(CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene
(CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene
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DOI：
10.1128/mcb.13.5.2802-2814.1993
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发表时间：
1993-05
影响因子：
5.3
通讯作者：
Qin Lu;Lori L. Wallrath;H. Granok;Sarah C. R. Elgin
中科院分区：
文献类型：
--
作者：
Qin Lu;Lori L. Wallrath;H. Granok;Sarah C. R. Elgin
先前对黑腹果蝇的hsp 26基因的分析表明，除了TATA盒和近端和远端热休克元件（HSE）（相对于转录起始位点，以-59和-340为中心）之外，完全热休克诱导还需要位于-135至-85的（CT）n重复片段（R.L.格拉泽，G.H.托马斯，E.S.齐格弗里德，S.C.R.埃尔金和J.T. Lis，J. Mol. 211：751-761，1990）。该（CT）n元件似乎有助于形成hsp 26的野生型染色质结构，这是一种有组织的核小体阵列，其将HSE留在无核小体的DNA酶I超敏感（DH）位点（Q. Lu，L.L. Wallrath，B.D. Allan，R.L.格拉泽，J.T. Lis和S.C.R.埃尔金，J. Mol. 225：985-998，1992）。对hsp 26上游序列的检查揭示了在远端HSE附近的-347至-341处的另外的（CT）n元件。我们分析了远端（CT）n元件（-347至-341）、近端（CT）n元件（-135至-85）和两个HSE对染色质结构形成和热休克诱导的贡献。含有这些序列元件的定点突变、缺失、取代或重排的hsp 26构建体已与大肠杆菌lacZ基因框内融合，并重新导入D.通过P-元件介导的生殖系转化法获得黑腹果蝇基因组。通过DNA酶I或限制酶处理分离的细胞核分析转基因的染色质结构（在基因活化之前），并通过测量β-半乳糖苷酶活性监测热诱导表达。结果表明，远端或近端（CT）n元件的突变、缺失或取代影响转基因的染色质结构和热诱导表达。这些（CT）n重复序列在体内与非组蛋白蛋白相关，在体外与纯化的果蝇蛋白GAGA因子结合。相反，HSE是热诱导表达所必需的，但在建立转基因的染色质结构中仅起次要作用。先前的分析表明，在热休克之前，这些HSE似乎不含蛋白质。我们的研究结果表明，GAGA因子，许多果蝇基因的正常表达所需的丰富的蛋白质因子，和热休克因子，热休克后激活的特异性转录因子，在基因调控中发挥不同的作用：GAGA因子建立和/或维持的DH网站之前，热休克诱导，而激活的热休克因子识别并结合位于DH网站内的HSEs触发转录。
Previous analysis of the hsp26 gene of Drosophila melanogaster has shown that in addition to the TATA box and the proximal and distal heat shock elements (HSEs) (centered at -59 and -340, relative to the start site of transcription), a segment of (CT)n repeats at -135 to -85 is required for full heat shock inducibility (R.L. Glaser, G.H. Thomas, E.S. Siegfried, S.C.R. Elgin, and J.T. Lis, J. Mol. Biol. 211:751-761, 1990). This (CT)n element appears to contribute to formation of the wild-type chromatin structure of hsp26, an organized nucleosome array that leaves the HSEs in nucleosome-free, DNase I-hypersensitive (DH) sites (Q. Lu, L.L. Wallrath, B.D. Allan, R.L. Glaser, J.T. Lis, and S.C.R. Elgin, J. Mol. Biol. 225:985-998, 1992). Inspection of the sequences upstream of hsp26 has revealed an additional (CT)n element at -347 to -341, adjacent to the distal HSE. We have analyzed the contribution of this distal (CT)n element (-347 to -341), the proximal (CT)n element (-135 to -85), and the two HSEs both to the formation of the chromatin structure and to heat shock inducibility. hsp26 constructs containing site-directed mutations, deletions, substitutions, or rearrangements of these sequence elements have been fused in frame to the Escherichia coli lacZ gene and reintroduced into the D. melanogaster genome by P-element-mediated germ line transformation. Chromatin structure of the transgenes was analyzed (prior to gene activation) by DNase I or restriction enzyme treatment of isolated nuclei, and heat-inducible expression was monitored by measuring beta-galactosidase activity. The results indicate that mutations, deletions, or substitutions of either the distal or the proximal (CT)n element affect the chromatin structure and heat-inducible expression of the transgenes. These (CT)n repeats are associated with a nonhistone protein(s) in vivo and are bound by a purified Drosophila protein, the GAGA factor, in vitro. In contrast, the HSEs are required for heat-inducible expression but play only a minor role in establishing the chromatin structure of the transgenes. Previous analysis indicates that prior to heat shock, these HSEs appear to be free of protein. Our results suggest that GAGA factor, an abundant protein factor required for normal expression of many Drosophila genes, and heat shock factor, a specific transcription factor activated upon heat shock, play distinct roles in gene regulation: the GAGA factor establishes and/or maintains the DH sites prior to heat shock induction, while the activated heat shock factor recognizes and binds HSEs located within the DH sites to trigger transcription.