Genomic ‘dark matter’ in the heart A clinical treasure trove Samir Ounzain, Ph.D CGRM Seminar...

Genomic ‘dark matter’ in the heart

A clinical treasure trove

Samir Ounzain, Ph.DCGRM Seminar Series, Maastricht – 5th December 2014

Pedrazzini LabDepartment of Medicine

Experimental Cardiology UnitUniversity of Lausanne Medical School

Centre Hospitalier Universitaire Vaudois (CHUV)Lausanne, Switzerland

@ispiyou about.me/ounzainsamir Samir Ounzain, 2014

Cardiovascular disease and heart failure – Heartbreaking statistics

Bill Cannon, Outlook - Heart Health, Nature, 31st January 2013

about.me/ounzainsamir @ispiyou

Cardiovascular disease and heart failure – More heart disease ahead


the projected annual number of worldwide

deaths from cardiovascular disease in 2030 23.6M

global deaths per minute from cardiovascular

disease in 2008 33


Cardiovascular disease and heart failure – Global burden



Response to biomechanical stress in the adult heart

Compensated FailingNormal

Biomechanical StressMyocardial InfarctionHypertension

Cardiomyocyte hypertrophy

Gradual loss of cardiomyocytes

Fibroblast proliferation

Massive loss of myocytes

Fibrosis

Electro-mechanicalRemodeling

Mobilization of immature cardiomyocytesand cardiac stem cells

Regeneration

What we have: Fewer myocytes

More fibrosis

What we want: More myocytes

Less fibrosis


Cardiac gene regulatory networks


Long non-coding RNAs – Characteristics and mechanisms of action


Hu, W et al (2012) Regulation of mammalian cell differentiation by long non-coding RNAs. EMBO Reports

Typically (A) >200bp (B) lack coding potential (C) PolyA(+) (D) Pol2 transcribed (E) multi-exonicHighly tissue/context specific – Thousands likely exist


Long non-coding RNAs – Characteristics and mechanisms of action

Functions in the heart are poorly characterized

Rinn, J and Chang, H al (2012) Genome regulation by long non-coding RNAs. AnnuRev-Biochem


Jerome DauvillierFrederic BurdetMark IbbersonIoannis Xenarios

Rory JohnsonRoderic Guigo

Blanche SchroenStephane Heymans

Alexandre Sarre

Keith Harshman

Rudi, Tal, Miki, Iole, Moh

Characterising the mouse long non-coding (lncRNA) transcriptome


Humanortholog identification

1110 lncRNAsMouse model

Human heart disease

Myocardialinfarction

14 d.

Borderzone

Poly A(+) RNA-Sequencing

ab initio reconstruction

Association with specific chromatin state

transitions

Heart specificity

UC

SC

ln

cRN

As

mR

NA

s

Nov

el ln

cRN

As

Correlation with cardiac physiology

Functional roles as Enhancer derived ncRNAs

p300

H3K4me1

H3K4me3

H3K27Ac

H3K27me3

Mouse enhancer

LVLA

RA

RV

UCSC mRNA UCSC lncRNA

Novel lncRNA

8.7% (1521)5.6%

85.7%

Novel lncRNA identification and characterisation


0.0

0.1

0.2

0.3

Den

sity

0 20 40 60 80

Coding potential (Gene ID score)

UCSC mRNAs UCSC lncRNAs

Novel lncRNAs

UCSC mRNA UCSC lncRNA

Novel lncRNA

8.7% (1521)5.6%

85.7%

Exons Introns Promoters

Den

sity

0.0-0.5-1.0-1.5-2.0 0.0-0.5-1.0-1.5-2.0 0.0-0.5-1.0-1.5-2.0

0.0

0.5

1.0

1.5

2.0

2.5

PhastCons (Normalized by size)

UCSC lncRNA

Novel lncRNA

UCSC mRNA

Random intergenic

Overlapping antisense

Overlapping sense

Intronic antisense

Intronic sense

Exonic sense

Divergent

Convergent

Same strand

UCSC lncRNAs

Novel lncRNAs

Transcript number

0.0

0.2

0.4

0.6

0.8

1.0

Den

sity

1 10 100 10000.10.01

Mean transcription (FPKM)

