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Ross Tellam Effects of maternal periconceptional overnutrition on the adipose tissue epigenome of offspring EpiSCOPE
Ross Tellam
Effects of maternal periconceptional overnutrition on the adipose tissue epigenome of offspring
EpiSCOPE
• Obesity: risk factor for a broad spectrum of adult diseases
• Increasing incidence for the last ~3-4 decades• Direct cost: AUD $8.3 b/yr Aust. Government Preventative Health Taskforce 2009
• Increasing incidence despite ‘eat less, exercise more’ campaign
• Obesity in US children has tripled in 50 years• Evidence for intergenerational influence acting on
developmental trajectories
Obesity
Increasingly overweight children
http://www.indiatalkies.com/2011/05/call-junk-food-ad-ban-tackle-childhood-obesity.html
Pre-preg. BMI
BMI ≥ 25 (Overwt)
BMI >30(Obese)
BMI >40(Morbidly Obese)
1991 25% 16% 2.7%
2001 35% 22% 5%
More women are starting pregnancyoverweight or obese
LaCoursiere et al American Journal of Obstetrics & Gynecology 2005
Obesity trends
Parsons et al, BMJ 2001
Heavy babies become heavy adults
Insulin sensitivity is decreased in
pregravid and early pregnancy obese women
An intergenerational cycle of obesity?
Risk of adult obesity
Pre-pregnancy
obesity
Risk of gestational
diabetes
Risk of heavy infant
Risk of childhood
obesity
Rattanatray et al Endocrinology 2010; Zhang, Rattanatray et al , PBMB 2011
Overweight mothers give birth to overweight offspring resulting in transfer and amplification of obesity risk across generations
McMillen and Morrison
Is there early life epigenetic priming of obesity risk?
• Epigenetics• The epigenomic architecture underpinning a rapid
developmental transition from brown to white adipose tissue in the ovine perirenal depot
• The impact of maternal periconceptional overnutrition on the offspring epigenome
• Conclusions
Role of epigenetics in the developmental origins of health and disease
• Development and epigenetics are intimately interwoven: one genome 100 cell types
• Response to environment• Evolutionary mechanism - short term adaptation to changing
environment without modification of the genetic code – plasticity + robustness fitness advantage
• Mitotic (& sometimes meiotic) memory in the absence of the initiating stimulus
• Epigenetic marks• DNAme• Chromatin • Small RNA
The epigenomic architecture underpinning a rapid developmental transition from brown to white adipose tissue in the perirenal depot
conception
birth
d 0 d 7-1 w-4 m d 147 3 m
LambFetus
d 140
Images of BAT and WAT
Fetus Lamb
MU
20 × 20 ×
1
10
100
1000
10000
Fold
cha
nge
(c)
0
500
1000
1500
2000
fetus lambRatio
mtD
NA
/ nD
NA
P < 0.05
A BAT to WAT transition in perirenal adipose tissue from late gestation fetus to lamb
P < 0.05
Brown adipose tissue
• Nonshivering thermogenesis• Young: protection from hypothermia• UCP1: Uncouples ox/phos from ATP
synthesis heat• UCP1: BAT specific expression• Small amount in adults• BMI negatively associated with BAT
activity (Robinson et al 2014)
• Energy expenditure after cold challenge higher in people with more BAT (Yoneshiroet al 2011, 2013)
• Obesity treatment: decrease energy intake or increase expenditureBAT deposition is very
different in mammals
Stephan Herzig, Univ. Heidelberg
PET
Lomax M A et al. Endocrinology 2007;148:461
RNA-Seq lamb 1
chr9chr2
chr1chr17
chr11 chr19ENSOART00000001272
