
determine if increased Af9 is coupled with corresponding changes in Dot1a-mediated H3 K79
296
methylation, ChIP with an antibody specific for di-methyl K79 (m2K79) was performed in parallel. We
297
found substantial association of H3 m2K79 in all subregions except Ra in WT mice, as we reported in
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IMCD3 cells and Af17-/- mouse kidney (7, 55-57). The association of H3 m2K79 was significantly
299
increased by 41% and 54% in R1, and R3 of the MR-/- mice. Although there was a trend toward 40%
300
and 22% increases in H3 m2K79 in the R0 and R2, respectively, these increases did not reach the
14
301
statistical significance (Fig. 6C).
302
The increased Af9 binding and H3 m2K79 association with the αENaC promoter may also result from
303
impaired Sgk1 and increased Af9 and Dot1a expression. To address this question, we examined their
304
mRNA levels by RT-qPCR and found that MR-/- vs. WT significantly downregulated Sgk1 to 60% and
305
upregulated Af9 to 170%, respectively (Fig. 5, D & E). There was a 40% increase in Dot1a mRNA
306
level, but it was not statistically significant (Fig. 5F). In brief, our data uncovered a new mode of MR
307
action. MR may activate transcription of αENaC partially by interacting with Af9 and preventing Af9
308
from recruiting Dot1a for targeted H3 hypermethylation at the αENaC promoter. MR may also execute
309
the same effects by increasing Sgk1 and decreasing Dot1a and Af9 expression. We believe that
310
these mechanisms are not mutually exclusive. They may be always mixed and coordinately
311
regulated. These mechanisms may also be applicable to βENaC, γENaC and other MR targets.
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Discussion.
313
In this report, we identify a novel interaction between MR and Af9, which are considered as the
314
positive and negative regulators of αENaC transcription, respectively. We demonstrate the interaction
315
by different approaches (yeast two-hybrid, Co-IP, co-localization, and GST pulldown competition
316
assays). These two players appear to regulate mRNA expression of ENaC genes in IMCD3 cells,
317
partially by mutually attenuating their opposing effects. Consistently, inactivation of MR in vivo in
318
mouse kidney results in decreased mRNA expression of all three ENaC subunits in 5-day-old
319
neonates. In case of αENaC, the decreased mRNA expression in the MR mutant mice appears to be
320
partially attributable to increased Af9 association with the αENaC promoter. The resulting increase in
321
Af9 binding is coupled with H3 K79 hypermethylation at the promoter, which is presumably achieved
322
through Dot1a-Af9 interaction. Therefore, these studies show that MR, like Sgk1, is a novel binding
323
partner and regulator of Af9. MR upregulates ENaC mRNA expression in part by relieving Dot1a-Af9-
15
324
mediated repression in IMCD3 cells and in mouse kidney. In terms of downregulation of Dot1a-Af9
325
complex, MR resembles Af17 and Sgk1, although the underlying mechanisms may vary.
326
MR is a nuclear receptor and plays a key role in the pathophysiology of hypertension and cardiac
327
fibrosis (52). In the epithelial cells of kidney and colon, MR is critical for controlling sodium and
328
potassium transport. This is clearly demonstrated by inactivation of MR in mice. MR-/- mice exhibited
329
PHA-1 symptoms including hyponatremia, hyperkalemia, and a strongly activated RAAS, with 440-
330
fold increase in renin, 50-fold increase in angiotensin II, and 65-fold increase in aldosterone by day 8
331
after birth, compared to WT littermates (2, 3). Our current study revealed that MR-/- mice have
332
decreased mRNA levels of ENaC genes at day 5 after birth, possibly due to less activated RAAS and
333
insufficient plasma aldosterone concentration to attenuate Dot1a-Af9-mediated repression and to
334
activate substantial amount of GR. The impaired ENaC expression may be at least partially
335
responsible for the progressively developed PHA-1 phenotype.
