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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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slide1

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

waddington s epigenetic landscape

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

waddington s epigenetic landscape1

Waddington, Conrad H. 1953. The Epigenetics of birds. Cambridge University Press

Waddington’s Epigenetic Landscape

  • What happens if an obstacle gets in the way?
fetal alcohol exposure
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

fetal alcohol exposure in an animal model
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

epigenetic mechanisms of interest
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

fetal alcohol exposure and methylation
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.

imprinted ncrna
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

fetal alcohol exposure and mirnas
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.

functional mechanisms
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.

hypothesis
Hypothesis

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

continuous preference drinking cpd
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.

slide14

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

pten s ignaling pathway
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

enriched biological functions
Enriched Biological Functions
  • Many have been previously implicated in FASD and all are highly compatible
top affected ipa network
Top Affected IPA Network

“Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65)

40% of hub-genes promoters investigated had CTCF binding sites

slide18

CTCFBSDB: a CTCF binding site database for characterization of vertebrate genomic insulators

CTCF

Bao Let al. Nucleic Acids Research2008

Gene

P

CTCF

Gene

P

methylation array results summary
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

whole brain ncrna expression array analysis
Whole Brain ncRNA Expression Array Analysis

PND 70

Gene Expression

microRNA

mirna array heatmaps
miRNA Array Heatmaps

p < 0.05

FC 1.2

Treated

Control

Treated

Control

Treated

Control

Treated

Control

ncrna venn diagram
ncRNA Venn Diagram

p < 0.05

FC 1.2

1

imprinted noncoding rna clusters
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
cluster of interest snrpn ube3a chr 7
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

mirna and gene expression results summary
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

whole brain bioinformatic analysis
Whole Brain Bioinformatic Analysis

PND 70

DNA Methylation

microRNA

Gene Expression

ipa mirna target filter
IPA miRNA Target Filter
  • 34 genes identified with reverse pairwise relationships to predicted miRNAs
  • 4 are highly compatible with FASD:
mirna target filter gene of interest
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

other target genes of interest
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

summary of results
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

proposed molecular cascade
Proposed Molecular Cascade

ncRNA

Gene Expression

Endophenotypes

Fetal Ethanol

Exposure

Methylation