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Fetal alcohol spectrum disorders (FASDs) are characterized by life-long changes in gene expression, neurodevelopment and behavior. What mechanisms initiate and maintain these changes are not known, but current research suggests a role for alcohol-induced epigenetic changes. We assessed alterations to adult mouse brain tissue by assaying DNA cytosine methylation and small noncoding RNA (ncRNA) expression, specifically the microRNA (miRNA) and small nucleolar RNA (snoRNA) subtypes. We found long-lasting alterations in DNA methylation as a result of fetal alcohol exposure, specifically in the imprinted regions of the genome harboring ncRNAs and sequences interacting with regulatory proteins. The findings of this study help to expand on the mechanisms behind the long-lasting changes in the brain transcriptome of FASD individuals. Webinar Link: http://www.youtube.com/watch?v=fzdc0GIdCnA
Citation preview
Long-lasting alterations to DNA methylation and ncRNAs could underlie the effects of fetal alcohol
exposure in mice.
PMID: 23580197
Ben LauferPI: Shiva M. Singh
Contributors: Katherine Mantha, Morgan L. Kleiber, Eric J. Diehl, Sean M.F. AddisonInstitution: Western University (formerly University of Western Ontario)
1
Above and Beyond the Genome
Welcome to The Post-Genomics Era
• We’ve sequenced the human genome
• It can now be done for only a few thousand dollars!– We can sequence thousands of human genomes!– Individual people can now go see their sequence!
• The benefits we’ve seen are…
Is this it?
• Shouldn’t cracking the code of life give us more breakthroughs than we can dream of?
• Can 4 base pairs really code for complex life?
• What’s missing?
What treasure is left?
www.biocomicals.com, Alper Uzun, PhD.
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“A mitotically (or meiotically) inheritable change in gene expression, independent of an alteration in DNA sequence”
– Berger et al. (2009) Genes Dev.
The Solution to the Post Genomics Era: Epigenetics
Epigenetic Mechanisms (Macmillan Publishers Ltd: Nature 441: 143-145. 11 May 2006)
A Few Epigenetic Mechanisms of Interest
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
6Images from Wikimedia Commons
Redefining the Central DogmaSaletore et al. Genome Biology 2012, 13:175
Epigenetic Mechanisms
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
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The Histone Code
http://eukaryoticgeneexpression.weebly.com/uploads/5/6/3/0/5630004/7502951_orig.jpg
Epigenetic Mechanisms
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
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microRNAs
• The second level of the epigenetic landscape
– Acts at translation, as opposed to Histone modifications and DNA methylation, which act transcription
• Act as fine-tuners, rather than on and off switches like histone modifications and DNA methylation.
– Typically result in low fold changes in gene expression• However, these changes are physiologically relevant.
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microRNA (miRNA)
http://www.wormbook.org/chapters/www_microRNA/celmicRfig5.jpg
Epigenetic Mechanisms
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
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DNA Methylation (and other modifications)
• Histones aren’t the only ones to enjoy modifications
• There aren’t just four base pairs anymore– Cytosine has made a few friends
http://www.atdbio.com/img/articles/epigenetic-base-modifications.png
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DNA Cytosine Methylation
. Metivier, R. et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 45–50 (2008).
Key Players of the Epigenetic Landscape
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
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The Epigenetic Landscape
http://cnx.org/content/m26565/latest/graphics35.jpg
It really is a landscape…
19
Waddington’s Epigenetic LandscapeWaddington, 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.
20
Environmental Conditions
21
Environmental Epigenetics
22
Environmentally Responsive Genome
http://learn.genetics.utah.edu/content/epigenetics/nutrition/images/pathway.jpg
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Waddington’s Epigenetic LandscapeWaddington, Conrad H. 1953. The Epigenetics of birds. Cambridge University Press
• What happens if an obstacle gets in the way?
24
Fetal Alcohol Exposure • Leading cause of preventable birth defects and mental deficits in North
America
• FASD– Fetal Alcohol Spectrum Disorders
– 2-5% of pregnancies!
