Iannello R C, Kola I, Dahl H H
Murdoch Institute, Royal Children's Hospital, Melbourne, Victoria, Australia.
J Biol Chem. 1993 Oct 25;268(30):22581-90.
We have determined by deletion analysis that the most proximal region of the Pdha-2 promoter between nucleotide position -187 to +22 harbors a transcriptionally active core. This "core" promoter directs high levels of CAT (chloramphenicol acetyltransferase) reporter gene transcription in HeLa cells. DNase I footprinting of the proximal promoter revealed four regions of protection. One of these contains the consensus sequence for the Sp1 binding site and another the ATF/CREB binding site. The cis-sequences of the remaining two protected regions (designated MEP-2 and MEP-3; Mouse E1 alpha Promoter site) show no apparent consensus homology with cis-elements of other known transcription factors. Results of electrophoretic mobility shift assays confirm that the ATF/CREB and MEP binding sites interact in a characteristic and specific manner with factors present in nuclei of both testis and somatic tissue. The factor which recognizes the MEP-3 motif appears to be ubiquitous, whereas the MEP-2-protein complexes were tissue-specific. Interestingly, formation of a complex involving MEP-2 and a putative testis-specific binding factor (tau-MEP-2BF) is first observed in the testis of 2-week-old mice, this correlates with the expression of Pdha-2. In contrast, the formation of complexes between the MEP-2 binding site and a somatic variant of MEP-2BF (sigma-MEP-2BF) decreases in the testis as spermatogenesis proceeds. Our results suggest that 1) the MEP-2 binding factors are temporally regulated during spermatogenesis, and 2) interactions involving these factors with the MEP-2 cis-element may be important for modulating Pdha-2 expression.
我们通过缺失分析确定，Pdha - 2启动子在核苷酸位置 - 187至 + 22之间的最近端区域含有一个转录活性核心。这个“核心”启动子在HeLa细胞中指导高水平的氯霉素乙酰转移酶（CAT）报告基因转录。近端启动子的DNase I足迹分析揭示了四个受保护区域。其中一个区域含有Sp1结合位点的共有序列，另一个区域含有ATF/CREB结合位点。其余两个受保护区域（命名为MEP - 2和MEP - 3；小鼠E1α启动子位点）的顺式序列与其他已知转录因子的顺式元件没有明显的共有同源性。电泳迁移率变动分析结果证实，ATF/CREB和MEP结合位点与睾丸和体细胞组织细胞核中存在的因子以特征性和特异性方式相互作用。识别MEP - 3基序的因子似乎普遍存在，而MEP - 2 - 蛋白质复合物具有组织特异性。有趣的是，在2周龄小鼠的睾丸中首次观察到涉及MEP - 2和假定的睾丸特异性结合因子（tau - MEP - 2BF）的复合物形成，这与Pdha - 2的表达相关。相反，随着精子发生的进行，睾丸中MEP - 2结合位点与MEP - 2BF的体细胞变体（sigma - MEP - 2BF）之间的复合物形成减少。我们的结果表明：1）MEP - 2结合因子在精子发生过程中受到时间调控；2）这些因子与MEP - 2顺式元件的相互作用可能对调节Pdha - 2的表达很重要。
