The broad goal of this research is to identify the molecular mechanisms underlying transcriptional control of the epithelial Na+ channel a-subunit (ENaCa) in kidney. ENaC .is a multi-subunit protein that plays a major role in control of epithelial Na+ transport, blood pressure, and the response to hyperaldosteronism. The ENaCa subunit is induced by aldosterone in the collecting duct, and appears to be rate-limiting for ENaC activity in this segment. Despite its importance, the mechanisms controlling ENaCa transcription and its induction by aldosterone are incompletely defined. We have characterized a novel histone methyltransferase, disrupter of telomeric silencing (Dot1), and show that it interacts with AF9a to form a chromatin-associated represser complex in the 5'-flanking region of the mouse ENaCa gene. Through SGK1-mediated phosphorylation of AF9a, aldosterone disrupts the complex, relieving the repression on the ENaCa and activating its transcription, independent of actions of the mineralocorticoid receptor. We now propose to use quantitative chromatin immunoprecipitation assays and promoter-reporter transient transfection assays to follow association of specific transcription factors and coregulatory proteins with the AF9a-Dot1 complex at the ENaCa promoter, to define patterns of binding, to test hypotheses regarding interactions among these factors, and to monitor changes in covalent histone modifications associated with transcriptional activation of the ENaCa gene under basal conditions and in response to aldosterone. The ability of defined nuclear proteins to alter the ENaCa promoter in trans will be tested in coexpression and RNA interference experiments. Studies in transgenic mice will test whether the candidate AF9a regulatory element identified is critical for faithful replication of the responses of the endogenous ENaCa gene.
Aim 1 will test the hypothesis that AF9a is a transcriptional represser that binds the 5' flanking region of ENaCa and nucleates the ordered recruitment of specific corepressors and chromatin remodeling proteins to the ENaCa promoter.
Aim 2 will test the hypothesis that AF9a, Dot1, SIRT1, Rad6 and other coregulator proteins promote histone hypermethylation and deacetylation and basal repression of ENaCa.
Aim 3 will test the hypothesis that aldosterone and SGK1 promote sequential and combinatorial recruitment and dismissal of coregulatory proteins at the AF9a element locus, dictating ENaCa gene activation. These studies will allow us to construct a novel, dynamic regulatory network to the overall model of ENaCa gene regulation in kidney, and to provide important insights into transcriptional control of complex genes and the molecular actions of mineralocorticoids. The proposed studies will also define new modes of function for SGK1, Dot1, and AF9a that may be more broadly applicable to other target proteins and to gene regulation in general.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
7R01DK075065-03
Application #
7456744
Study Section
Special Emphasis Panel (ZRG1-RUS-G (02))
Program Officer
Rasooly, Rebekah S
Project Start
2006-04-04
Project End
2010-03-31
Budget Start
2007-05-16
Budget End
2008-03-31
Support Year
3
Fiscal Year
2007
Total Cost
$291,616
Indirect Cost
Name
University of Florida
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Yu, Zhiyuan; Kong, Qun; Kone, Bruce C (2013) Aldosterone reprograms promoter methylation to regulate ?ENaC transcription in the collecting duct. Am J Physiol Renal Physiol 305:F1006-13
Singh, Rani; Kone, Bruce C; Gounni, Abdelilah S et al. (2013) Molecular regulation of Trypanosoma congolense-induced nitric oxide production in macrophages. PLoS One 8:e59631
Zhang, Wenzheng; Yu, Zhiyuan; Wu, Hongyu et al. (2013) An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the ýýENaC gene. Am J Physiol Renal Physiol 304:F367-75
Yu, Zhi-Yuan; Kong, Qun; Kone, Bruce C (2013) Physical and functional interaction of Rnf2 with Af9 regulates basal and aldosterone-stimulated transcription of the ?-ENaC gene in a renal collecting duct cell line. Biosci Rep 33:
Yu, Zhiyuan; Kong, Qun; Kone, Bruce C (2013) Sp1 trans-activates and is required for maximal aldosterone induction of the ?ENaC gene in collecting duct cells. Am J Physiol Renal Physiol 305:F653-62
Kone, Bruce C (2013) Epigenetics and the control of the collecting duct epithelial sodium channel. Semin Nephrol 33:383-91
Duarte, Julio D; Zineh, Issam; Burkley, Ben et al. (2012) Effects of genetic variation in H3K79 methylation regulatory genes on clinical blood pressure and blood pressure response to hydrochlorothiazide. J Transl Med 10:56
Yu, Zhiyuan; Kong, Qun; Kone, Bruce C (2010) CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells. Am J Physiol Renal Physiol 298:F617-24
Reisenauer, Mary Rose; Anderson, Marc; Huang, Le et al. (2009) AF17 competes with AF9 for binding to Dot1a to up-regulate transcription of epithelial Na+ channel alpha. J Biol Chem 284:35659-69
Zhang, Dongyu; Yu, Zhi-yuan; Cruz, Pedro et al. (2009) Epigenetics and the control of epithelial sodium channel expression in collecting duct. Kidney Int 75:260-7

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