1
Welcome to The Post-Genomics Era
• We’ve sequenced the human genome
• It can now be done for only a few thousand
dollars!
...
Is this it?
• Shouldn’t cracking the code of life give us
more breakthroughs than we can dream of?
• Can 4 base pairs real...
What treasure is left?
www.biocomicals.com, Alper Uzun, PhD.
“A mitotically (or meiotically)
inheritable change in gene
expression, independent of an
alteration in DNA sequence”
– Ber...
A Few Epigenetic Mechanisms of Interest
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
6
Images from Wikime...
Redefining the Central Dogma
Saletore et al. Genome Biology 2012, 13:175
Epigenetic Mechanisms
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
8
The Histone Code
http://eukaryoticgeneexpression.weebly.com/uploads/5/6/3/0/5630004/7502951_orig.jpg
Epigenetic Mechanisms
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
10
microRNAs
• The second level of the epigenetic landscape
– Acts at translation, as opposed to Histone
modifications and DN...
microRNA (miRNA)
12
http://www.wormbook.org/chapters/www_microRNA/celmicRfig5.jpg
Epigenetic Mechanisms
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
13
DNA Methylation (and other
modifications)
• Histones aren’t the only ones to enjoy
modifications
• There aren’t just four ...
DNA Cytosine Methylation
15
. Metivier, R. et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature ...
Key Players of the Epigenetic Landscape
• Histone Modifications
• MicroRNA
• DNA Cytosine Methylation
16
The Epigenetic Landscape
http://cnx.org/content/m26565/latest/graphics35.jpg
It really is a landscape…
19
Waddington’s Epigenetic Landscape
Waddington, Conrad H. 1953. The Epigenetics of birds. Cambridge University Press
• A ...
Environmental Conditions
20
Environmental Epigenetics
21
Environmentally Responsive Genome
22http://learn.genetics.utah.edu/content/epigenetics/nutrition/images/pathway.jpg
23
Waddington’s Epigenetic Landscape
Waddington, Conrad H. 1953. The Epigenetics of birds. Cambridge University Press
• Wh...
Fetal Alcohol Exposure
24
• Leading cause of preventable birth defects and mental deficits in
North America
• FASD
– Fetal...
Spectrum Disorders
Fetal Alcohol Spectrum Disorders
Fetal Alcohol Spectrum Disorders
Epigenetics
Where does an individual land in the
spectrum?
• Depends on:
– Genetic Background
– Timing of Exposure
– Dosage of Exposur...
28
Spectrum Meets Landscape
Alcohol-Related
Neurodevelopmental Disorder
(ARND)
Mouse Models
• Reasons for use:
– Useful for studies that would be impractical in humans
– Reach sexual maturity early (6–...
Fetal Alcohol Exposure in an Animal Model
• We have shown that Fetal Alcohol Exposure (FAE)
affects behaviour, learning an...
Epigenetic Mechanisms of Interest
• DNA Cytosine
Methylation
– Typically turns
expression on or
off
31
Metivier, et al 200...
Fetal Alcohol Exposure and Methylation
• FAE alters the methylation and expression of
genomically imprinted (uni-parental)...
Imprinted ncRNA
• Key role in neurodevelopment and
memory.
• Important for early life processes
– and functionally importa...
Fetal Alcohol Exposure and miRNAs
• miRNAs have been shown to be deregulated
by FAE in fetal mouse brain cell culture
• Co...
Functional Mechanisms
• An alteration of methylation in a transcription factor
binding site has the potential to affect ge...
Hypothesis
Alterations in DNA methylation and ncRNA
expression are associated with life-long
alterations in gene expressio...
Continuous Preference Drinking (CPD)
• Free choice
• Quantity monitored daily
• No Stress
• 70% preference for 10% EtOH
• ...
Experimental Design
• Everything done downstream of your workflow is
dependent on what has happened upstream.
• Errors wil...
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...
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 ...
Systems Biology
• The antithesis to a reductionist approach
• Reductionism is a philosophy that the
understanding of a com...
Independent Component Analysis
• Ingenuity Core Analysis
• A 1.2 fold increase in many genes of a pathway can
have a poten...
