Epigenetics~from mechanism to therapy~
Literature seminar
January 31, 2012
Soichi Ito (B4)1

Contents• Introduction• Central dogma• What is the Epigenetics?

• Topics
1.
2.
3.
4.
5.

DNA methylation
Histone modification
Epigenetic modifications and disease
Epigenetic therapy of cancer
Perspective2

Introduction
Central Dogma
Replication: DNA polymarase

DNA
Transcription: RNA polymerase

RNATranslation: Ribosome

Protein3

Chromatin structure

Stephen B. Baylin et al.
Nature Reviews Cancer , 2011,11, 726-734
4What is the epigenetics?

Waddingtonian epigenetics(1940s～) was located at the junction of genetics, developmental
Biology, and ecology, all of which were rooted in evolutionary biology.

Holliday’s definition“The study of the changes in gene expression, which occur in organisms with differentiated cells,
and the mitotic inheritance of given patterns of gene expression.”
5“Nuclear inheritance which is not based on differences in DNA sequence.”Epigenetic modification• DNA modifications• Histone modifications• Nucleosome positioning

regulate

・Gene and microRNA expression
・DNA-protein interactions
・suppression of transposable element mobility
・cellular differentiation
・embryogenesis
・X-chromosome inactivation
・genomic imprintingetc.

6Topics
1. DNA methylation and demethylation
2. Histone modification
3. Epigenetic modifications and disease
4. Epigenetic therapy of cancer
5. Perspective

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DNA methylation: What is methylated?

the fifth basethe sixth base

The canonical DNA nucleosides : dA, dG, dT, dC
Cytosine can be modified to mC and hmC in mammalian tissues.
DNMT : DNA methyltransferase
TET enzyme: ten eleven translocation enzyme

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Where is 5-methylcytosine?
••

DNA methylation mainly occurs in the context of CpG dinucleotides.
60～90% of CpG is methylated in mammals (corresponding to 3～8% of all cytosineresidues).
 The remaining unmethylated CpG dinucleotides are mostly found near genepromoters in dense clusters known as “CpG islands”.
 The definition of CpG island is “a sequence of more than 200 bases with a C+G
content of at least 50% and a ratio of observed to statistically expected CpG
frequencies of at least 0.6. (CpG dinucleotides are usually quite rare in mammaliangenomes (～1%))
 About 60% of human gene promoters are associated with CpG islands and are usuallyunmethylated in normal cells, although some of them (～6%) become methylated in atissue-specific manner during early development or in differentiated tissues.

 Unmethylated CpG islands at promoters of genes allow transcription.

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The function of DNA methylation• Methylated cytosine can block binding of transcriptional factors.
• Methylated cytosine can promote the recruitment of methyl-CpGbinding domain (MBD) proteins.
• MBD family members recruit histone-modifying and chromatinremodeling complexes to methylated sites.

• In general, methylation of CpG islands is associated with gene silencing.
*Genomic imprinting

Hypermethylation at one of the two parallel alleles leads to monoallelic expression.
A similar gene-dosage reduction is observed in X-chromosome inactivation in females.

10DNA methylation patterns

Anna Portela et al.Nature Biotechnology,2010, 28, 1057–1068.
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The types of methylation• De novo methylation
The DNA methylation pattern is largely established during early
embryogenesis, at around the time of implantation.
De novo DNMTs: DNMT3A, DNMT3B, DNMT3L(non-catalytic paralogue)*DNMT3A and DNMT3B may participate in maintenance methylaion.

• Maintenance methylation
Global DNA metylation patterns must be stably maintained during
replication to ensure that transposons remain in a silenced state and topreserve cell type identity.
Maintenance DNMT: DNMT1

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Maintenance ofDNA methylation

J. A. Law, S. E. Jacobsen, Nat. Rev. Genet. 2010, 11, 204 – 220.

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the mechanism of methylation

Drug Discovery Today, 2011, 16, 418-425.

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Summary of DNA methylation

Randy L. Jirtle , Michael K. Skinner, Nat. Rev. Genet. 2007, 8, 253 – 262.

