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.

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. Barker Hypothesis

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 Cause for biallelic expression was due to aberrant DNA methylation M Bi M un-M Whittens KSOM

SCNT(cloning) In vitro fertilization control Animals produced by Somatic Cell Nuclear Transfer in particular, can have very profound phenotypes… Tamashiro et al., Nat Med 8(3), 2002 Cezar et al., Biol Rep 68, 2003 Pace et al., Biol Rep 67, 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.

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. Are Imprinted Genes Differentially Expressed in Response to Maternal Under-Nutrition?

Facts about Imprinted Genes ~ 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 pcd5 pcd10pcd19 Mating 2 wks prior to mating C57B6 females fed a low protein Diet. 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: AlleleFunction (based on observed Expressedphenotypes found in mutations). Igf2PaternalGrowth Igf2rMaternalGrowth retardation H19Maternal?? Ata3PaternalAmino acid transport (Placenta) P57kip2 MaternalReduced survival; growth retardation Peg1/MestPaternalGrowth Peg3PaternalGrowth

Imprinted Genes: Liver Brain HeartKidney H19 Igf2 Igf2r Peg3 Ata3 P57kip2 ND

Igf2H19 26 CpG dinucleotides? Is Altered Expression of H19 Gene Expression Associated with a Change in Methylation of the DMD?

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. Eric Ravussin! Dr. Rob Koza: Bisulphite Sequencing Low Density Arrays Microarrays