Transcript - Haematopoietic Stem Cell Lab

Runx1+VE-CD41+
Runx1+VE+CD41+
Runx1+VE+CD41-
Runx1-VE+
Runx1+VE-CD41+
Runx1+VE+CD41+
Runx1+VE+CD41-
Runx1-VE+
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.
Supplementary Figure 1
A
Peak Numbers Sequence motifs
Population 2
67
Max, JunD, CRE, Runx
Population 3
51
Runx
Population 4
747
Runx, Gata, ETS,JunD
B
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:
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 2
Runx1-VE-cadherin+
Runx1+VE-cadherin+CD41Runx1+VE-cadherin+CD41
Runx1+VE-cadherin-CD41+
Myeloid
potential
Lymphoid
potential
++
+++
+
++
+++
+
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.
Supplementary Figure 3
8
Fold Enrichment
7
Runx1 +23kb (positive region)
Runx1 +30kb (negative region)
6
5
4
3
2
1
0
Runx1+
VE-cadherin+/CD41-
Runx1+
VE-cadherin+/CD41+
Runx1+
VE-cadherin-/CD41+
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)
Supplementary Figure 4
A
B
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.
Supplementary Figure 5
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 6
VE-cadherin
~LS
NP
EHF
LHF
CD41
ES(YS)
~4sp(YS)
Supplementary Figure 7:
Timecourse analysis of CD41 expression in early mouse embryos.
7sp(YS)
CD41 and VE-cadherin expression were analysed by flow cytometry between E7.0 and E8.25 of mouse embryonic
development. Seven distinct developmental stages between E7.0 and E8.5 were analysed. A distinct CD41 bright
population only becomes clearly separable by FACS at the ES stage. LS = late streak stage, NP = neural plate
stage, EHF = early head fold stage, LHF = late head fold stage, ES = early somite pair stage (0-1 smites pairs), 4sp
= approximately 3-4 somite pair stage, 7sp = 7 somite pair stage. The Runx1+/- and Runx1-/- embryos analyzed in
Figure 5 are approximately equivalent to the NP stage. Each FACS plot of LS-LHF stage embryos is from 8-10
pooled stage-matched whole embryos. Each FACS plot from somite pair stage embryos is from 4-6 pooled stagematched yolk sacs. All embryos are from ICR females crossed with GFP-ICR males to avoid contamination of
maternal cells.
Supplementary Figure 7
Runx1
Gata2
Unknown1
Unknown2
Unknown3
Unknown4
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.
Supplementary Figure 8