GSN FABP5
DIO1
PPARG
UCP1
38 kb 10 kb
23 kb10 kb
133 kb5.9 kb
RNA-Seq fetal 1
CpGiENSEMBL
Ref-Seq
RNA-Seq fetal 1
CpGiENSEMBL
Ref-Seq
RNA-Seq fetal 1
CpGiENSEMBL
Ref-Seq
RNA-Seq lamb 1
RNA-Seq lamb 1
Representative RNA-Seq profilesn=3/gp; ~30 m reads/ sample
F
F
F
L
LL
F1 F2 F3 L2 L1 L3
Strong transcriptional differences: 5354 DE genes at Padj < 0.1
-10
-5
0
5
-15 -10 -5 0 5
Log 2
fold
chan
ge
qRT-
PCR
Log2 fold change RNA-Seq
validation
Principal Components Plot
DIO1
Fetus Lamb
0 5 10 15 20 25 30 35
GO:0006091~generation of…
GO:0045333~cellular respiration
GO:0015980~energy derivation by…
GO:0022904~respiratory electron…
GO:0006119~oxidative…
-log10 Padj
BP
0 5 10 15
GO:0010033~response to…
GO:0042127~regulation of cell…
GO:0006928~cell motion
GO:0051270~regulation of cell…
GO:0016477~cell migration
-log10 Padj
BP
RNA-Seq functional enrichments
• Confirmed markers of BAT and WAT function in fetal and lamb PRAT• Fetal PRAT - strong mitochondrial and lipid metabolism themes• Lamb PRAT – ECM, cell- cell contact and TGF beta pathway themes • Genes encoding secreted growth & differentiation factors • Innervation ‘type’ changed (ADRB3 ADRA2A and ADRB2)• Functionally anonymous genes with large fold changes • No signatures of adipogenesis• Fetal PRAT - unlikely to be beige adipose tissue• Fetal PRAT – markers for proliferating preadipocytes• Lamb PRAT – markers of inflammation
Gene states
• H3K4me3 and H3K27ac - relaxed chromatin; constitutive gene expression
• H3K27me3 – condensed chromatin; gene repression
• H3K4me3 + H3K27me3 ‘Poised’ genes
7
10
X
1
H3K27me3 is different
0
250
500
750
1000
autosome1 2 3 4 5 6 7 8 9 1011121314151617181920212223242526X
Prom
oter
read
cou
ntChromosome
Xi in females
0
2000
4000
0 1000 2000
Gene
exp
ress
ion
(RPK
M)
H3K27me 3 promoter read count
100
1000
10000
0 500 1000 1500 2000H3K2
7me3
pro
mot
er re
ad
coun
t
Gene rank
Approximately 500 autosomal transcription factors are intensively modified by H3K27me3
0 20 40 60
IPR017970:Homeobox, conserved site
IPR001356:Homeobox
IPR012287:Homeodomain-related
0 25 50
GO:0003700~transcription factor activity
GO:0043565~sequence-specific DNA…
GO:0030528~transcription regulator activity
GO:0003677~DNA binding
-log10 Padj-log10 Padj
Intense H3K27me3 is conserved and silences genes involved in early life development
0
2000
4000
N L M HGroups
0
500
1000
N L M HGroups
0
200
400
N L M HGroups
Feta
l pro
mot
er c
ount
H3K4me3 H3K27ac H3K27me3
0
500
1000
1500
N L M HGroups
0
200
400
600
N L M HGroups
0
200
400
N L M HGroups
Lam
b pr
omot
er c
ount
Chromatin modifications regulate gene expression
LAMTOR5 (a) PAX6
KCNC2HOXD locus (d)(c)
(b)
Representative ChIP-Seq profiles
Differentially modified regions between fetal and lamb PRAT
• H3K4me3• 56,169 enriched regions (3.8% genome)• 10,447 differentially modified regions (Padj < 0.1)
• H3K27ac• 86,246 enriched regions (5.7% of the genome)• 18,853 DM regions (Padj < 0.1)
• H3K27me3• 83,493 enriched regions (4.8% of the genome)• 4372 DM regions (Padj < 0.1)
Intersection of DE and DM genes
P< 0.001
P<0.001
P<0.001
Enriched functions for differentially modified chromatin regions
Chromatin modification Fetal PRAT Lamb PRAT
H3K4me3 Mitochondria, lipid metabolism, ‘brown fat’, metabolic process, mitosis
ECM, cell/cell contact, inflammation, protein metabolism
H3K27ac Mitochondria function, chromatin assembly
n.s.
