epigenetics and obesity
DESCRIPTION
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. epigenetics. Factors contributing to the current obesity epidemic. genetics. environment. - PowerPoint PPT PresentationTRANSCRIPT
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
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
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