2015 07 09__epigenetic_profiling_environmental_health_sciences_v42
TRANSCRIPT
Next Generation Epigenetic Profiling for Environmental Health Sciences
wvcrieki
Prof. Wim Van Criekinge9th July 2015Vancouver
Overview
Next Generation Epigenetic Profiling for Environmental Health Sciences
Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion
Overview
Next Generation Epigenetic Profiling for Environmental Health Sciences
Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion
Lab for Bioinformatics and computational genomics
10 “genome hackers” mostly engineers (statistics)
42 scientiststechnicians, geneticists, clinicians
>100 people hardware engineers,
mathematicians, molecular biologists
Overview
Next Generation Epigenetic Profiling for Environmental Health Sciences
Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion
Introduction Biology: Epigenetics
Definition(s)Molecular BiologyFunction in normal and disease statesEvolution perspective
Technology: Next Generation Epigenetic profiling Applications: Environmental EpigeneticsConclusion
Overview
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)
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
Epigenetic modifications of histones and DNA include:– Histone acetylation and methylation, and DNA methylation
HistoneAcetylation
HistoneMethylation
DNA Methylation
MeMe
Ac
Me
Chromatin is a Key Component of Epigenetic Mechanisms
Geneexpression
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
Geneexpression
AcAc
Ac
Ac
Ac
Ac
Ac
Ac
Ac
TFTF
Jamie A. Hackett, and M. Azim Surani Phil. Trans. R. Soc. B 2013;368:20110328
(re)programming dynamics
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 ofmRNA/proteins
Alteredchromatin structure
X X
Oncogenesis
Tumor
Source: Schuebel et al 2007
SYNE1
APC2
GPNMB
MM
P2EVL
STARD8
PTPRDCD109
LGR6
RETCHD%
RNF182
ICAM5
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
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 enzymeLoss 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
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
25Median 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 2005352(10):1036-8.Study with 207 patients
Evolutionary perspectives
Doing multiple things with one genome …
When do we find epigenetic machinery ?
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 ?
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
Overview
Next Generation Epigenetic Profiling for Environmental Health Sciences
Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion
Introduction Biology: EpigeneticsTechnology: Next Generation Epigenetic profiling
from candidate genes …… to epigenome Wide
Applications: Environmental EpigeneticsConclusion
Overview
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)
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)
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
PCRUsing methylation specific primers
PCR Product No PCR Product
Methylated Un Methylated
Step II: Amplification and Detection
Methylation Specific PCR (MSP)
# 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 GenerationEpigenetic Profiling
# 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 GenerationEpigenetic Profiling
Immobilized Methyl binding domain
MgCl2
Next Gen SequencingGA Illumina: 100 million reads
MethylCap_Seq
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
Quality evaluation of Methyl Binding Domain based kits for enrichment DNA-methylation sequencing
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
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Next_next
# 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 GenerationEpigenetic Profiling
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
25% 50% 25%
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
Dense methylated needed for transcriptional silencingAre there alleles with all three positions methylated ?
GCATCGTGACTTACGACTGATCGATGGATGCTAGCAT
unmethylated alleles
less methylationmethylated alleles
more methylation
Deep Sequencing
Overview
Next Generation Epigenetic Profiling for Environmental Health Sciences
Introduction
Biology: Epigenetics
Technology: Next Generation Epigenetic profiling
Applications: Environmental Epigenetics
Conclusion
Introduction Biology: EpigeneticsTechnology: Next Generation Epigenetic profilingApplications: Environmental Epigenetics
Nutrition: nutri(epi)genomicsCircadian rhythm, temperatureSocial behaviourSmall molecules
Conclusion
Overview
Phyto-oestrogens
Structure
α- en β-receptors
isoflavonen 8-prenylnaringinine 17-β-estradiol.
Nutri-epi-genomics
EXPOSURE DNA-EXTRACTION
METHYLATION ARRAY
DATA-ANALYSIS
Programs on soy, broccoli, green tea, chocolate … REACH ?
Nutri-epi-genomics
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
Introduction Biology: EpigeneticsTechnology: Next Generation Epigenetic profilingApplications: Environmental Epigenetics
Nutrition: nutri(epi)genomicsCircadian rhythm, temperatureSocial behaviourSmall molecules
Conclusion
Overview
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
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)
Zebra Finch (Taeniopygia guttata)
• Focus of much research• Rapid maturation• Tendency to sing and breed in captivity
• Recent publication of complete genome sequence
Attractive model for investigating:• Genome-wide DNA methylation profile in brain regions of interest• DNA methylation reconfiguration during brain development and
ontogeny
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)
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
Introduction Biology: EpigeneticsTechnology: Next Generation Epigenetic profilingApplications: Environmental Epigenetics
Nutrition: nutri(epi)genomicsCircadian rhythm, temperatureSocial behaviour (gregarisation)Small molecules
Conclusion
Overview
Introduction Biology: EpigeneticsTechnology: Next Generation Epigenetic profilingApplications: Environmental Epigenetics
Nutrition: nutri(epi)genomicsCircadian rhythm, temperatureSocial behaviourSmall molecules
Conclusion
Overview
Introduction Biology: EpigeneticsTechnology: Next Generation Epigenetic profilingApplications: Environmental EpigeneticsConclusion
Overview
Epigenetic editing !
Beyond monitoring … from cellular reprogramming to environmental reprogramming (metastatic niche)
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