UCSC mRNAs UCSC lncRNAs

Novel lncRNAs

Response of the transcriptome to stress – 14 days post MI


Sham MI

NppaNppbMyh6Myh7Col1a1PostnTgfb1XLOC_007752XLOC_012723XLOC_010855XLOC_001225XLOC_007565XLOC_007852

Sham MI

UCSC mRNAs

1338 do

wn

2204 up

Sham MI

UCSC lncRNAs

67 up

66 do

wn

Sham MI

Novel lncRNAs

86 up

225 do

wn

Canonical Markers

Sham MI

Sh

am

MI

UCSC mRNAs

Sham MI

Sh

am

MI

UCSC lncRNAs

Sham MI

Sh

am

MI

Novel lncRNAs

Novel lncRNAs exhibit significant cardiac specific expression


All transcriptsH

eart

sp

ecif

icit

yH

eart

sp

ecif

icit

yH

eart

sp

ecif

icit

y

15075 UC

SC

mR

NA

s988 U

CS

C ln

cRN

As

1521 No

vel lncR

NA

s

Hea

rt

ShamHeart

MIHeart

17 non cardiac tissues

Cu

ffli

nks

Pre

dic

tio

ns

+

-

+

-

+

-

+

-

Heart

Testis

Kidney

Liver

Str

and

sp

ecif

ic

tran

scri

pti

on

Lung

Stomach

PhastConsVertebrates

+

-

+

-

Novlnc6

Proximal coding genes are heart specific and implicated in cardiac biology

P value (-log 10)

Heart specific transcripts (%)

Correlation score = 0.995

Cufflinks predictions

UCSC annotations

+-

ENCODE Heart

Sham

MI

PolyA(+)RNA-Seq

278 lncRNA/mRNA pairs with correlations > 0.9


Novel lncRNAs exhibit significant cell and sub-cellular specificity


Novlnc333

Novlnc174

Novlnc95

Novlnc86

Novlnc44

Novlnc90

Novlnc103

Novlnc96

Novlnc49

Novlnc11

Novlnc15

Novlnc32

Novlnc76

Novlnc61

Novlnc6

Novlnc35

Novlnc23

CMFibr.

0 0.05 0.10 0.15-0.05-0.10-0.15

Novlnc11

Novlnc95

Novlnc49

Novlnc76

Novlnc86

Novlnc44

Novlnc96

Novlnc90

Novlnc15

Novlnc6

Novlnc35

Novlnc333

Novlnc61

Novlnc174

Novlnc103

Novlnc32

Novlnc23

CMFibr.

0 2 4-2-4

Delta Fold Change Nuc. Cyto.Cyto/Nuc Ratio

Novel lncRNAs are highly correlated with cardiac physiological traits


LV

mas

s / B

WL

V m

ass

IVR

TS

A M

I tra

ceS

A L

V a

rea;

dL

A M

I tra

ceL

A L

V a

rea;

d

LV

ID;

sL

V v

olu

me;

sL

VID

; d

LV

vo

lum

e; d

LV

PW

; d

LV

PW

; s

EF

FS

IVS

; s

IVS

; d

Bo

dy

wei

gh

tH

eart

rat

e

LA

LV

are

a; s

Echocardiography derived traits

1.

2.

3.

4.

No

vel l

ncR

NA

s

Correlation

2.0

1.5

1.0

0.5

0.25 0.50 0.75 1.00

Den

sity

EF2.0

1.5

1.0

0.5

0.25 0.50 0.75 1.00

Correlation

Den

sity

MI Trace

UCSC lncRNAs

Novel lncRNAs0 0.5 1.0-1.0 -0.5

Correlation

Heart specific transcripts (%)

EF

0.25 0.50 0.75 1.00

1

2

3

4

5

Den

sit

y

Correlation

Cluster 1

Cluster 2

Cluster 3

Cluster 4


Novlnc35

mhy7

Novlnc6Novlnc15

Novlnc76

Novlnc61

Novlnc103

Novlnc174

Novlnc23

Novlnc333

Novlnc32

Novlnc49

Novlnc11

Novlnc95

Novlnc90

Novlnc86

Novlnc96

Novlnc44

col1actgfnppatgfb2

LV m

ass

/ B

W

LA M

I tr

ace

SA

MI

trac

e

LV m

ass

SA

LV

are

a; d

LA L

V a

rea;

d

LA L

V a

rea;

s

LVID

; s

LV v

olum

e; s

LVID

; d

LV v

olum

e; d

Hea

rt r

ate

Bod

y w

eigh

t

IVS

; s

LVP

W;

s

EF

FS

LVP

W;

d

IVS

; d

0 0.5-0.5

Border Zone

70

50

30

10

0-1-2-3

Novlnc6

EF

(%

)

