Aratani Satoko, Kageyama Yuji, Nakamura Akira, Fujita Hidetoshi, Fujii Ryoji, Nishioka Kusuki, Nakajima Toshihiro
Department of Locomotor Science, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Kanagawa 216-8512, Japan.
Int J Mol Med. 2008 Apr;21(4):469-76.
Transcription levels of the genes on X chromosome are regulated through the dosage compensation mechanisms. The dosage compensation complex (DCC) localizes to X chromosome and activates the transcription of target genes in male 2-fold more than in female. Drosophila maleless (MLE), an ATPase/helicase, is a component of the DCC and essential for the viability of male flies. However, the functions of MLE on gene expression are not clear. RNA helicase A (RHA) is a homologue of Drosophila MLE and mediates the expression of several genes. RHA recruits preinitiation complex via the minimal transactivation domain (MTAD), consisting of 50 amino acids to target promoters. The tryptophan residues in MTAD are important for transactivation via RHA. The amino acid sequence of MTAD is conserved in MLE. In this study, we assessed whether the functions of MTAD are conserved in fruit fly by investigating the transcriptional activity of MLE. Transactivation assay indicated the MTAD of MLE had transcriptional activity in Schneider's cells. In vitro binding assays revealed that MLE recruited RNA polymerase II (Pol II) complexes through MTAD. Reporter assays showed that the MTAD, especially tryptophan residues, is important for transcription from roX promoter, similar to RHA. The results confirm that MTAD of MLE mediates the expression of MLE target genes through recruitment of Pol II.
X染色体上基因的转录水平通过剂量补偿机制进行调控。剂量补偿复合体(DCC)定位于X染色体,并使雄性中靶基因的转录激活程度比雌性高两倍。果蝇无雄基因(MLE)是一种ATP酶/解旋酶,是DCC的一个组成部分,对雄蝇的生存力至关重要。然而,MLE在基因表达方面的功能尚不清楚。RNA解旋酶A(RHA)是果蝇MLE的同源物,介导多个基因的表达。RHA通过由50个氨基酸组成的最小反式激活结构域(MTAD)募集起始前复合体至靶启动子。MTAD中的色氨酸残基对于通过RHA进行的反式激活很重要。MTAD的氨基酸序列在MLE中是保守的。在本研究中,我们通过研究MLE的转录活性来评估MTAD的功能在果蝇中是否保守。反式激活分析表明,MLE的MTAD在施奈德细胞中具有转录活性。体外结合分析显示,MLE通过MTAD募集RNA聚合酶II(Pol II)复合体。报告基因分析表明,MTAD,尤其是色氨酸残基,对于从roX启动子进行转录很重要,这与RHA类似。结果证实,MLE的MTAD通过募集Pol II介导MLE靶基因的表达。