DNASE-I HYPERSENSITIVITY ANALYSIS OF THE L-TYPE PYRUVATE-KINASE GENE IN RATS AND TRANSGENIC MICE
DNASE-I HYPERSENSITIVITY ANALYSIS OF THE L-TYPE PYRUVATE-KINASE GENE IN RATS AND TRANSGENIC MICE
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DOI：
10.1111/j.1432-1033.1992.tb17013.x
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发表时间：
1992-07-01
期刊：
影响因子：
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通讯作者：
RAYMONDJEAN, M
中科院分区：
文献类型：
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作者：
BOQUET, D;VAULONT, S;RAYMONDJEAN, M
大鼠L型丙酮酸激酶基因具有两个相距500 bp的启动子。 L' 启动子对红系细胞具有特异性。 L启动子是肝脏特异性的，受饮食和激素的调节；葡萄糖和胰岛素呈正向作用，胰高血糖素通过 cAMP 呈负向作用。参与这种组织特异性和激素调节基因表达的 DNA 元件位于 5' 侧翼区域的 3.2 kbp 内，如先前通过转基因小鼠分析所证明的 [Tremp, G. L.、Boquet, D.、Ripoche, M. A.、Cognet, M.、Yu-Chun, L.、Jami, J.、Kahn, A. 和 Daegelen, D. (1989) J. Biol.化学。 264, 19904-19910]。此外，我们在这些小鼠中观察到基因表达依赖于转基因拷贝数而不依赖于整合位点。我们在此介绍内源性大鼠 L 型丙酮酸激酶基因和两种转基因构建体与发育、组织分化、营养和激素状态相关的 DNase-I 超敏性分析。在大鼠中，检测到内源基因的两组近端位点；过敏位点 (HSS) 成人肝脏中的 HSS-1 和胎儿肝脏（主要的红细胞生成组织）中的 HSS-A。两组都可能与 L 或 L' 启动子处的转录起始复合物有关。检测到另外两个远端组；成人肝脏中的 HSS-2 约为 3 kbp（相对于肝脏特异性帽位点），胎儿肝脏中的 HSS-B 约为 4 kbp。这些位点被认为对应于激活序列；在成人肝脏中，删除包含 HSS-2 的片段会引起从转基因的 L 启动子开始的转录急剧减少。在成年肝脏中，HSS-1 似乎是一个转录相关位点，在禁食大鼠中大大减弱，而 HSS-2 则与转录无关。大鼠内源基因和完全大鼠转基因的 DNase-I 超敏性模式相似；存在肝脏特异性 HSS-1 和 HSS-2，并且位点的强度与整合的拷贝数相关。有趣的是，HSS-1 仍然是可检测到的，并且其强度仍然与 HSS-2 缺失的截短转基因中的整合拷贝数成正比，而该转基因的表达非常弱（但仍然是组织特异性的）。这些结果强烈表明，每个转基因拷贝都拥有一套完整的特定核蛋白复合物，并且无论有或没有HSS-2，DNA都处于潜在的活性构型。
The rat L-type pyruvate kinase gene possesses two promoters located 500 bp apart. The L' promoter is specific to erythroid cells. The L promoter is specific to liver and is regulated by diet and hormones; positively by glucose and insulin and negatively by glucagon via cAMP. The DNA elements involved in this tissue-specific and hormone-regulated gene expression are located within 3.2 kbp of 5' flanking region as previously demonstrated by transgenic mice analysis [Tremp, G. L., Boquet, D., Ripoche, M. A., Cognet, M., Yu-Chun, L., Jami, J., Kahn, A. and Daegelen, D. (1989) J. Biol. Chem. 264, 19904-19910]. Moreover, we have observed in these mice that gene expression was dependent on the transgene copy number and independent of the integration site. We present here DNase-I-hypersensitivity analysis of the endogenous rat L-type pyruvate kinase gene and of two transgene constructs in relation to development, tissue differentiation, nutritional and hormonal status. In rats, two groups of proximal sites were detected on the endogenous gene; hypersensitive site (HSS) HSS-1 in adult liver and HSS-A in fetal liver (a major erythropoietic tissue). Both groups are probably related to the transcriptional initiation complexes at either the L or L' promoter. Two other distal groups were detected; HSS-2 at - 3 kbp (with respect to the liver-specific cap site) in adult liver and HSS-B around - 4 kbp in fetal liver. These sites are thought to correspond to activating sequences; in adult liver, deletion of a fragment encompassing HSS-2 provokes a dramatic reduction of transcription starting at the L promoter of the transgene. In adult liver, HSS-1 appears to be a transcription-associated site, being greatly weakened in fasted rats, while HSS-2 is transcription independent.The pattern of DNase-I hypersensitivity is similar for the rat endogenous gene and for the complete rat transgene; the liver-specific HSS-1 and HSS-2 are present and the intensity of the sites is correlated to the number of integrated copies. Interestingly, HSS-1 is still detectable and its intensity remains proportional to the number of integrated copies in a truncated transgene with HSS-2 deletion, while this transgene is very weakly (but nevertheless tissue-specifically) expressed. These results strongly suggest that each transgene copy possesses a complete set of specific nucleoprotein complexes and that, with or without HSS-2, the DNA is in a potentially active configuration.