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Suppl. Figure 1
A
C
UAS-lz
UAS-dPias-RNAi-#1
UAS-lz +
UAS-dPias-RNAi-#1
UAS-dPias
UAS-dPias-RNAi-#2
UAS-lz +
UAS-dPias-RNAi-#2
D
UAS-dPias
UAS-lz + UAS-dPias
B
UAS-Pias-C388A
UAS-dPias-RNAi-#1
UAS-dPias-RNAi-#2
rp-49
UAS-dPias-C388A
UAS-lz + UAS-dPias-C388A
hs
Figure S1. A large-scale genetic-modifier screen identified dPias as a regulator of
lz/RUNXs
(A) UAS-lz and/or UAS-dPias were expressed in eye using the eye-specific driver GMRgal4. Microscopic images (left) and scanning electron microscopic images (SEM, right) are
shown. GMR-driven overexpression of either lz or dPias result in an mild rough-eye
phenotype (UAS-lz and UAS-dPias). When both lz and dPias were overexpressed, the rougheye phenotype was more severe (UAS-lz + UAS-dPias).
(B) dPias with a defect in the SUMO E3 ligase activity (UAS-dPias-C388A) was expressed
with or without UAS-lz using the eye-specific driver GMR-gal4. Overexpression of dPiasC388A did not affect the eye-defect phenotype induced by lz.
(C) dPiasV29448 (UAS-dPias-RNA-i#1) and dPiasV31623 (UAS-dPias-RNAi-#2) bear UASdPias-RNAi. GMR-driven eye-specific knockdown of dPias upon expression of dPias-RNAi
resulted in eye-defect phenotypes in both lines (left). The defective phenotype was rescued
by overexpression of lz. Microscopic images are shown.
(D) UAS-dPias, UAS-dPias-C388A, UAS-dPias-RNAi-#1, and UAS-dPias-RNAi-#2 were
crossed with the heat-shock driver (hs). The flies were cultured at either 25°C or 37°C (heatshock) for 1 h, and the levels of dPias expression were measured by RT-PCR. rp49 was used
as an internal control.
Suppl. Figure 2
187 234
RX3-WT
Runt
RX3-1-187
Runt
RX3-1-234
Runt
RX3-325
Runt
325
451
IP : HA
+ +
WT
+
DRunt
+
1-234
1-325
Myc-RX3 +
HA-PIAS1 -
1-187
RX3-DRunt
+ +
IB : Myc
RX3/PIAS1
IB : HA
PIAS1
IB : Myc
RX3
Figure S2. RUNX3 interacts with PIAS1 through the Runt domain.
Schematic diagram showing the RUNX3 deletion constructs. Myc-tagged RUNX3-wild type
(WT), RUNX3(1–187), RUNX3(1–234), RUNX3(1–325) and RUNX3-DRunt were
coexpressed with HA-PIAS1, and the RUNX3-PIAS1 interaction was monitored by IP and IB.
Suppl. Figure 3
Figure S3. The crystal structure of Runt domain/CBFb/DNA (PDB accession code:
1H9D43). Amino acids in the Runt domain are labeled according to the primary sequence of
RUNX3. K129 and K148 are exposed on the surface.
Suppl. Figure 4
PIAS1/SUMO1
Myc-RX3
- +
- +
+ + +
+ + +
Myc-RX3
+
+
FLAG-AKT1
SUMO1-RX3
IB: Myc
RX3
IB: FLAG
IP: FLAG
IB: Myc
-
-
+
+ +
+
DRunt
-
325
-
187
234
FLAG-ERK
WT
ERK1 ERK2
WT
C
A
+ +
RX3/AKT1
ERK
IB: HA
PIAS1
IB: FLAG
IP: Myc
IB: Myc
RX3
SUMO1
b-tubulin
IB: Tub
IB: FLAG
IB: Tub
AKT1
b-tubulin
B
WT
FLAG-AKT1 -
KD
-
PIAS1/SUMO1 - +
Myc-RX3 + +
+ + +
+ + +
+
+
SUMO1-RX3
IB: Myc
RX3
IB: FLAG
AKT1
IB: HA
PIAS1
IB: FLAG
IB: Tub
SUMO1
b-tubulin
Figure S4. AKT1 pathway but not ERK pathway stimulates RUNX3 sumoylation
(A) Myc-RUNX3, HA-PIAS1, and FLAG-SUMO1 were coexpressed with ERK1 or ERK2
in HEK293 cells, and RUNX3 sumoylation was analyzed by IB.
(B) Myc-RUNX3, HA-PIAS1, and FLAG-SUMO1 were coexpressed with FLAG-AKT1WT or FLAG-AKT1-KD (kinase-dead mutant) in HEK293 cells, and RUNX3 sumoylation
was analyzed by IB.
(C) Myc-RUNX3-WT and serial deletion mutants were cotransfected along with FLAGAKT1 into HEK293 cells, and the RUNX3-AKT1 interaction was monitored by IP and IB.