MYB BINDING-SITES MEDIATE NEGATIVE REGULATION OF C-MYB EXPRESSION IN T-CELL LINES
MYB BINDING-SITES MEDIATE NEGATIVE REGULATION OF C-MYB EXPRESSION IN T-CELL LINES
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DOI：
10.1182/blood.v86.5.1873.bloodjournal8651873
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发表时间：
1995-09-01
期刊：
影响因子：
20.3
通讯作者：
BOXER, LM
中科院分区：
文献类型：
--
作者：
GUERRA, J;WITHERS, DA;BOXER, LM
在造血细胞发育过程中，c-myb转录因子起着重要作用，c-myb基因在未成熟的增殖细胞和白血病细胞中高水平表达。我们研究了Myb蛋白与人c-myb启动子结合的调节作用。已描述了三个Myb结合位点，位于帽状位点上游约600bp处。通过对造血细胞系的瞬时转染实验，我们发现，先前定义的最大5‘Myb结合位点的缺失对活性没有影响，而含有剩余两个Myb结合位点的区域的缺失导致T细胞系和髓系细胞系的活性增加。为了具体测试这两个Myb结合位点的重要性，我们测量了三点突变构建体的活性。与野生型启动子相比，Myb结合位点的突变导致T细胞中活性的增加。这两个位点的突变产生了更高的活性。将Myb位点突变体导入髓系细胞系，与野生型构建物相比，活性没有变化。凝胶位移分析、紫外光交联和Western blotts结果表明，c-Myb和B-Myb都与Myb I和II结合。我们得出的结论是，Myb家族蛋白在T细胞系中负向调节c-MYB的表达，而不是通过这些位点的正向调节，这在成纤维细胞中已经显示出来。此外，在髓系细胞系中，Myb结合位点是不起作用的。(C)1995年由美国血液病学会主办。
In hematopoietic cell development, the c-myb transcription factor plays an important role, c-myb mRNA is expressed at high levels in immature proliferating cells and in leukemic cells. We have investigated the regulatory role of Myb protein binding to the human c-myb promoter. Three Myb binding sites have been described at approximately 600 bp upstream of the cap site. By transient transfection assays in hematopoietic cell lines, we found that deletion of the previously defined most 5' Myb binding site had no effect on activity, whereas deletion of the region containing the remaining two Myb binding sites resulted in an increase in activity in both a T-cell line and a myeloid cell line. To specifically test the importance of these two Myb binding sites, the activity of three-point mutation constructs was measured. Mutation of either Myb binding site resulted in an increase in activity compared with the wild-type promoter in T cells. Mutation of both sites produced even higher activity. Transfection of the Myb site mutants into the myeloid cell line resulted in no change in activity compared with the wild type construct. Results from gel shift analysis, UV crosslinking, and Western blots showed that both c-Myb and B-Myb bound to the Myb I and II sites. We conclude that the Myb family proteins negatively regulate c-myb expression in T-cell lines in contrast to the positive regulation via these sites, which has been shown in fibroblasts. In addition, in a myeloid cell line, the Myb binding sites are nonfunctional. (C) 1995 by The American Society of Hematology.