336
However, the molecular mechanism by which MR controls transcription of its target genes including
337
αENaC is still incompletely defined. Like other members of the steroid hormone receptor family, MR
338
acts as a ligand-inducible transcription factor. Upon binding to its ligand such as aldosterone, the
339
receptor activates or represses the transcription of target genes by directly binding as monomers,
340
homodimers, or heterodimers with GR to the hormone responsive elements and produce long-lasting
341
physiological effects of aldosterone stimulation. It is commonly accepted that MR and GR are capable
342
of regulating transcription through a common hormone response element, normally presented in the
343
5’ flanking region of target genes (reviewed in (43)).
344
It has also been suggested that unique receptor complexes may target distinct cis-acting elements
345
(43). These modes of steroid hormone action involve the trans-activation and trans-repression via
346
interaction with cognate DNA-binding sites, such as GRE. In contrast to direct trans-activation and
16
347
repression, the steroid receptor may regulate gene expression by protein-protein interaction between
348
the receptor and other trans-acting factors. MR has been reported to interact with multiple
349
coactivators including steroid receptor coactivator 1 (SRC-1) and 2 (SRC-2), Peroxisome proliferator-
350
activated receptor gamma coactivator 1 (PGC-1α), RNA helicase A (RHA), eleven-nineteen lysine-
351
rich leukemia protein (ELL), FLICE-associated huge (FLASH), Fas-associated factor 1 (FAF-1),
352
Ubiquitin carrier protein 9 (Ubc9), transcription intermediary factor 1α (TIF-1α), and receptor-
353
interacting protein 140 (RIP140) (Reviewed in (51)). Interaction between MR and various
354
corepressors has also been documented. These corepressors are silencing mediator of retinoid and
355
thyroid hormone receptor (SMRT), nuclear receptor corepressor (NCoR), death-associated protein
356
(DAXX), and Protein inhibitor of activated STAT protein 1 (PIAS1) (Reviewed in (51)). Obviously,
357
before interacting with the cognate GRE, the ligand-receptor complex must have the accessibility to
358
the DNA, which is compacted into the chromatin.
359
Interestingly, some of these coregulators are enzymes that have either histone-modifying activities or
360
ATP-dependent chromatin remodeling activities to promote the accessibility of transcription factors to
361
GRE (25, 47). For example, MR binds RNA helicase A (RHA) upon aldosterone induction, which
362
recruits a complex with histone acetyltransferase (HAT) activity that contains cAMP-response
363
element-binding protein (CREB)-binding protein (CBP), leading to the cooperative potentiation of MR
364
transcriptional activity by RHA and CBP complex (19).
365
In addition to activation through MR/aldosterone, ENaC transcription is also controlled by Dot1a-Af9
366
complex-mediated repression. Our previous studies demonstrated that downregulation of Dot1a-Af9
367
complex can be achieved by aldosterone-dependent and -independent mechanisms. The former
368
includes reduction of Dot1a and Af9 expression and impairment of Dot1a-Af9 interaction through
369
Sgk1-mediated Af9 phosphorylation (55-57). The latter involves Af17 that competes with Af9 to bind
17
370
Dot1a and enhances Dot1a cytoplasmic expression at the expense of its nuclear expression (7, 30,
371
49).
372
The current study revealed a novel mechanism of MR action that couples activation with derepression
373
through competitive interactions between MR-Af9 and Dot1a-Af9. Af9 represses αENaC transcription
374
by modulating Dot1a-mediated H3 K79 hypermethylation at R0-R3, but not Ra subregions of the
375
αENaC promoter. Our most recent work demonstrated that Af9 directly binds an Af9 binding site in
376
the R3 subregions. Mutation of this site resulted in higher basal αENaC promoter activity and
377
impaired Dot1a-mediated inhibition in trans-repression assays. Consistently, ablation of MR resulted
378
in a significance increase in Af9 binding and in H3 K79 methylation in the R3. Similar results were
379
also observed in R0 and R1, suggesting that additional Af9 sites exist in these two subregions.
380
Identification of these Af9 sites requires future studies. In addition, similar patterns of transcriptional
381
control of β and γENaC by Dot1a-Af9 and MR were also observed. Future investigations are also
382
deserved to identify and characterize the potential elements responsible for recruiting Dot1a, Af9 and
383
MR directly or indirectly in the upstream sequences of the β and γENaC genes.
