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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

(a) Schematic features of the bsk promoter-luciferase fusion plasmid. The DRE-like sequence is indicated. (b) Western immunoblot analysis of S2 cells treated with dMLFdsRNA, YFPdsRNA or no dsRNA (Mock). Proteins were probed with anti-dMLF IgG and anti-α-tubulin IgG. (c) Effects of dMLFdsRNA treatment on bsk gene promoter activity in S2 cells. Mean activities with s.d. from three independent transfections are shown, with the P-value by Welch's t-test. (d) dMLF and DREF interact in vivo. Extracts of S2 cells were first immunoprecipitated with anti-dMLF IgG followed by immunoblotting with anti-DREF IgG.
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fig5: (a) Schematic features of the bsk promoter-luciferase fusion plasmid. The DRE-like sequence is indicated. (b) Western immunoblot analysis of S2 cells treated with dMLFdsRNA, YFPdsRNA or no dsRNA (Mock). Proteins were probed with anti-dMLF IgG and anti-α-tubulin IgG. (c) Effects of dMLFdsRNA treatment on bsk gene promoter activity in S2 cells. Mean activities with s.d. from three independent transfections are shown, with the P-value by Welch's t-test. (d) dMLF and DREF interact in vivo. Extracts of S2 cells were first immunoprecipitated with anti-dMLF IgG followed by immunoblotting with anti-DREF IgG.

Mentions: To further examine the requirement of dMLF for bsk gene promoter activity, we carried out dMLF RNA interference (RNAi) experiments in cultured Drosophila S2 cells (Figure 5). Measuring levels of dMLF proteins in S2 cells by western immunoblot analysis confirmed efficient knockdown of the dMLF gene after treatment with dMLFdsRNA (Figure 5b). We conducted transient luciferase expression assays with the wild-type bsk gene promoter-luciferase reporter gene after treating S2 cells with dMLFdsRNA, DREFdsRNA, control YFPdsRNA or no dsRNA (Mock). Treatment of S2 cells with dMLFdsRNA and DREFdsRNA reduced bsk gene promoter activity by 80% and 90%, respectively, whereas control YFPdsRNA treatment exerted no effect (Figure 5c). These results indicate that both DREF and dMLF are required for bsk promoter activity.


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)

(a) Schematic features of the bsk promoter-luciferase fusion plasmid. The DRE-like sequence is indicated. (b) Western immunoblot analysis of S2 cells treated with dMLFdsRNA, YFPdsRNA or no dsRNA (Mock). Proteins were probed with anti-dMLF IgG and anti-α-tubulin IgG. (c) Effects of dMLFdsRNA treatment on bsk gene promoter activity in S2 cells. Mean activities with s.d. from three independent transfections are shown, with the P-value by Welch's t-test. (d) dMLF and DREF interact in vivo. Extracts of S2 cells were first immunoprecipitated with anti-dMLF IgG followed by immunoblotting with anti-DREF IgG.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: (a) Schematic features of the bsk promoter-luciferase fusion plasmid. The DRE-like sequence is indicated. (b) Western immunoblot analysis of S2 cells treated with dMLFdsRNA, YFPdsRNA or no dsRNA (Mock). Proteins were probed with anti-dMLF IgG and anti-α-tubulin IgG. (c) Effects of dMLFdsRNA treatment on bsk gene promoter activity in S2 cells. Mean activities with s.d. from three independent transfections are shown, with the P-value by Welch's t-test. (d) dMLF and DREF interact in vivo. Extracts of S2 cells were first immunoprecipitated with anti-dMLF IgG followed by immunoblotting with anti-DREF IgG.
Mentions: To further examine the requirement of dMLF for bsk gene promoter activity, we carried out dMLF RNA interference (RNAi) experiments in cultured Drosophila S2 cells (Figure 5). Measuring levels of dMLF proteins in S2 cells by western immunoblot analysis confirmed efficient knockdown of the dMLF gene after treatment with dMLFdsRNA (Figure 5b). We conducted transient luciferase expression assays with the wild-type bsk gene promoter-luciferase reporter gene after treating S2 cells with dMLFdsRNA, DREFdsRNA, control YFPdsRNA or no dsRNA (Mock). Treatment of S2 cells with dMLFdsRNA and DREFdsRNA reduced bsk gene promoter activity by 80% and 90%, respectively, whereas control YFPdsRNA treatment exerted no effect (Figure 5c). These results indicate that both DREF and dMLF are required for bsk promoter activity.

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