A complex array of DNA-binding proteins required for pairing-sensitive silencing by a polycomb group response element from the Drosophila engrailed gene.
A complex array of DNA-binding proteins required for pairing-sensitive silencing by a polycomb group response element from the Drosophila engrailed gene.
复制标题
果蝇 engrailed 基因中的多梳基团反应元件进行配对敏感沉默所需的一系列复杂的 DNA 结合蛋白。
DOI：
10.1093/genetics/160.4.1561
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发表时间：
2002
期刊：
影响因子：
3.3
通讯作者：
Kassis,JudithA
中科院分区：
文献类型：
--
作者：
Americo,Jeffrey;Whiteley,Mary;Brown,JLesley;Fujioka,Miki;Jaynes,JamesB;Kassis,JudithA
来自果蝇基因的调节性DNA导致转基因果蝇中连锁报告基因(迷你白色)的沉默。这种沉默在转基因纯合子的果蝇中得到加强，被称为“配对敏感沉默”。对大片段(2.6kb)和小片段(181bp)的配对敏感沉默活性进行了研究。由于配对敏感的沉默通常与多梳基团反应元件(PreS)相关，我们在一个旨在检测胚胎中Pre活性的结构中测试了每个增强片段的活性。两个片段在abxd-Ubx-lacZReport构建中都表现为PreS，而较大的片段显示出额外的沉默能力。利用迷你转运蛋白基因，定义了139个碱基的最小配对敏感元件(PSE)。利用果蝇核提取液进行的DNA迁移率分析表明，在这个139个碱基的元件中有8个蛋白质结合位点。突变分析表明，这些位点中至少有五个对配对敏感的沉默很重要。其中一个必需的位点是多梳族蛋白PleioHome，另一个是GAGAG，这是一个由Gaga因子和PinSqueak蛋白结合的序列。其他蛋白质的特性尚不清楚。这些数据表明，PSE功能所需的DNA结合蛋白的复杂程度令人惊讶。
Regulatory DNA from the Drosophila geneengrailedcauses silencing of a linked reporter gene (mini-white) in transgenic Drosophila. This silencing is strengthened in flies homozygous for the transgene and has been called “pairing-sensitive silencing.” The pairing-sensitive silencing activities of a large fragment (2.6 kb) and a small subfragment (181 bp) were explored. Since pairing-sensitive silencing is often associated with Polycomb group response elements (PREs), we tested the activities of each of theseengrailedfragments in a construct designed to detect PRE activity in embryos. Both fragments were found to behave as PREs in abxd-Ubx-lacZreporter construct, while the larger fragment showed additional silencing capabilities. Using the mini-whitereporter gene, a 139-bp minimal pairing-sensitive element (PSE) was defined. DNA mobility-shift assays using Drosophila nuclear extracts suggested that there are eight protein-binding sites within this 139-bp element. Mutational analysis showed that at least five of these sites are important for pairing-sensitive silencing. One of the required sites is for the Polycomb group protein Pleiohomeotic and another is GAGAG, a sequence bound by the proteins GAGA factor and Pipsqueak. The identity of the other proteins is unknown. These data suggest a surprising degree of complexity in the DNA-binding proteins required for PSE function.