cancer, genetic predispositions and chromatin architecture · 6/29/2015 · e r l a p p i n g w i...

Cancer, Genetic Predispositions and Chromatin Architecture www.pmgenomics.ca/lupienlab"

June 29, 2015 Mathieu Lupien, PhD

Let’s all be leprechauns"

Three ways to exploit ENCODE data"

3- Understanding the three-dimensional architecture of the genome"

"2- Finding the targets of "genetic predispositions"

1- Annotating the functional genome using epigenetics"

Heintzman et al (2007) Nat Genet. 39: 311-318 Lupien et al (2008) Cell. 132: 958-970 Heintzman et al (2009) Nature. 1-5 Ernst et al. (2011) Nature, 2011 pp. 1-9

Epigenetic modifications annotate the functional elements across the genome"

Diff

eren

t cel

l typ

es"

A small snapshot of the human genome!

GENE 1! GENE 2! GENE 3! GENE 4!

Epigenetic reprogramming underlies breast cancer resistance to standard-of-care"

Magnani, et al. (2013). Proc. Natl. Acad. Sci. U.S.A. 110. pE1490-1499 Akthar-Zaidi et al (2012) Science. 11: 736-739

Active enhancer

Inactive enhancer

Inactive enhancer

Active enhancer

ERα PBX1

NOTCH "Effector"

Switc

h in

tr

ansc

riptio

nal r

egul

atio

n

New target

Endocrine therapy

Dru

g Tr

eatm

ent (

AI)

Drug Sensitive

Drug Resistant

Functional interpretation of genetic predispositions using epigenetics"

ENCODE Ernst et al. (2010). Nature. 1-9 Schaub et al. (2012). Genome Res. 22:1748-1759 Maurano et al. (2012). Science. 337: 1190-1195 and many more…

My contributions: Akthar-Zaidi et al (2012) Science. 11: 736-739 Zhang et al (2012) Genome Research. 22: 1427-1436 Cowper Sal lari et al (2012) Nat Genet. 44: 1191-1198 Corradin et al (2014) Genome Research. 24: 1-13 Zhang et al (2014) Trends in Genetics. 30: 140-149 Ghoussaini et al. (2014). Nat Commun. 4: 4999 Chadar et al. (2014) MCB. 34: 3291-3304 Bailey et al (2015) Nat Commun. 2: 6186

Variant Set Enrichment (VSE) approach – "BCa GWAS risk-variants target enhancers"

Cowper Sal-lari et al. (2012). Nature Genetics 44. p1191

BCa GWAS risk-variants target the ERa and FOXA1 transcription factors"


GTGTTTGCTA[T/C]GATTAGTTC

ChIP-seq signal for your favorite TF

Assessing the role of genetic variants on transcription factor DNA binding affinity"

IntraGenomic Replicates (IGR) approach"In sillico allele-specific DNA binding affinity predictor"

TTGCTAT


TTGCTAT TGCTATG


ChIP-seq signal for your favorite TF






TTGCTAT TGCTATG GCTATGA





TTGCTAT TGCTATG GCTATGA CTATGAT





TTGCTAT TGCTATG GCTATGA CTATGAT TATGATT





TTGCTAT TGCTATG GCTATGA CTATGAT TATGATT ATGATTA





TTGCTAT TGCTATG GCTATGA CTATGAT TATGATT ATGATTA TGATTAG





TTGCTAC TGCTACG GCTACGA CTACGAT TACGATT ACGATTA CGATTAG



IGR predicts changes in binding affinity imposed by SNVs for any transcription factor"

ChI

P-s

eq S

igna

l

Distance from SNV (bp)


