Drosophila Mi-2 negatively regulates dDREF by inhibiting its DNA-binding activity
Drosophila Mi-2 negatively regulates dDREF by inhibiting its DNA-binding activity
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DOI：
10.1128/mcb.22.14.5182-5193.2002
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发表时间：
2002-07-01
影响因子：
5.3
通讯作者：
Yamaguchi, M
中科院分区：
文献类型：
--
作者：
Hirose, F;Ohshima, N;Yamaguchi, M
果蝇DNA复制相关元件（DRE）因子（dDREF）是一种转录调控因子，对携带5 '-TATCGATA DRE的基因的表达起重要作用。据报道，dDREF与染色质边界元件中的序列结合，因此，dDREF可能在调节绝缘子活性中起作用。为了进一步了解dDREF功能，我们以DREF多肽为诱饵进行了酿酒酵母双杂交筛选，并鉴定Mi-2为DREF相互作用蛋白。生化分析表明，果蝇Mi-2（dMi-2）的C-末端区域特异性结合dDREF的DNA结合结构域。电泳迁移率变动分析表明，dMi-2从而抑制DNA结合活性的dDREF。异位表达的dDREF和dMi-2在眼睛的成虫盘导致严重和轻度粗糙的眼睛表型，分别，而苍蝇同时表达这两种蛋白质表现出几乎正常的眼睛表型。半剂量减少dMi-2基因增强DREF诱导的粗糙眼表型。唾液腺多线染色体的免疫染色表明，dDREF和dMi-2结合在相互排斥的方式。这些证据定义了dMi-2通过其DNA结合活性在dDREF的负调节中的新功能。最后，我们假设dDREF和dMi-2可能表现出相互调节其功能。
Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5'-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.