Van Dijck P, De Vos P, Winderickx J, Verhoeven G
Laboratory for Experimental Medicine and Endocrinology, Department of Developmental Biology, Gasthuisberg, Leuven, Belgium.
J Steroid Biochem Mol Biol. 1993 May;45(5):353-66. doi: 10.1016/0960-0760(93)90004-g.
In order to understand the tissue- and hormone-specific control of alpha 2u-globulin synthesis we isolated the 5'-upstream putative regulatory region of two alpha 2u-globulin genes: RAP 01 and RAO 01. Both clones seem to be expressed in rat liver. DNAseI footprinting analysis after incubation with rat liver nuclear extracts was used to identify regions of potential interest. Particular attention was paid to protected regions located in the neighbourhood of domains which, according to our previous studies, interact specifically with androgen- and glucocorticoid-receptor complexes. Fifteen DNAseI footprints could be mapped in clone RAO 01 (bp -758 up to the cap site). Nineteen footprints were observed in the corresponding region of RAP 01. Differences in the footprinting patterns were mainly observed in the more distal regions. Our data confirm the presence in both clones of two binding sites for the liver enriched factor pseudo-NF1 and one site for C/EBP previously observed in other alpha 2u-globulin genes. In addition we have been able to demonstrate, in RAP 01 only, a binding site for transferrin-liver factor 1. No differences in footprinting patterns could be demonstrated using liver nuclear extracts derived from animals with a high hepatic expression of alpha 2u-globulins (normal male rats) and animals with low to absent expression (prepubertal rats, female rats, rats with the testicular feminization syndrome, diabetic rats and hypophysectomized animals). Transfection experiments indicate that a fragment of RAP 01 (bp -643 up to -617) is able to act as a glucocorticoid and as an androgen response element. Larger fragments of RAP 01 and fragments of RAO 01 are ineffective. It is concluded that the expression of individual alpha 2u-globulin genes is probably the result of combinatorial interactions of several trans-acting factors with appropriate cis-acting elements. Moreover, important sites for tissue-specific and hormone-regulated expression may be situated outside the regions investigated.
为了了解α2u -球蛋白合成的组织特异性和激素特异性调控，我们分离了两个α2u -球蛋白基因RAP 01和RAO 01的5'-上游假定调控区域。这两个克隆似乎都在大鼠肝脏中表达。与大鼠肝脏核提取物孵育后的DNA酶I足迹分析用于鉴定潜在感兴趣的区域。特别关注位于结构域附近的受保护区域，根据我们之前的研究，这些结构域与雄激素和糖皮质激素受体复合物特异性相互作用。在克隆RAO 01（从 -758 bp到帽位点）中可以定位到15个DNA酶I足迹。在RAP 01的相应区域观察到19个足迹。足迹模式的差异主要在更远端的区域观察到。我们的数据证实，在这两个克隆中都存在肝脏富集因子假NF1的两个结合位点以及先前在其他α2u -球蛋白基因中观察到的一个C/EBP位点。此外，我们仅在RAP 01中证明了转铁蛋白 - 肝脏因子1的一个结合位点。使用来自α2u -球蛋白肝脏高表达动物（正常雄性大鼠）和低表达至无表达动物（青春期前大鼠、雌性大鼠、睾丸雌性化综合征大鼠、糖尿病大鼠和垂体切除动物）的肝脏核提取物，未发现足迹模式有差异。转染实验表明，RAP 01的一个片段（从 -643 bp到 -617 bp）能够作为糖皮质激素和雄激素反应元件起作用。RAP 01的较大片段和RAO 01的片段无效。得出的结论是，单个α2u -球蛋白基因的表达可能是几种反式作用因子与适当的顺式作用元件组合相互作用的结果。此外，组织特异性和激素调节表达的重要位点可能位于所研究区域之外。