15

DNA demethylation during development

S. C. Wu, Y. Zhang, Nat. Rev. Mol. Cell Biol. 2010, 11, 607 –620.

16

Locus-specific active demethylation
in somatic cells
Active demethylation at the brain-derived
neurotrophic factor (BDNF) promoter.

Active demethylation at nuclear receptor
Target promoters.

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DNA demethylation• Passive demethylation• Passive demethylation occurs during replication in the absence of
maintenance methyltransferase DNMT1.
• A rather slow process• Active demethylation• Active demethylation occurs through the enzymatic replacement of5meC with C.
Proposed mechanisms
 deamination of 5meC to T followed by BER(base excision repair) oxidative demethylation (adult mouse brain in vivo, mouse ES cell?) Radical SAM mechanism (zygotic paternal genome?) etc.
 A consensus has yet to be achieved.
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Passive demethylation

19

BER mechanisms for DNA demethylation

20

Oxidative demethylation

21

Radical SAM mechanism

22Topics
1. DNA methylation and demethylation

2. Histone modification
3. Epigenetic modifications and disease
4. Epigenetic therapy of cancer5. Perspective

23

Histones in chromatin

Brian D. Strahl et al. NATURE, 2000, 403,41-45.

• Core histones
An octamer consisting of H3/H4 tetramer and two H2A/H2B dimers.
147 bp of DNA wrap in 1.65 turns around it.
• Linker histone
Histone H1.
A Linker histone binds to the linker DNA(~50 bp of free DNA separating nucleosomes.)
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Histone modification(1)•The histone tails protrude out of nucleosomes and are subject to a number of posttranslational modifications.

 Acetylation (K:lys)
 Methylation (K:lys, R:arg)
 Phosphorylation (S:ser, T:thr)
 Ubiquitylation (K:lys) Sumoylation (K:lys)etc.

Acetyl groups neutralize the positive
charges on the basic histone tails.
As a result, electrostatic interactions
between the histones and the negatively
charged phosphate backbone of DNA.

Play important roles

 Transcriptional regulation
 Chromosome condensation
 DNA repair
 DNA replication
 Alternative splicing

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Histone modification(2)26Histone modification(3)• Euchromatin (actively transcribed)
 High levels of acetylation (K in H2A, H2B, H3, H4)
 High levels of trimethylated H3K4, H3K36 and H3K79• Heterochromatin (transcriptionally inactive) Low levels of acetylation
 High levels of H3K9, H3K27 and H4K20 methylation

Actively transcribed genes
In the promoter
 High levels of H3K4me3, H3K27ac, H2BK5ac and H4K20me1
Along the gene body High levels of H3K79me1, H4K20me1
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Histone methylation(A) Lysine is methylated by HKMTs(B) Type I and II PRMTs methylate arginines and generate MMA, ADMA, and SDMA.
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Histone demethylation(C) LSD demethylates lysine via an amine oxidation reaction using FAD as a cofactor.

(D) JMJD enzymes use aKGand Fe(II) as cofactors to demethylate the methylated lysines.
Keqin Kathy Li et al. Medicinal Research Reviews , 2010, DOI: 10.1002/med.20228

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Histone acetylation (by HATs)

Brian C. Smith et al. B. B. A, 2009, 1789, 45-47.

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Histone deacetylation (by class I/II/IV HDACs)

Ruibo Wu et al. J. Am. Chem. Soc., 2010, 132 (27), pp 9471–9479.

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Histone deacetylation (by class Ⅲ HDACs)

Brian C. Smith et al. B.B.A. 2009, 1789, 45-47
32‘histone code’ hypothesis‘Distinct histone modifications, on one or more tails, act sequentially or in
combination to form a ‘histone code’ that is read by other proteins tobring about distinct downstream events’A single histone mark does not determine outcome alone.
It is the combination of all marks in a nucleosome or region
that specifies outcome.
ExampleThe H3K4me3 active mark is found together with the H3K27me3
repressive mark at promoters of developmentally important genes withinthe ‘bivalent domains’ in ES cells. Bivalent domains enable ES cells totightly regulate and rapidly activate gene expression during different
developmental processes, but lost with cell commitment.
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Establishment of bimodal methylation
DNMT3L binds to chromatin by recognizing
H3K4, and recruit DNMT3.

