Transcript NORC Retreat Presentation

Epigenetic Programming in utero
and Later in Life Disease
CNRU Retreat
September 28, 2006
Ken Eilertsen, Ph. D.
Stem Cell Biology Group/Epigenetics and Nuclear
Reprogramming
Outline
• Brief overview of where we started and how we got
here
– How we got here is really a function of the CNRU enabling us
to ask new questions.
• A summary of our first experiment venturing into this
area
• Overview of the current P & F award
Past 10 years…
• My lab focused on assisted reproductive technologies
research
• Primarily somatic cell nuclear transfer (cloning)
– Nuclear reprogramming
• Upon arriving at The Pennington, we wanted to explore
new, but very related questions.
– Barker Hypothesis
– CNRU has been key to meeting this latter objective.
Barker Hypothesis
Fetal (Developmental) Origins of Adult Disease Hypothesis:
•Posits that a poor in utero environment elicited by maternal dietary
or placental insufficiency ‘programs’ susceptibility in the fetus
to later development of cardiovascular and metabolic disease.
Programming:
-commonly ascribed to any situation where a stimulus or
insult during development establishes a permanent physiological response.
-in the context of a predisposition to a later in life disease,
no mechanism described.
Some animal models lend support to
Developmental Origins of Disease Hypothesis
A.
B.
Maternal low protein diet
causes a significant
reduction in ICM
(embryonic stem cells)
and TE (placenta)
cell numbers at blastocyst
stage.
Assisted Reproductive Technologies (ART) in
humans have been linked to later in life disease:
• Angelman Syndrome (aberrant DNA
methylation)
• Beckwith-Weideman syndrome (aberrant
DNA methylation)
Suboptimal culture conditions may be a causative factor for
predisposing offspring to these syndromes
M Bi
M
un-M
Cause for biallelic
expression was due to aberrant DNA methylation
Animals produced by Somatic Cell Nuclear Transfer
in particular, can have very profound phenotypes…
Pace et al., Biol Rep 67, 2002
In vitro fertilization
Cezar et al., Biol Rep 68, 2003
SCNT(cloning)
control
Tamashiro et al., Nat Med 8(3), 2002
The general consensus is that the cause of these
phenotypes are EPIGENETIC in nature
• Epigenetics is a mechanism that ensures heritable
characteristics of cells and functional differences
between cell types
• Epigenetic mechanisms alter chromatin (DNA and
proteins) in ways that change the availability of genes
to transcription factors. Key components include:
– Addition of methyl group to CpG dinculeotides*
– Association of Polycomb and other DNA binding proteins that
modify histones.
DNA methylation patterns
• Known to be established during development and
subsequently maintained by DNA methyltransferases
• Traditionally thought that once established, the
methylation patterns were reliably maintained for the
life of the organism and irreversible.
– View is evolving a bit these days.
Are Imprinted Genes Differentially Expressed in
Response to Maternal Under-Nutrition?
What is an imprinted gene?
• Genes that are mono-allelically expressed
-Either maternal allele OR paternal allele is expressed
• Paternal genes are thought to extract maternal resources for the
benefit of the offspring
– Growth promoters
• Maternal genes are thought to allocate resources ‘equitably’
between offspring and mother.
– Growth suppressors
• Thus, imprinted genes have growth related functions with
antagonistic properties.
• Imprinted gene expression is epigenetically regulated.
Facts about Imprinted Genes
• ~70-200 exist in mammals
• Found in clusters (referred to as imprinted domains) at
~ 15 different chromosomal sites
• Imprinted domains are coordinately regulated by
epigenetic mechanisms:
– DMD, or Differentially Methylated Domain
H19 and Igf2: Examples of imprinted genes and coordinate
regulation of expression through a differentially methylated domain.
Experimental design: C57B6 female mice
Mating
pcd5
2 wks prior
to mating
C57B6 females
fed a low protein
Diet.
pcd10
pcd19
Pups delivered by C-section;
Organs harvested for mRNA
isolated to measure
expression levels of
imprinted genes
in each individual organs (n=10).
Controls fed normal chow diet throughout.
LPD diet
Normal chow diet
Imprinted Genes:
Igf2
Igf2r
H19
Ata3
P57kip2
Peg1/Mest
Peg3
Allele
Expressed
Function (based on observed
phenotypes found in mutations).
Paternal
Maternal
Maternal
Paternal
Maternal
Paternal
Paternal
Growth
Growth retardation
??
Amino acid transport (Placenta)
Reduced survival; growth retardation
Growth
Growth
Imprinted Genes:
Liver
H19
18
16
14
12
10
8
6
4
2
0
Con
Brain
Heart
Kidney
ND
ND
ND
Exp
3
0.8
3.5
3
Igf2
2.5
0.7
0.6
0.5
2
0.4
1.5
1.5
0.3
1
1
0.2
0.5
0.1
0
0
Con
0.5
0
Con
Exp
ND
2
2.5
Exp
Con
Exp
3.5
3
3
2.5
2.5
2
Igf2r
2
ND
1.5
1
0.5
ND
1.5
1
0.5
0
0
Con
Con
Exp
Exp
2
1.85
1.8
1.5
1.75
Peg3
ND
1.7
1.6
0.5
1.55
1.5
0
1.45
1.4
Con
Ata3
ND
1
1.65
ND
Exp
ND
Con
ND
3
Exp
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Con
Exp
2.5
2
P57kip2
ND
1.5
ND
1
0.5
0
Con
Exp
ND
Is Altered Expression of H19 Gene Expression
Associated with a Change in Methylation of the DMD?
Igf2
H19
26 CpG dinucleotides?
Summary
• Exposure to a maternal LPD during preimplantation period
appears to alter imprinted gene expression in organs of near born
pups.
• Expression appears to be tissue specific
• No detected differences in methylation patterns of DMD
• Encouraged by the preliminary data indicting imprinted gene
expression, known to be regulated epigenetically, could be altered
by maternal nutrition.
Submitted a Pilot and Feasibility Grant to the CNRU.
CNRU P & F Proposal
Weaning*
6 wks.
9 wks.*
12 weeks
Normal Chow
Low Protein
Pups weaned to
Hi fat
Low Protein
Pups weaned to
Low protein
Hi Fat
Pups weaned to
Hi Fat
Can we link later in life phenotypic characteristics such as weight, BP, glucose
tolerance, diabetes,etc. with differentially expressed genes and methylation
patterns of associated CpG islands and/or promoters?
Acknowledgements
Ms. Heather Kirk
Dr. Barry Robert (Kidney)
Ms. Regina Staten
Stem Cell Biology Lab:
Dr. Jeff Gimble
Dr. Randy Mynatt
Dr. Basia Kozak
Dr. Beth Floyd
Dr. Rob Koza:
Bisulphite Sequencing
Low Density Arrays
Microarrays
!Dr. Eric Ravussin!