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Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway.

Yanai H, Yoshioka Y, Yoshida H, Nakao Y, Plessis A, Yamaguchi M - Oncogenesis (2014)

Bottom Line: Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk).Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis.Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan.

ABSTRACT
Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis.

No MeSH data available.


Related in: MedlinePlus

Overexpression of dMLF in the wing imaginal discs induces programmed cell death. (A) Immunostaining of a wing disc from an en-GAL4/+ fly with anti-activated caspase-3 IgG. (a) Activated Caspase-3 is not apparent without overexpression of dMLF. (b) Phase contrast image. Scale bars, 100 μm. A, anterior; P, posterior. (B) Immunostaining of a wing disc from an en-GAL4, UAS-dMLF/+ fly with anti-dMLF IgG, anti-activated caspase-3 IgG. DNA was labeled with DAPI. (a) Expression of dMLF (green) driven by en-GAL4 in wing discs. (b) Activated Caspase-3 (magenta) is present in posterior of wing discs on overexpression of dMLF. (c) Merged image of anti-dMLF and anti-active Caspase-3 signals. (d) Phase contrast image. Scale bars are for 100 μm. (e–g) A higher magnification of the region marked with the square in (Bc). Fragmentation of DNA (blue) by apoptosis via caspase-3. Scale bars, 20 μm.
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fig3: Overexpression of dMLF in the wing imaginal discs induces programmed cell death. (A) Immunostaining of a wing disc from an en-GAL4/+ fly with anti-activated caspase-3 IgG. (a) Activated Caspase-3 is not apparent without overexpression of dMLF. (b) Phase contrast image. Scale bars, 100 μm. A, anterior; P, posterior. (B) Immunostaining of a wing disc from an en-GAL4, UAS-dMLF/+ fly with anti-dMLF IgG, anti-activated caspase-3 IgG. DNA was labeled with DAPI. (a) Expression of dMLF (green) driven by en-GAL4 in wing discs. (b) Activated Caspase-3 (magenta) is present in posterior of wing discs on overexpression of dMLF. (c) Merged image of anti-dMLF and anti-active Caspase-3 signals. (d) Phase contrast image. Scale bars are for 100 μm. (e–g) A higher magnification of the region marked with the square in (Bc). Fragmentation of DNA (blue) by apoptosis via caspase-3. Scale bars, 20 μm.

Mentions: As noted previously,26en-GAL4-driven overexpression of dMLF in the wing imaginal discs induced programmed cell death in the posterior region, although restricted in some areas (Figure 3). Altogether, these data indicate that overexpression of dMLF induces apoptosis through JNK activation during wing development, and consequently an atrophied phenotype of the wing posterior is exhibited in adults.


Drosophila myeloid leukemia factor acts with DREF to activate the JNK signaling pathway.

Yanai H, Yoshioka Y, Yoshida H, Nakao Y, Plessis A, Yamaguchi M - Oncogenesis (2014)

Overexpression of dMLF in the wing imaginal discs induces programmed cell death. (A) Immunostaining of a wing disc from an en-GAL4/+ fly with anti-activated caspase-3 IgG. (a) Activated Caspase-3 is not apparent without overexpression of dMLF. (b) Phase contrast image. Scale bars, 100 μm. A, anterior; P, posterior. (B) Immunostaining of a wing disc from an en-GAL4, UAS-dMLF/+ fly with anti-dMLF IgG, anti-activated caspase-3 IgG. DNA was labeled with DAPI. (a) Expression of dMLF (green) driven by en-GAL4 in wing discs. (b) Activated Caspase-3 (magenta) is present in posterior of wing discs on overexpression of dMLF. (c) Merged image of anti-dMLF and anti-active Caspase-3 signals. (d) Phase contrast image. Scale bars are for 100 μm. (e–g) A higher magnification of the region marked with the square in (Bc). Fragmentation of DNA (blue) by apoptosis via caspase-3. Scale bars, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Overexpression of dMLF in the wing imaginal discs induces programmed cell death. (A) Immunostaining of a wing disc from an en-GAL4/+ fly with anti-activated caspase-3 IgG. (a) Activated Caspase-3 is not apparent without overexpression of dMLF. (b) Phase contrast image. Scale bars, 100 μm. A, anterior; P, posterior. (B) Immunostaining of a wing disc from an en-GAL4, UAS-dMLF/+ fly with anti-dMLF IgG, anti-activated caspase-3 IgG. DNA was labeled with DAPI. (a) Expression of dMLF (green) driven by en-GAL4 in wing discs. (b) Activated Caspase-3 (magenta) is present in posterior of wing discs on overexpression of dMLF. (c) Merged image of anti-dMLF and anti-active Caspase-3 signals. (d) Phase contrast image. Scale bars are for 100 μm. (e–g) A higher magnification of the region marked with the square in (Bc). Fragmentation of DNA (blue) by apoptosis via caspase-3. Scale bars, 20 μm.
Mentions: As noted previously,26en-GAL4-driven overexpression of dMLF in the wing imaginal discs induced programmed cell death in the posterior region, although restricted in some areas (Figure 3). Altogether, these data indicate that overexpression of dMLF induces apoptosis through JNK activation during wing development, and consequently an atrophied phenotype of the wing posterior is exhibited in adults.

Bottom Line: Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk).Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis.Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan.

ABSTRACT
Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis.

No MeSH data available.


Related in: MedlinePlus