R=0.785

70

50

30

10

0-1-2-3-4

Novlnc15

R=0.811

70

50

30

10

0-1-2-3 1

Novlnc95

R=0.785

Fold change over Sham

70

50

30

10

10 21

Novlnc174

R=-0.538

Fold change over Sham

70

50

30

10

321-1 0

Myh7

R=-0.426

70

50

30

10

210-2 -1

Col1a

R=-0.484

70

50

30

10

210-1

Nppa

R=-0.788E

F (

%)

Novel lncRNAs Cardiac markers

Myocardialinfarction

BorderZone

1, 7 and 14 days

Novel lncRNAs are highly correlated with cardiac physiological traits

Novel lncRNAs are associated with cardiac specific active enhancers


H3K4me3+H3K27me3 bivalent/poised promoterH3K4me3+H3K27ac active/initiating promoterH3K4me3 initiating promoterH3K27me3+H3K4me1 poised developmental enhancerH3K4me1 poised enhancerH3K27ac+H3K4me1 active enhancer H3K27me3 Polycomb repressed

ENCODE Data SetsChIP-Seq data sets from 5 mouse whole tissues


Kidney Liver SpleenHeart Testis

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

1

2

3

Down

None

Up

H3K4me3+H3K27me3 bivalent/poised promoterH3K4me3+H3K27ac active/initiating promoterH3K4me3 initiating promoterH3K27me3+H3K4me1 poised developmental enhancerH3K4me1 poised enhancerH3K27ac+H3K4me1 active enhancer H3K27me3 Polycomb repressed

ENCODE Data SetsChIP-Seq data sets from 5 mouse whole tissues

No

vel l

ncR

NA

s

No

vel l

ncR

NA

s



H3K

4me1

H3K

4me3

H3K

27A

cH

3K27

me3


Heart

Kidney

Liver

Spleen

Testis

Cu

fflin

ksP

red

icti

on

s

Heart

Kidney

Liver

Spleen

Testis

Heart

Kidney

Liver

Spleen

Testis

Heart

Kidney

Liver

Spleen

Testis

Novlnc6

Enhancer


Mouse embryonic stem cell cardiac differentiation


Oct4Nanog

EomesBrachyury T

Mesp1 GATA4Nkx2.5

Cardiac genes(-MHC; -MHC)

d0 d2-3 d3 d3-d6 d6-12 d>12

Organizedsarcomeres

mES MES CPC CM

Integration of ChIP-sequencing data from Wamstad J.A. et al. 2012. Cell.

Embryonicstem cells

Mesodermalprecursors

Cardiacmesodermalprecursors

Cardiacprecursors

Immaturecardiomyocytes

Maturecardiomyocytes

Spontaneously beatingSpontaneously beatingcardiomyocytescardiomyocytes

Hanging dropsHanging drops Suspension cultureSuspension culture Adherent cultureAdherent culture

Embryoid bodiesEmbryoid bodies DifferentiatingDifferentiatingembryoid bodiesembryoid bodies