H3K27me3 Transcription factors, homeobox encoding genes, development
Gated ion channels,
H3K27ac+ / H3K4me3- TGFβ signalling pathway
Padj<0.05
H3K4me3+ / H3K27me3+ Nuclear receptors
CpG islands CpG island shores non-island CpG
Rela
tive
Freq
uenc
y
Methylation ratio
No genome-wide differences in DNAm
Only 660 DMRs identified despite large scale functional, transcriptional and chromatin differences
Whole genome bisulphite sequencing
Chr2:2,201,000-2,212,000
Chr2:234,351,847-234,451,887
Chr2:136,424,233-136,802,863
RefSeqENSEMBL
Me fetusCpGi
Me lambDMR fetusDMR lamb
UMR lambUMR fetus
RefSeqENSEMBL
Me fetus
CpGi
Me lamb
RNA-Seq fetus
DMV lamb
UMR lamb
UMR fetus
RefSeqENSEMBL
Me fetus
CpGi
Me lambDMV fetus
H3K27me3 fetus
H3K4me3 fetus
H3K27me3 fetus
H3K4me3 fetus
H3K4me3 fetus
RNA-Seq fetus
H3K4me3 fetus
Epigenetic modifications are intimately interlinked
Chromatin and DNAm features are intimately interlinked
UMR LMR DMV
active enhancer repressed
XGene Gene
• UMRs and LMRs reflect cell lineage and are acted upon by chromatin modification changes during development
• Only 660 DMRs despite large scale morphological, functional, transcriptional and chromatin changes
• Fetal and lamb PRAT DNAme profiles are very similar i.e. likely derived from the same progenitor cells
• Therefore, transdifferentiation not adipocyte replacement• no adipogenic functional signatures• very similar DNAme profiles
A hierarchy of epigenetic changes
DMRs and nuclear receptors• ~660; mean size 255 bp• Strong intersection with chromatin modifications, UMRs & LMRs• DMRs tend to populate the edges of UMRs & LMRs• Genes close to DMRs only explain 3% of de genes• Enrichment for transcription factors especially NRs involved in
regulation of metabolic processes (Padj < 0.05)• NRs activated by steroid hormones, thyroid hormone, cortisol,
lipids, vit A/D, retinoids, prostaglandins, phosphoinostitols, unknown ligands
• NRs act in conjunction with coactivators and corepressors - often scaffolds for epigenetic modifiers
NR4A2, NR1D1, NR5A1, NR5A2, NR2F2, ESRRB, RARB, NCOR2, FOXA2, FOXO1, NR4A3
Fetal brown adipose tissue Lamb white
adipose tissue
Birth(adrenalin, cortisol)
Thermogenesis
Lipid mobilization and storage
Interconversion
Adipokines
Low level inflammation
↑ Mitochondria
Secreted growth and differentiation factors
ECM change
T4
T4↓T3
UCP1
Sympathetic innervation Y Sympathetic
innervation
Cold
ECM
Uncoupling Ox/phos
Energy demand
Nuclear receptor activation Chromatin reprogramming
Gene expression and functional changes
Effects of maternal periconceptional overnutrition on the late gestation fetal epigenome - Is there early life epigenetic priming of fat cell development later in life?
