Epigenetics and Obesity

A potential role for the imprinted gene MEST in diet-induced obesity in mice

Pennington Biomedical Research CenterPennington Biomedical Research Center

Robert A. KozaDepartment of Molecular Genetics

genetics environment

Factors contributing to the current obesity epidemic

epigenetics

Experimental design used for feeding study with 112 male C57BL/6J mice

3 wks 12 wks8 wks

weaned; chow diet high fat diet, NMR NMR, tissue collection

Body weights measured weekly

10

15

20

25

30

35

2 3 4 5 6 7 8 9 10 11 12

Entire Cohort (112 mice)Lowest 10%Highest 10%

7.82 gInitiation of HFD

Weeks High fat diet

body

wei

ght (

g)

10

15

20

25

30

35

2 3 4 5 6 7 8 9 10 11 12

Entire Cohort (112 mice)Lowest 10%Highest 10%

7.82 gInitiation of HFD

Weeks High fat diet

body

wei

ght (

g)

0

2

4

6

8

10

25 30 35 40

fa

t mas

s(1

2wks

-8w

ks)

body weight (12 wks of age)

R= 0.80P= < 10-25

0

2

4

6

8

10

25 30 35 40body weight (12 wks of age)

R= 0.30P= 0.001

le

an m

ass

(12w

ks-8

wks

)

0

2

4

6

8

10

25 30 35 40

fa

t mas

s(1

2wks

-8w

ks)

body weight (12 wks of age)

R= 0.80P= < 10-25

0

2

4

6

8

10

25 30 35 40body weight (12 wks of age)

R= 0.30P= 0.001

le

an m

ass

(12w

ks-8

wks

)

Bodyweight variation primarily due to increased adiposity

Microarray Analysis

High versus low weight gaining mice; HFD for 2, 4 and 12 weeks

Identified a gene called mesoderm specific transcript(MEST/Peg1) to be more highly expressed in adipose tissue of high weight gaining mice. Microarray data was validated by qRT-PCR

Strong association of MEST with adiposity, but not lean mass(112 mice HEF 23)

MEST vs R pValue3 wk BWT 0.204 0.038 wk BWT 0.065 0.4912 wk BWT 0.303 0.001Lean mass (g) 8wk 0.004 0.96Lean mass (g) 12wk 0.010 0.92Lean mass (g) (12wk-8wk) 0.006 0.95Fat mass (g) 8wk 0.207 0.03Fat mass (g) 12wk 0.500 <0.0001Fat mass (g) (12wk-8wk) 0.498 <0.0001Fat (g)/Lean (g) 8wk 0.210 0.03Fat (g)/Lean (g) 12wk 0.553 <0.0001

Fat (g)/lean (g) at 12 weeks

05

1015202530354045

0.15 0.25 0.35 0.45 0.55

ME

ST m

RN

A (A

U/c

yclo

phili

n)

Fat (g)/lean (g) at 12 weeks

05

1015202530354045

0.15 0.25 0.35 0.45 0.55

ME

ST m

RN

A (A

U/c

yclo

phili

n)

Adipose tissue MEST is induced in some, but not all mice by dietary fat in as little as 2 days

0

1

2

3

4

5

6

7

0

5

10

15

20

25

ING

fat M

EST

mR

NA

EPI f

at M

EST

mR

NA

Chow 2d 7d Chow 2d 7dHFD HFD

0

1

2

3

4

5

6

7

0

5

10

15

20

25

ING

fat M

EST

mR

NA

EPI f

at M

EST

mR

NA

Chow 2d 7d Chow 2d 7dHFD HFD

2.91±

13.88±12.35±

2.21±1.80±

0.74± 0.09

0.29 0.30

0.36

1.622.03

a

b

b

a

b b

A B

0

1

2

3

4

5

6

7

0

5

10

15

20

25

ING

fat M

EST

mR

NA

EPI f

at M

EST

mR

NA

Chow 2d 7d Chow 2d 7dHFD HFD

0

1

2

3

4

5

6

7

0

5

10

15

20

25

ING

fat M

EST

mR

NA

EPI f

at M

EST

mR

NA

Chow 2d 7d Chow 2d 7dHFD HFD

2.91±

13.88±12.35±

2.21±1.80±

0.74± 0.09

0.29 0.30

0.36

1.622.03

a

b

b

a

b b

A BInguinal Epididymal

Mesoderm Specific Transcript (MEST)

AKA Peg1 (paternally expressed gene 1); mouse Chr 6 (7.5 cM); human 7q32; function unknown, putative soluble epoxide () hydrolase (lipid transport and metabolism?). Protein of 335 amino acids (~37 kDa)

Targeted mutation leads to abnormal maternal behavior (impaired placentophagia) and growth retardation (L. Lefebvre et al, 1998).

Overexpression in mouse adipose tissue and 3T3-L1 adipocytes leads to increased adipocyte size and and induced expression of adipogenic transcription factors (Takahashi et al, 2004)

Importance implicated in mammalian metanephrenic development (Kanwar et al. 2002) and oncofetal angiogenesis (Mayer et al. 2000).