d0 d3-6 d6-9 d9-12

UndifferentiatedUndifferentiatedembryonic stem cellsembryonic stem cells

Embryonic stem cellEmbryonic stem cellcoloniescolonies


MES CPC CMES

A

B

C

D

H3K27me3

H3K4me1 & H3K27Ac

H3K4me1

H3K4me1 & H3K27me3

H3K4me3

H3K4me3 & H3K27Ac H3K4me3 & H3K27me3

1

2

3

USCC mRNAs

USCS lncRNAs

Novel lncRNAs

MES CPC CMES

1 2 3 1 2 3 1 2 3 1 2 30

5

10

15

20

25

%

Cluster A Cluster B Cluster C Cluster D

%Heart specificy

Oct4Nanog

Mesp1 GATA4Nkx2.5 -MHC; -MHC


Novel lncRNA expression correlates with chromatin state


Novlnc6

Novlnc11

Novlnc15

Novlnc23

Novlnc32

Novlnc35Novlnc44Novlnc49

Novlnc61

Novlnc76Novlnc86Novlnc90

Novlnc95Novlnc96

Novlnc103

Novlnc174

Novlnc333

ES

ME

SC

PC

CM

H3K4me3 H3K27me3E

SM

ES

CP

CC

M

H3K4me1

ES

ME

SC

PC

CM

H3K27ac

ES

ME

SC

PC

CM

Fol

d ch

ange

ove

r d0

Novlnc61 Novlnc23 Novlnc49 Novlnc44

ES cell differentiation

Fol

d ch

ange

ove

r d0

ES cell differentiation

Novlnc11 Novlnc32 Novlnc41 Novlnc95 Novlnc333

MES CPC CMES

Oct4Nanog


d3 d6 d12d0

Inferring functions for novel lncRNAs based on chromatin state transitions


Embryonicstem cells


Cardiacprecursors

Cardiomyocytes

Exp

ress

ion

Genes

Lineage-specific gene expression

Stages of differentiation

x y z x y z x y z x y z

Based on Wamstad JA et al. 2012. Cell 151: 206


Embryonicstem cells


Cardiacprecursors

Cardiomyocytes

Exp

ress

ion

En

rich

men

tE

nri

chm

ent

En

rich

men

tE

nri

chm

ent

H3K4me3

H3K27me3

H3K4me1

H3K27Ac

Genes

Lineage-specific gene expression

Functional inference

Stages of differentiation

x y z x y z x y z x y z

Ch

rom

atin

e st

ate

tran

siti

on




1st pattern

2nd pattern

3rd pattern

Cluster 1(gene x, plus other geneswith similar pattern)

34 Patterns / Clusters

Cluster 2(gene y, plus other geneswith similar pattern)

Cluster 3(gene z, plus other geneswith similar pattern)

Wamstad JA et al. 2012. Cell 151: 206

Inferred function based on top

GO terms

Cluster

1 House keeping

2 House keeping

3 House keeping

4 Cellular homeostasis

5 Pluripotency, Metabolic process

6 Metabolic process, Transport

7 Cellular homeostasis, Matrix

8 Channel activity, Signaling

9 Development

10 Vascular homeostasis

11 Pluripotency, Development

12 Renal homeostasis

13 Catabolic process

14 Metabolic process

15 Enzymatic activity

16 Inflammatory response

17 Metabolic process



20 Contraction, Heart development

21 G-protein coupled receptors








29 House keeping

30 Contraction

31 Transcription, Translation

32 Mitochondrial activity

33 Translation

34 Translation



1

3

18

25 2623 24

28

USCS mRNAs

20

27

2

Novel lncRNAs

1

3

18

28

26

24

23

20

27

25

2

Definition of 34 clusters corresponding to 34 groupsof genes that are functionally related

E.g.: Cluster 1, 2, 3: House keeping function Cluster 18: Immune/Inflammatory response Cluster 20, 23-27: Cardiac development, muscle contraction Cluster 28: Calcium homeostasis, GPCR signaling

Wamstad JA et al. 2012. Cell 151: 206

- 5.70

- 4.00

- 2.00

0.00

2.00

4.00

7.25

Pearsonresidual

Down Unchanged Up

1

3

23

24

28

18

20

27

2

2526

Sham vs. MI

Cluster-associated novel lncRNAs are enriched in –up/-down regulated lncRNAs


Candidate cardiac enhancer derived lncRNA – Novlnc6


p300

H3K4me1

H3K4me3

H3K27Ac

H3K27me3


Mouse enhancer

LVLA

RA

RV

Novlnc6 Nkx2.5

Scrambled

Novlnc6 Gapmer 1

Bmp10 Gata4 Tbx20 Myh6 Myh7

Fol

d ch

ange

ove

r S

cram

ble

Novlnc6 Gapmer 2

** *** * * *

Remote ZoneBorder Zone

d1 d7

*

d1 d7

* *

Fol

d ch

ange

over

Sha

m d

1

Novlnc6

d1 d7

*

Nppa

Fol

d ch

ange

over

Sha

m d

1

*

d1 d7

*Col1a

d1 d7

*

EF

*

%

Novel lncRNAsChromatin State Clusters

1

3

18

28

26

24

23

20

27

252

Novlnc6

Bmp10

Cardiomyocytes

Novlnc6Gapmers

70

50

30

10

210-1

Nppa70

50

30

10

0-1-2-3

Novlnc6

EF

(%

)

