Disruption of Imprinted Regions in a Mouse Model of Fetal Alcohol Spectrum Disorders (FASD) Laufer, B., Diehl, E., Kleiber, M., Janus, K., Wright, E., and Singh, S. 1

Nature Vs. Nurture 2

3

Environmental Insults 4

What is Epigenetics? “A mitotically (or meiotically) inheritable change in gene expression, independent of an alteration in DNA sequence” – Berger et al. (2009) Genes Dev. 5

Epigenetic Mechanisms Histone Modifications DNA Cytosine Methylation MicroRNA 6

DNA Cytosine Methylation 7 5’------CpG ’ 3’ -----GpC ’ CH 3

DNA Cytosine Methylation 8. Metivier, R. et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 45–50 (2008).

Non Coding RNA 95% of the human genome is transcribed and produces a vast number of noncoding RNAs (ncRNAs). Two types of Interest: – MicroRNAs Degrade target mRNA or prevent translation – SnoRNAs Typically involved in mRNA editing – However, there are exceptions 9 Carnicini et al. 2008, Curr Opin Cell Biol

Environmental Epigenetics 10

Alcohol as Environmental Insult Fetal Alcohol Exposure (FAE) affects gene expression, behaviour and learning. Changes maintained for a lifetime – Even after exposure has ceased for months Mechanism unknown 11

Fetal Alcohol Exposure 12 Leading cause of preventable birth defects and mental deficits FASD – Fetal Alcohol Spectrum Disorder – 1 out of 100 pregnancies – Physical abnormalities, behavioural and intellectual problems

Continuous Preference Model Free choice Quantity monitored daily 70% preference for 10% EtOH – mg/dl per day – DBA/2J mice has only 10% No Stress 13 Young C, Olney JW. Neuroapoptosis in the infant mouse brain triggered by a transient small increase in blood alcohol concentration. Neurobiol Dis 2006;22:

Hypothesis Fetal alcohol exposure induces alterations in brain gene expression that last into adulthood and are correlated with epigenetic modifications. 14

Methods: Array Analysis 15 Gene Expression MicroRNA DNA Methylation

Results 3 Large Clusters of Brain Specific Imprinted noncoding RNA (ncRNA) 16

Genomic Imprinting 17 Genes are expressed in a parent of origin specific manner. Achieved by DNA Methylation. Maternal Paternal Normal Bialleic Gene Expression Maternally Expressed, Paternally Imprinted Paternally Expressed, Maternally Imprinted

Imprinted ncRNA 30% of parentally imprinted transcripts are ncRNA. Key role in neurodevelopment and memory. – Some appear to be fully imprinted in the adult brain only. Imprinted ncRNAs are not only important for early life processes, but are also functionally important for ‘online’ adult brain functions. 18 Morison et al. 2005, Trends Genet Wang et al 2004, Mol Cell Biol Davies et al. 2008, Adv Exp Med Biol

14q32.21 (Murine 12qF1) 19 Pten AKT-mTOR pathway Tempo of newborn neuron integration during adult neurogenesis Implicated in the developmental basis of major psychiatric disorders Maternally Expressed, Paternally Imprinted

15q11-q13 (Murine 7qC) 20 III 5htr2c Pre-mRNA III III IV Va Vb VI Receptor with a stronger serotonin response Inclusion of exon Vb without mRNA editing during alternative splicing snoRNA binds to mRNA H/MBII-52 (SNORD115) Paternally Expressed, Maternally Imprinted

Murine 2qA1 21 Intron 10 Sfmbt2 Sfmbt2-AS Over 130 miRNAs Mouse Specific No Imprinting Center Paternally Expressed, Maternally Imprinted

Future Directions 1.Confirmation of Results 2.Brain Region Specificity – Hippocampus 1.Trans-generational Effects 22

What are Trans-generational effects? 23 F0F0 F1F1 F2F2 Direct ExposureIndirect Exposure Great Grandchildren F 3

Conclusions The genome is environmentally responsive. The epigenome serves as the bridge between environment and genome. Thus, the environment shapes an individual significantly for a lifetime and beyond. The nature vs. nurture debate has been solved by epigeneticists. 24

Acknowledgements Supervisor Shiva Singh Funding: Advisory Committee NSERC Kathleen HillCIHR Anthony Percival-SmithOMHF Singh Lab: Raihan Uddin Morgan Kleiber Haroon Sheikh Christina Castellani Katarzyna Janus Eric Diehl Pat McDonald Kiran Kumar Sujit Maiti Rachel Danielson Randa Stringer Elizabeth Yan Aniruddho C. Hoque 25