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Epigenetics and FASD

Epigenetics and FASD. Alterations in genomically imprinted miRNA and snoRNA clusters in a mouse model of Fetal Alcohol Spectrum Disorders (FASD). Benjamin I. Laufer, Katherine Mantha , Morgan L. Kleiber , Eric J. Diehl, Sean M.F. Addison, and Shiva M. Singh University of Western Ontario.

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Epigenetics and FASD

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  1. Epigenetics and FASD Alterations in genomically imprinted miRNA and snoRNA clusters in a mouse model of Fetal Alcohol Spectrum Disorders (FASD) Benjamin I. Laufer, Katherine Mantha, Morgan L. Kleiber, Eric J. Diehl, Sean M.F. Addison, and Shiva M. Singh University of Western Ontario

  2. Waddington, Conrad H. 1953. The Epigenetics of birds. Cambridge University Press • A metaphor for biological development • Cell fates are established in development by epigenetic marks much like a marble rolls down to the lowest point • Increasing irreversibility of cell type differentiation as ridges rise between the valleys. Waddington’s Epigenetic Landscape

  3. Waddington, Conrad H. 1953. The Epigenetics of birds. Cambridge University Press Waddington’s Epigenetic Landscape • What happens if an obstacle gets in the way?

  4. Fetal Alcohol Exposure • Leading cause of preventable birth defects and mental deficits in North America • FASD • Fetal Alcohol Spectrum Disorders • 1 out of 100 pregnancies • Umbrella term for a number of physical abnormalities, behavioural and intellectual problems • Strongest manifestation is Fetal Alcohol Syndrome (FAS) Chudleyet al.CMAJ 2005

  5. Fetal Alcohol Exposure in an Animal Model • We have shown that Fetal Alcohol Exposure (FAE) affects behaviour, learningand related genes. • We have also recently shown that these changes are maintained for a lifetime • Even after exposure has ceased for weeks • Are epigenetic mechanisms responsible? Kleiberet al. Behav. Brain. Res. 2012 Kleiberet al. Brain. Res. 2012

  6. Epigenetic Mechanisms of Interest • DNA Cytosine Methylation • Typically turns expression on or off Metivier, et al 2008. Nature • Noncoding RNA • i.e: miRNA • Fine tuners of gene expression • Low fold changes http://www.wormbook.org/chapters/www_microRNA/celmicRfig5.jpg

  7. Fetal Alcohol Exposure and Methylation • FAE alters the methylation and expressionof genomically imprinted (uni-parental) genes in: • Whole embryo • Placenta Liu et al. 2009 Epigenetics. Shukla et al. 2011 Alcohol. Clin. Exp. Res • 30% of parentally imprinted transcripts are ncRNA. Morison et al. 2005,Trends. Genet.

  8. Imprinted ncRNA • Key role in neurodevelopment and memory. • Important for early life processes • and functionally important for adult brain functions. • Many are microRNAs Wang et al. 2009 PLoS One Davies et al. 2008Adv. Exp. Med. Biol

  9. Fetal Alcohol Exposure and miRNAs • miRNAs have been shown to be deregulated by FAE in fetal mouse brain cells • Co-incubation with folic acid prevents altered miRNA and target gene expression in mouse embryos • Folic Acid is involved in establishing DNA methylation. • Association between methylation and expression, but what is the mechanism behind this relationship? Sathyanet al.2007 J. Neurosci. Wang et al.2009 Hum. Reprod.

  10. Functional Mechanisms • An alteration of methylation in a transcription factor binding site has the potential to affect gene expression. • CTCF binding sites in the H19/Igf2 imprinting control region show differential methylation in FASD placental tissue. • CTCF is a highly conserved ubiquitous zinc-finger protein with multiple functions in chromatin organization and gene regulation • It binds in a methylation sensitive manner to target sequences • Is this the functional mechanism for the association between gene expression and DNA methylation in FASD? Haycock et al. 2009 Biol. Reprod. Williams et al 2008. J. Exp. Med. Filippova 2008 Curr. Top. Dev. Biol.

  11. Hypothesis Alterations in DNA methylation and ncRNAexpression are associated with life-long alterations in gene expression in the mouse brain after fetal alcohol exposure.

  12. Continuous Preference Drinking (CPD) • Free choice • Quantity monitored daily • 70% preference for 10% EtOH • In C57BL/6J mice • No Stress Young & Olney 2006 Neurobiol. Dis.

