Epigenetic regulation of cardiogenesis

INSERM UMR 910 Faculté de Médecine La Timone , Marseille , France Michel Pucéat Cours Réseau International des Instituts Pasteur 19 october 2016

Epigenetic regulation of cardiogenesis

Nuclear structure

Darkly stained and condensed Transcriptionally silent and silences adjacent genes

Present at centromeres and telomeres

Repressive structure can be propagated

Euchromatic gene placed in heterochromatin is repressed

Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-33

Chromatin fibers

+ charged N termini (bind DNA on neigboring nucleosomes)

highly acetylated core histones

(especially H3 and H4)

30 nm chromatin fiber

11 nm (beads)

• HIGH level of histone H1 • Reduced level of histone H1

• NO gene transcription • Gene transcription possible

Histone modifications

Histone modifications

Modified from Matharu et al Plos Gen 2015

Chromatin is organized in three dimensions

CTCF CTCF

LAD

euchromatin

Unicellular ancestor of metazoan

Metazoan

Complex gene repertoire involved in multicellular functions

Evolution is not corelated with new genes

Shift in genomic regulatory capabilities

Genetics does not fully account for cell lineage determination during embryogenesis

Enhancer diversity , Distal Enhancer-promoter loops: major evolutionnery innovation

Histones H3/H4 Modifications

CONSERVED

Transcription factors

Science 2006 311, 96 Davidson and Erwin Nature rev gen 2012, 13,233 Lenhart etal Genome Biol 14, R15, 2013, Fairclough et al Nature 2013 424, 147 Levine et al Cell 2011 144, 324 Buldger and Goudine Cell 2016, 165 1124 Sebe-Pedros et al

De Witt, De Laat, 2012

Technologies to be used to decrypt epigenetic regulation of gene transcription

BMP2 FGF TGFβ Wnt/βCat ⊥ Epiblast

Definitive Endoderm

Posterior

Anterior

Goosecoid Foxa2

Brachyury

Primitive Streak

Brachyury Flk1, FoxH1

Sox17 Hex

Cardiac Mesoderm

Wnt non Canonical

ICM

ESC

in vitro

Primitive Endoderm

Visceral Endoderm

Mesp1

BMP Cerberus Activin A

BMP2 FGF

Wnt-βCat

Sox17 Hex

Sox17 Hex

Notch ⊥ Wnt-βCat ⊥

Ectoderm

E5 E6 E6.5 E7.5 E13.5 E7.75 E8.25 E4 E4.25

FGF Notch Wnt/βCat ⊥

E10.5

SM-MHC SMA

Atrial Cardiomyocyte Atrioventricular Nodal Cell Endothelial Cell Right Ventricular Cardiomyocyte Sinostrial Nodal Cell Smooth Muscle Cell Vascular Smooth Muscle Cell

HCN4

Atrial and Left Ventricular Cardiomyocyte Smooth Muscle Cell

Aorta smooth muscle cells Autonomous nervous system

Epicardium Coronary vessels Fibroblasts

BMP2

FGF

Endocardium

Tbx18

Wt1

FHL

SHL

Nkx2.5 FGF8/10 Hand2 Isl1 Mef2c Tbx1 Tbx20 Nkx2.5 Hand1 Tbx5

Extraembryonic Ectoderm BMP

BMP2

Cardiac Neural Crest

Sox17 Hex

Sox17 Hex

cTNT

The cardiac developmental pathways

GATA5,Cx37 valves

1st Heart Field (Cardiac Crescent) 2nd Heart Field (SHF)

Modified from Vincent and Buckingham, 2010

Main steps of Heart morphogenesis in the mouse embryos

P A

Weber et al Birth Defects Res A Clin Mol Teratol. 2015.

Modified histone marks and cardiac congenital defects

Liu et al epigenomics 2015

Cell lineages ?

Physiological

Pathological (cohesinopathies, laminopathies)

?

