Environmental Epigenetics

1. Next Generation Epigenetic Profiling for
Environmental Health Sciences
wvcrieki
Prof. Wim Van Criekinge
9th July 2015
Vancouver

2. Overview
Next Generation Epigenetic Profiling for Environmental Health Sciences
Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion

3. Overview
Next Generation Epigenetic Profiling for Environmental Health Sciences
Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion

5. Lab for Bioinformatics and
computational genomics
10 “genome hackers”
mostly engineers (statistics)
42 scientists
technicians, geneticists, clinicians
>100 people
hardware engineers,
mathematicians, molecular biologists

7. These slides are available
bioinformatics.be

8. Overview
Next Generation Epigenetic Profiling for Environmental Health Sciences
Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion

9. Introduction
Biology: Epigenetics
Definition(s)
Molecular Biology
Function in normal and disease states
Evolution perspective
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion
Overview

10. Defining Epigenetics
• Reversible changes in gene
expression/function
• Without changes in DNA
sequence
• Can be inherited from
precursor cells
• Allows to integrate intrinsic
with environmental signals
(including diet)

11. Chromatin is a Key Component
of Epigenetic Mechanisms
 Chromatin organizes genes to be accessible for transcription, replication, and
repair
nucleosomehistone
DNA
chromatin
Cellular DNA is packaged into a structure called
chromatin
The unit of chromatin is the nucleosome, a complex
of a histone tetramer with approx. 125 bp of DNA
wound around it

12.  Epigenetic modifications of histones and DNA include:
– Histone acetylation and methylation, and DNA methylation
Histone
Acetylation
Histone
Methylation
DNA Methylation
MeMe
Ac
Me
Chromatin is a Key Component
of Epigenetic Mechanisms

13. Gene
expression
 Gene expression (transcription) requires DNA to be physically accessible to
transcription factors (TF)
 Epigenetic changes alter the structure of the chromatin, which determines
whether DNA is accessible
– Open chromatin allows gene expression
– Closed chromatin prevents gene expression
Epigenetic Changes can Alter Chromatin
Structure and Regulate Gene Expression
Gene
expression
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
TFTF

14. The histone code …

15. The histone code …

16. (re)programming
the DNA methylome
Active
demethylation
(5hmC+5fC)

17. Jamie A. Hackett, and M. Azim Surani Phil. Trans. R. Soc. B
2013;368:20110328
(re)programming
dynamics

18. DNA Methylation differs
Totipotency (ES) and Adult Cells

19. Epigenetic
(meta)information ~ stem cells250 different cell types

20. Genomic Aging

21. Genomic Aging

22. Disease ?

23. Epigenetic Changes are
Important in Causing Cancer
Example:
Replication errors
GENETIC EPIGENETIC
Example:
Chromatin modification errors
Altered
DNA/mRNA/proteins
Altered
DNA sequence
Altered levels of
mRNA/proteins
Altered
chromatin structure
X X
Oncogenesis
Tumor

24. Source: Schuebel et al 2007
0
20
40
60
80
100
120
Methylated Mutated
76-100 51-75 21-50 1-20
Dx
CDx
Example of Methylation
vs Mutation: Colon & Breast Cancer

25. MGMT
O6 Methyl-Guanine
Methyl Transferase
Essential DNA Repair Enzyme
Removes alkyl groups from damaged
guanine bases
Healthy individual:
- MGMT is an essential DNA repair enzyme
Loss of MGMT activity makes individuals
susceptible to DNA damage and prone to
tumor development
Glioblastoma patient on alkylator chemotherapy:
- Patients with MGMT promoter methylation
show have longer PFS and OS with the use of
alkylating agents as chemotherapy

26. MGMT Promoter Methylation Predicts
Benefit from DNA-Alkylating
Chemotherapy
Post-hoc subgroup analysis of Temozolomide Clinical trial with primary glioblastoma
patients show benefit for patients with MGMT promoter methylation
0
5
10
15
20
25
Median Overall Survival
21.7 months
12.7 months
radiotherapy
plus
temozolomide
Methylated
MGMT Gene
Non-Methylated
MGMT Gene
radiotherapy
Adapted from Hegi et al.
NEJM 2005
352(10):1036-8.
Study with 207 patients

27. Evolutionary perspectives
 Doing multiple things with one genome …
 When do we find epigenetic machinery ?

28. Evolutionary perspectives

29. Evolutionary perspectives
 Doing multiple things with one genome …
 When do we find epigenetic machinery ?
– Not systematically studied but in prokayotes mostly a
method to “tag” foreign DNA, in multicellular used for
differentiation
– Can we artificially introduce it ?

