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