Getting to the heart of genomic dark matter

Getting to the heart of genomic dark matter

Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart specific long non-

coding RNAs.

Samir Ounzain, Ph.D

Pedrazzini LabDepartment of Medicine

Experimental Cardiology UnitUniversity of Lausanne Medical School

Switzerland

@ispiyou@CardiolncRNA

Ounzain S, Micheletti R, Beckmann T, Schroen B, Alexanian M, Pezzuto I, Crippa S, Nemir M, Sarre A, Johnson R, Dauvillier J, Burdet F, Ibberson M, Guigó R, Xenarios I, Heymans S, Pedrazzini T. Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel

heart-specific long non-coding RNAs. Eur Heart J. 2014 Apr 30.

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

Functions in the heart are poorly characterized

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

Long non-coding RNAs – Characteristics and mechanisms of action

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

Characterizing 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

mR

NA

s

UC

SC

ln

cRN

As

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 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

Cufflinks

Pred

ictio

ns

+

-

+

-

+

-

+

-

Heart

Testis

Kidney

Liver

Str

and

sp

ecif

ic

tran

scri

pti

on

Lung

Stomach

PhastConsVertebrates

+

-

+

-

Novlnc6

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 m

ass

/ BW

LV 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

LVID

; s

LV v

olu

me;

sLV

ID;

dLV

vo

lum

e; d

LVP

W;

dLV

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

Novel lncRNAs are highly correlated with cardiac physiological traits

Myocardialinfarction

Borderzone

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

Novel lncRNAs are associated with cardiac specific active enhancers

H3K

4me1

H3K

4me3

H3K

27A

cH

3K27

me3


Heart

Kidney

Liver

Spleen

Testis

Cufflinks

Pred

ictio

ns

Heart

Kidney

Liver

Spleen

Testis

Heart

Kidney

Liver

Spleen

Testis

Heart

Kidney

Liver

Spleen

Testis

Novlnc6

Enhancer

Inferring functions for novel lncRNAs based on chromatin state transitions

Embryonicstem cells

Mesodermalprecursors

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: 206Paige SL et al. 2012. Cell 151: 221

Embryonicstem cells

Mesodermalprecursors

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

Based on Wamstad JA et al. 2012. Cell 151: 206Paige SL et al. 2012. Cell 151: 221

Inferring functions for novel lncRNAs based on chromatin state transitions

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

Novel lncRNAs are associated with cardiac specific chromatin state clusters

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

- 5.70

- 4.00

- 2.00

0.00

2.00

4.00

7.25

Down Unchanged Up

1

3

23

24

28

18

Pearsonresidual

20

27

2

2526

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

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

1Novlnc6

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

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

Rudi MichelettiTal BeckmannMichael AlexanianIole PezzutoStefania CrippaMohamed NemirPedrazzini Lab

@ispiyou@CardiolncRNA

Jerome DauvillierFrederic BurdetMark IbbersonIoannis Xenarios

Rory JohnsonRoderic Guigo

Blanche SchroenStephane Heymans

Alexandre Sarre Keith Harshman

Getting to the heart of genomic dark matter

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