Chromatin and epigenetics: from genotype to epigenotype to complex

diseases?

Dr Richard R MeehanMRC, Human Genetics Unit

Edinburgh, Scotland

Use of Emerging Science and Technologies to Explore Epigenetic Mechanisms Underlying the Developmental Basis for Disease.

Epi-genetics and Development

How is gene activity modulated during development?

Life begins

+ zygote adult?

Totipotent Unipotent

DNA is the same in cells but transcription profiles are different.

Arbeitman MN, Furlong EE, Imam F, Johnson E, Null BH, Baker BS, Krasnow MA, Scott MP, Davis RW, White KP . Gene expression during the life cycle of Drosophila melanogaster. Science. 2002 Sep 27;297(5590):2270-5

Stern CD. Conrad H. Waddington's contributions to avian and mammalian development, 1930-1940. Int J Dev Biol. 2000;44(1):15-22.

Robertson, Alan. 1977. Conrad Hal Waddington. 8 November 1905 — 26 September 1975. Biographical Memoirs of Fellows of the Royal Society 23, 575-622.

Cambridge 1926

Embryology 1920s-1940

World War II he was involved in operational research with the Royal Air Force and became scientific advisor to the Commander in Chief of Coastal Command from 1944 to 1945.

After the war he became Professor of Animal Genetics at the University of Edinburgh.

1905-1975

Waddington, C. H. The Strategy of the Genes (Geo Allen & Unwin, London, 1957).

A depiction of the epigenetic landscape. The ball represents a cell, and the bifurcating system of valleys represents the 'chreodes' or bundles of trajectories in state space.

Muscle Liver Leg Eye

1905-1975

Inherited at each generation

Epigenetic landscapesHe anticipated the difficulties of a too reductionist approach to biology, and proposed some solutions.

•‘Epigenetics’ was coined by Waddington as an English equivalent of Entwicklungsmechanik –the branch of science that undertakes a causal analysis of embryonic development by experimental means.

•‘Entwicklungsmechanik’ = experimental embryology’

“‘Epigenetics’. The science concerned with the causal analysis of development”. It was development (epigenesis) as clarified by the description of gene action.

In modern biology, epigenetics has two closely related meanings:

•The study of the processes involved in the unfolding development of an organism. This includes phenomena such as X-chromosome inactivation in mammalian females, and gene silencing within an organism.

•The study of heritable changes in gene function that occur without a change in the sequence of nuclear DNA.

Development

Chromatin

Medawar & Medawar (1983): ‘‘Genetics proposes: epigenetics disposes.’”

How do epigenetic mechanisms operate in development?

Xenopus : 1864 - 1905

Pieter Nieuwkoop 1963.

• Keen for experimental Epigenetics in 1960s.

Centre for Systems Biology at Edinburgh is a Centre for Integrative Systems Biology (CISB)

•http://www.robinholliday.com/

• It was proposed in 1975 by Robin Holliday and John Pugh, and independently by Arthur Riggs, that 5 methyl-cytosine may have an important role in controlling gene activity. It was also proposed that the pattern of methylated cytosine could be inherited through cell divisions. In 1975 there was no direct evidence for these proposals.

• Several years later this became of central importance in the new field of epigenetics.

• Almost all this work relates to the more narrow meaning of epigenetics. A full understanding of development undoubtedly depends on the broad meaning, and that is a very important field for future research.

Epigenetic mechanisms are intimately involved in setting up and maintaining the two major forms of chromosomal structure, heterochromatin and euchromatin, and in the structural switches of facultative heterochromatin between the two.