Enriched Biological Functions
45
Molecular and Cellular Functions
Name p-value # Genes
Cell Death 4.06E-04 - 4.97E-02 224
...
Using MeDIP and ChIP Data
• Super Simple Stuff
• Excel table with column 1 containing gene
name from upstream analysis
– S...
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 ha...
49
Top Affected IPA Network
“Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65)
40% of hub-genes...
GeneP
Gene Name Protein Name
H19 N/A (ncRNA)
Gtl2 (Meg3) N/A (ncRNA)
Npy Pro-neuropeptide Y
Akt1 RAC-alpha serine/threonin...
Pten Canonical Signaling Pathway
• Significantly affected canonical pathway
(p=1.9E-06)
• 54/95 molecules showed significa...
Methylation Array Results Summary
• Over 6000 genes with significant
differences in their promoters
• More than ½ of the m...
Whole Brain ncRNA Expression Array
Analysis
53
Gene Expression
PND 70
microRNA
miRNA Array Heatmaps
54
p < 0.05
FC 1.2
Treated
Treated
Treated
Treated
Control Control
Control Control
Laufer et al. Dise...
ncRNA Venn Diagram
55
p < 0.05
FC 1.2
1
Laufer et al. Disease Models & Mechanisms. 2013
Imprinted Noncoding RNA Clusters
• Localized to the brain
• Only 3 clusters in mouse genome
– Sfmbt2 (Chr 2), Snrpn-Ube3a ...
Cluster of Interest: Snrpn-Ube3a (Chr 7)
57
III5htr2c
Pre-mRNA
I II III IV Va Vb VI
Receptor with a stronger
serotonin res...
PND 70
microRNA
miRNA and Gene Expression Results
Summary
• Global Expression changes
• Individual miRNAs unique to
treatm...
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
techn...
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
o...
+ The Power of Ad-Hoc
IPA miRNA Target Filter
71
miRNA ID
miRNA Fold
Change
Gene ID
Gene Fold
Change
Confidence of
Interaction
mir-369-5p -1.336...
miRNA Target Filter Gene of Interest
• A novel role for Pten in FASD:
– Pten is a lipid phosphatase that suppresses Akt
ac...
Other Target Genes of Interest
• Otx2:
– Expressed in the brain and involved in mood disorders
– Identified in our previou...
Summary of Results
7
Mir-369
Pten
Impaired
behaviour,
learning and
memory
Chr 12 ICR
Mir-152 Otx2 Mood Disorders
Mir-25
Mi...
Proposed Molecular Cascade
75
Fetal Ethanol
Exposure
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 sp...
Resources
• researchgate.net and academia.edu
• Concise Summaries of Headline Epigenetic
Literature (some written by yours truly)
– http://epigenie.com/
79
et al.
About the presenter:
Visit
BenLaufer.com
Long-lasting alterations to DNA methylation and ncRNAs could underlie the effects of fetal alcohol exposure in mice
Long-lasting alterations to DNA methylation and ncRNAs could underlie the effects of fetal alcohol exposure in mice
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Long-lasting alterations to DNA methylation and ncRNAs could underlie the effects of fetal alcohol exposure in mice

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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.

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  • Insert Reference
  • Different Modifications
  • Different Modifications
  • A huge catalogue of histone modifications (100s) has been described But a functional understanding of most is still lacking. Collectively, it is thought that histone modifications may underlie a histone codeWhereby combinations of histone modifications have specific meanings.However, most functional data concerns individual prominent histone modifications that are biochemically amenable to detailed study.
  • Different Modifications
  • Different Modifications
  • Dynamic
  • Different Modifications
  • Today, the term refers to the combined epigenetic modifications of a given domain of DNA sequence. The specific combination of epigenetic modifications determines the conformation of the chromatin fibre into which the DNA and histones are packaged, and can thereby regulate the transcriptional potential of the underlying genes.
  • Highlight that this is our labs work
  • Box
  • Reduce
  • 120 - 180 mg/dl per dayBeer bottle and water bottle
  • Make numbers stick outPrecentage?
  • Cite IPAHighlight hubsHow it was done
  • Out of place
  • Fix cs and tsCut out other trimestersMention data analysis specifics
  • Emphasize MBII52ConsequencesMake it clear whats mine and what’s the literature
  • I’d like to thank my colleagues and the following institutions for their support. The work presented wouldn’t have been possible without them.
