Ess K C, Whitaker T L, Cost G J, Witte D P, Hutton J J, Aronow B J
Department of Pediatrics, Children's Hospital Medical Center, University of Cincinnati, Ohio 45229, USA.
Mol Cell Biol. 1995 Oct;15(10):5707-15. doi: 10.1128/MCB.15.10.5707.
Locus control regions (LCRs) are powerful assemblies of cis elements that organize the actions of cell-type-specific trans-acting factors. A 2.3-kb LCR in the human adenosine deaminase (ADA) gene first intron, which controls expression in thymocytes, is composed of a 200-bp enhancer domain and extended flanking sequences that facilitate activation from within chromatin. Prior analyses have demonstrated that the enhancer contains a 28-bp core region and local adjacent augmentative cis elements. We now show that the core contains a single critical c-Myb binding site. In both transiently cotransfected human cells and stable chromatin-integrated yeast cells, c-Myb strongly transactivated reporter constructs that contained polymerized core sequences. c-Myb protein was strongly evident in T lymphoblasts in which the enhancer was active and was localized within discrete nuclear structures. Fetal murine thymus exhibited a striking concordance of endogenous c-myb expression with that of mouse ADA and human ADA LCR-directed transgene expression. Point mutation of the c-Myb site within the intact 2.3-kb LCR severely attenuated enhancer activity in transfections and LCR activity in transgenic thymocytes. Within the context of a complex enhancer and LCR, c-Myb can act as an organizer of thymocyte-specific gene expression via a single binding site.
基因座控制区（LCRs）是顺式元件的强大组合，可组织细胞类型特异性反式作用因子的作用。人类腺苷脱氨酶（ADA）基因第一内含子中的一个2.3 kb LCR控制胸腺细胞中的表达，它由一个200 bp的增强子结构域和促进染色质内部激活的侧翼延伸序列组成。先前的分析表明，该增强子包含一个28 bp的核心区域和局部相邻的增强性顺式元件。我们现在表明，该核心包含一个关键的c-Myb结合位点。在瞬时共转染的人类细胞和稳定染色质整合的酵母细胞中，c-Myb强烈反式激活包含聚合核心序列的报告基因构建体。c-Myb蛋白在增强子活跃的T淋巴母细胞中强烈可见，并定位于离散的核结构内。胎鼠胸腺中内源性c-myb表达与小鼠ADA和人类ADA LCR指导的转基因表达表现出显著的一致性。完整的2.3 kb LCR内c-Myb位点的点突变严重减弱了转染中的增强子活性和转基因胸腺细胞中的LCR活性。在复杂的增强子和LCR背景下，c-Myb可通过单个结合位点作为胸腺细胞特异性基因表达的组织者。
