Upload
eustacia-burke
View
225
Download
0
Tags:
Embed Size (px)
Citation preview
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