Loss of imprinting (LOI) implicated in etiology of lung adenocarcinoma, colon cancerand metastatic breast cancer (Kohda et al. 2001; Nishihara et al. 2000; Pederson et al. 1999, 2002).

Several reports of alternative splicing, transcript variants and possibly an antisense transcript.

Is variability in adipose tissue MEST expression due to differences in methylation of the MEST gene?

ATCGTTTTTATTTCGATGTCGATT

hydroquinonesodium metabisulfite

+

ATCGTCTCTACCTCGACGCCGACTCH3 CH3CH3

ATCGTTTTTATTTCGATGTCGATT

hydroquinonesodium metabisulfite

+

ATCGTCTCTACCTCGACGCCGACTCH3 CH3CH3

ATCGTTTTTATTTCGATGTCGATT

hydroquinonesodium metabisulfite

+hydroquinone

sodium metabisulfite+

ATCGTCTCTACCTCGACGCCGACTCH3 CH3CH3

ATCGTCTCTACCTCGACGCCGACTCH3CH3 CH3CH3CH3CH3

Bisulfite Sequencing

Animals selected for CpG methylation analysis of inguinal fat DNA

Mouse# MEST (AU/ng RNA) 12 wk BWTFM/LM (12 wk)

496 0.45 29.10.19

501 1.15 32.10.25

530 1.20 32.90.25

549 0.55 28.80.18

552 1.62 30.60.19

587 0.68 30.80.20

Mean= 0.94 ± 0.19 30.72 ± 0.660.21 ± 0.013

498 38.31 31.10.38

524 30.84 34.00.39

537 30.75 32.00.41

560 42.76 34.60.38

562 42.83 34.40.38

573 32.33 33.00.36

Mean= 36.30 ± 2.34 33.18 ± 0.570.38 ± 0.006

p<10-7 p=0.02p<10-5

Methodology:

DNA isolated from mouse inguinal fat

DNA imbedded in agarose beads

Denaturation with NAOH

Bisulfite conversion of non-methylated cytosine to cytosine sulfonate

Hydrolytic deamination converts cytosine sulfonate to uracil sulphonate

Alkali desulphonation to convert uracil sulphonate to uracil

PCR with GSPs/TOPO-TA cloning/BDT sequencing

-CTCGGCGAACCC- -TTTGGCGAATTT-m

Methylation Analysis-analysis of 22 CpGs within a 318 bp DMR region 5’ from open reading frame

GGATTAGAGATCTATAAGGAAAGAGGGGGTAGCGGGTCAATACCCCTGGAGGCGCCCACGGGAAAGGCC

CCCCCTCTCTGCAGCGGTGGGGAGGGGCTCTGCGGCGGGAGCGAGAAGGAGGGGCGCTGCGGCAGGA

TGGGCGGGTTAGAGGCGGGGCTCAGTGGGCTTTAAAAGTCGGTGCCTGCTTGCTCCTCTCTGCTTCGGC

CACCAGCACATCCCGGTGCTTCTTCTCAGGCGCAGCAGCTTTCCTCTGCGGCAGCCGCACCTCGCCAAA

CGGCGTAGTGCTGCAGGCTCGCCCGAGTTGCTGCTTGCTGCCTCTGCTGCCGCTGCCGCG

Exon 1

AP-2

Sp1

Sp1 Sp1,GCF,APRT(-)

forward primer

reverse primer

…showed no differences in methylation of MEST gene in inguinal fat ofhigh vs low MEST expressing mice

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

CpG Island

% m

ethy

latio

n

low MESThigh MEST

4981 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 221 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

5241 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

537

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

5601 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

5621 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

573

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

4961 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

5011 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

530

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

5491 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

5521 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

1 2 3 4 98765 1413121110 201918171615 21 22

Exon 1Sp1GCFAPRT (-)Sp1AP-2 Sp1

587

LOW MEST EXPRESSION_Region B analyses

HIGH MEST EXPRESSION_Region B analyses

->98% of cytosines not found in CpGs were effectively converted to uracils

-clear patterns of methylation were observed in maternal vs paternal alleles

-no significant differences in adipose tissue MEST promoter (CpG island) DNA methylation were apparent between low and high MEST expressing mice

Summary

MEST is induced by dietary fat in adipocytes but not stromal-vascular fraction of fat depot

05

1015202530

AD SV AD SV

chowHFD

EPI ING

ME

ST m

RN

A

EPI INGEPI ING

ME

ST m

RN

A

05

1015202530

AD SV AD SV

chowHFD

05

1015202530

AD SV AD SV

chowHFD

EPI INGEPI ING

ME

ST m

RN

A

EPI INGEPI ING

ME

ST m

RN

A

Genomic methylation patterns of MEST in mature adipocytes and corresponding stromal-vascular cells

