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from B-cell Biology to Data Integration
1. From
B-‐cell
Biology
to
Data
Integra3on
Gopuraja
Dharmalingam
15-‐02-‐2013,
Barcelona
2. Transcrip3on
Factor
-‐
Ikaros
• Founding
member
of
a
family
of
transcrip3on
factor
• Expressed
in
haematopoie3c
stem
cells,
lymphoid
cells
and
some
myeloid
cells
• Required
for
Lymphocyte
development
• Muta3ons
in
IKZF1
observed
in
Human
Leukemia
Merkenschlager
review,
2010
3. B
lymphocyte
development
mul:-‐lineage
progenitors
lineage-‐restricted
lymphoid
progenitors
common
haematopoie:c
lymphoid
cycling
Res3ng
Immature
stem
cells
progenitors
pre-‐pro-‐B
cell
pro-‐B
cell
pre-‐B
cell
pre-‐B
cell
B
cell
MLP
Fr.
A
Fr.
B/C
Fr.
C’
Fr.
D
Fr.
E
HSC
MPP
CLP
LMPP
T
cell
progenitors
B3
Cell
line
4. Challenges!!
• Single
gene
to
genome
wide
-‐
no
high
throughput
technologies
were
available
• How
to
make
sense
of
the
data?
• Linking
with
published
resources
• Analysis
challenges:
Different
soVware
&
method
provides
different
results
(Ex:
different
peak
callers
provide
different
set
of
peaks
for
Ikaros)
• Comparing
and
Integra3ng
data
from
different
methods
(Ex:
expression
and
binding)
• Biological
relevance
5. B
lymphocyte
development
mul:-‐lineage
progenitors
lineage-‐restricted
lymphoid
progenitors
common
haematopoie:c
lymphoid
cycling
Res3ng
Immature
stem
cells
progenitors
pre-‐pro-‐B
cell
pro-‐B
cell
pre-‐B
cell
pre-‐B
cell
B
cell
MLP
Fr.
A
Fr.
B/C
Fr.
C’
Fr.
D
Fr.
E
HSC
MPP
CLP
LMPP
T
cell
progenitors
B3
Cell
line
mRNA expression (array counts)
Ikaros
target
genes?
differentiation
Vidal
et
al,
2013
6. Roles
of
Ikaros
in
B-‐cell
development?
• Global
iden3fica3on
of
Ikaros
targets
and
regulated
genes
• Iden3fying
pre-‐B-‐cell
differen3a3on
pathways
enriched
for
Ikaros
targets
• Co-‐occurrence
of
Ikaros
binding
with
other
hematopoie3c
transcrip3on
factors
• Integra3ng
Ikaros
binding
and
gene
expression
with
gene
expression
changes
during
hematopoie3c
development
(using
Immgen
data)
Vidal
et
al,
2013
7. Inducible
Ikaros
construct
HA
Ikaros IKAROS
ERt2
IRES
GFP
Cytoplasm
Nucleus
HA-‐IK-‐ER
fusion
protein
Heat-‐Shock
protein
in
cytoplasm
4-‐hydroxytamoxifen
(4-‐OHT)
9. Genome
wide
iden3fica3on
of
Ikaros
binding
sites
endogenous
Ikaros
ChIP-‐Seq
HA-‐tagged
Ikaros
ChIP-‐Seq
Common:
11349
high
confidence
peaks
11349
peaks
6746
genes
5732
in
affy
arrays
Isabel
Ferreiros
Vidal,
Anna
Terry,
Tom
Carroll
10. Integra3ng
Ikaros
binding
and
gene
expression
All
Upreg.
Downreg.
Ikaros-‐responsive
genes
at
2h
79
28
51
Ikaros
binding
observed
65
(82.3%)
22
(78.6%)
43
(84.3%)
Ikaros
binding
expected
28
10
18
p
value
9.90x10-‐19
4.55x10-‐7
5.08x10-‐14
odds
ra3o
8.55
6.75
9.9
Ikaros-‐responsive
genes
at
6h
401
159
242
Ikaros
binding
observed
286
(71.3%)
112
(70.4%)
174
(71.9%)
Ikaros
binding
expected
141
56
85
p
value
5.88x10-‐51
2.35x10-‐20
2.39x10-‐32
odds
ra3o
4.58
4.39
4.71
Ikaros-‐responsive
genes
at
48h
4471
1982
2486
Ikaros
binding
observed
2497
(55.8%)
1195
(60.3%)
1302
(41.5%)
Ikaros
binding
expected
1573
697
875
p
value
2.16x10-‐246
1.43x10-‐131
7.52x10-‐82
odds
ra3o
2.33
2.8
1.398
Vidal
et
al,
2013
12. Mo3fs
of
other
transcrip3on
factors
close
to
Ikaros
binding
sites
Vidal
et
al,
2013
13. Ikaros
co-‐localiza3on
with
other
hematopoie3c
transcrip3on
factors
Overlap
between
binding
sites
of
Overlap
between
genes
bound
by
karos
and
Ikaros
and
other
TFs(%)
other
TFs(%)
Vidal
et
al,
2013
14. Ikaros
targets
in
B-‐cell
differen3a3on
pathways
Pre-‐BCR
signaling
Cell
cycle
V(D)J
recombina3on
Vidal
et
al,
2013
15. Ikaros
target
genes
in
hematopoiesis
and
B-‐cell
lineage
specifica3on
Goals
of
Immunological
Genome
Project
(Immgen)
• To
study
whole-‐genome
expression
of
all
known
Immune
cell
types
in
Mouse
• To
predict
the
gene
regulatory
network
in
immune
cells
using
computa3onal
tools
• Data
deposited
to
GEO
/
ArrayExpress
www.Immgen.org
16. Immgen
Igll1
gene
expression
in
B-‐cell
popula:on
www.Immgen.org
17. Ikaros
target
genes
in
hematopoiesis
and
B-‐cell
development
gene
expression
Ikaros
binding
Cycling
to
resNng
pre-‐B-‐cell
transiNon
gene
expression
Ikaros
binding
Vidal
et
al,
2013
19. Conclusion
• Integra3ve
analysis
allowed
us
to
address
the
role
of
Ikaros
in
B-‐cell
biology
• Ikaros
targets
were
highly
enriched
in
key
pre-‐
B-‐cell
differen3a3on
pathways
• Inducible
Ikaros
expression
in
cycling
pre-‐B
cells
was
sufficient
to
drive
the
transcrip3onal
changes
that
resembled
the
differen3a3on
of
cycling
to
res3ng
pre-‐B
cells.
20. Acknowledgements
Lymphocyte
Development
Group:
University
of
Cambridge:
Manhias
Merkenschlager
Mikhail
Spivakov
Amanda
Fisher
Isabel
Ferreiros
Vidal
University
of
California,
Los
Tom
Carroll
Angeles
Ben
Taylor
Anna
Terry
Stephen
T.
Smale
Ziwei
Liang
Sanjay
Khadayate
Prashant
Srivastava
Enrico
Petreno
Lee
Cooper