30. Evolutionary perspectives
 Doing multiple things with one genome …
 When do we find epigenetic machinery ?
– Not systematically studied but in prokayotes mostly a
method to “tag” foreign DNA, in multicellular used for
differentiation
– Can we artificially introduce it ?
 What is the difference between species – thought
experiment mouse versus man

31. 31
Evolutionary perspectives

32. Transgenerational
inheritence

33. Epigenetic memory,
Soma-to-germline communication
Transgenerational
inheritence

34. Overview
Next Generation Epigenetic Profiling for Environmental Health Sciences
Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion

35. Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
from candidate genes …
… to epigenome Wide
Applications: Environmental Epigenetics
Conclusion
Overview

36. Genome-wide methylation by
methylation sensitive restriction enzymes

37. Genome-wide methylation
by probes

38. Unmethylated Cytosine is de-Aminated with Na Bisulfite
Cytosine Uracil
5-Methyl Cytosine 5-Methyl Cytosine
5’-Methyl Cytosine does not react
Methylation Specific PCR (MSP)

39. C G A C G C G C G C C G C C G A C G C G C G C C G C
C G A C G C G C G U C G U U G A U G U G U G U U G U
Na BiSulfite Treatment
Methylated Un Methylated
Step I: Bisulfite Treatment
Methylation Specific PCR (MSP)

40. C G A C G C G C G U C G U U G A U G U G U G U U G U
G C T G C G C G C A G C A G C T G C G C G C A G C A
PCR
Using methylation specific primers
PCR Product No PCR Product
Methylated Un Methylated
Step II: Amplification and Detection
Methylation Specific PCR (MSP)

41. Genome-wide methylation
by sequencing

42. # samples
# markers
Discovery Verification Validation
3 000 000
5
6 000
50
<50
only models
and fresh frozen
> 50
All sample types
Incl. FFPE
Enrichment Sequencing (RUO) Targeted Sequencing (IVD)
Next Generation
Epigenetic Profiling

43. # samples
# markers
MethylCap_Seq
Discovery Verification Validation
3 000 000
5
6 000
50
<50
only models
and fresh frozen
> 50
All sample types
Incl. FFPE
Enrichment Sequencing (RUO) Targeted Sequencing (IVD)
Next Generation
Epigenetic Profiling

44. MethylCap_Seq
DNA Sheared
Immobilized
Methyl Binding Domain
Condensed Chromatin
DNA Sheared

45. Immobilized
Methyl binding domain
MgCl2
Next Gen Sequencing
GA Illumina: 100 million reads
MethylCap_Seq

46. Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
Quality evaluation of Methyl Binding Domain based
kits for enrichment DNA-methylation sequencing

47. MGMT = dual core

48. Bock et al. Nature 2012
48
Bock et al, Nature, 2012

49. Data integration
Correlation tracks
methylation methylation
expression expression
Corr =-1 Corr = 1

50. Correlation track
in GBM @ MGMT
+1
-1

51. miRNA, (l)ncRNA, CIS/TRANS splicing, SV, fusion loci ,
bidirectional promoters ?
RNA_seq: sequence RNA molecules Next Gen Platform
Total RNA_seq: all RNA molecules (normalisation procedure)
Directional Total RNA_seq: before amplification use different
5’ and 3’ adaptors
Integrated Directional Total RNA_seq: Combine with other
datasets eg. enrichment sequencing data, visualise and query
in genome browser
51
Next_next

52. Direction RNAseq
bidirectional promoters
52

53. # samples
# markers
MethylCap_Seq
Deep_Seq
Discovery Verification Validation
3 000 000
5
6 000
50
EpiHealth
<50
only models
and fresh frozen
> 50
All sample types
Incl. FFPE
Methylation Specific Seq
Enrichment Sequencing (RUO) Targeted Sequencing (IVD)
Next Generation
Epigenetic Profiling

54. GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
25% 50% 25%
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
Dense methylated needed for transcriptional silencing
Are there alleles with all three positions methylated ?

