Peters B, Merezhinskaya N, Diffley J F, Noguchi C T
Laboratory of Chemical Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 Feb 15;268(5):3430-7.
The developmental control of expression of the human epsilon-globin gene appears to be mediated, at least in part, by a transcriptional silencer in the DNA 5' to the cap site of this gene. We have used site-directed mutagenesis and DNA-protein binding assays to define the active motifs of this epsilon-globin silencer. DNase I foot-printing of the silencer region with K562 cell nuclear extracts defined a sequence, which we designate as the epsilon-globin silencer motif or epsilon GSM (epsilon -278 to -258 base pairs (bp)) containing a region (epsilon -270 to -258) with 90% homology to the yeast mating type silencer, ABF-1 (autonomous replicating sequence binding factor one) and which also overlaps at (epsilon -269 to -262) with the human YY1 consensus sequence, an element which mediates transcription repression and activation of viral, mouse, and human genes. The DNase I footprint extended 5' in the silencer region to include an inverted repeat of a six-nucleotide motif (epsilon -267 to -278 bp) which shares 5 of 6 bases with the GATA-1 consensus sequence. In gel mobility shift assays, two specific proteins (A and B) in nuclear extracts from erythroleukemia K562 cells bound to the DNase I-footprinted region. Protein B, associated with epsilon-globin silencer activity in vitro, required an intact epsilon GSM sequence for binding. Mutation of 5 bases within the epsilon GSM in an epsilon-globin promoter-containing fragment extending upstream to 1400 bp in transient transfection assays increased activity by 3.0-fold compared with the native sequence, suggesting that the silencer activity was mediated by the epsilon GSM sequence. We found that protein A could be displaced by a competitor containing the GATA-1 consensus sequence, suggesting that protein A is a GATA-like protein. The region from -267 to -271 within the epsilon GSM and GATA-1 homology region was important for binding of both proteins A and B. These data suggest that protein binding to the epsilon GSM and GATA motifs mediate the negative effect of the silencer on transcription, possibly via direct competition for binding to this DNA region. Recombinant yeast ABF-1 and human YY1 bound to the epsilon GSM. Mutating three bases (epsilon -259, -262, -264) in the epsilon GSM decreased the binding affinity of protein B and recombinant human YY1 but increased the binding affinity of recombinant yeast ABF-1. Furthermore, competitor containing the YY1 consensus sequence competed for protein B binding, whereas competitor containing a perfect yeast ABF-1 consensus sequence did not.(ABSTRACT TRUNCATED AT 400 WORDS)
人类ε-珠蛋白基因表达的发育控制似乎至少部分是由该基因帽位点5'端DNA中的转录沉默子介导的。我们利用定点诱变和DNA-蛋白质结合试验来确定这个ε-珠蛋白沉默子的活性基序。用K562细胞核提取物对沉默子区域进行DNase I足迹分析，确定了一个序列，我们将其命名为ε-珠蛋白沉默子基序或ε-GSM（ε-278至-258碱基对（bp）），其中包含一个区域（ε-270至-258），与酵母交配型沉默子ABF-1（自主复制序列结合因子1）有90%的同源性，并且在（ε-269至-262）处也与人YY1共有序列重叠，YY1是一个介导病毒、小鼠和人类基因转录抑制和激活的元件。DNase I足迹在沉默子区域向5'端延伸，包括一个六核苷酸基序（ε-267至-278 bp）的反向重复序列，该序列与GATA-1共有序列有6个碱基中的5个相同。在凝胶迁移率变动分析中，红白血病K562细胞核提取物中的两种特异性蛋白质（A和B）与DNase I足迹区域结合。蛋白质B在体外与ε-珠蛋白沉默子活性相关，其结合需要完整的ε-GSM序列。在瞬时转染试验中，将含ε-珠蛋白启动子且向上游延伸至1400 bp的片段中的ε-GSM内的5个碱基突变，与天然序列相比，活性增加了3.0倍，这表明沉默子活性是由ε-GSM序列介导的。我们发现蛋白质A可以被含有GATA-1共有序列的竞争者取代，这表明蛋白质A是一种GATA样蛋白。ε-GSM和GATA-1同源区域内-267至-271的区域对蛋白质A和B的结合都很重要。这些数据表明，蛋白质与ε-GSM和GATA基序的结合介导了沉默子对转录的负面影响，可能是通过直接竞争结合该DNA区域。重组酵母ABF-1和人YY1与ε-GSM结合。在ε-GSM中突变三个碱基（ε-259、-262、-264）会降低蛋白质B和重组人YY1的结合亲和力，但会增加重组酵母ABF-1的结合亲和力。此外，含有YY1共有序列的竞争者会竞争蛋白质B的结合，而含有完美酵母ABF-1共有序列的竞争者则不会。（摘要截短至400字）