384
Acknowledgement. We thank Mary Rose Reisenauer for technical support. This work was funded by
385
National Institutes of Health grants R01 DK080236 (to W.Z.), U01 AI09090 (to XD. Z.), Scleroderma
386
Foundation (to XD. Z.), and The National Natural Science Foundation of China (NSFC) grant
387
81070552 (to Q.L.Z.). The authors have declared that no conflict of interest exists.
388
18
389
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563
564
Figure legends.
565
Fig. 1. MR and Af9 interacted in yeast two-hybrid assays. MR was expressed as GAL4-AD fusion
566
and tested for the ability to interact with GAL4-BD-Af9 fusions in yeast strain AH109. The
567
corresponding empty vectors were included as control. Interaction between the two fusions was
568
identified by the activation of the reporters, resulting in Ade+ His+ phenotype. Presence (+) or absence
569
(-) of interaction is shown.
26
570
Fig. 2. MR and Af9 co-immunoprecipitated and displayed partial nuclear colocalization in
571
IMCD3 cells. A. Co-IP. Whole kidney lysates of MR+/+ and MR-/- mice were immunoprecipitated (IP)
572
with a rabbit antibody specific for Af9 or normal rabbit IgG (as a negative control), using the Catch
573
and Release kit (Millipore) according to the manufacturer’s instruction. Immunoprecipitated proteins
574
were analyzed by immunoblot (IB) analysis in parallel with the antibodies as indicated. B.
575
Colocalization assay. MR was coexpressed as a GFP fusion along with RFP-Af9 in IMCD3 cells and
576
examined by deconvolution microscopy. Scale bar: 5 µm.
577
Fig. 3. MR competes with Dot1a to Af9. A. Diagrams showing how MR prevents its competitor
578
Dot1a from binding Af9. B. GST pulldown assay demonstrating the inhibitory effect of MR on Dot1a-
579
Af9 interaction. GST-Af9 397-557 was purified from E. coli and incubated with whole cell lysate of
580
293T cells expressing GFP-Dot1a 479-659. Alternatively, before incubation with the GST-Af9 fusion,
581
the whole cell lysate of 293T cells expressing GFP-Dot1a 479-659 was preincubated with various
582
amounts (μl) of 293T cells lysates harboring overexpressed MR. Input (In) of the lysates (5%) and
583
proteins bound to Glutathione Sepharose 4B beads were examined by immoblotting (IB) with the
584
antibodies indicated.
585
Fig. 4. MR and Dot1-Af9 complexes counterbalance ENaC mRNA expression. IMCD3 cells were
586
cotransfected with a mixture of constructs as indicated. The total amount of DNA (ng) was kept
587
constant across transfections by addition of an empty vector. Relative mRNA levels of ENaC subunits
588
were analyzed by real-time RT-qPCR and normalized to β-actin. For each subunit, the abundance of
589
the control was set to 1 and used to determine the relative level and the significance of the other
590
samples. *: P <0.05 versus control, n = 3. A. Overexpression of Dot1a and Af9 decreased ENaC
591
expression, which was counterbalanced by MR in a dose-dependent manner. B. In contrast, MR-
27
592
mediated stimulation of mRNA expression of ENaC was partially impaired by increasing amount of
593
Dot1a-Af9 DNA used for transfection.
594
Fig. 5. Transcriptional defects in 5-day-old MR-/- mice. A-F. Real-time RT-qPCR analysis. Total
595
kidney RNAs were isolated from MR+/+ (WT, n=5 mice) and MR-/- mice (MT, n=6 mice) at day 5 after
596
birth and examined for expression of genes as indicated by real-time RT-qPCR. β-actin was used as
597
internal control. For each gene, the abundance of the MR+/+ was set to 1 and used to determine the
598
relative level and the significance of the MR-/- samples. *: P <0.05.
599
Fig. 6. MR inactivation leads to increased Af9 binding and H3 K79 hypermethylation at the
600
αENaC promoter. A. Diagram of the 5’-flanking region of αENaC. Fragments designated Ra-R3 are
601
shown along with their relative positions to the major transcription start site (+1) of αENaC.