R=0.785

R=-0.788

Nkx2.5

Sham MI

Novlnc6 is modulated in Human heart disease


Hs Novlnc6

p < 0.001

Nkx2.5 Nppa Col1a

p < 0.05 p < 0.001 p < 0.001

p < 0.01 p < 0.01

TransMapped Human Orthologs


Mammalian Basewise PhyloP

Hs Novlnc6

p < 0.001

EF

p < 0.001

PW thickness

Dilated cardiomyopathy

Aortic stenosis

1100 human orthologs identified


Summary

Identified 1500 novel lncRNAs in the infarcted mouse heart

Novel lncRNAs are heart specific

Novel lncRNAs are highly correlated with physiological traits

Novel lncRNAs are enriched at heart specific active enhancers

Inferred novel lncRNA functions based on chromatin states

Conserved novel lncRNAs are modulated in human disease

Unique characteristics of novel lncRNAs render them ideal tissue specific therapeutic targets and biomarker candidates


Jerome DauvillierFrederic Burdet

Rory Johnson

Matthew BlowDalit MayLen Pennacchio

Alexandre Sarre

Keith Harshman

Rudi, Miki, Iole, Razan, Christine, Moh

Enhancers – Key information processing units within genome


Approx 1 million enhancers in all human cells (number increasing)Deregulation of enhancers = Disease

Most disease associated genetic variation occurs within them


Enhancer associated lncRNAs are emerging as integral functional components

Modulate chromatin loops/genome topology



Collaborate with transcriptional activators



Evict transcriptional repressors – Promoter pause release


-cis vs –trans activity of enhancer associated lncRNAs

Enhancer associated lncRNAs diversify enhancer function


Identification of bona fide developmental cardiac enhancers

• 3,000 candidate cardiac enhancers were identified in the embryonic heart at E11.5

• 130 were tested in a transgenic reporter assay in vivo (E11.5)

• 63% of these enhancers drive reproducible reporter gene expression in the developing heart

• We selected 7 bona-fide cardiac enhancers proximal to important cardiac genes


Developmental cardiac enhancers produce ncRNA


mm67 mm77 mm85 mm104 mm130 mm132 mm172

Forebrain p300

Midbrain p300

Limbs p300

Conservation

Heart p300

Enhancer region

Enr

ichm

ent

302302302302302

+RT -RT +RT -RT +RT -RTNkx2.5EpiprofinHesx1mm67mm77mm85mm104mm130mm132mm172

Forebrain Limbs Heart

Forebrain Limbs Heart

RNARNA RNA

E11.5 Micep300 ChIP-Seq

Enhancer associated ncRNAs are dynamically expressed


Rel

ativ

e R

NA

exp

ress

ion

(fol

d ov

er E

9.5)

mm67

*

* * *

*

mm85

* *

Myocardin

** * *

** * *

mm67 mm85 Myocd

d0 d3 d6 d9 d12 d0 d3 d6 d9 d12 d0 d3 d6 d9 d12Rel

ativ

e R

NA

exp

ress

ion

(fo

ld o

ver

d0

)

Oct4Nanog

EomesBrachyury T

Mesp1 GATA4Nkx2.5

Cardiac genes(-MHC; -MHC)

