Targeted Expression of the DNA Binding Domain of DRE-Binding Factor, a Drosophila Transcription Factor, Attenuates DNA Replication of the Salivary Gland and Eye Imaginal Disc
Targeted Expression of the DNA Binding Domain of DRE-Binding Factor, a Drosophila Transcription Factor, Attenuates DNA Replication of the Salivary Gland and Eye Imaginal Disc
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DOI：
10.1128/mcb.19.9.6020
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发表时间：
1999-09
影响因子：
5.3
通讯作者：
F. Hirose;M. Yamaguchi;A. Matsukage
中科院分区：
文献类型：
--
作者：
F. Hirose;M. Yamaguchi;A. Matsukage
摘要 果蝇编码 DNA 复制相关蛋白的基因启动子含有由 8 bp 回文 DNA 复制相关元件 (DRE) 序列 (5'-TATCGATA) 组成的转录调控元件。特异性DRE结合因子(DREF)是具有709个氨基酸残基的多肽同二聚体，是含有DRE的基因转录的正反式作用因子。 DRE 结合和二聚体形成均与 N 端区域的残基 16 至 115 相关。我们已经建立了在热休克启动子、唾液腺特异性启动子或眼成象盘特异性启动子的控制下表达全长DREF多肽或其N末端片段（氨基酸残基1至125）的转基因果蝇。在胚胎、幼虫或蛹阶段，N 末端片段的热激诱导导致大于 50% 的致死率。通过全长 DREF 的共表达克服了这种致命性。在表达N末端片段的转基因幼虫的唾液腺中，该片段与内源DREF形成同二聚体和异二聚体。唾液腺细胞中 N 末端片段的异位表达降低了 DNA 聚合酶 α 的 180 kDa 亚基和 dE2F 的 mRNA 含量以及 DNA 内复制的程度。眼睛成虫盘中 N 末端片段的异位表达显着减少了第二次有丝分裂波时细胞中的 DNA 复制。一系列证据表明，N 端片段可以以显性负向方式阻碍内源性 DREF 功能，并且 DREF 是有丝分裂细胞周期和内周期中正常 DNA 复制所必需的。
ABSTRACT The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory elements consisting of an 8-bp palindromic DNA replication-related element (DRE) sequence (5′-TATCGATA). The specific DRE-binding factor (DREF), a homodimer of the polypeptide with 709 amino acid residues, is a positive trans-acting factor for transcription of DRE-containing genes. Both DRE binding and dimer formation are associated with residues 16 to 115 of the N-terminal region. We have established transgenic flies expressing the full-length DREF polypeptide or its N-terminal fragment (amino acid residues 1 to 125) under the control of the heat shock promoter, the salivary gland-specific promoter, or the eye imaginal disc-specific promoter. Heat shock induction of the N-terminal fragment during embryonic, larval, or pupal stages caused greater than 50% lethality. This lethality was overcome by coexpression of the full-length DREF. In salivary glands of the transgenic larvae expressing the N-terminal fragment, this fragment formed a homodimer and a heterodimer with the endogenous DREF. Ectopic expression of the N-terminal fragment in salivary gland cells reduced the contents of mRNAs for the 180-kDa subunit of DNA polymerase α and for dE2F and the extent of DNA endoreplication. Ectopic expression of the N-terminal fragment in the eye imaginal discs significantly reduced DNA replication in cells at the second mitotic wave. The lines of evidence suggest that the N-terminal fragment can impede the endogenous DREF function in a dominant negative manner and that DREF is required for normal DNA replication in both mitotic cell cycle and endo cycle.