DNA modificationHistone modification

5mC stains heterochromatin

Active Repressed

Histone modification

In molecular terms; how is chromatin organised with respect to gene expression and repression?

euchromatin heterochromatin

Chromatin and epigenetics timeline

1953

DNA double helix

1974

Nucleosome chromatin subunit

1997

Nucleosome X-ray crystal structure

Association between histone acetylation and transcription

1964

Nucleosomesinvolved in gene regulation

1988

Chromatin remodellingcomplexes

1995

1942

The term epigenetics was proposed by C.Waddington

Develop-ment of genetics

Histone code of epigenetic information proposed

1993 2001

Dnmt1 essential in mice

1992

Dnmt1 cloned

1988

MeCPs and HDACs linked

1998

Role of paternal and maternal genomes in development

1984

Chromatin and epigenetics timeline

1871

Dolly cloned

1996

DNA methylation and gene switching

1975

1951 Barbara McClintock ‘the nature of the mutable locus’

1948

DNA cytosinemethylation

Chromatin and epigenetics timeline

DNA hypomethylation linked to environmental toxins and diet

DNA hyper-methylation linked to diet and phenotypic changes

Renaissance of hypomethylation and gene activation in cancer

Polycomb proteins hypothesized to account for widespread gene silencing in cancer

Epigenotype–phenotype analysis shows gatekeeper role of LOI in a human cancer syndrome

Altered histone lysine methylation at silenced tumour suppressor loci

Clinical trials of combined 5-aza-2′-deoxycytidine and trichostatinA therapy in patients with cancer

Cloned sheep has altered DNA methylation.

2001 2002 20031998 2004

2006-7 Induced Pluripotent Stem Cells

Cancer epigenetics linked to stem-cell programmes.

Epigenomics.

Double stranded RNA is introduced into a cell and gets chopped up by the enzyme dicer to form siRNA. siRNA then binds to the RISC complex and is unwound. The anitsense RNA complexedwith RISC binds to its corresponding mRNA which is the cleaved by the enzyme slicer rendering it inactive

Non-coding RNAs (ncRNA)

A variegated petunia. Upon injection of the gene responsible for purple coloring in petunias, the flowers became variegated or white rather than deeper purple as was expected. Due to the production of double stranded RNA.

Feed back recruiting chromatin-modifying enzymes, including the de novo cytosine methyltransferase, to adjacent DNA.

DNA methylation in vertebrates.

1. DNA methylation at the 5th position of cytosine in CpG (MeCpG) is the major modification found in vertebrate genomes.

2. Catalysed by DNA cytosine methyltransferases (Dnmt1).

3. Methylation patterns are heritable. (Development)

4. DNA methylation is associated with gene repression invitro and in vivo.

SAM

Dnmt

DNA methylation:

- Generating and maintenance of methyl-CpGs(DNA methyltransferases)

Zn-finger polybromo (KG)n catalytic

I IV VI IX X

1 1,602NLS

Dnmt1

R-foci targeting

Dnmt1 - maintenance methyltransferase

Dnmt3a2 de novo methyltransferases

6891

Ｉ IV VI IX XPWWP PHD-likecatalytic

Dnmt3a2 Rep

SAM SAM

S-adenosylmethionine(SAM) is a cofactor involved in methyl group transfers

The global pattern of DNA methylation can be dynamic during development.

Hum Mol Genet. 2005 Apr 15;14 Spec No 1:R47-58. Epigenetic reprogramming in mammals. Morgan HD, Santos F , Green K, Dean W, Reik W.

DNA

5mC

Merge

Oct3/4 expMe-H3(K4)Ac-H3(K9,14)Me-H3(K9)5mC

Oct

3/4

Exp

/H3

Mod

/ DN

A m

eth

0 24 48 72 96

20

40

60

80

100

Retinoic Acid (h)

Changes in histone modification precede Oct-3/4 promoter methylation in RA treated ES cells

Nat Cell Biol. 2006 Feb;8(2):188-94.G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis. Feldman N, Gerson A, Fang J, Li E, Zhang Y , Shinkai Y , Cedar H, Bergman Y .

Zygote

Embryonal stem cell

Germ layer stem cell

Lineage stem cell

Tissue determinedstem cell

Terminal cell

TotipotentTotipotent

pleuripotentpleuripotent

multipotentmultipotent

oligopotentoligopotent

Tri or biTri or bi--potentpotent

unipotentunipotent

Developmental potential is governed by transcription competence

Epig

enet

icEp

igen

etic

[Oct4, Nanog] switched off

[Poised, lineage] specific genes on

‘Direct reprogramming’

Viral transfection

Reprogramming

Sox2 Klf4

cMyc

Oct4

Somatic cell

iPS cell

Efficiency <0.01%Time- several days-weeks

iPS = induced pluripotency cells.