Bases

−200 0 200

4

8

12 C37336.41

T332510.48

10.89

−200 0 200

10

14

18 C35097.45

T41407.78

0.26

rs4784227Fo

xA1

IGR

scor

eTc

f4 IG

R sc

ore

Freq

uenc

y

20 40 60 800

20

40

60

80

BCaP = 0.009

C A A G G G T C C A0.0

0.5

1.0

1.5

0.90

0.95

1.00A

CT

CACG

ACGCT AGT

A

G AG

A

CG

AG

GA

C

GC

C

0.2

0.6

1.0

1.4

FoxA

1 Ch

IP-q

PCR

AC

G

A

CT CG ACG

AC

G

CGT AG

A

GA

G CGT

CC

A

A

2 4 6 8 101 3 5 7 9

0

1

2Fo

xA1

IGR

scor

eFK

H PW

M sc

ore

Bits

FKH position

10

20

30

40

4

6

8

10

FoxA

1 IG

R sc

ore

FoxA

1 IG

R sc

ore

−200 0 200

4

8

12 G25514.97

T31495.36

0.42

−200 0 2006

10

14

18

Bases

G447816.13

T532110.15

10.56

rs6983267

908024.42

−200 0 200Bases

1439210.49

T

−200 0 200

2010440.49

−200 0 200Bases

149326.41

C

−200 0 200

0.6 0.8 1.0 1.2 1.4

0.2

0.4

0.6

0.8

1.0

1.2

r = 0.89

FoxA1 ChIP-qPCR

FoxA

1 IG

R sc

ore

0.88

1.00r = 0.45

0.6 1.4

FKH position 8

FKH position 6

A

G

2

2

% of clusters with affinity modulating SNPs

FKH motifa

b

c

d

e

f

g

h

6

Figure 4 Single column (89mm)

Bases

−200 0 200

4

8

12 C37336.41

T332510.48

10.89

−200 0 200

10

14

18 C35097.45

T41407.78

0.26

rs4784227

FoxA

1 IG

R sc

ore

Tcf4

IGR

scor

eFr

eque

ncy

20 40 60 800

20

40

60

80

BCaP = 0.009

C A A G G G T C C A0.0

0.5

1.0

1.5

0.90

0.95

1.00A

CT

CACG

ACGCT AGT

A

G AG

A

CG

AG

GA

C

GC

C

0.2

0.6

1.0

1.4

FoxA

1 Ch

IP-q

PCR

AC

G

A

CT CG ACG

AC

G

CGT AG

A

GA

G CGT

CC

A

A

2 4 6 8 101 3 5 7 9

0

1

2

FoxA

1 IG

R sc

ore

FKH

PWM

scor

eBi

ts

FKH position

10

20

30

40

4

6

8

10

FoxA

1 IG

R sc

ore

FoxA

1 IG

R sc

ore

−200 0 200

4

8

12 G25514.97

T31495.36

0.42

−200 0 2006

10

14

18

Bases

G447816.13

T532110.15

10.56

rs6983267

908024.42

−200 0 200Bases

1439210.49

T

−200 0 200

2010440.49

−200 0 200Bases

149326.41

C

−200 0 200

0.6 0.8 1.0 1.2 1.4

0.2

0.4

0.6

0.8

1.0

1.2

r = 0.89

FoxA1 ChIP-qPCR

FoxA

1 IG

R sc

ore

0.88

1.00r = 0.45

0.6 1.4

FKH position 8

FKH position 6

A

G

2

2

% of clusters with affinity modulating SNPs

FKH motifa

b

c

d

e

f

g

h

6

Figure 4 Single column (89mm)

IGR

PWM

ChIP-‐qPCR

IntraGenomic Replicate (IGR) tool is a better predictor of DNA binding affinity than PWM"


IGR predictions are agnostic to cell-type or origin of ChIP-seq datasets"

ChIP-‐seq: FoxA1


Allele-‐specific ChIP-‐qPCR: FoxA1

IGR based allele-‐specific FoxA1 binding

IGR reveals that most BCa genetic risk-loci modulate FoxA1 binding to the DNA "


Genetic alterations

Transcription factors IGR based analysis >2000 Genetic alterations in DHS

0 2 4 6 8 10 Absolute fold change in binding

affinity (allele1/allele2)

Nature Gene*cs AOP. (2015) | doi:10.103/ng.3335.

Combining IGR with ENCODE: all genetic variants versus all transcription factors"

"What are the target gene(s) "

of genetically or epigenetically altered regulatory elements?"