If H3K4 is methylated, DNMT3L cannot bindand the underlying DNA region is protectedfrom de novo methylation.

This may be one of the mechanisms used to generate a bimodal methylation patterncharacterized by methylation over most of the genome, but not at CpG islands.
Howard Cedar et al. Nat. Rev. Gen. 2009, 10, 295-304.

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Turning off pluripotency genes

G9a: histone methyltransferase
HDAC: histone deacetylase
HP1: heterochromatin protein 135

Questions of Epigenetic inheritance Can histone modifications serve as templates for autonomously
reproducing these same structures on newly incorporated
nucleosomes following replication?
• It is known that the presence of methyl group in DNA affects
chromatin packaging.
 How is the DNA methylation pattern actually translated to
produce the correct histone modification profile?
Hypothetical model

H3K9me inS. pombe heterochromatin

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Nucleosome positioning• Nucleosome positioning plays an important role intranscriptional regulation.
 Nucleosomes block access of activators and transcription factors totheir sites on DNA.
 Nucleosomes inhibit elongation of the transcripts by engaged
polymerases.
 Transcriptionally active gene promoters possess a nucleosome-free
region at the 5’ and 3’ untranslated region, providing space forassembly and disassembly of the transcription machinery.

 The nucleosome remodeling machinery is influenced by
DNA methylation and has been linked with specific histonemodifications.
37Topics
1. DNA methylation and demethylation
2. Histone modification

3. Epigenetic modifications and disease
4. Epigenetic therapy of cancer
5. Perspective38

Aberrant epigenetic mark and disease• Cancer
Global changes in DNA methylation, histone modification patterns andchromatin-modifying enzyme-expression profiles• Neurodevelopmental disorders
 Rett syndrome: point mutations in the MBD protein MeCP2
 Rubinstein-Taybi syndrome: dysfunction of a HAT
 Coffin-Lowry syndrome: loss-of-function mutations in RSK2
*RSK2 promotes gene transcription through chromatin opening• Neurodegenerative and neurological diseases
Hyper- and hypomethylation of DNA, histone hypoacetylation• Autoimmune diseases
Hyper- and hypomethylation of DNA39

Epigenetic modifications in cancer

Anna Portela et al. Nature Biotechnology, 2010, 28, 1057–1068. 40

Epigenetic modifications in
Neurological disorders and Autoimmune diseases

41Topics
1. DNA methylation and demethylation
2. Histone modification
3. Epigenetic modifications and disease

4. Epigenetic therapy of cancer
5. Perspective

42

Cancer epigenetics: DNA methylation• Hypermethylation at the CpG islands of certain promoters→transcriptional inactivation of genes involved in the main cellular pathway
DNA repair, vitamin response, Ras signaling, cell cycle control,p53 network,apoptosis among others
DNMT1 and DNMT3B are overexpressed in many tumor types• Global hypomethylation (20-60% less overall 5meC)
 At repetitive sequences→ reactivation of endoparasitic sequeneces→chromosomal instability, translocations and gene disruption

 At specific promoters→aberrant expression of oncogenes and loss of imprinting (LOI)*LOI of IGF2 is the most common LOI event across the widest range oftumor types.
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Altered DNA-methylation patterns
in tumorigenesis

Manel Esteller Nature Reviews Genetics,2007, 8, 286-298.

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Epigenetic therapy of cancer (1)1. DNMT inhibitor

Christine B. Yoo et al.
Nat. Rev. Drug Discovery, 2006, 5, 37-50.

