Cell biological and genetical studies on Drosophila myeloid leukemia factor (dMLF) and DRE/DREF transcriptional regulatory pathway

ショウジョウバエ骨髄性白血病因子dMLFとDRE/DREF転写制御経路の細胞生物学的及び遺伝学的研究

2015-03-25

2015-03-25

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 a 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, I 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, I provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, I show that dMLF interacts with DREF in vivo. Together, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis. In addition, the DRE/DREF transcriptional regulatory system has been demonstrated to activate a wide variety of genes with various functions. In Drosophila, the Hippo (Hpo) pathway is known to suppress cell proliferation by inducing apoptosis and cell cycle arrest through inactivation of Yorkie, a transcription co-activator. Here, I also carried out analyses of transcriptional regulation of the hippo (hpo) gene in Drosophila. By this analysis, I revealed that DREF is required for transcriptional activation of the hpo gene to positively control the Hippo pathway. Altogether, the DRE/DREF pathway is involved in both the JNK and the Hpo pathway, and appears to play an important role in induction of apoptosis.

博士（学術）

京都工芸繊維大学

14303甲第731号

http://hdl.handle.net/10212/2251

en

ETD

Thesis or Dissertation

Thesis