Singh M, Birshtein B K
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4392-7. doi: 10.1073/pnas.93.9.4392.
The transcription factor, B-cell-specific activator protein (BSAP), represses the murine immunoglobulin heavy-chain 3' enhancer 3' alpha E(hs1,2) in B cells. Analysis of various 3'alpha E deletional constructs indicates that sequences flanking a and b BSAP-binding sites are essential for appropriate regulation of the enhancer. An octamer motif 5' of the a site and a specific G-rich motif 3' of the b site were identified by competition in electrophoretic mobility-shift assays and methylation-interference foot-printing analysis. Site-directed mutagenesis of either the octamer or G-rich sites resulted in the complete release of repression of 3' alpha E(hs1,2), implicating these two motifs in the repression of this enhancer in B cells. However, when both BSAP-binding sites were mutated, the octamer and G-rich motifs functioned as activators. Moreover, in plasma cells, when BSAP is not expressed, 3' alpha E(hs1,2) is active, and its activity depends on the presence of the other two factors. These results suggest that in B cells, 3' alpha E (hs1,2) is down-regulated by the concerted actions of BSAP, octamer, and G-rich DNA-binding proteins. Supporting this notion of concerted repression, a physical interaction between BSAP and octamer-binding proteins was demonstrated using glutathione S-transferase fusion proteins. Thus, concerted repression of 3' alpha E (hs1,2) in B cells provides a sensitive mechanism by which this enhancer, either individually or as part of a locus-controlling region, is highly responsive to any of several participating factors.
转录因子B细胞特异性激活蛋白（BSAP）在B细胞中抑制小鼠免疫球蛋白重链3'增强子3'αE（hs1,2）。对各种3'αE缺失构建体的分析表明，a和b BSAP结合位点侧翼的序列对于增强子的适当调控至关重要。通过电泳迁移率变动分析中的竞争和甲基化干扰足迹分析，鉴定出a位点5'的八聚体基序和b位点3'的特定富含G的基序。对八聚体或富含G的位点进行定点诱变导致3'αE（hs1,2）的抑制完全解除，表明这两个基序参与了B细胞中该增强子的抑制。然而，当两个BSAP结合位点都发生突变时，八聚体和富含G的基序起激活剂的作用。此外，在浆细胞中，当不表达BSAP时，3'αE（hs1,2）是活跃的，其活性取决于其他两个因子的存在。这些结果表明，在B细胞中，3'αE（hs1,2）通过BSAP、八聚体和富含G的DNA结合蛋白的协同作用而下调。使用谷胱甘肽S-转移酶融合蛋白证明了BSAP与八聚体结合蛋白之间的物理相互作用，这支持了协同抑制的观点。因此，B细胞中3'αE（hs1,2）的协同抑制提供了一种敏感机制，通过该机制，该增强子无论是单独还是作为基因座控制区的一部分，都对几种参与因子中的任何一种高度敏感。