I.
II.
III.
IV.
V.

Inhibition of DNMTs
Inhibition of methylation of DNA
Passive demethylation
Reactivation of genes including apoptotic genes and cell-cycle
regulators
Cell death and cell-cycle arrest
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DNMT inhibitors (1)Nucleoside analogues• They have a modified cytosine ring that is attached to either ribose or
deoxyribose.
 They are metabolized by kinases and converted into nucleotides forincorporation into DNA and/or RNA.
 Ribonucleotide diphosphates can be reduced by ribonucleotide
reductase into a deoxy-diphosphate, and can then be incorporated intoDNA.
• Aniti cancer activity is mediated by
I. Cytotoxicity resulting from incorporation into the RNA and/or DNA
II. Restoring normal growth and differentiation by demethylation oftumor suppressor genes
 Cytosine analogues form a covalent complex with DNMTs.
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DNMT inhibitors (2)
Cyt

FdC

AzaC

Zeb

L. Zhou et al. J Mol Biol. 2002, 321(4), 591–599.

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DNMT inhibitors (3)•Nucleoside analogues carry a considerable concern on cytotoxicity, which isprobably associated with the drugs’ incorporation into DNA.

→Non-nucleoside DNMT inhibitors•These small-molecule inhibitors inhibit DNA methylation by binding directly to thecatalytic region of the DNMTs, without incorporation into DNA.

Hydralazine

Procainamide

EGCG

Psammaplin A

MG98: 20-bp anti-sense oligonucleotide of human DNMT1, which
prevents the translation of DNMT1 mRNA
→ Despite promising results in preclinical studies, the clinical use of
MG98 has not been validated
RG10848Cancer epigenetics: histone modification• A global loss of H4K16ac
 mediated by overexpressed or mutated HDACs in different tumor types
 Several cancer types (e.g., colon, uterus, lung and leukemia) also bear translocations
leading to the formation of aberrant fusion proteins, mutations, or deletions in HATs
and HAT-related genes.
→global imbalance of histone acetylation
 Loss of acetylation does not only result in gene silencing, but can also lead todecreased DNA repair→accelerating molecular events leading to the development of cancer• A global loss of H4K20me3→loss of the constitutive heterochromatin structure• A global loss of H3K4me3 and a gain in H3K9me andH3K27me3→transcription repression

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Epigenetic therapy of cancer (2)2. Histone deacetylase inhibitor
1.
2.
3.
4.
5.

Inhibition of histone deacetylase enzyme
Accumulation of acetylation in histones
Chromatin remodelling
Transcriptional activity
Restoration of malignant cells to a morenormal state

3. Histone methylase or demethylase inhibitor→ not ideal
The methyl marks exist as both active and inactive markers.
Histone demethylase such as LSD1 demethylates both H3K4 (active) and H3K9 (inactive).
The loss of histone acetylation seems to be the primary event leading to gene silencing,whereas the accumulation H3K9 methylation plays a secondary role.

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Histone deacetylase(classⅠ/Ⅱ/Ⅳ) inhibitors (1)• Short-chain fatty acids
 The first known HDAC inhibitors
 Generally, they are not very potent in inhibiting HDACs.
Butyrate

Valproic acid

• Hydroxamic acids
 They are potent inhibitors of HDACs.
 It is postulated that hydroxamates inhibit HDAC by binding to a zinc ion in thecatalytic domain of the enzyme, thereby inhibiting the deacetylation of histones.

m-Carboxy cinnamic
Acid bishydroxamic
Acid (CBHA)

Trichostatin A (TSA)

Suberoylanilide hydroxamicacid (SAHA)
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Histone deacetylase(classⅠ/Ⅱ/Ⅳ) inhibitors (2)• Cyclic tetrapeptides
 They are potent inhibitors of HDACs.
 Their target is the zinc ion in the HDAC catalytic domain.

Apicidin

Depsipeptide

TPX-HA analogue• Benzamides
 The benzamide group of MS275 inhibits HDACs by binding to the catalytic zinc ion.

MS-275

CI-99452Combination therapy• Reactivation of tumor-suppressor genes and/or genes that are crucial
for normal functioning cells is the target of epigenetic drugs.
 DNA methylation and histone modification represent the two most important
pathways involved in gene silencing.

→combination of DNMT inhibitor and HDAC inhibitor• Combination therapies are expected to achieve the following goals
1.
2.

To enhance or extend the molecular effects of the inhibitors
To reduce the side effects through applying lower dosages of one or both drugs.

