Epigenetic Control Transcription, Na

and Blood Pressure

Wenzheng Zhang

Department of Internal Medicine

The University of Texas Medical School at Houston

International Conference on

Geriatrics & Gerontology

Chicago, July 08

Epigenetic Control of ENaC, Na+ Metabolism

Blood Pressure

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

Epithelial Na Channel (ENaC)

� Consists of 3 partially homologous subunits

� Expressed in the apical membrane of salt

epithelia of kidney, colon, and lung.

� Critical for Na+ absorption in the lung, colon, and kidney.� 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 aldoster

protein, and subcellular distribution).

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.

absorption in the lung, colon, and kidney.absorption in the lung, colon, and kidney.

Mutations associated with two genetic diseases: Liddle’s

syndrome (Hypertension) and pseudohypoaldosteronism

1, Hypotension).

terone at multiple levels (mRNA,

protein, and subcellular distribution).

Liddle’s Syndrome (Hypertension)

ββββ

� Described by G. Liddle in 1963

� Autosomal dominant fashion

� Early and severe hypertension

� Hypokalemia

� Metabolic alkalosis� Metabolic alkalosis

� Low plasma aldosterone

concentration

γγγγConstitutive hyperactivity of ENaC

Liddle’s Syndrome (Hypertension)

M1 M2 PPxY

R566*

A576fr Y620H

Bonny et al Kidney International (2000) 57, 1313–1318

A576fr

Q591*

T594fr

P596fr

R597fr

Y620H

P618L/S

P617S

M1 M2 PPxY

W574*

Pseudohypoaldosteronism type 1 (PHA-1, Hypotension)

αααα

� Two forms:

Autosomal dominant renal

form by mutations in MR

Autosomal recessive form by

mutations in ENaC subunits

R56*

I68fr

� Salt wasting

� Hyperkalemia

� Metabolic acidosis

� High plasma aldosterone

concentration

ββββ

G37S

γγγγ

Loss-of-function mutations in ENaC

Pseudohypoaldosteronism type 1 1, Hypotension)

M1 M2 PPxY

F435fr

Bonny et al Kidney International (2000) 57, 1313–1318

R56*

I68fr

C133Y

R139D

T168fr

R508*

T168fr

M1 M2 PPxY

G37S T216fr D305fr

M1 M2 PPxY

KYS106-108 N

NR

HRE

NR

The Classical model for Molecular Action of Aldosterone

NR

Nucleus

HRE

NR

HRE αααα

The Classical model for Molecular Action of Aldosterone

Nucleus

SGK1

ENaCαααα

Dot1a

1 416 479

NSL1

MD Af9 /

A.

B.1 29 112

YEATSAf9

Af17

1

PHD-ZF

20 180

B.

C.

479 659

Af9 / Af17

NSL2 NSL3

1540

LZ

397 557

Dot1a / MR / Hsp90

Dot1a

635 789 1079

S435: Sgk1 phosphorylation site

Histone Methyltransferase Gene Dot1

• Identified in yeast as a disruptor

• Encodes a methyltransferase specific

• Expressed in kidney and otherstage.

• Five alternative splicing variantsbeing the most highly expressedbeing the most highly expressedhDot1L.

• Dot1L-deficient mouse embryosabnormalities, including growthdefects in the yolk sac, and cardiac9.5 and 10.5 days post coitum.

• Involved in cell cycle regulation,remodeling, transcription regulation

Histone Methyltransferase Gene Dot1

isruptor of telomeric silencing.

specific for histone H3 K79.

other tissues and as early as 2-cell

variants in mouse (Dot1a-e), with Dot1aexpressed and most closely related toexpressed and most closely related to

embryos show multiple developmentalgrowth impairment, angiogenesis

cardiac dilation, and die between

regulation, DNA repair, chromosomeregulation and leukemogenesis.

Putative Transcription Factor AF9

• Identified as one of the mostof the mixed-lineage leukemia

• A putative transcription• A putative transcriptionregulated genes have been

• Expressed in kidney and other

• AF9 deletion leads to perinatal

Putative Transcription Factor AF9

most common fusion partnersleukemia protein (MLL or ALL-1)

factor, although, no AF9-factor, although, no AF9-been well documented

other tissues

perinatal lethality

Dot1a and AF9 Colocalize in the Nucleus of mIMCD3 Cells

AF9Dot1a AF9Dot1a

Dot1a and AF9 Colocalize in the Nucleus of mIMCD3 Cells

DAPIAF9Dot1a

DAPIAF9

Zhang et al. JBC. 2006.

AF9 Contains a Putative Phosphorylation Site for SGK1

AF9

Mouse AF9

Human AF9

SGK1

AF9

P

AF9

Consensus

Horse AF9

Rat AF9

Mouse AF9

AF9 Contains a Putative Phosphorylation Site for SGK1

430…RSRRVS…435Mouse AF9

441…RSRRVS…446Human AF9

RXRXX(S/T)Consensus

35…RSRRVS…. 40Horse AF9

243…RSRRVS…248Rat AF9

430…RSRRVS…435Mouse AF9

Zhang et al. JCI. 2007.