TSS

0-175 +92 +116 +334

ORF

-1100 -818

15 CpGs 31 CpGs22 CpGs

TSS

0-175 +92 +116 +334

ORF

-1100 -818

15 CpGs 31 CpGs22 CpGs

A B C

01020304050607080

ADSV

CpG Islands1 15

(N=37)(N=28)

Region C

01020304050607080

ADSV

CpG Islands1 15

(N=37)(N=28)

Region A

01020304050607080

ADSV

CpG Islands1 31

(N=38)(N=46)

Region B

01020304050607080

ADSV

CpG Islands1 31

(N=38)(N=46)

Region C

CpG Islands1 22

01020304050607080 AD

SV(N=62)(N=65)

Region B

% m

ethy

latio

n

MEST Promoter

TSS

0-175 +92 +116 +334

ORF

-1100 -818

B CA15 CpGs 31 CpGs22 CpGs

TSS

0-175 +92 +116 +334

ORF

-1100 -818

15 CpGs 31 CpGs22 CpGs

TSS

0-175 +92 +116 +334

ORF

-1100 -818

B CA15 CpGs 31 CpGs22 CpGs

TSS

0-175 +92 +116 +334

ORF

-1100 -818

15 CpGs 31 CpGs22 CpGs

Adipose tissue (ING fat) from low vs high MEST expressing mice

0

20

40

60

80

100low mesthigh mest

0

20

40

60

80

100low mesthigh mest

0

20

40

60

80

100low mesthigh mest

A CB

15 221 3111CpG CpGCpG

% m

ethy

latio

n

MEST Promoter

0

20

40

60

80

100 low MESThigh MEST

0

20

40

60

80

100 low MESThigh MEST

% m

ethy

latio

n

% m

ethy

latio

n

1 115 31

Region A Region C

TSS

0-175 +92 +116 +334

ORF

-1100 -818

B CA15 CpGs 31 CpGs22 CpGs

TSS

0-175 +92 +116 +334

ORF

-1100 -818

15 CpGs 31 CpGs22 CpGs

TSS

0-175 +92 +116 +334

ORF

-1100 -818

B CA15 CpGs 31 CpGs22 CpGs

TSS

0-175 +92 +116 +334

ORF

-1100 -818

15 CpGs 31 CpGs22 CpGs

MEST Promoter

CpG methylation in adipocytes isolated from epididymal fat of mice with high or low MEST expression after 7 days on HFD

Summary Loss of imprinting is observed in genomic regions of adipocyte DNA that are ~1 kb upstream from, and immediately distal to the characterized imprinted genomic region of the MEST promoter.

No differences in methylation patterns were observed in total inguinal fat DNA, or epididymal fat adipocyte DNA from mice with high or low MEST mRNA expression.

These data suggest that the MEST promoter in adipocytes is ‘poised’ for activation by an undefined mechanism.

Future studies will examine genes that are highly associated with MEST expression in adipose tissue. These will include several genes involved in Wnt signaling.

0

10

20

30

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50

0 10 20 30 40 50

MEST (Au/cyclo)

Sfr

p5 (A

U/c

yclo

)

R=0.93p=2.86E-18

0

5

10

15

20

25

0 10 20 30 40 50

Sfrp5 (Au/cyclo)

Nak

ed (A

U/c

yclo

)

R=0.80p=6.92E-10

0

5

10

15

20

25

0 10 20 30 40 50

MEST (Au/cyclo)

Nak

ed (A

U/c

yclo

)

R=0.80p=3.84E-10

0

10

20

30

40

50

0 10 20 30 40 50

MEST (Au/cyclo)

Sfr

p5 (A

U/c

yclo

)

R=0.93p=2.86E-18

0

10

20

30

40

50

0 10 20 30 40 50

MEST (Au/cyclo)

Sfr

p5 (A

U/c

yclo

)

R=0.93p=2.86E-18

0

5

10

15

20

25

0 10 20 30 40 50

Sfrp5 (Au/cyclo)

Nak

ed (A

U/c

yclo

)

R=0.80p=6.92E-10

0

5

10

15

20

25

0 10 20 30 40 50

Sfrp5 (Au/cyclo)

Nak

ed (A

U/c

yclo

)

R=0.80p=6.92E-10

0

5

10

15

20

25

0 10 20 30 40 50

MEST (Au/cyclo)

Nak

ed (A

U/c

yclo

)

R=0.80p=3.84E-10

0

5

10

15

20

25

0 10 20 30 40 50

MEST (Au/cyclo)

Nak

ed (A

U/c

yclo

)

R=0.80p=3.84E-10

MEST vs Sfrp5 MEST vs Nkd1 Sfrp5 vs Nkd1

Les Kozak

Larissa NikonovaJessica HoganJong-Seop RimTamra MendozaChris Faulk

Ken Eilertsen

Jihad Skaf

Acknowledgements

PBRC

Applied Biosystems

HEF- L. Kozak, D. YorkCNRU- E. Ravussin