Epigenetics: Impact of DNA methylation

transcript

Max Planck Institute for Molecular Genetics

Epigenetics:

Impact of DNA methylation

Christine Steinhoff

Berlin, Germany

• Introduction and Definitions

• Historical aspects

• Overview: Impact of DNA methylation on cellular regulation

•Accepted issues

•Controversial issues

• The DNA methylation enzymes

• Interaction with „the histone epigenetics component“

• Overview: Experimental methods

• Human epigenomics projects

Outline

Introduction and DefinitionsEpigenetics

You can inherit something beyond the DNA sequence.

That‘s where the real excitement in genetics is now (2003)

Introduction and DefinitionsTwo Components of Epigenetics

DNA MethylationMost times: Addition of methyl marks leads to repression of gene activity

Histone modificationCombination of molecules attached to histones alters DNA activity in different ways

Introduction and DefinitionsTwo Components of Epigenetics

DNA Methylation• Established during embryonic development

• Stable through multiple cell divisions (cellular memory)

• Differs between tissues, developmental stages

• Normally associated with gene silencing

• Methylation state-sensitive transcription factor binding

• up to 70% of CG dinucleotides methylated in human somatic cell

Introduction and Definitions:Chemistry

Figure 1.4b Genomes 3 (© Garland Science 2007)

Introduction and DefinitionsChemistry

5‘ Methylcytosine

Introduction and DefinitionsDeamination

Introduction and DefinitionsDefinitions

5‘CpG3‘

phosphate

„CG/GC are either completely methylated or completely unmethylated“(Bird 1978)

5‘ 3‘

5‘3‘

Gardiner-Garden, M. & Frommer, M. (1987)

> 200-bp stretch DNA G+C content > 50% observed CpG/expected CpG > 0.6

Takai D, Jones PA (2002)

> 500-bp stretch DNAG+C content >= 55% observed CpG/expected CpG > 0.65

CpG Islands - Definition

• in mammalian genomes typically 300-3,000 base pairs

• mammals: in/near approximately 40% of gene promoters

• humans: 56% genes associated with CpG Islands

• mouse: 47% genes associated with CpG Islands

• normally not methylated

CpG Islands some properties

Introduction and DefinitionsWhere?

no no no

yes yes

yes yes yes yes

Historical AspectsWhat is Epigenetics

“… the branch of biology which studies the causal interactions between genes and their products, which bring the phenotype into being”

“… Epigenesis … development of individual organic form from unformed ”

“… The study of mitotically and/or meiotically heritable changes in genes function that cannot be explained by changes in DNA sequence” Current „working definition“

Epi: greek: above, over, on, upon, besides

Impact of DNA methylation on cellular regulationOverview

Regulatory Involvement of DNA methylation

• Repeat elements• Imprinted Genes• X Chromosome Inactivation• „Diseases“

DNA methylation specific patterns

• Tissue• Development• Cancer

Impact of DNA methylation on cellular regulationAccepted Issues: Repeat elements

Genomes 3 (© Garland Science 2007)

LINE 1 Element

Methylation of LINE-1 promoter

• densely methylated in normal somatic tissues

• contained in inactive chromatin

• reactivation by hypomethylation (cancer, autoimmune diseases)

• demethylated -> chromosomal instability

Impact of DNA methylation on cellular regulationAccepted Issues: Imprinting

• Genes that are expressed in a parent-of-origin specific manner

• in diploid cells

• demonstrated in insects, mammals and flowering plants

• Not necessarily imprinted in all developmental stages

• Not necessarily imprinted in all tissues.

• in human and mouse probably 100-600 genes

• in mammals so far all genes DNA methylation involvement

Impact of DNA methylation on cellular regulationAccepted Issues: Imprinting

Impact of DNA methylation on cellular regulationAccepted Issues: X Chromosome Inactivation

Kinetics of random X chromosome inactivation

Impact of DNA methylation on cellular regulationAccepted Issues: Diseases

Example: RETT Syndrome

• Neurodevelopmental disorder

• Initial development normal

• Autism

• Learning disability

• X linked mental retardation

• Unusual stereotyped hand movements

Example:

