Lee B S, Underhill D M, Crane M K, Gluck S L
Department of Medicine/Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1995 Mar 31;270(13):7320-9. doi: 10.1074/jbc.270.13.7320.
Monocyte-macrophage differentiation was used as a model system for studying gene regulation of the human vacuolar H(+)-ATPase (V-ATPase). We examined mRNA levels of various V-ATPase subunits during differentiation of both native monocytes and the cell line THP-1, and found that transcriptional and post-transcriptional mechanisms could account for increases in cell V-ATPase content. From nuclear runoff experiments, we found that one subunit in particular, the B2 isoform (Mr = 56,000), was amplified primarily by transcriptional means. We have begun to examine the structure of the B2 subunit promoter region. Isolation and sequencing of the first exon and 5'-flanking region of this gene reveal a TATA-less promoter with a high G + C content. Primer extension and ribonuclease protection analyses indicate a single major transcriptional start site. We transfected promoter-luciferase reporter plasmids into THP-1 cells to define sequences that mediate transcriptional control during monocyte differentiation. We found that sequences downstream from the transcriptional start site were sufficient to confer increased expression during THP-1 differentiation. DNase I footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5'-untranslated and proximal coding regions.
单核细胞-巨噬细胞分化被用作研究人类液泡H(+)-ATP酶（V-ATP酶）基因调控的模型系统。我们检测了天然单核细胞和细胞系THP-1分化过程中各种V-ATP酶亚基的mRNA水平，发现转录和转录后机制可以解释细胞V-ATP酶含量的增加。通过核转录实验，我们发现，特别是一种亚基，即B2同工型（Mr = 56,000），主要通过转录方式得到扩增。我们已开始研究B2亚基启动子区域的结构。该基因第一个外显子和5'-侧翼区域的分离与测序揭示了一个高G + C含量的无TATA启动子。引物延伸和核糖核酸酶保护分析表明有一个主要转录起始位点。我们将启动子-荧光素酶报告质粒转染到THP-1细胞中，以确定在单核细胞分化过程中介导转录控制的序列。我们发现转录起始位点下游的序列足以在THP-1分化过程中赋予增加的表达。DNA酶I足迹法和序列分析揭示在5'-非翻译区和近端编码区存在多个AP2和Sp1结合位点。
