Hematopoietic development, Stem cells, Transcription 3D

Transcription

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2 Hematopoietic development, Stem cells, Transcription 3D Moderator Pieter Sonneveldt 1st author / speaker Frank Grosveld Co-authors Ralph Stadhouders, Elaine Dzierzak, Kerstin Wendt Alireza Ghamari

3 Belangenverklaring In overeenstemming met de regels van de Inspectie van de Gezondheidszorg (IGZ) Naam: Organisatie: Frank Grosveld ErasmusMC Ik heb geen 'potentiële' belangenverstrengeling Ik heb de volgende mogelijke belangenverstrengelingen: Type van verstrengeling / financieel belang Ontvangst van subsidie(s)/research ondersteuning: Ontvangst van honoraria of adviseursfee: Lid van een commercieel gesponsord speakersbureau : Financiële belangen in een bedrijf (aandelen of opties): Andere ondersteuning (gelieve te specificeren): Wetenschappelijke adviesraad: Naam van commercieel bedrijf X X X Harbour Antibodies BV NWO, NGI, EU Clinical Sciences Center UK; Cell Biology Wellcome Institute Edinburgh UK.

4 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?

5 Erythroid development

6 Driving erythropoiesis: erythropoiesis? the LDB1-complex Erythropoiesis 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

7 Hematopoietic specification occurs multiple times in the embryo Primitive Definitive Progenitors HSC Transient ES/iPS cell differentiation Permanent No Adult HSC yet Dzierzak and Speck, Nat Imm, 2008

8 Hemangioblast development ESC GATA2 MESODERM LDB1 HEMANGIOBLAST SCL ENDOTHELIUM HEMOGENIC ENDOTHELIUM PRIMITIVE ERYTHROID PROGENITORS DEFINITIVE HEMATOPOIETIC PROGENITORS

9 Embryonic stem cells to embryonic bodies (ESC/Ebs) system 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 D4 EBs Magnetic-activated cell sorting (MACS) Flk1+ cells (BL-CFC = Hemangioblast) Hemangioblast is an undifferentiated mesodermal precursor cell that gives rise to endothelial and hematopoietic cells

10 Flk1+ population within EBs Day0 D2 D4 D4.5 EBs Differentiation D6 Flk1+ population D4 EBs CVPs (Flk1+PDGFRα+) PDGFRα Flk1 BL-CFCs (Flk1+PDGFRα-)

11 Gata-switching Hemogenic endothelium HSC Erythroid progenitor cells Gata2 Gata1 Expression level Ldb1complex LDB1 GATA2 LDB1 Ldb1complex GATA1 Hemangioblast Hemogenic endothelium Erythroid progenitor cells Dynamics of LDB1 complex (GATA switch)

12 LDB1 GATA2 vs LDB1 GATA1 BL-CFCs (Flk1+ cells) definitive cells Erythroid progenitor cells

13 What is the genetic program controlled by LDB1 in BL-CFCs? ChIP-sequencing for LDB1 in Flk1+ cells Flk1+ cells wt Flk1+ cells Ldb1-/- Genome-wide mapping of sequences bound by LDB1 Genome-wide transcripts RNA-seq List of candidate genes regulated by LDB1 in Flk1+ cells

14 Role of LDB1regulates essential hematopoietic transcription factors Signal transduction pathways regulated by LDB1 A. Mylona et al. Blood. 2013; 121(15):

15 Testing the involvement of MAPK/ERK and IGFR1 pathways in development by inhibition at mesodermal or hemangioblast stage Add Inhibitor Add Inhibitor FACS analysis Flk1+ PDGFRa- : BL-CFCs Flk1+ PDGFRa+ : CVPs CD41+: Hematopoietic progenitors CD31+ : Endothelial cells

16 MAPK/ERK pathway inhibition (mesodermal stage) FACS at D4 EBs cardiac hemogenic Flk1+ PDGFRa- : BL-CFCs Flk1+ PDGFRa+ : CVPs Inhibition of MAPK-ERK pathway at the mesodermal stage results in a reduction of BL-CFCs and increase in CVPs

17 MAPK/ERK pathway inhibition (mesodermal stage) FACS at D6 EBs A CD41+: Hematopoietic progenitors B CD31+ : Endothelial cells Inhibition of MAPK-ERK pathway at the mesodermal stage results in a reduction of hematopoieitic progenitors and increase in endothelial cells

18 Roles of MAPK/ERK and IGFR1 pathways in early hematopoiesis

19 LDB1 TAL1 GATA2 GATA1 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

20 Target genes for LDB1, TAL1, GATA2 in Flk1+ cells Cytoskeleton organization, microtubule dynamics Cardiac and endothelial development Cellular growth and proliferation, Cardiac and endothelial development Ldb1 complex target genes in Flk1+ cells Functions enrichment -log(p-value) 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 differentiation of blood cells quantity of blood cells quantity of hematopoietic progenitor cells differentiation of phagocytes differentiation of myeloid cells Cardiovascular System Development and Function hematopoiesis of blood cells differentiation of hematopoietic progenitor cells Cellular Movement development of cardiovascular system differentiation of red blood cells myelopoiesis of hematopoietic cells 6.136

21 LDB1 complex : overlapping binding events in Flk1+ cells vs MEL cells FOX LDB1 GATA2 LDB1 GATA2

22 LDB1 complex : target genes in Flk1+ cells vs MEL cells LDB1 complex target genes in MEL cells LDB1 complex target genes in Flk1+ cells Endothelial and cardiac development Differentiation of hematopoietic cells (key hematopoietic transcriptional factors) Proliferation of blood cells, morphology of red blood cells

23 Hemangioblast stage (No GATA1 expression) intermediate stage of hematopoietic development (Low GATA1 expression) Proerythroblast stage (High GATA1 expression) LDB1 GATA1 LDB1 GATA1 LDB1 LDB1 FOX GATA2 GATA2 Repression of cardiac program Commitment to endothelial lineage Commitment to hematopoietic lineage Proerythroblast differentiation

24 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

25 Generating definitive /adult hemapoietic stem cells 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 What factors drive HCS generation?