Sanyal, et al (2011) Current Opinion in Cell Biology. 23 p325-‐331

Chromatin interactions define the social genome"

The social genome needs matchmakers"

Chromatin interactions bring regulatory elements to their target gene(s)"

LF1

LF2

Sanyal, et al (2013) Nature, 489 p109-‐113

Detecting chromatin interactions through Chromatin Conformation Capture (3C) assays"

ZNF143 and a few more transcription factors preferentially binds chromatin loop anchors"

BH

LHE4

0

P300

ZN

F143

C

TCF

SMC

3 ZN

F384

MA

Z M

XI1

TBP

CO

RES

T TB

LR1

STAT

5 PO

L2

CD

P R

AD

21

MA

X C

HD

2 YY

1 C

EBPB

EL

K1

NR

F1

PML

RFX

5 N

FKB

EL

F1

SRF

NFY

B

TAF1

U

SF2

E2F4

PU

1 C

REB

1 EG

R1

USF

1 N

RSF

N

FYA

ZBTB

33

−2

02

46

8

●

●●●

●

Zbtb33

Nfya

Nrsf

Usf1

Egr1

Creb1

Pu1

E2f4

Usf2

Taf1

Nfyb

Srf

Elf1

Nfkb

Rfx5

Pml

Nrf1

Elk1

Cebpb

Yy1

Chd2

Max

Rad21

Cdp

Pol2

Stat5

Tblr1

Corest

Tbp

Mxi1

Maz

Bhlhe40

Znf384

Smc3

Ctcf

Znf143

P300

●

●

●

●

●●●

●●

●●●

●

●

●●

●

●●

●

●●●●●●

●

●●

●●

●

●

●●

●

●

Enrichment of transcription factor binding sites at 5C anchors

-2

0

2

4

6

8

Z-Score

GM12878 cells

Bailey et al 2015. Nat Commun. 2: 6186

ZNF143 enrichment at chromatin loop anchors “validated” by others"

Heidari et al. Genome Research. Sept 2014 Rao et al. Cell. December 2014

−1000 TSS +100001020304050

POL2ZNF143CTCFSMC3

ZNF143 preferentially binds promoters next to RNA Pol II"

ZNF143 SMC3 CTCF POL2

0.0

0.2

0.4

0.6

0.8

1.0PromoterEnhancerTxnInsulatorHeterochrom/loRepressedRepetitive/CNV

% o

f ZN

F143

bin

ding

s ov

erla

ppin

g w

ith e

ach

chro

mat

in s

tate

s

0.0

0.2

0.4

0.6

0.8


% o

f ZN

F143

bin

ding

s ov

erla

ppin

g w

ith e

ach

chro

mat

in s

tate

s

0.0

0.2

0.4

0.6

0.8


% o

f ZN

F143

bin

ding

s ov

erla

ppin

g w

ith e

ach

chro

mat

in s

tate

s

0.0

0.2

0.4

0.6

0.8


% o

f ZN

F143

bin

ding

s ov

erla

ppin

g w

ith e

ach

chro

mat

in s

tate

s

0.0

0.2

0.4

0.6

0.8


% o

f ZN

F143

bin

ding

s ov

erla

ppin

g w

ith e

ach

chro

mat

in s

tate

s

0.0

0.2

0.4

0.6

0.8


% o

f ZN

F143

bin

ding

s ov

erla

ppin

g w

ith e

ach

chro

mat

in s

tate

s

0.0

0.2

0.4

0.6

0.8


% o

f Z

NF

14

3 b

ind

ing

s o

ve

rla

pp

ing

w

ith

ea

ch

ch

ro

ma

tin

sta

te

s

0.0

0.2

0.4

0.6

0.8


% of ZN

F143 bi

ndings o

verlapp

ing with

each ch

romatin

states

0.0

0.2

0.4

0.6

0.8


% o

f Z

NF

14

3 b

ind

ing

s o

ve

rla

pp

ing

w

ith

ea

ch

ch

rom

atin

sta

tes

0.0

0.2

0.4

0.6

0.8


% of ZN

F143 bi

ndings o

verlapp

ing with

each ch

romatin

states

-5kb +5kb

ZNF1

43 b

indi

ng s

ites

0.0 0.2 0.4 0.6 0.8 1.0

Top 10%

Remaining

Fraction of sites with ZNF143 motif

ChI

P-se

q si

gnal

inte

nsity

Distance (bp)

0 >30 ChIP-seq signal

intensity

POL2 Bound Promoters

GM12878 cells


Disrupting ZNF143 binding to the chromatin antagonizes chromatin loop formation"

rs2232015

ZNF143

CTCF

RAD21

PRMT6

2kb

PRMT6 NTNG1 ZNF143

SMC3

RAD21

CTCF

HindIII anchor nc1 nc2 nc3 nc4 test nc5

A C T A C A A G T C C C A G A

T

5’

