Department of Hematology
Model system
SCF
Epo
Epo
GR
Identification of translationally controlled genes
critical for the balance between expansion and
differentiation of erythroid progenitors
terminal
LY294002 erythroid
differentiation
renewal/proliferation
Epo,
SCF,
glucocorticoids
Godfrey Grech
Marieke von Lindern
Bob Lowenberg
Gene expression profiling
using polysome bound mRNA
Epo
ES
E
E
-
+
-
+
LY
(15μM)
Gene expression profiling
using polysome bound mRNA
SCF
cell lysate
cell lysate
sucrose
gradient
sucrose
gradient
free mRNA
ES
polysomal mRNA
free mRNA
NF
Epo
SCF
SCF Epo
polysomal mRNA
Nm23-M2
Nucleoside diphosphate
kinase (NDPK)
NDPK-B mRNA expression is regulated
at the level of translation initiation
Total mRNA
Polysomal mRNA
free
- E S E/S
PI3K pathway regulates eIF4E
availability
SCF
Epo
bound
c-Kit
PTEN
NF
PIP2
p110
Pim-1
SCF
C-Jun
C-MYB
5
p8
-Y-P
PIP3
PH
E
S
ES
P-4E-BPT37/T46
4E-BP
PI3K
E/S
eIF4E 4EBP
PIP3
PH
PKB
Epo
P-Y-
PDK1/2
EpoR
Nm23-M2
mTOR
Pi
Pi 4EBP
Pi Pi
eIF4E
Pi
Pi 4EBP
Pi Pi
1
PTEN
PIP2
EpoR
P-Y-
PIP3
PH
5
p8
PI3K
LY294002
rapamycine
eIF4E 4EBP
mTOR
SCF presents active eIF4E
for translation initiation
+SCF
1. SCF, but not Epo, transduce the
final phosphorylation of 4EBP at Ser64
needed to release eIF4E.
P-4E-BPT37/T46
PKB
PDK1/2
p110
-Y-P
PIP3
PH
U0126
c-Kit
LY294002
SCF
Epo
rapamycine
PI3K pathway regulates eIF4E
availability
4E-BP
Pi
Pi 4EBP
Pi Pi
2. Cap-pulldowns show increased
levels of eIF4G to form eIF4F complex
by SCF.
eIF4E
Pi
Pi 4EBP
Pi Pi
Objectives
• Identify the role of eIF4E in expansion and
eIF4E overexpression results in
delay in differentiation
differentiation of erythroid progenitors.
PTEN
PIP2
EpoR
P-Y-
PIP3
PH
PIP3
PH
PKB
p110
-Y-P
5
p8
have a role in SCF dependent expansion of
erythroid progenitors.
c-Kit
PDK1/2
• Identify translationally controlled genes that
Epo
4 days under Epo conditions
PI3K
eIF4E
eIF4E
eIF4E
eIF4E
eIF4E 4EBP
eIF4E 4EBP
eIF4E 4EBP
Cell Numbers
Cell Size
Hemoglobin
800
Cell Numbers
Cell morphology
600
0
0
0
24 48 72 96 120 144
24 48 72 96 120 144
10
1
600
400
200
0
0
24 48 72 96 120 144
hours
200
0
0
24 48 72 96 120 144
hours
0
24 48 72 96 120 144
hours
200
0
24 48 72 96 120 144
24 48 72 96 120 144
hours
hours
600
1000
400
400
0
0
600
hemoglobin (Ab units)
Mean Cell Volume
Epo
ESD
ES
100
24 48 72 96 120 144
hours
800
1000
10
1
0
hours
hours
eIF4E
overexpression
0
Epo
ESD
ES
cell number
1
200
100
Hemoglobin (Ab units)
200
Epo
ESD
ES
hemoglobin (Ab units)
400
400
cell number
600
Vector control
Hemoglobin (Ab units)
10
Mean Cell Volume
100
cell number
Vector control
Hemoglobin
1000
Epo
ESD
ES
cell number
eIF4E
overexpression
eIF4E
600
1000
eIF4E
overexpression
empty vector
100
10
1
400
200
0
0
24 48 72 96 120 144
hours
0
24 48 72 96 120 144
hours
2
Translation efficiency
Ectopic eIF4E promotes eIF4F
complex formation
NF
eIF4E
overexpressor
ss
ss
NF
Protein Synthesis Rate
eV
α -eIF4G
α -4E-BP
α -eIF4E
α-myc
Competitive mRNAs (β-actin, Fli1)
Non-Competitive mRNAs (VEGF,
Cyclin D1, c-myc, MMP-9)
limiting
increased
eIF4E level
Figure adapted
fromJR
Clinical
& Experimental
Adapted
from Graff
& Zimmer
SG, ClinicalMetastasis
& Experimental
(2003) 20: 265-273,
Graff JR & Zimmer SG
Metastasis
(2003) 20:265-273
Conclusion 1
Gene expression profiling
polysome bound vs Total mRNA
• Regulation of translation initiation by eIF4E
is an important pathway stimulated by SCF to
delay
differentiation
of
erythroid
progenitors.
