Major points:

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• Major points
• 1. Maintenance of chromatin architecture remains
  obscure
• 2. Interplay of epigenetic regulators (major
  players-Box 1, large protein complexes (Fig1)
• 3. Pivotal role in mammalian development (KO,
  Table I)
• 4. Paternal/maternal genomes (alleles) are
  differentially expressed due to imprinting
  (several dozen genes)
• 5. Major role for DNMT3Lneural tube deformaties
  in embryos of DNMT3L-/- moms
• 6. DNMT3L acts via DNMT3s (oocyte development
  Fig 2) distinct steps in spermatogenesis
• 7. Embryogenesis rapid methylation changes (Fig.
  3)most but not all sites demethylated
• 8. KO studiesembryonic lethal at different
  stages
• 9. Maintenance vs de novo patterns DNMT1 vs 3
  Dnmt1 needed for monoallelic expression of
  imprinted genes
• 10. Regulating X inactivationdosage compensation
• 11. Reprogamming the somatic nucleus nuclear
  cloning (Fig. 5) improper reprogramming of
  cloned embryos? Aberrant expression of imprinted
  genes (Oc4).
• Questions
• How can epigenetics help explain current problems
  in nuclear cloning?
• Give an example of each of these (structure,
  enzyme, etc.) DNA cytosine methylation histone
  modification ATP-dependent chromatin remodeling
• What do the chromatin remodeling enzymes do?

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• Major points
• Molecular biomarkers, DNA methylation
  markersspecific, sensitive, high information
  content, different clinical samples
• Good for scarce samples
• MSP, fluorogenic probes
• Applications to serum, plasma, urine, sputum,
  lavage
• Needed new developmentsearly detection
• Questions
• Why is methylation analysis less vulnerable to
  sample treatment artifacts?

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Looking for a signal Vs LOH, expression
changes DNA info is stable Methylation is
persistent Insensitivity of methylation analysis
to artifacts
Why is comethylation of all CpG sites needed in
MSP?
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Does serum from normal patients have circulating
DNA? What is the relationship between plasma,
serum and blood? How does Laird define
specificity and sensitivity for a biomarker?
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What is upper band in third lane?
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Questions How to identify DNA methylation
markers of greatest clinical use among millions
of CpG dinucleotides? How can methylation
markers be helpful in the use of
therapeutics? How can tumor-specific methylation
markers in serum be of prognostic value?
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• Major points
• (Summary slide) Epigenetics, AdoMet, DNMTs, CpG
  islands, DNA methylation as therapeutic target,
  molecular diagnostics
• Detection of DNA methylation
• a. Fig. 1 tissue source generates complexity
  methylation sensitive restriction enzymes
• b. bisulfite (Fig 2) primer design in silico
  bisulfite conversion
• 3. Marker discovery different stages of cancer
  compared to normal (rarely done) identical
  sample variability
• 4. Candidate gene approach vs systematic
  screening using genome wide scanning methods
  (Table 1a, content/levels Table 1b,
  patterns/profiles)
• 5. MSP-based CpG island hypermethylation/candidate
  genes in cancer (Table 3/4).
• 6. CIMP CpG island methylator phenotype far end
  of the spectrum or a distinct grouping?

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• Major points
• Immunocapturing microarray chromosome
  resolution of 80 kb island methylation
• Questions

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What is meant by the statement that only 3.9 of
all CpGs in the human genome reside at the sites
recognized by the frequently used enzyme
HpaII? How does IP work?
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How does the polymorphic SacI site allow one to
distinguish maternal and paternal alleles (d)?
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MeDIP and input genomic differentially
labeledQhow does this work?????? Submegabase
resolution tiling array 34K overlapping BAC
clones/1.5 fold human genome coverage Most
non-repetitive sequences
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