Moderator - hematologiecongres.nl · Moderator . Pieter Sonneveldt. 1st author / speaker . Frank...
Transcript of Moderator - hematologiecongres.nl · Moderator . Pieter Sonneveldt. 1st author / speaker . Frank...
Moderator Pieter Sonneveldt
1st author / speaker Frank Grosveld
Co-authors
Ralph Stadhouders, Elaine Dzierzak, Kerstin Wendt Alireza Ghamari
Hematopoietic development, Stem cells,
Transcription 3D
Belangenverklaring In overeenstemming met de regels van de Inspectie van de Gezondheidszorg (IGZ)
Naam: Frank Grosveld Organisatie: ErasmusMC
☐
☐
Type van verstrengeling / financieel belang Naam van commercieel bedrijf
Ontvangst van subsidie(s)/research ondersteuning: X
Ontvangst van honoraria of adviseursfee: X
Lid van een commercieel gesponsord ‘speakersbureau’: X
Financiële belangen in een bedrijf (aandelen of opties): Harbour Antibodies BV
Andere ondersteuning (gelieve te specificeren): NWO, NGI, EU
Wetenschappelijke adviesraad: Clinical Sciences Center UK; Cell Biology Wellcome Institute Edinburgh UK.
Ik heb geen 'potentiële' belangenverstrengeling
Ik heb de volgende mogelijke belangenverstrengelingen:
Molecular Control of Erythropoiesis EU Marie Curie Research Training Network Eurythron
The search to find therapeutics to increase fetal hemoglobin expression in patients using defined molecular targets?
Erythroid development
Driving erythropoiesis…? Driving erythropoiesis: the LDB1-complex
LDB1-complex: essential for
hematopoietic developement
LDB1-complex: main regulator of terminal
erythroid differentiation
Activates the late erythroid transcriptional
program (e.g. Globins, Membrane proteins)
LDB1-complex: important for looping
regulatory regions to the gene
Erythropoiesis
Primitive Definitive Progenitors HSC
Hematopoietic specification occurs multiple times in the embryo
Transient Permanent
ES/iPS cell differentiation No Adult HSC yet
Dzierzak and Speck, Nat Imm, 2008
Hemangioblast development
ESC
MESODERM
HEMANGIOBLAST
HEMOGENIC ENDOTHELIUM
PRIMITIVE ERYTHROID PROGENITORS
DEFINITIVE HEMATOPOIETIC PROGENITORS
ENDOTHELIUM
SCL
GATA2 LDB1
Embryonic stem cells to embryonic bodies (ESC/Ebs) system
D4 EBs
Magnetic-activated cell sorting (MACS)
Flk1+ cells (BL-CFC = Hemangioblast)
ESC/EBs system is a perfect model for yolk sac hematopoiesis
It provides access to relatively large number of precursor at early stages of development
Hemangioblast is an undifferentiated mesodermal precursor cell that gives rise to endothelial and hematopoietic cells
EBs Differentiation D4 D4.5 Day0 D2
Flk1+ population within EBs
Flk1+ population
D6
Flk1
PD
GFR
α
D4 EBs
BL-CFCs (Flk1+PDGFRα-)
CVPs (Flk1+PDGFRα+)
Hemogenic endothelium
Gata-switching
GATA2
LDB1
GATA1
LDB1
Ldb1complex Ldb1complex
Gata2 Gata1
Expr
essio
n le
vel
HSC Erythroid progenitor cells
Hemangioblast Erythroid progenitor cells Hemogenic endothelium
Dynamics of LDB1 complex (GATA switch)
GATA2
LDB1
BL-CFCs (Flk1+ cells)
Erythroid progenitor cells
vs
definitive cells
GATA1
LDB1
ChIP-sequencing for LDB1 in Flk1+ cells
Genome-wide mapping of sequences bound by LDB1
List of candidate genes regulated by LDB1 in Flk1+ cells
What is the genetic program controlled by LDB1 in BL-CFCs?
