Download - Bruce Richardson Epigenetics
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Epigenetics
Bruce Richardson MD PhDBruce Richardson MD PhD
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EpigeneticsHeritable changes in gene function that
occur without a change in the DNA sequence
Mechanisms:
DNA methylation Histone modifications
microRNAmicroRNA Histone variantsHistone variants
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Transcriptional regulation:
Prokaryotes
Signaling
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Transcriptional regulation: Eukaryotes
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Histone octamer core
~146 bp DNA
Histone Modifications
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Histone acetylation and gene
expressionAcetylated
Active
transcription
Unacetylated
Transcriptional
suppression
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Transcriptional regulation:
Vertebrates use DNAmethylation
ONH
N
NH2
ONH
N
NH2
H3C
-C5
N6
H4
-C5
N6
H4
N N
CH2
CH2S
C3
H5
NH2
COOH
H3C S
C3
H5
NH2
COOH
DNA Methyltransferase
Cytosine 5-methylcytosine
SAM SAH
1
2
3
4
5
6
1
2
3
4
5
6
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DNA methylation and gene
expression
CpG Island
Transcription
Promoter
CpG Island
Promoter
Suppression
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De Novo Methylation and
Demethylation
m
mC
Dnmt 3a, 3b
Demethylase
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DNA methylation can be dynamic
ILIL--2 promoter demethylates on T cell2 promoter demethylates on T cell
stimulationstimulation pS2/TFF1 promoter undergoes cyclicpS2/TFF1 promoter undergoes cyclic
methylation/demethylation on activation bymethylation/demethylation on activation byestrogens.estrogens.
Mechanism may involveMechanism may involve deaminationdeamination ofof
dC followed by base excision repair.dC followed by base excision repair.
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Ac Ac Ac
Ac Ac
Ac Ac
Ac Ac Ac
MeCP2 MeCP2
Ac
HDAC
HDAC
Unmethylated: transcriptionallyaccessible DNA
Methylated: transcriptionallyinaccessible DNA
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Importance of DNA
methylation
CELL FUNCTIONCELL FUNCTION
DifferentiationDifferentiation
ImprintingImprinting
X chromosomeX chromosome
inactivationinactivation
Suppression ofSuppression ofparasitic DNAparasitic DNA
SuppressSuppress
transcriptional noisetranscriptional noise
DISEASE STATESDISEASE STATES
Fragile X syndromeFragile X syndrome
ICF syndromeICF syndrome
Rett syndromeRett syndrome
Cancer (LOH,
instability, silencing) AutoimmunityAutoimmunity
Mental disordersMental disorders
AgingAging
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Copyright 2005 by the National Academy of Sciences
Fraga, Mario F. et al. (2005) Proc. Natl. Acad. Sci. USA 102,-
In development: Chromosome painting
Green =
hypermethylation
Red =
hypomethylation
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DNA Methylation Reaction
O
NH
N
NH2
O
NH
N
NH2
H3C
-C5N6H4 -C5N6H4
N N
CH2 CH2S
C3H5NH2COOH
H3C S
C3H5NH2COOH
DNA Methyltransferase
Cytosine 5-methylcytosine
SAM SAH
1
2
3
4
5
6
1
2
3
4
5
6
V = k[SAM][Dnmt]
[SAH]
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Maternal Diet Determines Agouti
Coat Color Through DNAMethylation
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DNA Methylation: Inhibitors
5-azacytidine*
2-deoxy-5-azacytidine*
siRNA (Dnmt1, 3a, 3b) Decitabine (MDS)
Zebularine (Cancer)
Procainamide (Dnmt1) Hydralazine (PKC)
Diet
UV light**Also DNA synthesis inhibitors, narrow window between
DNA methylation inhibition and DNA synthesis inhibition.
*Unstable in aqueous media.
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5-Azacytidine
Inhibits methylation ofInhibits methylation ofnewly synthesized DNAnewly synthesized DNA
Induces geneInduces gene
expressionexpression Alters differentiationAlters differentiation
N
NH
NH2
O
N
Requisite transcription factors must
be present and activated!