55. GCATCGTGACTTACGACTGATCGATGGATGCTAGCAT
unmethylated alleles
less methylationmethylated alleles
more methylation
Deep Sequencing

56. Deep MGMT
Heterogenic complexity

57. Data integration with
DEEP Sequencing, Infinium, Reactivation, (directional) Expression …

58. Overview
Next Generation Epigenetic Profiling for Environmental Health Sciences
Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion

59. Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Nutrition: nutri(epi)genomics
Circadian rhythm, temperature
Social behaviour
Small molecules
Conclusion
Overview

60. Nutri-epi-genomics

61. Nutri-epi-genomics

62. Nutri-epi-genomics

63. Nutri-epi-genomics

64. Phyto-oestrogens
 Structure
 α- en β-receptors
isoflavonen 8-prenylnaringinine 17-β-estradiol.
Nutri-epi-genomics

65. EXPOSURE
DNA-
EXTRACTION
METHYLATION
ARRAY
DATA-
ANALYSIS
Programs on soy, broccoli, green tea, chocolate … REACH ?
Nutri-epi-genomics

66. EXPOSURE
DNA-
EXTRACTION
METHYLATION
ARRAY
DATA-
ANALYSIS
Programs on soy, broccoli, green tea, chocolate … REACH ?
From age as confouder to … measuring age
Nutri-epi-genomics

67. Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Nutrition: nutri(epi)genomics
Circadian rhythm, temperature
Social behaviour
Small molecules
Conclusion
Overview

68. Neuroplasticity
• Formation of new neural connections throughout
ontogeny and adult life
• Changes in brain morphology, physiology and organization
during development
• Memory formation
• Regrowth of spinal nerves after damage
• …
• Epigenetic control of brain plasticity?
• Stimulus-induced de novo CpG methylation
• or active DNA demethylation associated with brain-specific
genes
• Expression of BDNF regulated by promoter methylation
• Following fear conditioning, DNMT inhibition blocks the general
process of memory formation

69. Brain research & songbirds
• Song learning ~ human speech learning
• Behavioural parallels
• Sensory phase
> memorize vocalizations early in life by imitating adults
• Sensorimotor phase
> trial and error, develop and refine own speech/song
• Anatomical/physiological parallels
• Striking homologies between the brains of birds and mammals
• Common neuronal and molecular substrates
=> widely used model system for neuroscientists
(behavioral neurobiology of learning, memory formation, sexual
differentiation of the brain and neuroplasticity)

70. Zebra Finch (Taeniopygia guttata)
• Focus of much research
• Rapid maturation
• Tendency to sing and breed in captivity
• Recent publication of complete genome sequence
non- invasive functional brain imagi
James Lee,with G.Gratton & M.Fabiani
Attractive model for investigating:
• Genome-wide DNA methylation profile in brain regions of interest
• DNA methylation reconfiguration during brain development and
ontogeny

71. Epigenetic analysis
• Two ZF cell lines
• ZFTMA
• Female, tetraploid
• Origin: Tumor of left thigh
• G266
• Male, diploid
• Origin: Tumor-like tissue under skin of the forehead
• Two treatments
• DMSO ~ control
• AZA ~ demethylation
• Two sequencing techniques
• MethylCap-seq
• RNA-seq
• (RRBS -> quality control)

72. Pathway analysis
• Ingenuity Pathway Analysis (IPA) software (UA)
• 358 putatively epigenetic regulated genes
• i.e. upregulated expression and down-methylated promoter after
AZA-treatment
• All 1,966 genes featured by a significantly down-methylated
promoter region, independent of their expression data
• Nervous system development and function
• Clear association with neurological disease
• including a schizophrenia and
• a speech/language disorder network
• Major contributing genes in the obtained neuronal networks

73. Temperature stress

74. Temperature stress

75. Temperature stress

76. Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Nutrition: nutri(epi)genomics
Circadian rhythm, temperature
Social behaviour (gregarisation)
Small molecules
Conclusion
Overview

77. Social behaviour

78. Social behaviour

79. Social behaviour

80. Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Nutrition: nutri(epi)genomics
Circadian rhythm, temperature
Social behaviour
Small molecules
Conclusion
Overview

82. Fungus
Sponge
Crinoid
Bacteria
Algae

83. M. Schnekenburger et al.
Cancer Letters 351 (2014) 182–197

84. M. Schnekenburger et al.
Cancer Letters 351 (2014) 182–197

85. M. Schnekenburger et al.
Cancer Letters 351 (2014) 182–197

86. Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic
profiling
Applications: Environmental Epigenetics
Conclusion
Overview

87. Enviromental Epigentics

88. Enviromental Epigentics

89. Epigenetic editing !
Beyond monitoring …
from cellular reprogramming to environmental reprogramming (metastatic niche)
89

90. 90
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