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首页> 外文期刊>Molecular and Cellular Biology >(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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(CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene.

机译：（CT）n（GA）n重复序列和热休克元件在果蝇hsp26基因的染色质结构和转录激活中具有独特的作用。

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摘要

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.
机译：先前对果蝇的hsp26基因的分析表明，除了TATA框和近端和远端热休克元件（HSE）（相对于转录起始位点位于-59和-340处）之外，为了完全产生热冲击，需要在-135至-85重复（CT）n（RL Glaser，GH Thomas，ES Siegfried，SCR Elgin和JT Lis，J。Mol。Biol。211：751-761，1990）。这种（CT）n元素似乎有助于形成hsp26的野生型染色质结构，hsp26是一种有组织的核小体阵列，使HSE处于无核小体的DNase I超敏感（DH）位点（Q. Lu，LL Wallrath， BD Allan，RL Glaser，JT Lis和SCR Elgin，J。Mol。Biol。225：985-998，1992）。对hsp26上游序列的检查发现，在-347至-341处有一个与远端HSE相邻的（CT）n元素。我们分析了该远端（CT）n元件（-347至-341），近端（CT）n元件（-135至-85）和这两种HSE对染色质结构形成和热休克诱导性。包含这些序列元件的定点突变，缺失，取代或重排的hsp26构建体已在框架内与大肠杆菌lacZ基因融合，并通过P元素介导的种系转化而重新引入了黑腹果蝇D. melanogaster基因组。通过DNase I或限制性酶处理分离的细胞核分析转基因的染色质结构（在基因激活之前），并通过测量β-半乳糖苷酶活性来监测热诱导表达。结果表明，远端或近端（CT）n元件的突变，缺失或替代都会影响染色质结构和转基因的热诱导表达。这些（CT）n重复序列在体内与非组蛋白相关，并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，在热激之前，这些HSE似乎不含蛋白质。我们的结果表明，GAGA因子是许多果蝇基因正常表达所必需的丰富蛋白质因子，而热激因子则是在热激后激活的特定转录因子，在基因调控中起着不同的作用：GAGA因子建立和/或维持了DH位点在热激诱导之前，而活化的热激因子则识别并结合位于DH位点内的HSE以触发转录。

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