Epigenetics:

1
Epigenetics From Phenomenon to Field and
Impact on Plastic Surgery Research
Basic Science Seminar Series University of
Michigan Department of Surgery Section of Plastic
Surgery Brent M. Egeland, MD October 13, 2007
2
Outline

• Importance
• History
• Definitions
• Chemistry
• Laboratory Techniques
• Implications in Plastic Surgery

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Why?
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Definition Epigenetics

• Waddington 1942
• Originally a combination of the words "genetics"
  and "epigenesis"
• Epigenesis differentiation of cells from a
  totipotent state in embryonic development (used
  in contrast to "preformationism").

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Definition Epigenetics

• 69th CSH Symposia (2004)
• Conference Dedicated to Epigenetics
• No definition could be agreed upon
• An epigenetic phenomenon is a switch-like change
  in phenotype that is heritable, but does not
  involve DNA mutation. C. David Allis

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Why Epigenetics?

• Post-Genomic Era
• ENCODE
• Genomics and proteomics is not enough
• Contol of above beyond promoters and regulators
  is of utmost importance in
• Oncogenesis
• Stem Cell Biology
• Genetic therapy
• Aging

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History

• Coalesced into field of study that affects all
  areas of biology
• Collection of apparently disparate phenomena
• Over 1000 manuscripts on topic
• First observations dates to 1941
• HJ Mueller eversporting displacement
• McClintock noted that these positional effects
  were the basis of mutible loci

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History

• X-Chromosome Inactivation
• Pre-32 cell stage
• mosaic cell population

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History

• 1980's Molecular Biology and Genetics
• 1990's Genomics
• 2003 Human Genome Complete
• 2004 ENCODE
• 2005 NCI announce
• Human Epigenome Project

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Definition Epigenome
Sum of genome-wide epigenetic patterns,
distinguishes and defines one tissue from
another, stem cells from somatic cells, and aged
from young cells.
National Cancer Institute (NCI), Division of
Cancer Biology (DCB)? Workshop on Defining the
Epigenome Addressing the Value and Scope of a
Human Epigenome Project November 2829,
2005 Andrew P. Feinberg and Peter A. Jones
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Chromatin

• Euchromatin
• Uncompacted
• Active
• Heterochromatin
• Compacted
• Untranscribed
• Changes with cell cycle

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30nm Fiber
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Beads on a String

• H1 Linker histone contacts the exit/entry of
  the DNA strand on the nucleosome.
• Linker DNA between Histones

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Nucleosome
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Histone
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DNA Packaging

• Varys greatly with Cell Cycle
• DNA never completely naked

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Scaffold or Dynamic?

• Is chromatin simply a scaffold for packaging DNA?

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Dynamic

• Histones are Modified
• DNA is Modified
• Heritable transcription states

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Histone Modifications
K
K
Acetylation Transcriptional Competence
Methylation Transcriptionally Silenced
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DNA Cellular Differentiation

• All cells inherit the same DNA sequences
• Cellular differentiation processes rely strongly
  on epigenetic rather than genetic inheritance.

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DNA Modification

• Chemical modification of DNA
• Can be inherited without sequence change
• Part of the epigenetic code
• Methylation is most characterized epigenetic
  mechanism
• Commonly occurs at junk DNA

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DNA Methylation
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DNA Methylation
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CpG Islands
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Laboratory Techniques

• Chromatin Immunoprecipitation
• Inhibition of Deacetylases (HDAC-I)?
• Site-specific analysis of histone methylation
• Multigenerational assesment of above
• Methylation-specific PCR in-situ Hybridization
• Q-PCR in Combination with ChIP

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Consequences

• Genome is static
• Genotype
• Germ Cell Inheritance
• Epigenome is dynamic
• Phenotype
• Somatic epigenetic inheritance
• Cellular "memory"
• Most terminally (irreversibly) differentiate
• Morphogens activate/silence genes DNA is not
  altered

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Consequences

• Mammals cannot respond to loss of some tissues
• Inability to regenerate limbs
• Plant cells do not terminally differentiate

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Evolution

• Short-term adaptation
• allowing for reversible phenotype
• Allows multigenerational revisible phenotypic
  switch to express and repress that particular
  gene
• DNA message is unchanged
• Differential mutation rates associated with
  epigenetic features to control the mutation rates
  of particular genes

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Teratogens

• Exert effects on fetus by epigenetic means
• Epigenetic effects may preserve effect of
  teratogen throughout life of child
• Can a teratogen exert influence offspring?
• Diethylstilbestrol Second and possibly Third
  generation effects

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Research
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Why Do I Care?
?
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Plastic Surgery

• Fundamentally involved in clinical epigenetics
• Aging
• Oncology
• Wound Healing
• Basic Science in Plastic Surgery
• Regenerative medicine
• Stem Cell biology
• Organogenesis
• Neural plasticity

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Oncology
Clin Cancer Res 200713(13) July 1, 2007
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Aging

• Chronological or Epigenetically Defined?
• Free-Radicals
• Oxidants
• Telomeres
• methylation

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Aging
Change in phenotype with methylation of DNA.
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Werner Syndrome
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Breast Cancer
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Stem Cells
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Organogenesis
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Neuroal Plasticity

• Why does the CNS lack plasticity?
• What are the possibilitys of increasing this
  plasticity towards regenerative capacity?

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Hernia
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Male Pattern Baldness
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The list goes on....

• Are there differing methylation signatures of
  fibroblasts in normal, hypertrophic, keloid
  scarring.
• Can fibrotic tissue (fibroblasts) be reversed to
  form skin, bone, muscle?
• Is cleft lip epigenetically inhereited?

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Can we Modify Epigenetics?

• Can methylation be reversed?
• Rate decreased?
• Environment?
• Diet?
• Pharmacologically?

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Diet
Agouti Gene Yellow fur, obesity, diabetes,
tumorogenesis
Methyl Donor Diet (onions, garlic, beets, folic
acid, B12, genestein)?
No Supplements
.
46
HCC Histone deacetylase inhibitors (HDAC-I)
Demethylating agents
Journal of Clinical Oncology, 2006 ASCO Annual
Meeting Proceedings Part I. Vol 24, No. 18S (June
20 Supplement), 2006 10066
47
Decitabine Azacitidine

• Mechanism of action
• Phosphorylation and direct insertion into DNA
• Blocks effects of DNA methyltransferase
• Causing hypomethylation fo DNA.
• May reverse inactivation of supressor genes
• Can lead to cytotoxicity, bone marrow suppression

5-aza-2'-deoxycytidine
aza-
Azacitidine
48
References
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Control of Senescence and Aging Entirely new
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-- Rauscher 65 (24) 11229 -- Cancer
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Age-related differences in leukemia biology and
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First Annual Iberian Congress on Anti-Aging
Medicine and Biomedical Technologies Proteome
analysis of human hair shaft from protei...Mol
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of Maternal Programming of Stress Responses ...
Altering Epigenetic Marking Later in Life --
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0168-9525 Fraga yr2007 vol23 iss8 pg413
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Nutritional Effects on Epigenetic Gene Regulation
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Molecular and Cellular Biology Transposable
Elements Targets for Early Nutritional Effects
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Jirtle 23 (15) 5293 -- Molecular and Cellular
Biology NCI Epigenome Project