Gen

Biological questions and clinical relevance

Genome- Wide Association Studies

Not a single TRANSCRIPTION FACTOR is specific of a cell lineage

Oct-4 targets the cohesin complex and change 3D chromatin structure to specify cardiogenesis: from basic science to a rare disease

OCT4 a reprogramming transcription factor more than a stem cell marker

• A gatekeeper for ICM/ES cell pluripotency and a reprogramming factor • A key player in early cardiogenesis • An inducer of MesP1, and in turn of EMT of epiblast cells into mesodermal

cells

E7.5

OCT4 SOX2

NANOG

Pluripotency network

SALL4

A siRNA approach to down regulate Oct-4 in the blastocyst

Pseudopregnant mouse

96h cultured blastocyst

Cardiac defects in embryos developed from Oct-4 downregulated blastocysts

Zeinedinne et al Dev Cell 2006

Embryonic stem cells: a powerful cell model to carry out mechanistic studies in early embryonic development

Mouse Human

Oct-4, a dual function conserved in Human cells

OCT4 increase in expression gives rise to mesendodermal cells

Oct-4 improves cardiac differentiation of HUES cells within embryoid bodies

Sox2/17 enhancer /promoter

Chromatin Anti-Oct4

Oct4

An increased level of Oct-4 leads to a loss in its interaction with Sox2 enhancer/promoter and a gain in its interaction with Sox17 enhancer/promoter

Stefanovic et al J Cell Biol 2009

BMP2 Wnt3a

The cardiogenic scenario…

How Oct-4 switches from Sox2 to Sox17 regulatory regions ?

The search for Oct4 targets: ChIP on chip analysis

OCT4 binds SALL4 promoter

Abboud, Moore-morris et al Nature Com 2015

SALL4 mediates the switch of OCT4 from Sox2 to Sox17 enhancers

Abboud, Moore-morris et al Nature Com 2015

SALL4 is required for the cardiogenic function of OCT4

Abboud, Moore-morris et al Nature Com 2015

The Epigenetic code Resolution of bivalent chromatin domains during ES cell differentiation.

Schuettengruber B , and Cavalli G Development 2009;136:3531-3542

OCT4 induced changes in epigenetic marks on Soxs promoters

Abboud, Moore-morris et al Nature Com 2015

SALL4-targeted Polycombs in OCT4 switch from Sox2 to Sox17 enhancers

se

Sequential ChIP

Abboud, Moore-morris et al Nature Com 2015

SALL4-targeted Polycombs in OCT4 switch from Sox2 to Sox17 enhancers

Abboud, Moore-morris et al Nature Com 2015

Cohesin and CTCF organise higher order chromatin structure in the genome

Dorsett, 2011

Cohesin

OCT4 targets the cohesin complex

Abboud, Moore-morris et al Nature Com 2015

Chromosome conformation capture (3C) reveals Sox2/Sox17 enhancers interactions

Abboud, Moore-morris et al Nature Com 2015

Chromosome conformation capture (3C) reveals Sox2/Sox17 enhancers interactions

Abboud, Moore-morris et al Nature Com 2015

Abboud, Moore-morris et al Nature Com 2015

Cohesin is required for Oct-4 mediated cardiogenic function

Abboud, Moore-morris et al Nature Com 2015

Abboud, Moore-morris et al Nature Com 2015

Mutations in the cohesin complex and partner proteins lead to a cohesinopathy

Chatfield et al Am J Gen 2012

E17.5

E17.5

Haploinsufficiency of Nipbl exacerbates the phenotype of Nk2.5 haploinsufficient mice and leads to cardiac dilatation

Nutrition and epigenetics: What is the link ?

Vitamin D : a proof of concept ?

Fetahu et al Frontiers in physiology 2014

Vitamin D deficiency is becoming a public health problem ( malnutrition, obesity, in North Europe countries with short days… (WHO) )

Vit D-R : In Cis-modulatory modules enriched in TF binding sites (superenhancers) Enriched in H3K4me1, K4me2, H3K27ac ( enhancers marks) Co-occupied by cohesin or cohesin:CTCF

VitD starvation in female mice lead to cardiac hypertrophy in the offsprings

Acquisition and Maintenance of locus –specific 3D configuration of chromatin is key for normal cardiac cell lineage determination and possibly for adult cardiac homeostasis

Haploinsuficiency of cohesin complex genes leads to developmental diseases and accelerated ageing in Cornelia de Lange patients Fetal life may prime for healthy ageing or cardiovascular pathologies

Haploinsuficiency in other players in the 3D structure of chromatin such as mediators also leads do developmental disease with cardiac malformations ( FL Lujan syndrome)

Take Home messages

Environmental factors including nutrition affects epigenetic regulation of gene transcription and are a source of developmental diseases

THANK YOU !

Former students: Nesrine Abboud Sonia Stefanovic Dana Zeinedinne Corinne Grey

Thomas Moore-Morris Imen Jebeniani Batoul Fahrat Fanny Boulet Eva Seipelt Julien Boissadier

Sylvia Evans, UCSD, La Jolla, CA

Valerie Cormier Daire Necker Hospital Paris

Henry Yang Bioinformatics, Biopolis , Singapore