Flk1+ cells wt
Flk1+ cells Ldb1-/- Genome-wide transcripts RNA-seq
Role of LDB1regulates essential hematopoietic transcription factors
Signal transduction pathways regulated by LDB1
A. Mylona et al. Blood. 2013; 121(15):2902-2013
FACS analysis Flk1+ PDGFRa+ : CVPs
Flk1+ PDGFRa- : BL-CFCs CD41+: Hematopoietic progenitors
CD31+ : Endothelial cells
Add Inhibitor
Add Inhibitor
Testing the involvement of MAPK/ERK and IGFR1 pathways in development by inhibition at mesodermal or hemangioblast stage
MAPK/ERK pathway inhibition (mesodermal stage) – FACS at D4 EBs
Inhibition of MAPK-ERK pathway at the mesodermal stage results in a reduction of BL-CFCs and increase in CVPs
Flk1+ PDGFRa- : BL-CFCs Flk1+ PDGFRa+ : CVPs
cardiac
hemogenic
MAPK/ERK pathway inhibition (mesodermal stage) – FACS at D6 EBs
CD41+: Hematopoietic progenitors
CD31+ : Endothelial cells
Inhibition of MAPK-ERK pathway at the mesodermal stage results in a reduction of hematopoieitic progenitors and increase in endothelial cells
A B
Roles of MAPK/ERK and IGFR1 pathways in early hematopoiesis
GATA2 GATA1
LDB1 TAL1
Genes encoding for key transcriptional regulators of hematopoietic development (Runx1, Gata1, Gata2, Tal1, PU.1,
Lmo2, Hex, Ets1 ..ect)
At the hemangioblast stage LDB1, TAL1 and GATA2 regulate a genetic program for the emergence of hematopoietic cells from
hemangioblast
Ldb1 complex target genes in Flk1+ cellsFunctions enrichment
differentiation of blood cells 10.21quantity of blood cells 9.735quantity of hematopoietic progenitor cells 9.197differentiation of phagocytes 8.042differentiation of myeloid cells 7.636Cardiovascular System Development and Function 7.476hematopoiesis of blood cells 7.332differentiation of hematopoietic progenitor cells 7.206Cellular Movement 7.188development of cardiovascular system 7.032differentiation of red blood cells 6.971myelopoiesis of hematopoietic cells 6.136
-log(p-value)
Cytoskeleton organization, microtubule dynamics
Cardiac and endothelial development
Cellular growth and proliferation, Cardiac and endothelial development
Target genes for LDB1, TAL1, GATA2 in Flk1+ cells
TAL1 target genes (Flk1+ cells) LDB1 target genes (Flk1+ cells) GATA2 target genes (Flk1+ cells) LDB1 complex target genes
LDB1 complex in hemangioblast controls the hematopoietic program
LDB1 complex : overlapping binding events in Flk1+ cells vs MEL cells
FOX GATA2
LDB1
GATA2
LDB1
LDB1 complex : target genes in Flk1+ cells vs MEL cells
Endothelial and cardiac development
LDB1 complex target genes in MEL cells LDB1 complex target genes in Flk1+ cells
Differentiation of hematopoietic cells (key hematopoietic transcriptional factors)
Proliferation of blood cells, morphology of red
blood cells
FOX GATA2
LDB1
GATA2
LDB1
Hemangioblast stage Proerythroblast stage
GATA1
LDB1
GATA1
LDB1
Repression of cardiac program Commitment to endothelial lineage
Commitment to hematopoietic lineage
Proerythroblast differentiation
intermediate stage of hematopoietic development
(Low GATA1 expression)
(High GATA1 expression) (No GATA1 expression)
Conclusion
• LDB1, TAL1 and GATA 2/1 play a different and stage-specific function during hematopoietic development
• At hemangioblast stage, LDB1, TAL1 and GATA2 work independently to control functions essential for hemangioblast development, however, as a complex, they regulate genes encoding for key transcriptional regulators of hematopoietic development
• Existence of a genomic wide GATA-switch between hemangioblast and proerythroblast stage
• Ldb1 complex directly controls signaling pathways essential for hemangioblast development
• GATA2 plays an essential role in the emergence of hematopoietic cells
What factors drive HCS generation?
HSCs and clusters generated first in aorta-vasculature Dieterlen-Lievre, 1975, Medvinsky, Cell, 1996, de Bruijn et al, EMBO, 2000
Specialized hemogenic endothelium Jaffredo et al, 2005, Development, Chen et al, 2009, Nature
Limited window of developmental time Zovein et al. Cell Stem Cell, 2008
Generating definitive /adult hemapoietic stem cells
Ly6A GFP marks HSCs and hemogenic endothelial cells
Ly6A
Ly6A GFP transgenic mice
Ma, Stem Cells, 2002 De Bruijn, Immunity, 2002
0
25
50
75
100
1 ee 2 ee % re
cipi
ents
repo
pula
ted
GFP- GFP+
0/6 0/4
3/4 4/5
AGM
0
10
20
30
40
50
1 ee 2 ee
1/5
0/5
3/9
0/8 %
reci
pien
ts re
popu
late
d Aorta/mesenchyme
GFP
2.0 % 96.9 %
AGM Ly6A GFP CD31
CD31
Ly6A-GFP
HSCs arise via a natural transdifferentiation of endothelium Boisset et al. Nature, 2010
E10.5, 35sp Ly-6A GFP
e
m
h h
h he
Transcription regulators
Hemogenic Endothelial Cell
Niche growth factors: Hh, BMP4
Flow/stress
HSC fate determination cluster formation loss of tight junctions/adhesion morphologic changes mobilization/migration
Transcription factors such as Runx1, Gata2, Tal1 etc.!