embryo transferconception
birth
d 0 d 7-1 w-4 m d 147 4 m
LambFetus
d 140
Donor ewes Recipient ewes
Ad libitum (170-190% MER)
100% MER
Rosiglitazone
Lisa Nicholas, Janna Morrison, Song Zhang, Caroline McMillen
Strong effects of overnutrition on donor ewes
45.00
55.00
65.00
75.00
85.00
- 23
Wks
- 21
Wks
- 19
Wks
- 17
Wks
- 15
Wks
- 13
Wks
- 11
Wks
- 9 W
ks- 7
Wks
- 5 W
ks- 3
Wks
- 1 W
k+
1 da
y+
3 da
ys+
5 da
ys
Donor ewe weights
CONT-ROSI
CONT-VEH
PCON-ROSI
PCON-VEH
conception
PCON affects plasma metabolites
1H-NMR plasma metabolomics
Lisa Nicholas, Janna Morrison, Caroline McMillen, Song Zhang Daisy Li
Periconceptional overnutrition results in increased fat mass in fetal and lamb female offspring
0
1,000
2,000
3,000
4,000
Control PCON
Tota
l fat
mas
s (g)
0
250
500
750
1000
Control PCONPe
rirne
alfa
t mas
s (g)
Rattanatray et al. Endocrinology2010, 151:5195-5205
P<0.05P<0.05lamb lamb
fetus
0
1
2
3
4
5
6
Control PCON
Feta
l wt (
kg)
0
5
10
15
20
25
Control PCON
Perir
enal
fat m
ass (
g)ns P<0.05fetus
Donor ewe conception wt (kg)
Lam
b fa
t mas
s (g)
Caroline McMillen, Janna Morrison, Lisa Nicholas, Daisy li
Lamb plasma metabolome at 18 wks
Effects of PCON on late gestation fetus
• No effect of PCON on fetal (H1-NMR) plasma metabolome
• Weak effect on transcriptome (RNA-Seq, 24 genes DE at q<0.05; n= 5/gp)
• 30 DM H3K4me3 regions (q<0.05; FC>1.5; n=4-5/gp)
• 58 DM H3K27me3 regions (q<0.05; FC>1.5; n=4/gp)
• 5846 DM H3K27ac regions (q<0.05; FC>1.5; n=4 /gp)
partial least squares discriminant analysis
DM H3K27ac functional enrichments (q<0.05; not highest rankings)
0 2 4 6 8
mitochondrial membrane organizationregulation of oxidative phosphorylation
regulation of mitochondrial membrane permeabilityabnormal white fat cell number
Type I diabetes mellitusMinimal subcutaneous fat
Nuclear Receptors in Lipid Metabolism and ToxicityRXR and RAR heterodimerization with other nuclear receptor
Genes involved in Insulin receptor recyclingGenes involved in Degradation of the extracellular matrix
Genes involved in Chondroitin sulfate biosynthesisTop 20 positive significant genes associated with liposarcomas,…
Genes up-regulated in adipose tissue from obese mouse strains…
-log10 Padj
BP
BP
BP
MP
HP
HP
MSigDB pathway
MSigDB pathway
MSigDB pathway
MSigDB pathway
MSigDB pathway
MSigDB perturbation
MSigDB perturbation
ConclusionsFetal – lamb development
• Transition from BAT to WAT• Chromatin and DNAme features define regulatory elements,
gene state, cell ontogeny and potential for pluripotency• Hierarchy of epigenetic modifications: discordant changes in
chromatin and DNAm despite an intimate relationship between chromatin marks and DNA hypomethylation features
PCON• PCON affects fetal and lamb PRAT deposition, insulin signalling,
lamb plasma metabolites• Few effects on mRNA, H3K4me3 and H3K27me3• Significant but weak effects on H3K27ac• Lipid metabolism, NR and ECM• Potential for early life epigenetic priming of increased fat
deposition in later life
Many thanks to a great group of people!
Mike BuckleyDenis BauerSean McWilliam
Peter MolloySusan van DijkStephen BradfordTim Peters
Tony VuocoloRoss Tellam
Caroline McMillenJanna Morrison Song ZhangLisa Nicholas
Shalima NairAaron StathamSue Clark
Daisy LiHorst Schirra
EpiSCOPE
