Bruce Richardson Epigenetics

8/14/2019 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

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Bruce Richardson Epigenetics