602
represent the putative GRE site (-811) and GRE half sites (-983, -416, -325, -241, and -234),
603
respectively.
604
of Af9 (B) and histone H3 dimethyl K79 (H3 m2K79) (C) to αENaC promoter. ChIP analyses with an
605
antibody against Af9 or H3m2K79 were performed with chromatin isolated from kidneys of the MR-/-
606
mice and their WT littermates at day 5 after birth. Relative ChIP efficiency was defined as
607
immunoprecipitated amount of materials to that of the initial input sample. The signals in R0 of WT
608
mice were set to 1 and used to calculate the relative levels of all other samples. *: P<0.05 vs. WT for
609
each region. n=4-7 mice/genotype.
and
indicates the Af9 binding site (+78) (55, 56, 58). B-C. MR deletion increases binding
610
28
611
612
613
614
615
616
617
618
619
620
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
Fig. 1. MR and Af9 interacted in yeast two-hybrid assays. MR was expressed as
GAL4-AD fusion and tested for the ability to interact with GAL4-BD-Af9 fusions in
yeast strain AH109. The corresponding empty vectors were included as control.
Interaction between the two fusions was identified by the activation of the reporters,
resulting in Ade+ His+ phenotype. Presence (+) or absence (-) of interaction is shown.
29
636
637
638
639
640
641
642
643
644
645
646
647
648
649
650
651
652
653
654
655
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660
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664
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666
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669
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672
673
674
675
676
677
Fig. 2. MR and Af9 co-immunoprecipitated and displayed partial nuclear
colocalization in IMCD3 cells. A. Co-IP. Whole kidney lysates of MR+/+ and MR-/mice were immunoprecipitated (IP) with a rabbit antibody specific for Af9 or normal
rabbit IgG (as a negative control), using the Catch and Release kit (Millipore)
according to the manufacturer’s instruction. Immunoprecipitated proteins were
analyzed by immunoblot (IB) analysis in parallel with the antibodies as indicated. B.
Colocalization assay. MR was coexpressed as a GFP fusion along with RFP-Af9 in
IMCD3 cells and examined by deconvolution microscopy. Scale bar: 5 µm.
30
678
679
680
681
682
683
684
685
686
687
688
689
690
691
692
693
694
695
696
697
698
699
700
701
702
703
704
705
706
707
708
709
710
711
712
713
714
715
716
717
718
719
720
721
Fig. 3. MR competes with Dot1a to Af9. A. Diagrams showing how MR prevents its
competitor Dot1a from binding Af9. B. GST pulldown assay demonstrating the
inhibitory effect of MR on Dot1a-Af9 interaction. GST-Af9 397-557 was purified from
E. coli and incubated with whole cell lysate of 293T cells expressing GFP-Dot1a 479659. Alternatively, before incubation with the GST-Af9 fusion, the whole cell lysate of
293T cells expressing GFP-Dot1a 479-659 was preincubated with various amounts
(μl) of 293T cells lysates harboring overexpressed MR. Input (In) of the lysates (5%)
and proteins bound to Glutathione Sepharose 4B beads were examined by
immoblotting (IB) with the antibodies indicated.
31
722
723
724
725
726
727
728
729
730
731
732
733
734
735
736
737
738
739
740
741
742
743
744
745
746
747
748
749
750
751
752
753
754
755
756
757
758
759
760
761
762
763
764
765
Fig. 4. MR and Dot1-Af9 complexes counterbalance ENaC mRNA expression.
IMCD3 cells were cotransfected with a mixture of constructs as indicated. The total
amount of DNA (ng) was kept constant across transfections by addition of an empty
vector. Relative mRNA levels of ENaC subunits were analyzed by real-time RT-qPCR
and normalized to β-actin. For each subunit, the abundance of the control was set to 1
and used to determine the relative level and the significance of the other samples. *: P
<0.05 versus control, n = 3. A. Overexpression of Dot1a and Af9 decreased ENaC
expression, which was counterbalanced by MR in a dose-dependent manner. B. In
contrast, MR-mediated stimulation of mRNA expression of ENaC was partially impaired
by increasing amount of Dot1a-Af9 DNA used for transfection.
