Download Slide 1

Epigenetics and Obesity
A potential role for the imprinted gene MEST in diet-induced obesity in mice
Robert A. Koza
Department of Molecular Genetics
Pennington Biomedical Research Center
Factors contributing to the current obesity epidemic
genetics
environment
epigenetics
Experimental design used for feeding study with 112 male C57BL/6J mice
Body weights measured weekly
3 wks
8 wks
12 wks
weaned; chow diet
high fat diet, NMR
NMR, tissue collection
body weight (g)
35
Initiation of HFD
 7.82 g
30
25
20
Entire Cohort (112 mice)
Lowest 10%
Highest 10%
15
10
2
3
4
5
6
Weeks
7
8
9
10 11 12
High fat diet
Bodyweight variation primarily due to increased adiposity
8
6
4
(12wks-8w ks)
10
R= 0.80
P= < 10-25
 lean mass
(12wks-8w ks)
 fat mass
10
6
4
2
2
0
0
25
30
35
40
body weight (12 wks of age)
R= 0.30
P= 0.001
8
25
30
35
40
body weight (12 wks of age)
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)
3 wk BWT
8 wk BWT
12 wk BWT
Lean mass (g) 8wk
Lean mass (g) 12wk
Lean mass (g)  (12wk-8wk)
Fat mass (g) 8wk
Fat mass (g) 12wk
Fat mass (g)  (12wk-8wk)
Fat (g)/Lean (g) 8wk
Fat (g)/Lean (g) 12wk
R
0.204
0.065
0.303
0.004
0.010
0.006
0.207
0.500
0.498
0.210
0.553
pValue
0.03
0.49
0.001
0.96
0.92
0.95
0.03
<0.0001
<0.0001
0.03
<0.0001
45
MEST mRNA (AU/cyclophilin)
MEST vs
40
35
30
25
20
15
10
5
0
0.15
0.25
0.35
0.45
Fat (g)/lean (g) at 12 weeks
0.55
Adipose tissue MEST is induced in some, but not all mice
by dietary fat in as little as 2 days
A
B
Inguinal
6
b
5
4
3
a
2
1
0
1.80±
0.74±
0.29
2.21±
0.30
15
12.35±
13.88±
1.62
2.03
10
a
5
2.91±
2d
7d
HFD
b
20
0.09
Chow
b
25
b
EPI fat MEST mRNA
ING fat MEST mRNA
7
Epididymal
0
0.36
Chow
2d
7d
HFD
Mesoderm Specific Transcript
(MEST)
AKA Peg1 (paternally expressed gene 1); mouse Chr 6 (7.5 cM); human 7q32;
function unknown, putative soluble epoxide (a/b) 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 cancer
and 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?
Bisulfite Sequencing
CH3
CH3
CH3
ATCGTCTCTACCTCGACGCCGACT
sodium metabisulfite
+
hydroquinone
ATCGTTTTTATTTCGATGTCGATT
Animals selected for CpG methylation analysis
of inguinal fat DNA
Mouse#
MEST (AU/ng RNA)
12 wk BWT
FM/LM (12 wk)
496
501
530
549
552
587
Mean=
0.45
1.15
1.20
0.55
1.62
0.68
0.94 ± 0.19
29.1
32.1
32.9
28.8
30.6
30.8
30.72 ± 0.66
0.19
0.25
0.25
0.18
0.19
0.20
0.21 ± 0.013
498
524
537
560
562
573
Mean=
38.31
30.84
30.75
42.76
42.83
32.33
36.30 ± 2.34
p<10-7
31.1
34.0
32.0
34.6
34.4
33.0
33.18 ± 0.57
p=0.02
0.38
0.39
0.41
0.38
0.38
0.36
0.38 ± 0.006
p<10-5
Methodology:
DNA isolated from mouse inguinal fat
Denaturation with NAOH
DNA imbedded in agarose beads
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
m
-CTCGGCGAACCC-
-TTTGGCGAATTT-
Methylation Analysis
-analysis of 22 CpGs within a 318 bp DMR region 5’ from open reading frame
forward primer
AP-2
GGATTAGAGATCTATAAGGAAAGAGGGGGTAGCGGGTCAATACCCCTGGAGGCGCCCACGGGAAAGGCC
Sp1
CCCCCTCTCTGCAGCGGTGGGGAGGGGCTCTGCGGCGGGAGCGAGAAGGAGGGGCGCTGCGGCAGGA
Sp1,GCF,APRT(-)
Sp1
TGGGCGGGTTAGAGGCGGGGCTCAGTGGGCTTTAAAAGTCGGTGCCTGCTTGCTCCTCTCTGCTTCGGC
Exon 1
CACCAGCACATCCCGGTGCTTCTTCTCAGGCGCAGCAGCTTTCCTCTGCGGCAGCCGCACCTCGCCAAA
CGGCGTAGTGCTGCAGGCTCGCCCGAGTTGCTGCTTGCTGCCTCTGCTGCCGCTGCCGCG
reverse primer
…showed no differences in methylation of MEST gene in inguinal fat of
