Zhang X Y, Ni Y S, Saifudeen Z, Asiedu C K, Supakar P C, Ehrlich M
Department of Biochemistry, Tulane Medical School, New Orleans, LA 70112, USA.
Nucleic Acids Res. 1995 Aug 11;23(15):3026-33. doi: 10.1093/nar/23.15.3026.
A closely related family of ubiquitous DNA binding proteins, called MDBP, binds with high affinity to two 14 base pair (bp) sites within the human cytomegalovirus immediate early gene 1 (CMV IE1) enhancer and with low affinity to one site beginning 5 bp downstream of the CMV IE1 transcription start point (+5 site). Unlike several cap position downstream MDBP sites in mammalian genes, these MDBP sites do not require cytosine methylation for optimal binding. Mutation of one of the enhancer MDBP sites to prevent MDBP recognition modestly increased the function of a neighboring CREB binding site in a transient transfection assay in the context of one promoter construct. A much larger effect on reporter gene expression (a 10-fold reduction) was seen when the low affinity MDBP recognition sequence at position +5 was converted to a high affinity site in a plasmid containing the CMV IE1 promoter upstream of the reporter gene. Evidence that the increased binding of MDBP at the mutant site is largely responsible for the observed results was provided by transfection experiments with this high affinity MDBP +5 site re-mutated to a non-binding site and by in vitro transcription assay.
一类密切相关的普遍存在的DNA结合蛋白家族，称为MDBP，它与人巨细胞病毒立即早期基因1（CMV IE1）增强子内的两个14碱基对（bp）位点具有高亲和力结合，而与CMV IE1转录起始点下游5 bp处开始的一个位点（+5位点）具有低亲和力结合。与哺乳动物基因中几个帽位点下游的MDBP位点不同，这些MDBP位点不需要胞嘧啶甲基化即可实现最佳结合。在一种启动子构建体的背景下，通过瞬时转染实验，将增强子MDBP位点之一进行突变以阻止MDBP识别，适度增加了相邻CREB结合位点的功能。当在报告基因上游含有CMV IE1启动子的质粒中，将+5位置的低亲和力MDBP识别序列转换为高亲和力位点时，对报告基因表达产生了更大的影响（降低了10倍）。用该高亲和力MDBP +5位点重新突变为非结合位点的转染实验以及体外转录实验提供了证据，表明突变位点处MDBP结合增加在很大程度上导致了观察到的结果。
