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DNA Methylation

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Mice mated a/a dam X Avy/a sire while the dam is on control ... to 100 y of age) and in some inbred rodent strains (18 to 33 mo of age, scales are approximate) ... – PowerPoint PPT presentation

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Title: DNA Methylation


1
Mice mated a/a dam X Avy/a sire while the dam is
on control or 3SZM diet produce Avy/a and a/a
offspring. Avy/a offspring have different
distributions of epigenetic phenotypes in the
population due to maternal diet.
2
Methyl metabolism showing some major metabolic
intermediates, cofactors and dietary sources of
methyl groups and an intersection with
antioxidant metabolism through homocysteine,
cysteine and glutathione.
3
Survival declines over a considerable range in
adult humans (40 to 100 y of age) and in some
inbred rodent strains (18 to 33 mo of age, scales
are approximate). (Lower panel) Epigenetic traits
are probably distributed unevenly and over a
considerable range in populations.
4
Agouti gene expression in the Avy allele. TOP
When the LTR is active, it drives expression of
the agouti gene in Avy mice. Transcription is
constant, rather than cyclical (indicated by the
bold arrow) and is ectopic (i.e., in all tissues
studied). BOTTOM When the LTR is suppressed in
Avy mice, expression of the agouti gene is
regulated by its normal promoters, is cyclic and
occurs only in the hair follicles (indicated by
the narrow arrow). LTR, long term repeat.
5
Map of the Avy allele showing the LTR, agouti
exons, primer positions and HaeII (H) and HinP1I
(P) sites. IAP, intracisternal A particle LTR,
long terminal repeat.
6
More efficient DNA isolation techniques
  • Based on magnetic silica beads
  • High throughput manually
  • 96 well plates and multichannel pipets
  • No ethanol precipitation
  • Takes about 30 minutes
  • Can be automated

7
  • DNA Methylation of Specific Sequences
  • Methylation Specific PCR Safar et al. (2005)
    Methylation Profiling of Archived Non-Small Cell
    Lung Cancer A Promising Prognostic System.
    Clinical Cancer Research 11, 4400-4405.
  • Methylation Sensitive PCR Kurmasheva et al.
    (2005) Upstream CpG Island Methylation of the
    PAX3 Gene in Human Rhabdomyosarcomas. Pediatric
    Blood and Cancer 44, 328-337
  • Cooney et al. (2002) Maternal Methyl Supplements
    in Mice Affect Epigenetic Variation and DNA
    Methylation of Offspring. Journal of Nutrition
    132, 2393S-2400S
  • Bisulfite Genomic Sequencing Grunau et al.
    (2001) Bisulfite genomic sequencing systematic
    investigation of critical experimental
    parameters. Nucleic Acids Res. 29, e65

8
Bisulfite Modification of DNA
  • Unmodified sequence
  • GTCmGAACCGTTCATGTTGCmGAGCTG

Bisulfite Modification
GTCmGAAUUGTTUATGTTGCmGAGUTG
PCR
GT CG AATTGTTTATGTTG CG AGTTG
9
Bisulfite Genomic Sequencing
10
Solvent percentage does not affect measurement of
percent methylation using completely volatile
buffer.
11
Methylation of locus 224 in several tissues (A-E)
in two types of epigenetically different mice
12
  • Global DNA Methylation
  • HPLC Cooney et al. (1997) An Improved Sample
    Preparation Method for the Quantitative HPLC
    Determination of 5-Methyldeoxycytidine in Animal
    Tissue DNA. Journal of Liquid Chromatography 20,
    1279-1293
  • Methylation Levels on Repetitive Elements Yang et
    al. (2004) A simple method for estimating global
    DNA methylation using bisulfite PCR of repetitive
    DNA elements. Nucleic Acids Res. 32, e38

13
Typical Chromatogram for Global Methylation by
HPLC
Deoxycytidine
5-methyldeoxycytidine
14
Global DNA Methylation in a Range of Eukaryotes
Lyko et al 2000
Cooney et al unpubl
15
Metabolic Efficiency
  • Respirometry (Core B).
  • MRI of metabolites.
  • Relate to ongoing diabetes studies.
  • Studies of genotype (Avy/a versus A/a as
    surrogates for epigenetic phenotypes) already
    done. These relate AL and CR to body weight and
    to liver gene expression (Kaput et al. 2004).

16
TOP Y2 (heavily mottled Avy/a) mouse. BOTTOM
black (a/a) mouse
17
TOP black (a/a) mouse BOTTOM Y0 (pseudoagouti
Avy/a) mouse.
18
LEFT Y5 (clear yellow Avy/a) mouse. RIGHT Y4
(slightly mottled Avy/a) mouse.
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