– Umbrella term for a number of physical abnormalities, behavioural and intellectual problems
– Strongest manifestation is Fetal Alcohol Syndrome (FAS)
Chudley et al. CMAJ 2005
Spectrum Disorders
Fetal Alcohol Spectrum Disorders
Fetal Alcohol Spectrum Disorders
Fetal Alcohol Syndrome (FAS)
Partial Fetal Alcohol Syndrome (PFAS)
Alcohol-Related Neurodevelopmental Disorder
Alcohol-Related Birth Defects
Fetal Alcohol Effects
Epigenetics
Where does an individual land in the spectrum?
• Depends on:
– Genetic Background
– Timing of Exposure
– Dosage of Exposure
– Other Epigenetic and Environmental Factors, either:• Inherited• Experienced
28
Spectrum Meets Landscape
Fetal Alcohol Syndrome (FAS)
Partial Fetal Alcohol Syndrome
Alcohol-Related Neurodevelopmental Disorder (ARND)
Alcoho
l-Rela
ted
Birth
Defe
cts (A
RBD)
Feta
l Alco
hol E
ffect
s (FA
E)
Mouse Models• Reasons for use:
– Useful for studies that would be impractical in humans
– Reach sexual maturity early (6–8 wk)
– Birth multiple offspring
– Abbreviated gestational period (18–21 days)
• Many generations can be analyzed within a relatively short timeframe (1–2 yr)
– Allows for studying long-term changes
– Allow for analysis in vivo, which is essential for epigenetic studies.
• Cell cultures do not exhibit natural epigenetic properties– See: Embryo culture and epigenetics. Velker BA, Denomme MM, Mann MR. Methods in Molecular Biology.
© Disney
Fetal Alcohol Exposure in an Animal Model
• We have shown that Fetal Alcohol Exposure (FAE) affects behaviour, learning and 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?30
Kleiber et al. Behav. Brain. Res. 2012
Kleiber et al. Brain. Res. 2012
Epigenetic Mechanisms of Interest
• DNA Cytosine Methylation– Typically turns
expression on or off
31
Metivier, et al 2008. Nature
http://www.wormbook.org/chapters/www_microRNA/celmicRfig5.jpg
• Noncoding RNA• i.e: miRNA• Fine tuners of gene
expression• Low fold changes
Fetal Alcohol Exposure and Methylation
• FAE alters the methylation and expression of genomically imprinted (uni-parental) genes in cell cultures derived from:
– Whole embryo
– Placenta
Liu et al. 2009 Epigenetics.
32
Shukla et al. 2011 Alcohol. Clin. Exp. Res
Morison et al. 2005, Trends. Genet.
• 30% of parentally imprinted transcripts are ncRNA.
33
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. 2008 Adv. Exp. Med. Biol
http://4.bp.blogspot.com/_Ik_ovkt6ICg/SfjaLNt1ehI/AAAAAAAAAF4/4ZzazV4xx3Y/s320/imprinted+brain.jpg
Fetal Alcohol Exposure and miRNAs• miRNAs have been shown to be deregulated
by FAE in fetal mouse brain cell culture
• 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?
Sathyan et al. 2007 J. Neurosci.
Wang et al. 2009 Hum. Reprod.
34
35
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?
Williams et al 2008. J. Exp. Med.
Filippova 2008 Curr. Top. Dev. Biol.
Haycock et al. 2009 Biol. Reprod.
36
Hypothesis
Alterations in DNA methylation and ncRNA expression are associated with life-long alterations in gene expression in the mouse brain after fetal alcohol exposure.
Continuous Preference Drinking (CPD)• Free choice
• Quantity monitored daily
• No Stress
• 70% preference for 10% EtOH
• C57BL/6J mice
• Metabolize alcohol much quicker than humans
• Blood Alcohol Concentration (BAC)
• Represents moderate drinking
• = pregnant human mother who has a drink or two every now and then.
37Young & Olney 2006 Neurobiol. Dis.
Experimental Design• Everything done downstream of your workflow is
dependent on what has happened upstream.