NF
NF
NF
NF
ES
ES
ES
ES
cell lysate
What are the translationally controlled genes
that have a role in the delay of Epo-induced
differentiation?
polysomal
sucrose
gradient
free
polysomal
ES
NF
NF
NF
ES
NF
NF
NF
ES
ES
ES
ES
ANOVA
comparison
of individual
replicates
Cluster analysis
Total mRNA
Gene expression profiling
Identification of translational targets
Selection Criteria
cluster analysis
394 genes – ANOVA signature
A (249)
B (89) C
D
1
ANOVA signatures
2
Translationally upregulated
polysome
bound RNA
3
down regulated in differentiation
early
differentiation
4
Classified based on function/s
late
differentiation
5
Polysome recruitment dependent on
eIF4E availability (signaling and/or
overexpression)
total RNA
3
Classification based on function
Translation upreg genes
[fc >1.5]
Translation downreg genes
[fc <0.75]
12%
Unknown function
15%
Signal transduction
Cell cycle regulation
Apoptosis
1%
15%
Classification based on function
2%
18%
chaperon proteins
Enzyme
3%
5%
4%
Cytoskeleton
3%
39%
3%
1%
Proteolysis
7%
4%
Metabolism
10%
Ribosomal proteins
6%
Translational factors
3%
4%
mRNA processing
DNA binding
17%
6%
15%
4%
1%
15%
Membrane
Cytoskeleton
1%
Membrane
4%
Translational
factors
6%
Unknown function
Unknown function
15%
25
Targets with
fold change >1,5 :
9
Cell cycle
regulation
1%
Apoptosis
2%
Ribosomal
proteins
15%
Enzyme
Metabolism 7%
17%
chaperone
proteins
5%
Chromatin modeling
Transcription complexes
% of Group A :
10%
No. of genes/EST :
23
Targets with
fold change >1,5 :
10
• Is the recruitment to polysomes by
SCF, PI3K dependent?
DNA binding
10%
Membrane
4%
• Does
eIF4E overexpression alone
result in polysome recruitment of the
selected transcripts.
Cell cycle
regulation
1%
Apoptosis
2%
mRNA
processing
3%
Translational
factors
6%
12%
No. of genes/EST :
Polysome recruitment
DNA binding
Signal
transduction
12%
Cytoskeleton
3%
% of Group A :
DNA binding
10%
mRNA
processing
3%
4%
Classification based on function
Signal
Signal
transduction
transduction
12%
12%
Signal
Transduction
Enzyme
Metabolism 7%
17%
Ribosomal
proteins
15%
chaperone
proteins
5%
Polysome recruitment of selected
transcripts is PI3K dependent
Non-competitive mRNA
Competitive mRNA
alpha-4 Polysome association
Fli-1 Polysome association
100
Percent polysome association
Empty vector
eIF4E
overexpression
40
30
hnrpa1
rnf138
rbms1
Cnih
20
10
Empty vector
S
NF
S+LY
E+LY
0
E+rap
S
NF
S+LY
E+LY
E+rap
S+rap
S
S+LY
E
ES
NF
0
50
S+rap
10
60
S
20
70
S+LY
30
80
E
40
90
ES
50
NF
60
ss
70
ss
Percent polysome association
80
eIF4E
overexpression
PI3K dependent
polysome recruitment of
13 transcripts shown by
RT-PCR.
Confocal microscopy
by
Bart Aarts
4
I11 cell line
a4 #2_myc
a4 #7_myc
Empty vector
mycAb
DAPI
overlay
acp1
acp1
800
100
700
600
5730466P16Rik
αα4
Stable overexpression of target genes in I11 cells. The proteins
are myc tagged – western using myc antibody. The sizes of the
tagged protein are expected as follows:
1. 5730466P16Rik - 27kD
2. acp1
- 23kD
3. a4
- 43kD
Cell number
p16Rik
4
500
400
10
300
200
ev
acp1
eV
eV
eV
a4
mEd2
acp1
a4
0
24
48
19 transcripts involved in signal
transduction and DNA binding –
regulated at translation level
T24
Translation regulation of 13 / 17
transcripts are PI3K dependent and
eIF4E level dependent
T72
6 proteins
successfully
overexpressed
3 proteins
not ectopically
expressed:
2 show
phenotype
96
RT-PCR of
2 transcripts not
successful
3 proteins
awaited:
4 do not show
phenotype
Translationally controlled genes
Identify UTR sequencies
- databases
- RACE
Future plans and ongoing experiments
72
Phenotypic Analysis of
Target genes – conclusion
5730466P16Rik
Department of Hematology
100
p16rIK
1
0
Phenotypic Analysis of
Target genes
Mean Cell Volume
Empty vector
acp1#1 _myc
acp1#2 _myc
Empty vector
p16Rik #9 _myc
kD
75
50
37
25
20
Phenotypic Analysis of
Target genes
Identify regulatory elements
within UTRs contributing to
translation control.
Overexpression of targets
9 delay in differentiation in I/11 cells
Mechanism of differentiation block by α4:
9 cap-pull downs
9 siRNA of a4 in eIF4E overexpressors.
• Luciferase constructs to identify regulatory
elements in 5’ UTR
• REMSA to show that proteins bind to the 5’UTR
RNA probe.
• Cross-linking assays to extract proteins binding to
specific UTRs
• Mass spec. to identify proteins.
• Y3H assay to confirm specific binding.
5