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Topics
1. DNA methylation and demethylation
2. Histone modification
3. Epigenetic modifications and disease
4. Epigenetic therapy

5. Perspective54

Perspective (1)• Combination of epigenetic therapy and other therapeutic modalities,
such as chemotherapy, immunotherapy or radiotherapy
 The reactivation of tumor-suppressor genes and restoration of DNA-repair pathways
by epigenetic drugs results in more chemosensitive cells.

• Biomarker for cancer
 Epimutations , or aberrant DNA methylation and histone modification patterns, areobserved in individuals with no history of malignancy, and can be used as an indicatorof the likelihood of developing cancer.

• Drugs designed to target a specific region of the genome
 The lack of specificity is a disadvantage of current epigenetic drugs.
DNMTi results in demethylation of many regions of the genome.
HDACi have a very broad specificity.

• The detailed map of specific epigenetic patterns in each tissue type in
their normal and in cancerous states

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Perspective (2)• ‘Artificial de novo DNA-methylating agents’ and‘artificial histone-acetylating agents’ as drugs?
 Current epigenetic drugs inhibit enzymes that are responsible for DNA-methylation orhistone-deacethylation, resulting in ‘passive’ DNA-demethylation or histone-acetylation.
 However, ‘active’ DNA-demethylation and histone-acetylation of silenced genes seemto be very difficult because the chromatins of the silenced regions are highly compacted.

 Sequence-specific artificial de novo DNA-methylation and/or histonedeacetylation of aberrantly expressed oncogenes and imprinted genes
would result in silencing of the unwanted genes, and this might be aneffective cancer treatment.
56•••••••••••••••••••••••••

Reference

R. Bonasio, S. J. Tu, D. Reinberg, Science 2010, 330, 612 – 616.
P. A. Jones, D. Takai, Science 2001, 293, 1068 – 1070.
J. A. Law, S. E. Jacobsen, Nat. Rev. Genet. 2010, 11, 204 – 220.
Albert Jeltsch, Chembiochem 2002, 3, 274-293.
S. C. Wu, Y. Zhang, Nat. Rev. Mol. Cell Biol. 2010, 11, 607 –620.
A. Portela and M. Esteller, Nat. Biotechnol., 2010, 28, 1057–1068.
Yoo CB, Jones PA, Nat. Rev. Drug Discov. 2006, 5, 37-50.
Cedar, H. & Bergman, Nat. Rev. Genet. 2009, 10, 295–304.
Andreas Lennartsson, Karl Ekwall, B.B.A. 2009, 1790, 863-868.
L. Zhou et al. J Mol Biol. 2002, 321(4), 591–599.
Christine B. Yoo et al. Nat. Rev. Drug Discovery, 2006, 5, 37-50.
Manel Esteller Nat. Rev. Genet. 2007, 8, 286-298.
Brian C. Smith, John M. Denu, B.B.A. 2009, 1789, 45-47.
Ruibo Wu et al. J. Am. Chem. Soc., 2010, 132 (27), 9471–9479.
Keqin Kathy Li et al. Medicinal Research Reviews , 2010, DOI: 10.1002/med.20228
Brian D. Strahl et al. NATURE, 2000, 403,41-45.
Randy L. Jirtle , Michael K. Skinner, Nat. Rev. Genet. 2007, 8, 253 – 262.
Jose´ L. Medina-Franco and Thomas Caulfield, Drug Discovery Today, 2011, 16, 418-425.
Stephen B. Baylin and Peter A. Jones, Nature Reviews Cancer , 2011, 11, 726-734.
EVA JACOBLONKA AND MARION J. LAMB, Ann. N.Y. Acad. Sci. 2002, 981, 82-96.
T. Pfaffeneder et al. Angew. Chem. Int. Ed. 2011, 50, 7008-7012.
Juan Ren et al. Cellular Signalling, 2011, 23, 1082-1093.
Munzel, M. et al. Angew. Chem. Int. Ed. 2011, 50, 6450-6468.
Keith D. Robertson, Nat. Rev. Genet. 2005, 6, 597-610.
Junjie U. Guo et al. Cell 2011, 145, 423-434.

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