  • Long-lasting alterations to DNA methylation and ncRNAs could underlie the effects of fetal alcohol exposure in mice

    1. 1. 1
    2. 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. 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. 4. What treasure is left? www.biocomicals.com, Alper Uzun, PhD.
    5. 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. 6. A Few Epigenetic Mechanisms of Interest • Histone Modifications • MicroRNA • DNA Cytosine Methylation 6 Images from Wikimedia Commons
    7. 7. Redefining the Central Dogma Saletore et al. Genome Biology 2012, 13:175
    8. 8. Epigenetic Mechanisms • Histone Modifications • MicroRNA • DNA Cytosine Methylation 8
    9. 9. The Histone Code http://eukaryoticgeneexpression.weebly.com/uploads/5/6/3/0/5630004/7502951_orig.jpg
    10. 10. Epigenetic Mechanisms • Histone Modifications • MicroRNA • DNA Cytosine Methylation 10
    11. 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. 12. microRNA (miRNA) 12 http://www.wormbook.org/chapters/www_microRNA/celmicRfig5.jpg
    13. 13. Epigenetic Mechanisms • Histone Modifications • MicroRNA • DNA Cytosine Methylation 13
    14. 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. 15. DNA Cytosine Methylation 15 . Metivier, R. et al. Cyclical DNA methylation of a transcriptionally active promoter. Nature 452, 45–50 (2008).
    16. 16. Key Players of the Epigenetic Landscape • Histone Modifications • MicroRNA • DNA Cytosine Methylation 16
    17. 17. The Epigenetic Landscape http://cnx.org/content/m26565/latest/graphics35.jpg
    18. 18. It really is a landscape…
    19. 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. 20. Environmental Conditions 20
    21. 21. Environmental Epigenetics 21
    22. 22. Environmentally Responsive Genome 22http://learn.genetics.utah.edu/content/epigenetics/nutrition/images/pathway.jpg
    23. 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. 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. 25. Spectrum Disorders
    26. 26. Fetal Alcohol Spectrum Disorders Fetal Alcohol Spectrum Disorders Epigenetics
    27. 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. 28 Spectrum Meets Landscape Alcohol-Related Neurodevelopmental Disorder (ARND)
    29. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 39. Whole Brain Methylation Array Analysis 39DNA Methylation PND 70
    40. 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. 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. 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. 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.
    44. 44. 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
    45. 45. 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)
    46. 46. 47 Top Affected IPA Network “Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65)
    47. 47. 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.
    48. 48. 49 Top Affected IPA Network “Behaviour, Neurological Disease, and Psychological Disorders” (IPA Score 65) 40% of hub-genes promoters investigated had CTCF binding sites
    49. 49. 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
    50. 50. 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
    51. 51. 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
    52. 52. Whole Brain ncRNA Expression Array Analysis 53 Gene Expression PND 70 microRNA
    53. 53. 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
    54. 54. ncRNA Venn Diagram 55 p < 0.05 FC 1.2 1 Laufer et al. Disease Models & Mechanisms. 2013
    55. 55. 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
    56. 56. 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
    57. 57. 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
    58. 58. Whole Brain Bioinformatic Analysis 59 Gene ExpressionmicroRNADNA Methylation PND 70
    59. 59. 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
    60. 60. Importing and formatting miRNA Expression
    61. 61. microRNA Analysis
    62. 62. Adding Gene Expression
    63. 63. Expression Pairing
    64. 64. High Quality Regulatory Relationships
    65. 65. Filtering
    66. 66. End Result!
    67. 67. 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!
    68. 68. + The Power of Ad-Hoc
    69. 69. 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:
    70. 70. 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
    71. 71. 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
    72. 72. 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
    73. 73. Proposed Molecular Cascade 75 Fetal Ethanol Exposure Methylation ncRNA Gene Expression Endophenotypes
    74. 74. 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/
    75. 75. Resources • researchgate.net and academia.edu
    76. 76. • Concise Summaries of Headline Epigenetic Literature (some written by yours truly) – http://epigenie.com/
    77. 77. 79 et al.
    78. 78. About the presenter: Visit BenLaufer.com

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