high vs low MEST expressing mice
% methylation
100
90
low MEST
80
high MEST
70
60
50
40
30
20
10
0
1
2
3
4
5
6
7
8
9
10
11
12
13
CpG Island
14
15
16
17
18
19
20
21
22
HIGH MEST EXPRESSION_Region B analyses
498
Sp1
AP-2
1
2 3
Sp1
GCF
Sp1 APRT (-)
4
5 6
7
8 9
560
1
2 3
4
11
12
13
14
15
16 17 18 19 20
5 6
7
8 9
10
11
21 22
1
2 3
4
5 6
14
15
16 17 18 19 20
8 9
21 22
1
2 3
4
10
11
5 6
7
8 9
10
11
537
Exon 1
12
13
14
15
16 17 18 19 20
21 22
1
2 3
4
5 6
14
15
16 17 18 19 20
8 9
21 22
1
2 3
4
10
11
Exon 1
12
13
14
15
16 17 18 19 20
13
14
15
16 17 18 19 20
Sp1
GCF
Sp1 APRT (-)
Sp1
AP-2
13
7
573
Exon 1
12
Sp1
GCF
Sp1 APRT (-)
Sp1
AP-2
Sp1
GCF
Sp1 APRT (-)
Sp1
AP-2
13
7
562
Exon 1
12
Sp1
GCF
Sp1 APRT (-)
Sp1
AP-2
Sp1
GCF
Sp1 APRT (-)
Sp1
AP-2
10
524
Exon 1
5 6
7
8 9
10
11
21 22
Exon 1
12
21 22
LOW MEST EXPRESSION_Region B analyses
496
Sp1
AP-2
1
2 3
Sp1
GCF
Sp1 APRT (-)
4
5 6
7
8 9
549
1
2 3
4
5 6
11
12
13
14
15
7
8 9
10
11
12
16 17 18 19 20
21 22
1
2 3
4
15
16 17 18 19 20
7
8 9
21 22
1
2 3
4
5 6
10
11
12
7
8 9
10
11
12
530
Exon 1
13
14
15
16 17 18 19 20
21 22
1
2 3
4
15
16 17 18 19 20
7
8 9
21 22
1
2 3
4
5 6
10
11
Exon 1
12
13
14
15
16 17 18 19 20
12
13
14
15
16 17 18 19 20
Sp1
GCF
Sp1 APRT (-)
Sp1
AP-2
14
5 6
587
Exon 1
13
Sp1
GCF
Sp1 APRT (-)
Sp1
AP-2
Sp1
GCF
Sp1 APRT (-)
Sp1
AP-2
14
5 6
552
Exon 1
13
Sp1
GCF
Sp1 APRT (-)
Sp1
AP-2
Sp1
GCF
Sp1 APRT (-)
Sp1
AP-2
10
501
Exon 1
7
8 9
10
11
21 22
Exon 1
21 22
Summary
->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
MEST is induced by dietary fat in adipocytes but
not stromal-vascular fraction of fat depot
MEST mRNA
30
chow
HFD
25
20
15
10
5
0
AD
SV
EPI
AD
SV
ING
Genomic methylation patterns of MEST in mature adipocytes
and corresponding stromal-vascular cells
TSS
MEST Promoter
A
22 CpGs
15 CpGs
% methylation
-1100
80
70
60
50
40
30
20
10
0
-175
-818
AD(N=37)
SV (N=28)
Region A
C
1
C ORF
B
CpG Islands
15
AD(N=62)
SV(N=65)
80
70
60
50
40
30
20
10
0
CpG Islands
+92 +116
22
+334
AD(N=38)
SV(N=46)
80
70
60
50
40
30
20
10
0
Region B
1
0
31 CpGs
Region C
B
1
CpG Islands
31
Adipose tissue (ING fat) from low vs high MEST expressing mice
TSS
MEST Promoter
A
22 CpGs
15 CpGs
-1100
-175
-818
A
0
B
100
% methylation
C ORF
B
31 CpGs
+92 +116 +334
100
low mest
low mest
80
low mest
80
high mest
80
60
60
40
40
40
20
20
20
0
0
1
CpG
15
high mest
high mest
60
0
C
100
1
CpG
22
1
CpG
31
CpG methylation in adipocytes isolated from epididymal fat of mice with
high or low MEST expression after 7 days on HFD
TSS
MEST Promoter
A
22 CpGs
15 CpGs
-1100
-175
-818
80
high MEST
+92 +116 +334
low MEST
100
% methylation
low MEST
% methylation
100
0
31 CpGs
Region C
Region A
60
40
20
0
C ORF
B
high MEST
80
60
40
20
0
1
15
1
31
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.
MEST vs Sfrp5
R=0.93
p=2.86E-18
R=0.80
p=3.84E-10
20
Naked (AU/cyclo)
Sfrp5 (AU/cyclo)
40
MEST vs Nkd1
25
30
20
10
0
Naked (AU/cyclo)
50
15
10
5
0
0
10
20
30
40
MEST (Au/cyclo)
50
25
Sfrp5 vs Nkd1
20
R=0.80
p=6.92E-10
15
10
5
0
0
10
20
30
40
MEST (Au/cyclo)
50
0
10
20
30
40
Sfrp5 (Au/cyclo)
50
Acknowledgements
PBRC
Les Kozak
Larissa Nikonova
Jessica Hogan
Jong-Seop Rim
Tamra Mendoza
Chris Faulk
Ken Eilertsen
Applied Biosystems
Jihad Skaf
HEF- L. Kozak, D. York
CNRU- E. Ravussin