Takahshi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006 Aug 25;126(4):663-76. GRN is primary?

‘The epigenetic landscape reversed’

Lineage-specific genes

Pluripotency genes

[Oct4, Nanog] switched on

In induced Pluripotency cells (iPS) and in cancer

Molecular Mechanisms for setting up and reinforcing (epigenetic memory) alternative chromatin states.

DNA methylation-modification machinery:DNA methyltransferasesMethyl-CpG Binding proteinsAssociated chromatin remodeling machinery (MeCP1/NuRD, MeCP2/Sin3a/Lsh)

Histone-modification machinery:Histone acetyltransferasesHistone deacetylasesHistone methyltransferasesHistone demethylasesHistone phosphorylasesNon-Histone proteins (HP1 etc)Associated chromatin remodeling machinery

Non-coding RNAs and heterochromatin (RNAi machinery)RNAi (inhibition) was initially defined by Andrew Fire and colleagues as a process that is triggered by double strand RNA (dsRNA) and silences the expression of genes complementary to the dsRNA in Caenorhabditis elegans.

Function:

Imprinting

X inactivation

Developmental gene expression

Germ cell specific gene expression

Repeat gene silencing

Centromericheterochromatin

Model organisms for the study ofchromatin and epigenetics

HeterochromatinPosition Effect VariegationDosage effectRemodelling complexesRNA based silencing

CentromericheterochromatinRNA based silencing

Chromatin remodellingHistone acetylation

complexesSir silencing

ImprintingX inactivationLocus controlCpG islands

DNA and histone methylation

RNA based silencing

Chromatin structure

Domains

Development

Population studies

Epidemiology

Twin studies

Assisted reproduction technologies

Potential transgenerational effects in humans resulting from the nutritional conditions experienced in previous generations (Overkalix, Sweden).

These timings suggest that information is being captured at key stages of egg and sperm formation, and is passed on to the offspring, possibly in form of epigenetic patterns, but no molecular evidence (unproven).

If there was food deprivation during the father's slow growth period (9 and 12 yrs) before reaching puberty, then cardiovascular disease mortality of the children was low. The lifespan of the grandchildren seemed to be influenced by their paternal grandfathers' access to food during the grandfather's slow growth period.

A food surplus during the paternal grandfathers slow growth period was associated with an increase in diabetes mortality in grandchildren.

Paternal grandfather's food supply was only linked to the mortality rates of grandsons, paternal grandmother's food supply (foetus and infant) was only linked to the mortality rates of granddaughters.

Kaati et al. (2002) Cardiovascular and diabetes mortality determined by nutrition during parents' and grandparents' slow growth period. Eur J Hum Genet 10, 682- 688; Pembrey et al. (2006) Sex-specific, male-line transgenerational responses in humans European Journal of Human Genetics 14, 159-166).

Thomas Jenuwein

Are stem cells ‘epigenetic’?

Dnmt1

Dnmt2

Dnmt3a

Dnmt3b

Dnmt3L

MeCP2

MBD1

MBD2

MBD3

MBD4

Kaiso

Mouse ES null cells Viable

HDAC1

G9a

Suvar39h

EZ2H

DNA methyltransferases

Methyl-CpG binding proteins

Histone modifying enzymes.

They can no longer maintain differentiation.

Dnmt1

(maintenance) (de novo)

Transcription repression.

(direct) (indirect)

Methylase

DNA replication

Apoptosis

Cell cycle checkpoint

Repair

The functions of DNMT1.

What is the range of DNMT1 mutant phenotypes?How do these properties of DNMT1 converge in

development and disease?

p53PCNA

? ?