d0 d2-3 d3 d3-d6 d6-12 d>12

Organizedsarcomeres

Embryonicstem cells


Cardiacmesodermalprecursors

Cardiacprecursors

Immaturecardiomyocytes

Maturecardiomyocytes

Enhancer associated ncRNA expression correlates with enhancer activity


mm67

AnnotationUCSC Genes

Enhancer

Phospho-Ser5RNA Pol II


mES

MES

CPCCM

mES

MES

CPCCM

mES

MES

CPCCM

mES

MES

CPCCM

mES

MES

CPCCM

mES

MES

CPCCM

H3K4me3

H3K4me1

H3K36me3

H3K27Ac

H3K27me3


mm67

d0 d3d6 d9d12

MES CPC CMES

Oct4Nanog


d3 d6 d12d0


mm85

AnnotationUCSC Genes

Enhancer

Phospho-Ser5RNA Pol II


mES

MES

CPCCM

mES

MES

CPCCM

mES

MES

CPCCM

mES

MES

CPCCM

mES

MES

CPCCM

mES

MES

CPCCM

H3K4me3

H3K4me1

H3K36me3

H3K27Ac

H3K27me3


Enhancer associated ncRNA expression correlates with enhancer activity

MES CPC CMES

Oct4Nanog


d3 d6 d12d0

mm85

d0 d3d6 d9d12

Enhancer associated ncRNAs are enriched in cardiac progenitor cells


101 102 103 104 105

EmGFP Intensity (a.u.)

1%

d0

SS

C

101 102 103 104 105


29%

d6

SS

C

101 102 103 104 105


23%

d10

SS

C

d0 d6 d10

%* *

Nkx2.5-positivecardiac progenitors

Nkx2.5-positivecardiomyocytes

UndifferentiatedES cells

Specification Differentiation

d6 d10

ES14 RP11-88L12

Nkx2.5 promoter GFP

GFP-positiveGFP-negative

mm67 Myocdmm85

***

**

*

mm67 Myocdmm85Rel

ativ

e R

NA

exp

ress

ion

(fol

d ov

er G

FP

-neg

ativ

e) Day 6 Day 10

Orthologous human enhancers are transcribed and functional


Conservation

Fetal heart

Human orthologof mm67

Adult heart



Enr

ichm

ent30

230

2

Enhancer region

p300


Fetal heart

s

mm67

mm85

mm130

Human orthologsof

s

Adult & Fetal Human Left Ventriclep300 ChIP-Seq


Human fetal heart

(12 weeks of gestation)

Enzymaticdissociation

Differentialplating

Control

Differentiation

Cardiacprecursors

Cardiomyocytes

d7 and d14

Rel

ativ

e R

NA

exp

ress

ion

(fol

d ov

er d

0)

*

*

d0 7 14

Myh6Mesp1

*

*

d0 7 14

Rel

ativ

e R

NA

exp

ress

ion

(fol

d ov

er d

0)mm67 ortholog

**

mm85 ortholog Myocardin

**

d0 7 14d0 7 14 d0 7 14

Orthologous human enhancers are transcribed and functional CPCs

tissue

Gata4Nkx2.5Mef2C

Cx43-MHC-MHC

GAPDHTroponin I

-actinin Nkx2.5


Oct4+

Nanog+

GATA4+

Nkx2.5+

d6ESC CPC

Paired-end RNA-seqpolyA+ RNA; 400-500 x 106 PE reads per

sample

Map reads to mouse genome (mm9)(Tophat)

Ab inition reconstruction(Cufflinks)

Filtering: >200 nucleotides; multiexonic

UCSC mRNAs UCSC lncRNAs Novel lncRNAs

elncRNAs plncRNAs

13.9 %(2608)

8.4 %(1537)

77.7 %(14376)

UCSC mRNA UCSC lncRNA Novel lncRNA

100

102

104

10-2

10-4

FPKM

H3K4me1 H3K27Ac (elncRNAs)

H3K4me3 (plncRNAs)