26 Ly6A GFP marks HSCs and hemogenic endothelial cells Ly6A GFP transgenic mice Ly6A CD31 AGM 96.9 % 2.0 % GFP % recipients repopulated AGM 4/5 3/4 0/6 0/4 1 ee 2 ee Ly6A GFP % recipients repopulated Aorta/mesenchyme 3/9 1/5 0/5 0/8 1 ee 2 ee GFP - GFP + Ma, Stem Cells, 2002 De Bruijn, Immunity, 2002

27 HSCs arise via a natural transdifferentiation of endothelium Boisset et al. Nature, 2010 CD31 Ly6A-GFP E10.5, 35sp Ly-6A GFP

28 HSC generation is a multi-step dynamic process Hypoxia h h h he Flow/stress e Niche growth factors: Hh, BMP4 m Hemogenic Endothelial Cell Transcription regulators Transcription factors such as Runx1, Gata2, Tal1 etc.! HSC fate determination cluster formation loss of tight junctions/adhesion morphologic changes mobilization/migration

29 What is the genetic program driving endothelial to hematopoietic transition? Ly6Aa-GFP (34sp) Purify the different cell types and determine which genes are expressed differently in which cell type. Aorta lumen HC H HSC E HEC HC: Hematopoietic HSC: Emerging hematopoietic* EC: Endothelial HEC: Hemogenic endothelium* Unpublished data

30 Cell sorting & RNAseq of EHT Ly6a-gfp CD31 c-kit HC HSC EC HEC CD31 gated HC HSC Progenitors HSC c-kit EC HEC Ly6a GFP Dzierzak group

31 Transcription factors - 14 endothelial vs hemogenic endothelial 21 hemogenic endothelial vs emerging hematopoietic cells Q1 Q2 c-kit Hematopoietic cells Emerging HSCs 15.1 Q3 endothelial cells hemogenic endothelium Q4 1.9 Ly6A 5.2 Gata2 transcription factor - expressed in hemogenic endothelial & emerging hematopoietic cells Dzierzak group

32 Genome wide LDB1 binding in Flk1+ cells vs adult hematopoietic progenitor cells Flk1+ cells 3,384 5, Bone marrow hematopoietic progenitor cells 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

33 HSCs are derived from a special subset of endothelial cells Relatively small set of genes differentially expressed between endothelial, hemogenic endothelial & emerging cells Somatic cell repair?? Reprogram ips cell Transplantation Differentiation Patient HSC

34 Function of the Ldb1 complex: initiation/elongation LMO E2.2 Lyl1 E2A Tal1 MTGR1 Eto2 Ldb1 Ldb1 Cdk9 LMO2 HEB Gata1 2 types of looping events: -Structural loops (CTCF, Cohesin) -Functional loops (Ldb1 complex)

35 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?

36 The human α- and β-globin loci The balance of γ/β is determined by a number of factors, e.g. BCL11a, MYB, KLF1

37 Human myb gene locus binds LDB1 complex and KLF1

38 Chromosome Conformation Capture (3C)-seq Viewpoint Illumina 10kb avg HiSeq2000 Y-axis: Interaction frequency (=bars with sequence counts) X-axis: Genome position Soler et al, G&D 2010 Stadhouders et al, EMBO J 2011 Thongjuea et al. Submitted 2 flowcells, 8 lanes each = 16 lanes per run 10 different 3C-Seq per lane = 160 viewpoints sorted cells

39 The regulatory regions contact the myb gene

40 The SNPs that increase fetal Hb expression

41 The SNP lowers LDB1 complex binding and looping to the myb gene

42 Model of looping in the myb locus

43 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

44 Chromatin folding Structural factors CTCF Cohesins Transcription factors Ldb1/Gata1/Fog1/EKLF Brg1 Nuclear receptors: ERα, AR Structural loops (e.g. locus insulation) Exclusion by looping Functional loops (e.g. enhancerpromoter interactions) No Transcriptional Activity Transcriptional Activation From Wallace & Felsenfeld, Curr Opin Genet Dev From Palstra et al., Nat Genet. 2003

45 HiC and T2C

46 Cleavage of cohesin

47 Cleavage of cohesin

48 Structural loops Functional loops within Structural loops

49 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

50 Acknowledgments Eric Soler Petros Kolovos Tobias Knoch Charlotte Andrieu Andrea Martella Athina Mylona Alireza Ghamari Mariette van de Corput Ernie de Boer Ruud Jorna Ralph Stadhouders Kerstin Wendt Jessica Zuin ESI Elaine Dzierzak Stem Cell Institute Edinburgh Catherine Robin Hubrecht laboratory Reinier van der Linden Gert van Cappellen Wilfred van Ijcken NWO, NIH (NIDDK), NGI

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