  13. Whole Brain Methylation Array Analysis PND 70 DNA Methylation

  14. Over 6,600 genes with differences in 1 or more promoter regions • More than half of imprinted genes in genome • p < 0.01 • Subjected to Ingenuity Pathway Analysis Control Treated

  15. PtenSignaling Pathway • Significantly affected canonical pathway (p=1.9E-06) • 54/95 molecules showed significant differential methylation in their promoters • Controls the tempo of the process of newborn neuron integration during adult neurogenesis • including correct neuron positioning, dendritic development, and synapse formation. Porteouset al. 2009 Neuron

  16. Enriched Biological Functions • Many have been previously implicated in FASD and all are highly compatible

  17. Top Affected IPA Network “Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65) 40% of hub-genes promoters investigated had CTCF binding sites

  18. CTCFBSDB: a CTCF binding site database for characterization of vertebrate genomic insulators CTCF Bao Let al. Nucleic Acids Research2008 Gene P CTCF Gene P

  19. Methylation Array Results Summary PND 70 • Over 6000 genes with significant differences in their promoters • More than ½ of the molecules involved Pten Signalingaffected • Not a random sample • Enriched for relevant functions • Many CTCF binding sites in important neurodevelopmental genes showed differences in methylation DNA Methylation

  20. Whole Brain ncRNA Expression Array Analysis PND 70 Gene Expression microRNA

  21. miRNA Array Heatmaps p < 0.05 FC 1.2 Treated Control Treated Control Treated Control Treated Control

  22. ncRNA Venn Diagram p < 0.05 FC 1.2 1

  23. Imprinted Noncoding RNA Clusters • Localized to the brain • Only 3 clusters in mouse genome • Sfmbt2 (Chr 2), Snrpn-Ube3a (Chr 7), Dlk1-Dio3 (Chr 12) • 20% of altered miRNAs in all exposure paradigms • Associated with FASD related endophenotypes

  24. Cluster of Interest: Snrpn-Ube3a (Chr 7) • Showed significant up-regulation in: • all 4 treatment paradigms • CPD and injections • both array types • miRNA and gene H/MBII-52 (SNORD115) snoRNA binds to mRNA III I II IV Va Vb VI III 5htr2c Pre-mRNA Inclusion of exon Vb without mRNA editing during alternative splicing Receptor with a stronger serotonin response

  25. miRNA and Gene Expression Results Summary PND 70 • Global Expression changes • Individual miRNAs unique to treatment paradigm • 20% of affected miRNAs belong to imprinted clusters • H/MBII-52 only ncRNA (and gene) affected in all paradigms and arrays Gene Expression microRNA

  26. Whole Brain Bioinformatic Analysis PND 70 DNA Methylation microRNA Gene Expression

  27. IPA miRNA Target Filter • 34 genes identified with reverse pairwise relationships to predicted miRNAs • 4 are highly compatible with FASD:

  28. miRNA Target Filter Gene of Interest • A novel role for Pten in FASD: • Pten is a lipid phosphatase that suppresses Aktactivation. • Akt: • Regulates neuronal development, including morphogenesis, dendritic development, synapse formation, and synaptic plasticity. • Showed a gain of methylation at a predicated CTCF binding site in its promoter. • More than half of the Pten signaling genes were significantly enriched for on the methylation arrays Porteouset al. 2009 Neuron

  29. Other Target Genes of Interest • Otx2: • Expressed in the brainand involved in mood disorders • Identified in our previous study on long-term brain gene expression changes in FASD. • Nmnat1: • Protects against axonal degeneration following mechanical or toxic insults by delaying axonal degeneration. • Slitrk2: • Significant expression is detected only in the adult brain. • Uniquely expressed in immature neurons • Inhibitory effect on neurite outgrowth. Kleiberet al. Brain. Res. 2012

  30. Summary of Results Chr 12 ICR Chr 12 ICR Mir-369 Impaired behaviour, learning and memory Pten Mir-495 Mir-25 Mir-25 Promoter Mir-152 Otx2 Mood Disorders Mir-1224 Mir- 743a Reduced ability to protect axons Nmnat1 Mir-431 Mir-431 Promoter Nmnat1 Promoter

  31. Proposed Molecular Cascade ncRNA Gene Expression Endophenotypes Fetal Ethanol Exposure Methylation

  32. Acknowledgements

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