DNA methylation inhibition requires dividing
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DNA methylation inhibition requires dividing
cells
C
G
G
C
m
CG
GA
m
m
A
G
G
C m
C
G
G
C
m
m
C
G
G
C
m
m
5-azaC
A
G
G
C
C
G
G
A
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5-azaC and T Cell Differentiation
55
--azaC activates:azaC activates:
IFNIFN-- in Th2 cellsin Th2 cells
ILIL--4 and IL4 and IL--6 in Th1 cells6 in Th1 cells
Perforin in CD4 cellsPerforin in CD4 cells
KIR on all T cellsKIR on all T cells
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5-azaC and PerforinCD4 CD4 + 5-azaC
CD8 CD8 + 5-azaC
MFI 11.05 MFI 36.67
MFI 29.6 MFI 223.5
D th l t d CD4+ ll
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Demethylated CD4+ cells cause
lupus in mice (5-azacytidine,procainamide, hydralazine, U0126)
CD4++
1. Immune complex glomerulonephritis
2. Anti-dsDNA antibodies
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DNA Methylation and Sex
Female Male
One female X inactivated in part by DNA methylation
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CD40L and Lupus
NormalNormal::
ExpressedExpressed
transiently on CD4+transiently on CD4+
T cellsT cells
B cell costimulatoryB cell costimulatorymoleculemolecule
Encoded on the XEncoded on the X
chromosomechromosome
LupusLupus::
AberrantlyAberrantly
overexpressed onoverexpressed on
CD4+ / CD8+ T cellsCD4+ / CD8+ T cells
and B cellsand B cells Contributes to B cellContributes to B cell
overstimulationoverstimulation
AntiAnti --CD40L treatsCD40L treatslupus in micelupus in mice
5 AzaC demethylates CD40L on the
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5-AzaC demethylates CD40L on the
inactive X in CD4+ T cells from womenbut not men
0 1 2.5 5
1.0
1.5
2.0
2.5
Male CD4+
Female CD4+
5-azaC (M)
CD40L
MFI
Treate
d/Un
trea
ted
CD40L protein level
p=0.034
n=5
C
D40LMFI
Trea
ted/Un
trea
ted
5-azaC (M)
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5-azaC and Parasitic DNA
55--azaC reactivates latent retroviralazaC reactivates latent retroviral
elementselements . (. (Molecular mimicry andMolecular mimicry andimmunomodulationimmunomodulation by the HRESby the HRES--1 endogenous1 endogenousretrovirus in SLE. Perl et al,retrovirus in SLE. Perl et al,Autoimmunity.Autoimmunity. 20082008
May;41(4):287May;41(4):287--9797)) 55--azaC reactivates EpsteinazaC reactivates Epstein--Barr virus.Barr virus.
(Regulation and dysregulation of Epstein(Regulation and dysregulation of Epstein--Barr virusBarr virus
latency: Implications for the development of autoimmunelatency: Implications for the development of autoimmunediseases.diseases. NillerNilleret al, Autoimmunity. 2008 May;et al, Autoimmunity. 2008 May;
41(4)29841(4)298--328)328)
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0-15 16-35 36-70
3.00
3.25
3.50
3.75
4.00
Group
%d
mC
Percent dmC vs Age
%d
mC
0-15 16-35 36-70
4.0
3.5
3.0
Age
T cell DNA demethylates in
aging
Isolate DNA
Digest to nucleosides
(Dnase1,
phosphodiesterase,
alkaline phosphatase)
Reverse phase HPLC
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Isoschizomeric Restriction
Endonuclease Analysis
HpaHpaIIII cleaves Ccleaves C--CC--GG--G but not CG but not C--mmCC--GG--GG
MspMspII cleaves Ccleaves C--CC--GG--GG andand CC--mmCC--GG--GG
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5-azacytidine demethylates
Aci I (CCGC) sites
200 400 600 800 1000 1200 1400 1600 1800 2000 2200
Probe
Transcription start site
Translation start site
AciI
AciI
AciI
AciI
BglII
AciI
CD11a promoter
BglII +AciI digestion
BglII + AciI
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CD11a promoter
demethylates with age
y m o
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Age
Re
lative
intens
ity
1.0 kb
1.2 kb
B.