Bits

2

rs2232015

ZNF143 motif 1

3’

0

80 Signal file

Peak file

0

30

0

30

0

80

0

Signal file

Peak file Signal file


Peak file

SMC3

80

30

30

80 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15



0 50000 1000000

2

4

6

FrequencyPRMT6 NTNG1

ZNF143

SMC3

RAD21

CTCF

HindIII anchor

107574000 107594000 107614000 107634000 107654000 107674000 107694000

r

1.0

0

DNaseI

nc1 nc2 nc3 nc4 test nc5

0

6

3C fr

eque

ncy

80 Signal file

Peak file

0

30

0

30

0

80

0

Signal file



Peak file



−30 −20 −10 0 10 20 30

−140−130−120−110−100−90−80−70−60−50−40−30−20−10

00102030405060708090

100110120130140150160170180190200210220230240

Alternate Allele

Reference Allele

Position Relative to SNP

Depth

of S

equence

Reads

Conta

inin

g S

NP

(n)

Dep

th o

f Rea

ds C

onta

inin

g SN

P (n

)

0

240

140 0 30 -30

Reference Allele (A)

Alternate Allele (T)

Position Relative to SNP (bp) −30 −20 −10 0 10 20 30

−140−130−120−110−100−90−80−70−60−50−40−30−20−10

00102030405060708090

100110120130140150160170180190200210220230240

Alternate Allele

Reference Allele


Depth

of S

equence

Reads

Conta

inin

g S

NP

(n)

A

T

0

0.5

1

Allele ratio

Con

trol

3C

ZNF1

43 C

hIP

Gen

omic

DN

A

rs2232015

ZNF143 ChIP-seq

qPCR



Figure 3

rs13228237 D

epth

of R

eads

Con

tain

ing

SNP

(n)

Position Relative to SNP (bp)

Reference Allele (G)

−30 −20 −10 0 10 20 30

−10

00

10

20

Reference Allele

Alternate Allele


Dep

th o

f Seq

uenc

e R

eads

Con

tain

ing

SN

P (

n)

-10

0

20

0 30 -30 −30 −20 −10 0 10 20 30

−140−130−120−110−100−90−80−70−60−50−40−30−20−10

00102030405060708090

100110120130140150160170180190200210220230240

Alternate Allele

Reference Allele


Depth

of S

equence

Reads

Conta

inin

g S

NP

(n)

Alternate Allele (C)

G

C

0

0.5

1

Allele ratio

Con

trol

3C

ZNF1

43 C

hIP

Gen

omic

DN

A

Loop1 Loop2

Con

trol

3C

ZNF143 ChIP-seq

qPCR


Allele-specific Binding from ChIP-seq (ABC)"https://github.com/mlupien/ABC"

−35 0 35

60

50

40

30

20

10

00

10

20

30


Dep

th o

f Seq

uenc

e R

eads

Con

tain

ing

SN

P (n

) Reference Allele

Alternate Allele

PosiOon RelaOve to SNV

Reference Allele

Reference

Variant

A A A A A A

2x read length

G G G G G

G

SNV De

pth of Seq

uence Re

ads C

ontaining SN

V (n)

Variant Allele

150

0

rs4784227 and FOXA1

p=3.18x10-3

Max=128

Bailey et al 2015. Bioinformatics. btv321

ZNF143 secures chromatin interactions at promoters"

Enhancer Promoter Gene

RNA

DNA

CTCF

ZNF143

TF cell type-spec.

POL2

Cohesin

Nucleosome

Mediator/ coactivator

www.pmgenomics.ca/lupienlab"Thank you!"Lupien’s Lab"Swneke D. Bailey"Genevieve Deblois!Kinjal Desai"Alexandra Fedor!Paul Guilhamon!Ken Kron!Parisa Mazrooei"Alexander Murison!Nadia M. Penrod!Aislinn Treloar!"

Alumni"Elizabeth Ballantyne!Luca Magnani "Richard Sallari"Xue Wu!Xiaoyang Zhang "Nahiyan Malik!Luna Xiaowan Lu!!!

Princess Margaret Cancer Centre!Benjamin Haibe-Kains "

Others!Jason H. Moore (Dartmouth College)!Michael Cole (Dartmouth College)!Peter Scacheri (CWRU)!!

cancer, genetic predispositions and chromatin architecture · 6/29/2015 · e r l a p p i n g w i...

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