epigenetic regulation of cardiogenesis
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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
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