Jenkins T D, Nakagawa H, Rustgi A K
Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA.
J Biol Chem. 1997 Sep 26;272(39):24433-42. doi: 10.1074/jbc.272.39.24433.
We previously employed 782 base pairs of the Epstein-Barr virus ED-L2 early lytic cycle promoter in a transgenic mouse model to target cyclin D1 to the stratified squamous epithelium of the tongue and esophagus. This promoter is located 5' to the transcriptional start site of a short open reading frame BNLF-2A and is immediately 3' to the BNLF-1 (LMP-1 oncogene) open reading frame. We studied transcriptional regulation of the ED-L2 promoter by phorbol 12-myristate 13-acetate (PMA) as a means of understanding the tissue specificity of this promoter. The transcriptional activity of the ED-L2 promoter was stimulated 40-fold by PMA and could be blocked with the compound H7 through antagonism of protein kinase C. 5' deletion analysis of the 782-base pair promoter demonstrated that the sequences necessary for PMA-stimulated trans-activation were located in two separate cis-regulatory regions of the promoter: -187 to -164 and -144 to -114 base pairs from the transcription start site of BNLF-2A. Importantly, mutation of critical base pairs in each region was sufficient to abolish PMA-stimulated trans-activation in the native ED-L2 promoter. Region -187 to -164 contains a CACCTG (E-box) motif, and region -144 to -114 contains a CACACCC motif. Both of these motifs are necessary for trans-activation by PMA. These regions do not, however, demonstrate enhancer characteristics when tested in a heterologous minimal promoter system. Variations of the CACACCC motif are found in other keratinocyte-specific promoters, as well as in the DNA binding motifs of the Krüppel-like family of transcription factors. Electrophoretic mobility shift assays with specific competitors and factor-specific antibody supershift assays demonstrated that one complex binding the -187 to -164 region containing the CACCTG nucleotides has characteristics of the helix-loop-helix protein upstream stimulatory factor, whereas a factor binding the CACACCC motif may be a member of the Krüppel-like family. These experiments show how ubiquitous and tissue-specific transcription factors induced by PMA regulate the ED-L2 promoter in squamous epithelial cells.
我们之前在转基因小鼠模型中使用了782个碱基对的爱泼斯坦-巴尔病毒ED-L2早期裂解周期启动子，将细胞周期蛋白D1靶向舌和食管的复层鳞状上皮。该启动子位于短开放阅读框BNLF-2A转录起始位点的5'端，紧挨着BNLF-1（LMP-1癌基因）开放阅读框的3'端。我们研究了佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）对ED-L2启动子的转录调控，以此来了解该启动子的组织特异性。ED-L2启动子的转录活性被PMA刺激了40倍，并且可以通过化合物H7拮抗蛋白激酶C来阻断。对782个碱基对启动子进行的5'端缺失分析表明，PMA刺激的反式激活所需序列位于启动子的两个独立顺式调控区域：从BNLF-2A转录起始位点起的-187至-164以及-144至-114碱基对。重要的是，每个区域中关键碱基对的突变足以消除天然ED-L2启动子中PMA刺激的反式激活。-187至-164区域包含一个CACCTG（E盒）基序，-144至-114区域包含一个CACACCC基序。这两个基序都是PMA反式激活所必需的。然而，当在异源最小启动子系统中测试时，这些区域并未表现出增强子特征。在其他角质形成细胞特异性启动子以及Krüppel样转录因子家族的DNA结合基序中也发现了CACACCC基序的变体。用特异性竞争剂进行的电泳迁移率变动分析和因子特异性抗体超迁移分析表明，一种结合包含CACCTG核苷酸的-187至-164区域的复合物具有螺旋-环-螺旋蛋白上游刺激因子的特征，而结合CACACCC基序的因子可能是Krüppel样家族的成员。这些实验展示了PMA诱导的普遍存在且组织特异性的转录因子如何调控鳞状上皮细胞中的ED-L2启动子。