• Errors will amplify.
• For perspective see:
– Fundamentals of experimental design for cDNA microarrays. Churchill GA. Nature Genetics.
– Probe set algorithms: is there a rational best bet? Seo J, Hoffman EP. BMC Bioinformatics.
– Tackling the widespread and critical impact of batch effects in high-throughput data. Leek JT. Nature Reviews Genetics.
Whole Brain Methylation Array Analysis
39DNA Methylation
PND 70
40
• 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
TreatedControl
Looking through the noise• What is a p-value?
• Is a p=0.01 stringent enough for data from 2.1 Million probes?
• There’s going to be some false positives!
– But do we want to get rid of all the useful data caught up in those p-values?
• Particularly relevant for spectrum disorders as they are riddled with heterogeneity since we expect large amounts of variation within the experimental group
Systems Biology
• The antithesis to a reductionist approach
• Reductionism is a philosophy that the understanding of a complex system can be achieved in full by understanding its simpler component parts.
• Systems biology, on the other hand relies on examining the entirety of cellular processes and interactions in concert .
Independent Component Analysis• Ingenuity Core Analysis
• A 1.2 fold increase in many genes of a pathway can have a potentially greater physiological impact than a 20-fold increase in a single gene.
• “Project microarray data into statistically independent components that correspond to putative biological processes, and to cluster genes according to over- or under-expression in each component.”– Further Perspective: Application of independent component analysis to microarrays. Lee SI,
Batzoglou S. Genome Biology.
45
Enriched Biological FunctionsMolecular and Cellular Functions Name p-value # GenesCell Death 4.06E-04 - 4.97E-02 224Cellular Development 6.24E-04 - 4.52E-02 166Cellular Function and Maintenance 9.57E-04 - 4.97E-02 86Cellular Movement 4.12E-03 - 4.52E-02 41Cell Signaling 8.43E-03 - 4.97E-02 26Physiological System Development and Function Name p-value # GenesNervous System Development and Function 3.86E-05 - 4.97E-02 273Tissue Morphology 1.64E-04 - 4.23E-02 97Behavior 1.62E-03 - 1.58E-02 24Embryonic Development 1.23E-02 - 4.23E-02 29Organismal Development 1.23E-03 - 4.23E-02 25
• Many have been previously implicated in FASD and all are highly compatible
Using MeDIP and ChIP Data
• Super Simple Stuff
• Excel table with column 1 containing gene name from upstream analysis
– Subsequent columns for metrics of interest (optional)
47
Top Affected IPA Network“Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65)
Network Biology• The distribution of nodes (i.e: genes) in cellular
networks is highly non-uniform– Most of the nodes having only a few links to other
nodes.
• However, there are a few nodes with a very large number of links called hubs– The importance of the relationship between a system
and single gene is highlighted in these cases.
• While a network can tolerate many disruptions to its lesser-connected nodes, a similar disruption to a single hub can be catastrophic to the networks it connects.
• Further Insight:– Network biology: understanding the cell's functional organization. Barabási AL,
Oltvai ZN. Nature Reviews Genetics.
