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

1. 1

2. 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…

3. 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?

4. What treasure is left?
www.biocomicals.com, Alper Uzun, PhD.

5. “A mitotically (or meiotically)
inheritable change in gene
expression, independent of an
alteration in DNA sequence”
– Berger et al. (2009) Genes Dev.
5
The Solution to the Post Genomics Era:
Epigenetics
Epigenetic Mechanisms (Macmillan Publishers Ltd: Nature 441: 143-145. 11 May 2006)

6. A Few Epigenetic Mechanisms of Interest
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
6
Images from Wikimedia Commons

7. Redefining the Central Dogma
Saletore et al. Genome Biology 2012, 13:175

8. Epigenetic Mechanisms
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
8

9. The Histone Code
http://eukaryoticgeneexpression.weebly.com/uploads/5/6/3/0/5630004/7502951_orig.jpg

10. Epigenetic Mechanisms
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
10

11. 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.

12. microRNA (miRNA)
12
http://www.wormbook.org/chapters/www_microRNA/celmicRfig5.jpg

13. Epigenetic Mechanisms
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
13

14. 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

15. DNA Cytosine Methylation
15
. Metivier, R. et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 45–50 (2008).

16. Key Players of the Epigenetic Landscape
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
16

17. The Epigenetic Landscape
http://cnx.org/content/m26565/latest/graphics35.jpg

18. It really is a landscape…

19. 19
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.

20. Environmental Conditions
20

21. Environmental Epigenetics
21

22. Environmentally Responsive Genome
22http://learn.genetics.utah.edu/content/epigenetics/nutrition/images/pathway.jpg

23. 23
Waddington’s Epigenetic Landscape
Waddington, Conrad H. 1953. The Epigenetics of birds. Cambridge University Press
• What happens if an
obstacle gets in the way?

24. Fetal Alcohol Exposure
24
• 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

25. Spectrum Disorders

26. Fetal Alcohol Spectrum Disorders
Fetal Alcohol Spectrum Disorders
Epigenetics

27. 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. 28
Spectrum Meets Landscape
Alcohol-Related
Neurodevelopmental Disorder
(ARND)

29. 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

30. 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

31. 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

32. 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
33
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

34. 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?
35
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.
36

37. 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.
37
Young & Olney 2006 Neurobiol. Dis.

38. 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.

39. Whole Brain Methylation Array Analysis
39DNA Methylation
PND 70

40. 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

41. 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

42. 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 .

43. 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 Functions
45
Molecular and Cellular Functions
Name p-value # Genes
Cell Death 4.06E-04 - 4.97E-02 224
Cellular Development 6.24E-04 - 4.52E-02 166
Cellular Function and Maintenance 9.57E-04 - 4.97E-02 86
Cellular Movement 4.12E-03 - 4.52E-02 41
Cell Signaling 8.43E-03 - 4.97E-02 26
Physiological System Development and Function
Name p-value # Genes
Nervous System Development and Function 3.86E-05 - 4.97E-02 273
Tissue Morphology 1.64E-04 - 4.23E-02 97
Behavior 1.62E-03 - 1.58E-02 24
Embryonic Development 1.23E-02 - 4.23E-02 29
Organismal Development 1.23E-03 - 4.23E-02 25
• Many have been previously implicated in FASD and all are highly
compatible

46. 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. 47
Top Affected IPA Network
“Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65)

48. 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. 49
Top Affected IPA Network
“Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65)
40% of hub-genes promoters investigated had CTCF binding sites

50. GeneP
Gene Name Protein Name
H19 N/A (ncRNA)
Gtl2 (Meg3) N/A (ncRNA)
Npy Pro-neuropeptide Y
Akt1 RAC-alpha serine/threonine-protein kinase
Ghr Growth hormone receptor
Ntrk1 High affinity nerve growth factor receptor
Apoe Apolipoprotein E
Grin2c Glutamate [NMDA] receptor subunit epsilon-3
Gene
Name
Protein Name
App
Amyloid beta A4 protein
Mbp
Myelin basic protein
Atp1a2 Sodium/potassium-transporting
ATPase subunit alpha-2
Grin1 G protein-regulated inducer of
neurite outgrowth 1
GeneP
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.
51
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 methylation
52DNA Methylation
PND 70

53. Whole Brain ncRNA Expression Array
Analysis
53
Gene Expression
PND 70
microRNA

54. miRNA Array Heatmaps
54
p < 0.05
FC 1.2
Treated
Treated
Treated
Treated
Control Control
Control Control
Laufer et al. Disease Models & Mechanisms. 2013

55. ncRNA Venn Diagram
55
p < 0.05
FC 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
56

57. Cluster of Interest: Snrpn-Ube3a (Chr 7)
57
III5htr2c
Pre-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
58
Gene
Expression

59. Whole Brain Bioinformatic Analysis
59
Gene ExpressionmicroRNADNA Methylation
PND 70

60. 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

62. Importing and formatting miRNA Expression

63. microRNA Analysis

64. Adding Gene Expression

65. Expression Pairing

66. High Quality Regulatory Relationships

67. Filtering

68. End Result!

69. 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!

70. + The Power of Ad-Hoc

71. IPA miRNA Target Filter
71
miRNA ID
miRNA Fold
Change
Gene ID
Gene Fold
Change
Confidence 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
72
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.
73
Kleiber et al. Brain. Res. 2012

74. Summary of Results
7
Mir-369
Pten
Impaired
behaviour,
learning and
memory
Chr 12 ICR
Mir-152 Otx2 Mood Disorders
Mir-25
Mir-495
Mir-1224
Mir- 743a
Nmnat1
Reduced ability to
protect axonsMir-431
Chr 12 ICR
Mir-25 Promoter
Mir-431 Promoter
Nmnat1 Promoter
Laufer et al. Disease Models & Mechanisms. 2013

75. Proposed Molecular Cascade
75
Fetal Ethanol
Exposure
Methylation ncRNA Gene Expression Endophenotypes

76. 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/

77. Resources
• researchgate.net and academia.edu

78. • Concise Summaries of Headline Epigenetic
Literature (some written by yours truly)
– http://epigenie.com/

79. 79
et al.

80. About the presenter:
Visit
BenLaufer.com