Epigenetic Control of ENaC

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1 Epigenetic Control of ENaC Transcription, Na + Metabolism and Blood Pressure Wenzheng Zhang, Ph.D. Department of Internal Medicine The University of Texas Medical School at Houston International Conference on Geriatrics & Gerontology Chicago, July 08-10, 2014

2 Epithelial Na Channel (ENaC) Consists of 3 partially homologous subunits α, β and γ. Expressed in the apical membrane of salt-absorbing epithelia of kidney, colon, and lung. Critical for Na + absorption in the lung, colon, and kidney. Mutations associated with two genetic diseases: Liddle s syndrome (Hypertension) and pseudohypoaldosteronism type 1 (PHA-1, Hypotension). Tightly regulated by aldosterone at multiple levels (mrna, protein, and subcellular distribution).

3 Liddle s Syndrome (Hypertension) Described by G. Liddle in 1963 Autosomal dominant fashion Early and severe hypertension Hypokalemia Metabolic alkalosis Low plasma aldosterone concentration Constitutive hyperactivity of ENaC β γ Bonny et al Kidney International (2000) 57, M1 M2 PPxY R566* A576fr Q591* T594fr P596fr R597fr Y620H P618L/S P617S M1 M2 PPxY W574*

4 Pseudohypoaldosteronism type 1 (PHA-1, Hypotension) Two forms: Autosomal dominant renal form by mutations in MR Autosomal recessive form by mutations in ENaC subunits Salt wasting Hyperkalemia Metabolic acidosis High plasma aldosterone concentration Loss-of-function mutations in ENaC α β γ Bonny et al Kidney International (2000) 57, M1 M2 PPxY R56* C133Y F435fr R508* I68fr R139D T168fr M1 M2 PPxY G37S T216fr D305fr M1 M2 PPxY KYS N

5 The Classical model for Molecular Action of Aldosterone NR NR HRE ENaCα NR NR HRE Nucleus SGK1

6 A MD Af9 / Af17 NSL1 LZ NSL2 NSL3 B Af9 YEATS / MR / Hsp90 S435: Sgk1 phosphorylation site C Af17 PHD-ZF

7 Histone Methyltransferase Gene Dot1 Identified in yeast as a disruptorr of telomeric silencing. Encodes a methyltransferase specific for histone H3 K79. Expressed in kidney and other tissues and as early as 2-cell stage. Five alternative splicing variants in mouse (-e), with being the most highly expressed and most closely related to hdot1l. Dot1L-deficient mouse embryos show multiple developmental abnormalities, including growth impairment, angiogenesis defects in the yolk sac, and cardiac dilation, and die between 9.5 and 10.5 days post coitum. Involved in cell cycle regulation, DNA repair, chromosome remodeling, transcription regulation and leukemogenesis.

8 Putative Transcription Factor Identified as one of the most common fusion partners of the mixed-lineage leukemia protein (MLL or ALL-1) A putative transcription factor, although, no - regulated genes have been well documented Expressed in kidney and other tissues deletion leads to perinatal lethality

9 and Colocalize in the Nucleus of mimcd3 Cells DAPI Zhang et al. JBC

10 Contains a Putative Phosphorylation Site for SGK1 SGK1 Human Mouse AF F9 Rat Horse 441 RSRRVS RSRRVS RSRRVS RSRRVS. 40 P Consensus RXRXX(S/T) Zhang et al. JCI

11 SGK1 Phosphorylates in vitro Active SGK1 - GST + - GST In vitro Assay SGK1 Inactive SGK1 32 P P Coomassie Zhang et al. JCI

12 Aldosterone Induces SGK1 and Phos- in mimcd3 cells NR Hrs 1 Aldo In vivo Assay SGK1 anti-sg GK1 P anti-phos anti-actin Zhang et al. JCI

13 Phosphorylation by SGK1 Impairs - Interaction in vitro Active SGK1 - + GST Pulldown SGK1 anti-phos Ser Phos P anti EGFP Retained Coomassie Input Zhang et al. JCI

14 AF17 facilitates Nuclear Export AF 9? GFP-MR GFP- RFP-AF17 Merge Reisenauer et al JBC 2009

15 A. -Aldo Af17 Af9 Af17 Af9 Af9 Meth K 79 αenac Af17 HYPOmethylated H3 K79 Degradation?

16 Characterization of AF17 -/- Phenotype Tissue Collection and Analyses

17 +/+ n= 57 mice -/- n= 65 mice Urinary Na + Excretion (µmol/24 h/bw) * /+ -/- BP (mmhg) * * * Diastolic Systolic Mean Chen et al JASN 2011

18 αenac βenac γenac +/+ +/+ -/- -/- +/+ -/- Chen et al JASN 2011

19 +/+ -/ /+ + -/- Open Probability * +/+ -/ /+ -/- Active Channels Effective Activity * * Chen et al JASN 2011

20 Chen et al JASN 2011

21 AF17 AF17 K 79 Meth αenac H3 m2k79 in the cells and at the αenac promoter α, β, γenac expression at mrna and protein levels Na + excretion in urine [Na + ] in blood Blood pressure AF17 HYPOmethylated H3 K79 Degradation?

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23 In one cohort, a polymorphismm in DOT1L (rs ) was strongly associated with great ter systolic (P = ) and diastolic (P = ) blood pressure response to hydrochlorothiazide in Caucasians. However, this association was not replicated in the other cohort. When untreated blood pressure levels were analyzed, we found directionally similar associations between a polymorphism in MLLT3 (rs ) and greater untreated systolic (P< 0.01 in both cohorts) and diastolic (P < 0.05 in both cohorts) blood pressure levels in both cohorts.

24 Mutated in Kidney Biopsies from CKD Patients

25 Acknowledgment

26 Acknowledgment Bruce C. Kone (University of Florida) Charles C. Hemenway (Loyola University) David Pearce (UCSF) Volker Vallon (UCSD) Florian Lang (Free Univer rsity, Germany) Dietmar Khul (Free University, Germany) Günther Schütz (University of Freiburg, Germany) Funding: NIH, AHA, ASN

27 SGK1 and Colocalize in mimcd3 Cells SGK1 SGK1 P RFP- GFP-SGK1 Merge Unpublished.

28 Af9 binds to +78/+92 of αenac in vitro Zhang et al. Am J Physiol Renal Physiol

29 Chen et al JASN 2011