Run

x1-V

E+

Run

x1+

VE

+C

D41

-

Run

x1+

VE

+C

D41

+

Run

x1+

VE

-CD

41+

Supplementary Figure 1

Supplementary Figure 1: Validation of cell populations for gene expression assays. Marker gene expression was analysed in populations 1, 2, 3 and 4 using quantitative RT-PCR. The following genes were tested and displayed expected expression patterns: Runx1, Gata1, b Major, B H1, Gfi1b and Cldn5.

Run

x1-V

E+

Run

x1+

VE

+C

D41

-

Run

x1+

VE

+C

D41

+

Run

x1+

VE

-CD

41+

Peak Numbers Sequence motifs

Population 2 67 Max, JunD, CRE, Runx

Population 3 51 Runx

Population 4 747 Runx, Gata, ETS,JunD

GO overrepresentation (p value < 0.001)

Population 2 Chitin binding, membrane, ABC transporters

Population 3 Chitin binding, membrane, ABC transporters, cytosceleton

Population 4 Phosphoprotein, acetylation, transcription regulation, myeloid cell differentiation, endomembrane system

Supplementary Figure 2

A

B

Supplementary figure 2:Motif and Gene Ontology analysis of Runx1 ChIP-Seq results for individual populations. A) Overrepresented motifs in Runx1 ChIP-Seq peaks from populations 2, 3 and 4. Shown is the number of peaks found for each population as well as the names of overrepresented motifs.B) Gene ontology functional enrichment analysis for genes next to Runx1 ChIP-Seq peaks from populations 2, 3 and 4. Shown are all Gene Ontology categories enriched with a p value < 0.001.

Supplementary Figure 3

Supplementary figure 3:Haematopoietic potential of sorted populations.In vitro hematopoietic differentiation potential of flow-sorted populations was assayed by culture on OP9 feeder cells. 500 cells from each subsets were seeded on OP9 feeder cells in the presence of SCF, Flt3L and IL-7. After 14 days of culture, the cells were harvested and analyzed by FACS for the expression of Mac1 and CD19 to check the production of myeloid and lymphoid lineages.

Myeloid potential

Lymphoid potential

Runx1-VE-cadherin+ - -Runx1+VE-cadherin+CD41- ++ ++Runx1+VE-cadherin+CD41 +++ +++Runx1+VE-cadherin-CD41+ + +

Supplementary Figure 4

Supplementary Figure 4: Validation of Runx1 ChIP assays. Runx1 ChIP assays in populations 2, 3 and 4 were validated using quantitative PCR with primer pairs for a region known to be bound by Runx1 (+23 kb, blue bars) and a negative control region (+30 kb, green bars). Primer pairs are the same as in Wilson et al (Blood. 2009. 113(22): 5456-65)

0

1

2

3

4

5

6

7

8

Runx1+VE-cadherin+/CD41-

Runx1+VE-cadherin+/CD41+

Runx1+VE-cadherin-/CD41+

Runx1 +23kb (positive region)Runx1 +30kb (negative region)

Fo

ld E

nri

chm

ent

Supplementary Figure 5

Supplementary Figure 5: Gene ontology (GO) analysis for Runx-1 bound correlated (A) and anti-correlated (B) genes. Shown are the full GO trees generated by the FATIGO program as part of the BABELOMICS suite of analysis tools (Medina et al, Nucleic Acids Res. 2010. 38(Web Server issue):W210-3). The degree of shading corresponds to the significance of overrepresentation.

A B

Supplementary Figure 6

Supplementary Figure 6: The CD41 promoter is bound by both the Scl/Gata2/Fli1 triad and Runx1 in haematopoietic progenitor cells. Raw ChIP-Seq read data were transformed into density plots and displayed as custom tracks within the UCSC genome browser above the UCSC track for gene structure (data obtained from Wilson et al Cell Stem Cell. 2010. 7(4):532-44). All four transcription factors show very significant enrichment over the CD41 promoter region.

Supplementary Figure 7

~L

SN

PE

HF

LH

FE

S(Y

S)

7sp(Y

S)

~4sp

(YS

)

VE-cadherin

CD

41

Su

pp

lemen

tary Fig

ure 7:

Tim

ecou

rse analysis o

f CD

41 expressio

n in

early mo

use em

bryo

s.C

D41

and V

E-cadherin e

xpressio

n were ana

lysed by flow

cytom

etry betwee

n E7.0 a

nd E8.25 of m

ouse embryonic

developm

ent. S

even distinct developm

ental stages b

etween

E7

.0 and E

8.5 w

ere analysed. A

distinct CD

41 bright

population only becom

es clearly separab

le by FA

CS

at the E

S stag

e. LS =

late streak sta

ge, NP

= n

eural plate sta

ge, EH

F =

early head fold stage, LH

F =

late head fold stage, E

S =

early somite

pair stage (0-1 sm

ites pairs),

4sp = a

pproximate

ly 3-4 som

ite pair stage, 7sp =

7 som

ite pair stage. T

he Run

x1+

/- and Runx1

-/- embryos an

alyzed

in Figure 5 are appro

ximately equivalen

t to the N

P stag

e. Ea

ch FA

CS

plot of LS

-LHF

stage embryos is from

8-10 poo

led stage-m

atched whole em

bryos. Ea

ch FA

CS

plot from

somite pair sta

ge emb

ryos is fro

m 4-6 poo

led stage-

matche

d yolk sacs. All em

bryos are from IC

R fem

ales crossed w

ith GF

P-IC

R m

ales to avoid

contamination of

maternal cells.

Unknown3

Runx1

Gata2

Unknown1

Unknown2

Supplementary Figure 8

Supplementary figure 8:De novo motif discovery on Runx1 peaks from all 3 populations analysed by ChIP-Seq.In addition to Runx1 and Gata consensus motifs, a novel motif (unknown 1) was identified both as a shorter 15 bp motif and also embedded in a longer 27 bp motif (both the motif and its reverse complement were found by MEME). The same motif was also recently found overrepresented in regions bound by GABPα in human blood stem/progenitor cells (Yu et al BLOOD 2011). Three other motifs (Unknown 2-4) were also found over-represented, with motif Unknown 4 representing the typical false-positive long motifs found by MEME, and caused by repetitive DNA sequences.

Unknown4