2015 07 09__epigenetic_profiling_environmental_health_sciences_v42

Next Generation Epigenetic Profiling for Environmental Health Sciences

wvcrieki

Prof. Wim Van Criekinge9th July 2015Vancouver

Overview


Introduction

Biology: Epigenetics

Technology: Next Generation Epigenetic profiling

Applications: Environmental Epigenetics

Conclusion

Overview


Introduction




Conclusion

Lab for Bioinformatics and computational genomics

10 “genome hackers” mostly engineers (statistics)

42 scientiststechnicians, geneticists, clinicians

>100 people hardware engineers,

mathematicians, molecular biologists

These slides are available bioinformatics.be

Overview


Introduction




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

The histone code …

The histone code …

(re)programming the DNA methylome

Active demethylation

(5hmC+5fC)

Jamie A. Hackett, and M. Azim Surani Phil. Trans. R. Soc. B 2013;368:20110328

(re)programming dynamics

DNA Methylation differsTotipotency (ES) and Adult Cells

Epigenetic (meta)information ~ stem cells250 different cell types

Genomic Aging

Genomic Aging

Disease ?

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 ?




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 ?



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

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Transgenerational inheritence

Epigenetic memory,Soma-to-germline communication

Transgenerational inheritence

Overview


Introduction




Conclusion

Introduction Biology: EpigeneticsTechnology: Next Generation Epigenetic profiling

from candidate genes …… to epigenome Wide

Applications: Environmental EpigeneticsConclusion

Overview

Genome-wide methylation by methylation sensitive restriction enzymes

Genome-wide methylation by probes

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


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


Genome-wide methylation by sequencing

# 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


3 000 000

5

6 000

50

<50 only models

and fresh frozen


Incl. FFPE



MethylCap_Seq

DNA Sheared

Immobilized Methyl Binding Domain

Condensed Chromatin

DNA Sheared

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

MGMT = dual core

Bock et al. Nature 2012

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Bock et al, Nature, 2012

Data integrationCorrelation tracks

methylation methylation

expression expression

Corr =-1 Corr = 1

Correlation trackin GBM @ MGMT

+1

-1

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

Direction RNAseq bidirectional promoters

52

# samples

# markers

MethylCap_Seq

Deep_Seq


3 000 000

5

6 000

50

EpiHealth

<50 only models

and fresh frozen


Incl. FFPE

Methylation Specific Seq



GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

25% 50% 25%



Dense methylated needed for transcriptional silencingAre there alleles with all three positions methylated ?

GCATCGTGACTTACGACTGATCGATGGATGCTAGCAT

unmethylated alleles

less methylationmethylated alleles

more methylation

Deep Sequencing

Deep MGMTHeterogenic complexity

Data integration withDEEP Sequencing, Infinium, Reactivation, (directional) Expression …

Overview


Introduction




Conclusion

Introduction Biology: EpigeneticsTechnology: Next Generation Epigenetic profilingApplications: Environmental Epigenetics

Nutrition: nutri(epi)genomicsCircadian rhythm, temperatureSocial behaviourSmall molecules

Conclusion

Overview

Nutri-epi-genomics

Nutri-epi-genomics

Nutri-epi-genomics

Nutri-epi-genomics

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



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

Temperature stress

Temperature stress

Temperature stress


Nutrition: nutri(epi)genomicsCircadian rhythm, temperatureSocial behaviour (gregarisation)Small molecules

Conclusion

Overview

Social behaviour

Social behaviour

Social behaviour



Conclusion

Overview

Fungus

Sponge

Crinoid

Bacteria

Algae

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



Introduction Biology: EpigeneticsTechnology: Next Generation Epigenetic profilingApplications: Environmental EpigeneticsConclusion

Overview

Enviromental Epigentics

Enviromental Epigentics

Epigenetic editing !

Beyond monitoring … from cellular reprogramming to environmental reprogramming (metastatic niche)

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2015 07 09__epigenetic_profiling_environmental_health_sciences_v42