SGK1 Phosphorylates AF9 in vitro

SGK1

AF9

Active SGK1

Inactive SGK1In vitroSGK1

AF9

P

AF9 Coomassie

In vitroAssay

SGK1 Phosphorylates AF9 in vitro

GST GST-AF9

Active SGK1

Inactive SGK1

-

-

-

+

+

-

-

-

+

-

-

+

Coomassie

32P

Zhang et al. JCI. 2007.

Aldo

anti-SGK1

Aldosterone Induces SGK1 and PhosAF9 in mIMCD3 cells

NR

AF9

In vivo anti-SGK1

SGK1

AF9

P

AF9

In vivoAssay

anti-Actin

anti-Phos AF9

Aldo - +

1

- +

1.5

- +

2Hrs

SGK1

Aldosterone Induces SGK1 and Phos-AF9 in mIMCD3 cells

SGK1

Actin

Phos AF9

Zhang et al. JCI. 2007.

Active SGK1

AF9 Phosphorylation by SGK1 Impairs AF9Dot1a Interaction in vitro

AF9Dot1a

anti-Phos SerSGK1

Dot1a

AF9

P

AF9

anti-Phos Ser

anti−EGFP

Coomassie

- +

GST

Pulldown

AF9 Phosphorylation by SGK1 Impairs AF9-Dot1a Interaction in vitro

Phos AF9Phos AF9

Retained Dot1a

Input AF9

Zhang et al. JCI. 2007.

AF17 facilitates Dot1a Nuclear Export

Dot1a

AF9

GFP

GFP-Dot1a RFP

AF17 facilitates Dot1a Nuclear Export

?Dot1aAF

GFP-MR

RFP-AF17 Merge

Reisenauer et al JBC 2009

Af9

Dot1a

Dot1a

Af17

Dot1a

A. -Aldo

Af9

Dot1a

HYPOmethylated H3 K79

Af9

Dot1a

K79

MethααααENaC

Af17

Dot1a

Af17

Dot1a

Degradation?

Characterization of AF17Characterization of AF17-/- Phenotype

Tissue Collection and Analyses

+/+ n= 57 mice

16*

0

4

8

12

16

+/+ -/-

Uri

nary

Na

+E

xcre

tio

n

( µµ µµm

ol/

24 h

/BW

)

+/+ n= 57 mice -/- n= 65 mice

60

90

120

150

Diastolic Systolic Mean

BP

(m

mH

g)

*

**

Chen et al JASN 2011

+/+ -/-

ααααENaC

+/+

ββββENaC

-/-

ENaC

+/+ -/-

γγγγENaC

Chen et al JASN 2011

+/+

-/-

0

0.1

0.2

0.3

0.4

0.5

+/+

Op

en

Pro

bab

ilit

y

0

0.6

1.2

1.8

2.4

3

3.6

+/+ -/-

Acti

ve C

han

nels

*

+/+ -/-

0

0.2

0.4

0.6

0.8

+/+ -/-

Eff

ecti

ve A

cti

vit

y

**

Chen et al JASN 2011

Chen et al JASN 2011

AF9

Dot1a

Dot1a

AF17

Dot1aAF9

Dot1a

HYPOmethylated H3 K79

AF9

Dot1a

K79

MethααααENaC

AF17

Dot1a

AF17

Dot1a

Degradation?

H3 m2K79 in the cells

and at the αENaC promoter

α, β, γENaC expression at mRNA and protein levels

✓

✓at mRNA and protein levels

Na+ excretion in urine

[Na+] in blood

Blood pressure

✓

✓

✓

✓

In one cohort, a polymorphismstrongly associated with greaterstrongly associated with greaterdiastolic (P = 0.0016) bloodhydrochlorothiazide in Caucasianswas not replicated in the otherpressure levels were analyzed,associations between a polymorphismand greater untreated systolicdiastolic (P < 0.05 in both cohorts)cohorts.

polymorphism in DOT1L (rs2269879) wasgreater systolic (P = 0.0002) andgreater systolic (P = 0.0002) and

blood pressure response toCaucasians. However, this association

cohort. When untreated bloodwe found directionally similar

polymorphism in MLLT3 (rs12350051)(P< 0.01 in both cohorts) and

cohorts) blood pressure levels in both

Mutated Dot1a in Kidney Biopsies from CKD Patients Mutated Dot1a in Kidney Biopsies from CKD Patients

Acknowledgment Acknowledgment

Acknowledgment

Bruce C. Kone (University of Florida)

Charles C. Hemenway (Loyola University)

David Pearce (UCSF)

Volker Vallon (UCSD)

Florian Lang (Free University, Germany)Florian Lang (Free University, Germany)

Dietmar Khul (Free University, Germany)

Günther Schütz (University of Freiburg, Germany)

Funding:

NIH, AHA, ASN

Acknowledgment

Bruce C. Kone (University of Florida)

Charles C. Hemenway (Loyola University)

(Free University, Germany)(Free University, Germany)

Dietmar Khul (Free University, Germany)

Günther Schütz (University of Freiburg, Germany)

GFP

SGK1 and AF9 Colocalize

RFP-AF9

SGK1AF9

GFP-SGK1

Colocalize in mIMCD3 Cells

Merge

AF9

P

AF9AF9

SGK1

Unpublished.

Af9 binds to +78/+92 of Af9 binds to +78/+92 of αENaC in vitro

Zhang et al. Am J Physiol Renal Physiol. 2013.

Chen et al JASN 2011