32
766
767
768
769
770
771
772
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774
775
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778
779
780
781
782
783
784
785
786
787
788
789
790
791
792
793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
808
809
810
811
Fig. 5. Transcriptional defects in 5-day-old MR-/- mice. A-F. Real-time RT-qPCR
analysis. Total kidney RNAs were isolated from MR+/+ (WT, n=5 mice) and MR-/- mice
(MT, n=6 mice) at day 5 after birth and examined for expression of genes as
indicated by real-time RT-qPCR. β-actin was used as internal control. For each gene,
the abundance of the MR+/+ was set to 1 and used to determine the relative level and
the significance of the MR-/- samples. *: P <0.05.
33
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
857
Fig. 6. MR inactivation leads to increased Af9 binding and H3 K79
hypermethylation at the αENaC promoter. A. Diagram of the 5’-flanking region of
αENaC. Fragments designated Ra-R3 are shown along with their relative positions to
the major transcription start site (+1) of αENaC.
and
represent the putative GRE
site (-811) and GRE half sites (-983, -416, -325, -241, and -234), respectively.
indicates the Af9 binding site (+78) (55, 56, 58). B-C. MR deletion increases binding of
Af9 (B) and histone H3 dimethyl K79 (H3 m2K79) (C) to αENaC promoter. ChIP
analyses with an antibody against Af9 or H3m2K79 were performed with chromatin
isolated from kidneys of the MR-/- mice and their WT littermates at day 5 after birth.
Relative ChIP efficiency was defined as immunoprecipitated amount of materials to that
of the initial input sample. The signals in R0 of WT mice were set to 1 and used to
calculate the relative levels of all other samples. *: P<0.05 vs. WT for each region. n=4-7
mice/genotype.
34
Interaction
pBD-Af9 / pAD-MR
+
pBD-Af9 397-557 / pAD-MR
+
pBD-Af9 2-406 / pAD-MR
-
pBD-Af9 / pAD
-
pBD / pAD-MR
-
pBD/ pAD
WT
B.
MT
tiA
an
ut
IP
In
p
Ig f9
G
tiA
an
IB:
anti-Af9
In
p
ut
IP
Ig f9
G
A.
anti-MR
GFP-MR RFP-Af9 Merge
DAPI
A.
MR
Dot1a
Dot1a
+
AF9
AF9
B.
GST-AF9 397-557
1000
500
0
In
MR (μl)
αGFP
GFP-Dot1a
479-659
αGST
GST-AF9
397-557
αMR
MR
2
3
4
Binding (%) 100
55
30
Lane
1
*
D.
A.
*
4
*
3
*
2
*
5
*
ENaC mRNA
ENaC mRNA
5
*
1
0
*
4
*
*
3
2
1
0
E.
B.
*
5
*
4
ENaC mRNA
ENaC mRNA
5
*
3
2
1
0
*
4
*
*
*
3
2
1
0
F.
C.
4
*
*
3
2
*
*
*
5
*
ENaC mRNA
ENaC mRNA
5
1
0
4
*
3
*
*
*
2
1
0
MR
Dot1a
0
0
0
50
50 100
50 50
Af9
0
50
50
50
MR
Dot1a
0
0
100
0
Af9
0
0
100 100
25 50
25
50
B.
A.
1
*
0.5
0
1
0.5
0
WT MT
E.
*
*
2
1.5
1
0.5
0
WT MT
*
WT MT
F.
2.5
Af9 mRNA
1.2
1
0.8
0.6
0.4
0.2
0
1.2
1
0.8
0.6
0.4
0.2
0
WT MT
2
Dot1a mRNA
D.
Sgk1 mRNA
*
ENaC mRNA
1.5
ENaC mRNA
ENaC mRNA
1.5
C.
1.5
1
0.5
0
WT MT
WT MT
A.
-1372
Ra
-988
-965
R0
-735
-713
R1
-414
-415
R2
-57
+80
R3
+494
αENaC
B.
Af9 binding
4
WT
*
MT
3
2
*
*
R0
R1
1
0
Ra
H3 m2K79
C.
5
WT
4
MT
R2
R3
*
*
3
2
1
0
Ra
R0
R1
R2
R3