Novel lncRNA

UCSC lncRNA

236

442

630

477

Global discovery of enhancer-associated lncRNAs during cardiac differentiation


ES MES CPC

H3K4me1 H3K27Ac

H3K4me3 PhastConsVertebrates

ESC

MES

CPC

ESC

MES

CPC

ESC

MES

CPC

Cufflinks Predictionsand enhancer

H3

K27

Ac

H3

K4m

e1

H3

K4m

e3

Ch

rom

atin

5 Kb

mm85

Chr. 11 65460157 - 65466563

UCSC mRNA

3222 up

2881 do

wn

d0 d6

UCSC lncRNA

244 up

297 do

wn

d0 d6

Novel lncRNA

311 up

806 do

wn

d0 d6

0+

1+

2-1

-2

Global discovery of enhancer-associated lncRNAs during cardiac differentiation

Mm85 – Poly(A)+, multi-exonic lncRNA expressed in adult heart


Heart

Kidney

Liver

Lung

Small Intestine

Spleen

Stomach


Cu

ffli

nks

Pre

dic

tio

ns

Str

an

d s

pe

cif

ic

tran

sc

rip

tio

n

+

-

+-

+

-

+-

+

-

+

-

+

-

mm85

Mm85 lncRNA is required for transcription of target gene


mm85mm67 Myocardin Map2k4 Gene / Enhancer ID

Genomic loci

sh mm85-1; sh mm85-2

MM85-Promoter distance381 kbp 138 kbp

Rel

ativ

e R

NA

exp

ress

ion

(fol

d ov

er s

hCon

)mm85 Myocardin Map2k4

* ** *

P19CL6

mm85shRNAi


Rel

ativ

e R

NA

exp

ress

ion

(fol

d ov

er s

hCon

)

mm85 Myocardin Map2k4

**

Cardiomyocytes

mm85Gapmers

mm85mm67 Myocardin Map2k4 Gene / Enhancer ID

Genomic loci

Mm85 GapmeR

MM85-Promoter distance381 kbp 138 kbp

Mm85 lncRNA is required for transcription of target gene

SMAD7-enhancer associated lncRNA is required for its transcription


Heart

Kidney

Liver

Lung

Small Intestine

Spleen

Stomach

Str

and

sp

ecif

ic

tran

scri

pti

on


+-

+-

+-

+-

+-

+-

+

-

Cu

ffli

nks

Pre

dic

tio

ns

SMAD7-lncRNA

SMAD7

ES MES CPC

H3K4me1 H3K27Ac

H3K4me3


Rel

ativ

e R

NA

exp

ress

ion

(fol

d ov

er C

on G

apm

eR)

SMAD7-lncRNA SMAD7

**

SMAD7-lnc SMAD7 Gene / Enhancer ID

Genomic loci

SMAD7-lncRNA GapmeR

SMAD7-lnc-Promoter distance<1 kbp

Cardiac Fibroblasts

SMAD7-lncGapmers

SMAD7-enhancer associated lncRNA is required for its transcription


Summary

Developmental cardiac enhancers generate ncRNAs

Enhancer ncRNA expression correlates with enhancer activity

Enhancer ncRNA expression is conserved in Humans

Identified 630 novel Poly(A)+ and multi-exonic elncRNAs

elncRNA is specifically required for target gene expression

Developmental elncRNAs could represent interesting therapeutic targets for modulating cardiac GRN activity and

providing insight in state of GRN


Nu

mb

er o

f n

ove

l ln

cRN

As

dis

cove

red

Average number of paired-end readsSequencing depth per sample

Ounzain S et al, EHJ, 2014

Ounzain S et al, JMCC, 2014

Why you need to sequence very deep to discover novel lncRNAs...........Typical sequencing depth of most studies in the heart

(50million PE-reads per sample)We sequenced to a depth of at least 300 million PE-reads per sample

(Identified 100s of novel cardiac specific lncRNAs with unique characteristics)

Missed novel lncRNAs at 50mil PE-reads

Detected novel lncRNAs at 50mil PE-reads



Future therapeutic approaches for repair

Differentiation

Reprogramming

iPSC

ESC

CPC

CM

Fibroblasts

DirectReprogramming

CPC

lncRNAs

lncRNAs

lncRNAs


CM

Fibroblasts

DirectReprogramming

2

LncRNA cocktail

CM

CPC

CPC MobilizationCM Proliferation

1

Future therapeutic approaches for repair


Rudi MichelettiTal BeckmannMichael AlexanianIole PezzutoMohamed NemirPedrazzini Lab

@ispiyou@CardiolncRNA


Rory JohnsonRoderic Guigo

Blanche SchroenStephane Heymans

Alexandre Sarre Keith Harshman


Genomic ‘dark matter’ in the heart A clinical treasure trove Samir Ounzain, Ph.D CGRM Seminar...

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Transcript of Genomic ‘dark matter’ in the heart A clinical treasure trove Samir Ounzain, Ph.D CGRM Seminar...