Prader Willi /Angelman Syndrome

• 1/10,000 births

• developmental delay

• PWS: obsessive compulsive behavior

• AS: „happy disposition“

• 70% deletions: 15q11-q13

Impact of DNA methylation on cellular regulationControversial Issues: Tissue Specificity

Impact of DNA methylation on cellular regulationControversial Issues: Developmental Specificity

Impact of DNA methylation on cellular regulationControversial Issues

Arguments against regulatory impact of DNA methylation

• CpG Islands in expressing and nonexpressing tissues are unmethylated

• Demethylation can be interpretated as consequence of gene activation

• Loss of methylation as consequence of TF binding

• Developmental and tissue specific genes with methylation profile are not CpG

Island genes and thus few (random?) CpG sites

• Knock out defects are „too late“

• Microarray experiments only identify few genes with high fold changes

• conserved developmental processes also in non-methylation organisms

Impact of DNA methylation on cellular regulationControversial Issues

a) Methylatedb) Non expressing in some tissue

Transfer to tissue, where it is normally expressed

Arguments against regulatory impact of DNA methylationProposed experiment

The enzymesMammals

DNMT 1

DNMT 2

DNMT 3a

DNMT 3b

DNMT 3L

DNMT=DNA methyltransferase

The enzymes

The enzymesDNMT1 = Maintenance methylation

• Preferred substrate: hemimethylated DNA

• Inactivation leads to genomewide loss of

CpG methylation

• Lethal E8.5

• X chromosome activation

• Activation of silent retrotransposons

• Abnormal imprinting expression

The enzymesDNMT3a = De novo methylation

• no preference for hemimethylated DNA

• Postnatal lethal 4-8 weeks

• Male sterility

• Methylation imprint fails in germ cells

The enzymesDNMT3b = De novo methylation

• no preference for hemimethylated DNA

• ko: Demethylation of satellite DNA

• Lethal at E14.5

• Vascular and liver defects

The enzymesDNMT3L = De novo methylation

• Establishment of imprinting patterns

The enzymesDNMT2 = weak DNA methylation activity

• The most strongly conserved, most widely

distributed

• no change in CpG methylation

• no obvious developmental phenotypes

• unusual case: DNMT by sequence and

structure, BUT no evidence of such a function in

genetic and biochemical tests lacking

• methylates small RNAs (tRNA)

Interaction with histones

Meissner et al 2008

Interaction with histones

Experimental methodsBisulfite sequencing

PCRSequencing

Experimental methodsHighthroughput

Weber et al, 2005, 2007 Nature meDIP

Sample + EcoR1

MspI (not meth specific)

Bisulfite treatment

Small DNA Pieces

C->U, Cm->C

C‘CGG cut

Fill in ends and extend

sequence

C C G G AAAAAAAAAAAAG G C C TTTTTTTTTTTTT

High throughput sequencing

Experimental methodsHighthroughput

Human epigenomics projects

Eckhardt et al. (2006), Rakyan et al. (2004)

HEP Project Consortium

Barski et al. (2007)National Heart, Lung and Blood Institute of the NIH

Mikkelsen et al. (2007)Broad Institute of MIT and Harvard

HEROIC Project Consortium (2005)HEROIC Project Consortium

ENCODE Project Consortium (2007, 2004)

ENCODE Project Consortium

Alliance for Human Epigenomics and Disease (2007); Jones and Martienssen (2005)

AHEAD Task Force

ReferenceInitiator

10/2007

Eckhardt et al. (2006), Rakyan et al. (2004)

HEP Project Consortium

ReferenceInitiator

• Differential methylation ~ evolutionary conservation

• 17% differentially methylated in 5‘UTR:

only 1/3 anticorrelation with transcription

• CpG density inversely correlated with methylation

ENCODE Project Consortium (2007, 2004)

ENCODE Project Consortium

ReferenceInitiator

Thanks

Thank you for your attention

Introduction and DefinitionsWhere?

Lecture: Introduction to DNA methylation and its impact on gene regulation V

Evolution CpG -> TpGBisulfite Sequencing

Spontaneous deamination followed

by substitution

Historical AspectsWhat is Epigenetics, Conrad Waddington

The enzymes

Epigenetics: Impact of DNA methylation

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