Phinney D G, Keiper C L, Francis M K, Ryder K
Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Oncogene. 1994 Aug;9(8):2353-62.
We have identified four DNAase I-hypersensitive regions (DHRs) at the junB locus. DHR1 is located between sequences -100 and +250, DHR2 is centered at -1000, DHR3 at -1650, and DHR4 at +2040 relative to the junB transcriptional start site. Sequence analysis of these DHRs revealed two serum response elements at -1452 and +2091, two cyclic AMP response elements at +2071 and +2116, and a 12-O-tetradecanoylphorbol-13-acetate (TPA) response element at -949. To study the contribution made by these cis-elements to junB transcriptional regulation, we stably transfected a recombinant mouse junB gene (JBSV4) containing the intact junB coding sequences, 6.3 kb of 5'-flanking DNA, and 2.0 kb of 3'-flanking DNA into Rat1A cells. The pattern of DHRs identified at the mouse junB locus was re-established at the JBSV4 locus. By directly comparing JBSV4 and rat junB mRNA levels, we found that these genes were induced to equivalent levels by serum, TPA, cyclic AMP, platelet-derived growth factor, epidermal growth factor, and basic fibroblastic growth factor. These results established that JBSV4 resides in a physical environment within chromatin that closely mimics that of the junB locus, and contains the necessary sequence information to recapitulate the transcriptional regulation of junB. By analysing a series of recombinant mouse junB genes containing deletion mutations in 5'-flanking and 3'-flanking sequences, we provide a quantitative assessment of the contribution these sequences make to junB induction by different regulatory agents.
我们在junB基因座处鉴定出四个脱氧核糖核酸酶I高敏区域（DHRs）。相对于junB转录起始位点，DHR1位于-100至+250序列之间，DHR2以-1000为中心，DHR3位于-1650，DHR4位于+2040。对这些DHRs的序列分析揭示，在-1452和+2091处有两个血清反应元件，在+2071和+2116处有两个环磷酸腺苷反应元件，以及在-949处有一个12-O-十四烷酰佛波醇-13-乙酸酯（TPA）反应元件。为了研究这些顺式元件对junB转录调控的作用，我们将一个重组小鼠junB基因（JBSV4）稳定转染到Rat1A细胞中，该基因包含完整的junB编码序列、6.3 kb的5'侧翼DNA和2.0 kb的3'侧翼DNA。在JBSV4基因座处重新建立了在小鼠junB基因座处鉴定出的DHRs模式。通过直接比较JBSV4和大鼠junB的mRNA水平，我们发现这些基因在血清、TPA、环磷酸腺苷、血小板衍生生长因子、表皮生长因子和碱性成纤维细胞生长因子的作用下被诱导至相同水平。这些结果表明，JBSV4存在于染色质内的一个物理环境中，该环境与junB基因座的环境密切相似，并包含重现junB转录调控所需的序列信息。通过分析一系列在5'侧翼和3'侧翼序列中含有缺失突变的重组小鼠junB基因，我们对这些序列在不同调节因子诱导junB过程中的作用进行了定量评估。