49
Top Affected IPA Network“Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65)
40% of hub-genes promoters investigated had CTCF binding sites
Gene P
CTCF
Gene Name Protein Name
H19 N/A (ncRNA)
Gtl2 (Meg3) N/A (ncRNA)
Npy Pro-neuropeptide YAkt1 RAC-alpha serine/threonine-protein kinaseGhr Growth hormone receptor
Ntrk1 High affinity nerve growth factor receptorApoe Apolipoprotein E
Grin2c Glutamate [NMDA] receptor subunit epsilon-3
Gene Name Protein Name
AppAmyloid beta A4 protein
MbpMyelin basic protein
Atp1a2 Sodium/potassium-transporting ATPase subunit alpha-2
Grin1 G protein-regulated inducer of neurite outgrowth 1
Gene P
CTCF
CTCFBSDB: a CTCF binding site database for characterization of vertebrate genomic insulators
Bao L et al. Nucleic Acids Research 2008
51
Pten Canonical Signaling 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.Porteous et al. 2009 Neuron
52
Methylation Array Results Summary• Over 6000 genes with significant differences
in their promoters
• More than ½ of the molecules involved Pten Signaling affected
• Not a random sample– Enriched for relevant functions
• Many CTCF binding sites in important neurodevelopmental genes showed differences in methylationDNA Methylation
PND 70
Whole Brain ncRNA Expression Array Analysis
53
Gene Expression
PND 70
microRNA
54
miRNA Array Heatmapsp < 0.05FC 1.2
Treated
Treated
Treated
Treated
Control Control
Control Control
Laufer et al. Disease Models & Mechanisms. 2013
55
ncRNA Venn Diagram
p < 0.05FC 1.2
1
Laufer et al. Disease Models & Mechanisms. 2013
56
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)
57
III5htr2cPre-mRNA
I II 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)
• Showed significant up-regulation in:• all 4 treatment paradigms
• CPD and injections• both array types
• miRNA and gene
Laufer et al. Disease Models & Mechanisms. 2013
58
PND 70
microRNA
miRNA and Gene Expression Results Summary
• 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
Whole Brain Bioinformatic Analysis
59
Gene ExpressionmicroRNADNA Methylation
PND 70
Creating a miRNA Target Filter
What types of Data can be used?
• Any Gene Expression Data!
• Most arrays and RNA-Seq technologies assay miRNAs
• Just create two separate files:
1. microRNA Expression
2. Gene Expression
Importing and formatting miRNA Expression
microRNA Analysis
Adding Gene Expression
Expression Pairing
High Quality Regulatory Relationships
Filtering
End Result!
Wasn’t that Easy?
• I really enjoyed the user interface of this program.
• It makes accessing a large annotated database of genetic information a breeze.
• Letting you get back to the biology!
+ The Power of Ad-Hoc
IPA miRNA Target Filter
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miRNA ID miRNA Fold Change Gene ID Gene Fold
ChangeConfidence of
Interaction
mir-369-5p -1.336 Pten 1.377 High
mir-25 -1.224 Pten 1.377 High
mir-495 -1.232 Pten 1.377 High
mir-152 1.208 Otx2 -1.27 High
mir-1224 1.528 Nmnat1 -1.237 Moderate
mir-431 1.366 Nmnat1 -1.237 Moderate
mir-743a 1.341 Nmnat1 -1.237 Moderate
mir-17* 1.451 Slitrk2 -1.202 High
mir-200a* 1.178 Slitrk2 -1.202 Moderate
• 34 genes identified with reverse pairwise relationships to predicted miRNAs
• 4 are highly compatible with FASD:
72
miRNA Target Filter Gene of Interest• A novel role for Pten in FASD:
– Pten is a lipid phosphatase that suppresses Akt activation.
– 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
Porteous et al. 2009 Neuron
73
Other Target Genes of Interest• Otx2:
– Expressed in the brain and 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.
Kleiber et al. Brain. Res. 2012
Summary of Results
74
Mir-369
PtenImpaired behaviour, learning and memory
Chr 12 ICR
Mir-152 Otx2 Mood Disorders
Mir-25
Mir-495
Mir-1224Mir- 743a
Nmnat1Reduced ability to protect axonsMir-431
Chr 12 ICR
Mir-25 Promoter
Mir-431 Promoter
Nmnat1 PromoterLaufer et al. Disease Models & Mechanisms. 2013
75
Proposed Molecular Cascade
Fetal EthanolExposure
Methylation ncRNA Gene Expression Endophenotypes
Take Home Message
• Don’t drink when you’re pregnant.
• There’s no safe time or safe amount.
• This knowledge should be spread by informed experts (like you!)
• These findings should be considered a public health guideline for future generations.
For more info on FASD see: http://www.nofas.org/
Resources• researchgate.net and academia.edu
• Concise Summaries of Headline Epigenetic Literature (some written by yours truly)– http://epigenie.com/
79
et al.
Questions?
About the presenter:VisitBenLaufer.com