DNA demethylation and methylation

X inactivationEffects of ART

Developmentally regulated genes

Rox, Igf2

Intrauterine conditionsNutrient supplyMaternal Diet

Epigenetic perturbation (drugs)Valproic Acid

Developmentally regulated genes

Adult globins

Childhood phenotypes - epimutationsSilver-Russell syndromeBeckwith-Wiedemann Syndrome

Environmental effects on epigenome?Somatic mutations

Germline epimutations

DNA demethylation and methylationGenomic imprinting & germ cell specific gene expression

Bla

stoc

yst

Embr

yoC

hild

Adu

ltG

amet

e

Cancer

Meiotic sex chromosome inactivation (spermatogenesis only).

Epigenetics of complex disease

Genotype + Environment Epigenotype

Phenotype

Cancer Schizophrenia and bipolar disorder

Autism

Anxiety and depression

Lupus Metabolic syndrome

Altered DNA methylation and

toxins

Low folate and maternal

malnutrition

Impaired methylation capacity

Glucocorticoidmetabolism

T-cells hypomethylated

Nutrition; Transgenerational

J. van Vliet, N. A. Oates and E. Whitelaw (2007).

Dex Low birth weight

AdultGlucose tolerance

Increased PEPCK activity

Increased hepatic GR

mRNA

Hypertension

The dexamethasone-programmed rat

Am J Physiol Regul Integr Comp Physiol. 2005 Jan;288(1):R34-8. Intergenerational consequences of fetalprogramming by in utero exposure to glucocorticoids in rats. Drake AJ, Walker BR, Seckl JR.

By the third generation, the pattern has re-set to untreated animals

Transmitted through germ cells

Environmentally induced epi-mutation Epi-mutation

The global pattern of DNA methylation can be dynamic during development.

Hum Mol Genet. 2005 Apr 15;14 Spec No 1:R47-58. Epigenetic reprogramming in mammals. Morgan HD, Santos F , Green K, Dean W, Reik W.

DNA

5mC

Merge

What is the gene expression profile?

What is the chromatin profile?

What is the DNA methylation profile?

What aspects of epigenetic regulation are conserved?

SiRNA and MiRNA profile?

In Development and in germ cells

How are patterns re-imposed at each generation? What is there sensitivity to perturbation?

PRACTICAL IMPORTANCE

Cancer

Hereditary Disease

Epigenetic Epidemiology

Epigenetic Defense Mechanisms

Remodelling & Cloning

Agriculture

Ecology

Cell-heritable patterns of DNA methylation that are often associated with the silencing of tumor-suppressor genes

Imprinting, Aging and genetic penetrance.

Maternal environment

Transpostion, virion exposure

Frequency of successful animal clones

Transgenics and “epigenetically engineered” crops.

Epigenetic modulation as a result of environmental interactions

The epi-genotype is remarkably pliable (soft genetics).

Phenotypes can be shaped by the environment throughout gestation and adulthood.

An epigenome can potentially provide a readout of an individual’s recent, and ancestral, environmental history.

Epigenetic profiling is possible.

“Epigenetic epidemiology”, is on the way.

Transgenerational epigenomics will follow.

Bjornsson HT, Fallin MD, Feinberg AP . An integrated epigenetic and genetic approach to common human disease. Trends Genet. 2004 Aug;20(8):350-8.

Conclusions

Acknowledgments

Dr. Sari Pennings

Molecular Physiology, University of Edinburgh, 47 Little France Crescent,

Edinburgh EH16 4TJ, UK

Alexey RuzovDonncha Dunican

Jamie HackettJames Reddington

Colm NestorKatie Appleby

Meng Huan

Dr Mandy Drake

Centre for Cardiovascular Science, 47 Little France

Crescent, Edinburggh EH16 4TJ.

http://www.epigenome-noe.net/index.php

Genetic Epigeneticvs

Cell lineages

Gametogenesis

Patches of cells (signalling)

Clonal (Xi)

Stable (rarely reversed)

Reversible (imprinting)

Resistant to environmental

influences (except

mutatgens).

Susceptible to environmental perturbation.

Acquired characteristics