HSC generation is a multi-step dynamic process Hypoxia
What is the genetic program driving endothelial to hematopoietic transition?
Unpublished data
Aorta lumen
H HSC
HEE
Aorta lumen
H HSC
HEE
Ly6Aa-GFP (34sp)
HC: Hematopoietic HSC: Emerging hematopoietic* EC: Endothelial HEC: Hemogenic endothelium*
HC
HEC
Purify the different cell types and determine which genes are expressed differently in which cell type.
Ly6a-gfp CD31 c-kit
HC
HEC EC
HSC
Ly6a GFP
c-ki
t
CD31 gated
HC HSC
EC HEC
Cell sorting & RNAseq of EHT
Progenitors HSC
Dzierzak group
Q1
Q2
Q3
Q4 1.9 5.2 Ly6A
c-Ki
t
EmergingHSCs
Hem
atop
oiet
icce
lls
hemogenicendothelium
endothelialcells
15.1
Gata2 transcription factor - expressed in hemogenic endothelial & emerging hematopoietic cells
Transcription factors - 14 endothelial vs hemogenic endothelial 21 hemogenic endothelial vs emerging hematopoietic cells
Dzierzak group
Genome wide LDB1 binding in Flk1+ cells vs adult hematopoietic progenitor cells
Bone marrow hematopoietic progenitor cells
Flk1+ cells
3,384 5,505 868
Comparative analysis between the different cell types to: Find clues what the difference is between embryonic and adult HSC generation
Find the genes that change while the hemogenic endothelium undergo the transition to HSC
Somatic cell
iPS cell Reprogram
Differentiation
HSC
Transplantation
Patient
HSCs are derived from a special subset of endothelial cells Relatively small set of genes differentially expressed between endothelial, hemogenic endothelial & emerging cells
repair ? ?
Function of the Ldb1 complex: initiation/elongation
2 types of looping events:
-Structural loops (CTCF, Cohesin)
-Functional loops (Ldb1 complex)
LMO Cdk9 E2.2
MTGR1
Eto2
HEB Lyl1 LMO2
E2A Tal1
Ldb1
Ldb1
Gata1
Molecular Control of Erythropoiesis EU Marie Curie Research Training Network Eurythron
The search to find therapeutics to increase fetal hemoglobin expression in patients using defined molecular targets?
The human α- and β-globin loci
The balance of γ/β is determined by a number of factors, e.g.
BCL11a, MYB, KLF1
Human myb gene locus binds LDB1 complex and KLF1
Chromosome Conformation Capture (3C)-seq
Soler et al, G&D 2010
Stadhouders et al, EMBO J 2011
Thongjuea et al. Submitted
•Y-axis: Interaction frequency (=bars with sequence counts)
•X-axis: Genome position
Viewpoint
10kb avg
Illumina
HiSeq2000
• 2 flowcells, 8 lanes each = 16 lanes per run
• 10 different 3C-Seq per lane = 160 viewpoints
• 1.106 sorted cells
The regulatory regions contact the myb gene
The SNPs that increase fetal Hb expression
The SNP lowers LDB1 complex binding and looping to the myb gene
Model of looping in the myb locus
Conclusions • The SNP at -84 in the myb locus affects the binding of the LDB1 complex • The lower level of binding destabilizes the interaction of the regulatory sequence with the gene
Structural factors
• CTCF
• Cohesins
Transcription factors
• Ldb1/Gata1/Fog1/EKLF
• Brg1
• Nuclear receptors: ERα, AR
Structural loops (e.g. locus insulation)
‘Functional’ loops (e.g. enhancer- promoter interactions)
Chromatin folding
Transcriptional Activation
Exclusion by looping
No Transcriptional Activity
From Wallace & Felsenfeld, Curr Opin Genet Dev. 2007 From Palstra et al., Nat Genet. 2003
HiC and T2C
Cleavage of cohesin
Cleavage of cohesin
Structural loops
Functional loops within Structural loops
Conclusions
• TADs (topological domains) are stable and maintained in different tissues
• CTCF appears to have different functions, localization versus interaction
• Interactions within domains are dynamic
• The domain structures can be solved at high resolution
• High resolution allows the analysis of the function of individual complexes
Acknowledgments
NWO, NIH (NIDDK), NGI
Reinier van der Linden Gert van Cappellen Wilfred van Ijcken
Eric Soler Ruud Jorna Petros Kolovos Ralph Stadhouders Tobias Knoch Charlotte Andrieu Kerstin Wendt Andrea Martella Jessica Zuin Athina Mylona Alireza Ghamari Mariette van de Corput Ernie de Boer
ESI Elaine Dzierzak Stem Cell Institute Edinburgh Catherine Robin Hubrecht laboratory