O'Reilly M A, Geiser A G, Kim S J, Bruggeman L A, Luu A X, Roberts A B, Sporn M B
Laboratory of Chemoprevention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1992 Oct 5;267(28):19938-43.
Transforming growth factor TGF-beta 2 is encoded by multiple mRNA transcripts of 5.8, 5.1, 4.0, 3.8, and 2.8 kilobase pairs (kb) that are expressed in various human and monkey cells. Northern blot analysis using genomic fragments of DNA was used to demonstrate that some of this size heterogeneity is due to differences in the length of the 5'-untranslated region. Probes that were colinear with the first 600 nucleotides of the 5'-untranslated region detected only the 5.8-, 4.0-, and 3.8-kb transcripts. In order to identify DNA elements that regulate the transcription of these mRNA transcripts, deletion constructs of 5'-flanking DNA were ligated to the coding region for chloramphenicol acetyltransferase (CAT) and analyzed for promoter activity in several cell lines. Sequences responsible for putative enhancer and silencer regions were identified between -778 and -40 relative to the transcription initiation site. Addition of a cyclic AMP-responsive element/activating transcription factor-like element at -74 resulted in a 5-10-fold increase in CAT activity over that expressed with a construct that contained only the TATA box. This increase in CAT activity was suppressed by the addition of DNA sequences between -257 and -187, whereas sequences between -778 and -257 stimulated CAT activity. Point mutations within the ATF binding site at -74 resulted in a marked decrease in CAT expression. Cotransfection with ATF-1 or ATF-2 expression plasmids resulted in both dose-dependent stimulatory and inhibitory activities that were cell type-dependent. These studies identify multiple transcription initiation sites for TGF-beta 2 and demonstrate that transcription from one of these promoters is dependent upon an ATF binding site located 5' of the TATA box.
转化生长因子β2（TGF-β2）由5.8、5.1、4.0、3.8和2.8千碱基对（kb）的多个mRNA转录本编码，这些转录本在多种人类和猴细胞中表达。使用基因组DNA片段进行的Northern印迹分析表明，这种大小异质性部分是由于5'非翻译区长度的差异所致。与5'非翻译区前600个核苷酸共线的探针仅检测到5.8 kb、4.0 kb和3.8 kb的转录本。为了鉴定调节这些mRNA转录本转录的DNA元件，将5'侧翼DNA的缺失构建体连接到氯霉素乙酰转移酶（CAT）的编码区，并在几种细胞系中分析其启动子活性。在相对于转录起始位点的-778至-40之间鉴定出了负责假定增强子和沉默子区域的序列。在-74处添加一个环磷酸腺苷反应元件/激活转录因子样元件导致CAT活性比仅包含TATA盒的构建体所表达的活性增加5至10倍。添加-257至-187之间的DNA序列可抑制CAT活性的这种增加，而-778至-257之间的序列则刺激CAT活性。-74处ATF结合位点内的点突变导致CAT表达明显降低。与ATF-1或ATF-2表达质粒共转染导致剂量依赖性的刺激和抑制活性，且这些活性依赖于细胞类型。这些研究确定了TGF-β2的多个转录起始位点，并证明这些启动子之一的转录依赖于位于TATA盒5'端的ATF结合位点。