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Bisulfite Sequencing-C-C-
mC-G-
Bisulfite treatment
-U-U-m
C-G-
PCR
-T-T-C-G-
Clone and sequence 5-10 fragments/sample
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1257525-25-75-125
0
2
4
6
8
10
Male CD4+ Pattern
bp
Fragm
en
t
1257525-25-75-125
0
2
4
6
8
10
Female CD4+ pattern
bp
Fragm
en
t
promoter
Transcription start site
NF-AT
NF-AT NF-AT
Bisulfite Sequencing - CD40L Promoter
CD40L promoter is demethylated in
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CD40L promoter is demethylated in
CD4+ cells of women with lupus
2001000-100-200-300-4000
1
Female Promo ter
Frac
tion
me
thy
lated
2001000-100-200-300-4000
1Lupus Promoter
SLEDAI = 4
Frac
tion
Me
thy
lated
2001000-100-200-300-4000
1Lupus PromoterSLEDAI = 8.7
Frac
tionme
thy
lated p=0.006
Fra
ction
met
hy
lated
Fractio
n
me
thyla
ted
Frac
tion
m
ethy
lated
1
1
1
0
0
0
Control women (n=5)
SLEDAI = 4
(n=5)
SLEDAI = 8.7
(n=3)
CD40L MFI on CD4+ T cells from
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Men (n=6) Women (n=7)0
2
4
6
SLEDAI Scores
SLEDAI
Men (n=6) Women (n=7)1.00
1.25
1.50
CD40L Expression
CD40LMFI(Pa
tient
/Con
tro
l)
p = 0.009
CD40L MFI on CD4+ T cells from
men and women with lupus
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Methylation
Specific
PCR
KIR2DL2 and 2DL4 MS PCR
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KIR2DL2 and 2DL4 MS-PCR
2DL2 CD4 2DL2 CD8 2DL4 CD4 2DL4 CD80
20
40
60
80
UntreatedTreated
Me
thy
latio
nIndex
X100
Methylation index = [Methylated/Control] / [Methylated/Control + Unmethylated/Control]
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Step 1
Step 2
Step 3
DNA methylation
by pyrosequencing
Effect of 5-azaC on Perforin promoter methylation in
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Effect of 5 azaC on Perforin promoter methylation in
human T cells by pyrosequencing
12 CpGs
0
10
20
30
40
50
60
70
80
90
CD4 CD8
Control
5-azaC
Methylation and Promoter
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y
Function
Methylated Mock methylated
0
2
4
6
8
10
12
Vector
Luc
iferase
/b-gal
Luciferase/b-gal
Methylated Mock
methylated
Patch Methylation
Clone promoter intoreporter construct
Excise region of interest
Methylate with SssI andSAM
Religate back into
reporter constructTransfect into T cells
Control: omit SssI
ITGAL promoter
Where to look for methylation
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y
changes?
Promoters (obvious)Promoters (obvious)
Enhancers (not so obvious)Enhancers (not so obvious) When in doubt, try DNaseI hypersensitivityWhen in doubt, try DNaseI hypersensitivity
analysesanalyses
cells
I l t N l i
A
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Isolate Nuclei
Limited DNase I Digestion
DNA Purification
Restriction Digest
Blot and Probe
(1)(2)
(3)(4)
B DNAse 1
Hypersensitivity
Perforin DNase1 Hypersensitivity
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3.2 kb
1.4 kb
Fibroblasts T cells
1.4 kb
Probe
SacI
SacI
A.
B.
3.2 kb
Perforin DNase1 Hypersensitivity
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Chromatin Immunoprecipitation
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PRF1 acetylation increases with
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age
-1400 0
0-200-400-600-800-1000-1200-1400
0
10
20
30
40CD4+ H3-oAc
Re
lativeace
tylatio
n Old (54 +/- 7)
Young (29 +/- 3)
p=0.005n=3
0-200-400-600-800-1000-1200-1400
0
10
20
30
CD4+ H4-oAc
Re
lativeace
tylation
Old (54 +/- 7)
Young (29 +/- 3)
p
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NIH Roadmap Epigenomics Program
REFERENCE EPIGENOME MAPPINGREFERENCE EPIGENOME MAPPINGCENTERSCENTERS
EPIGENOMICS DATA ANALYSIS ANDEPIGENOMICS DATA ANALYSIS ANDCOORDINATION CENTERCOORDINATION CENTER
TECHNOLOGY DEVELOPMENT INTECHNOLOGY DEVELOPMENT INEPIGENOMICSEPIGENOMICS
DISCOVERY OF NOVEL EPIGENETIC MARKSDISCOVERY OF NOVEL EPIGENETIC MARKS
IN MAMMALIAN CELLSIN MAMMALIAN CELLS EPIGENOMICS OF HUMAN HEALTH ANDEPIGENOMICS OF HUMAN HEALTH AND
DISEASEDISEASE
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Epigenetics and Rheumatology
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Epigenetics and Rheumatology
DNA methylationDNA methylation
LupusLupus SclerodermaScleroderma
Rheumatoid arthritisRheumatoid arthritis
OsteoarthritisOsteoarthritis
Histone ModificationsHistone Modifications
LupusLupus Rheumatoid arthritisRheumatoid arthritis
SclerodermaScleroderma