Transcriptional regulation of the human nonmuscle myosin II heavy chain-A gene - Identification of three clustered cis-elements in intron-1 which modulate transcription in a cell type- and differentiation state-dependent manner
Transcriptional regulation of the human nonmuscle myosin II heavy chain-A gene - Identification of three clustered cis-elements in intron-1 which modulate transcription in a cell type- and differentiation state-dependent manner
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DOI：
10.1074/jbc.273.15.9168
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发表时间：
1998-04-10
影响因子：
4.8
通讯作者：
Kawamoto, S
中科院分区：
文献类型：
--
作者：
Beohar, N;Kawamoto, S
在试图确定人类非肌肉肌球蛋白II重链（MHC）-A基因的转录调控的顺式作用元件，从转录起始位点，其中包括整个37-kb内含子1，上游延伸20个碱基（kb）和下游延伸40 kb的区域进行了检查。使用荧光素酶报告基因构建体的瞬时转染分析，已经发现位于转录起始位点下游23 kb的内含子1中的100个碱基对（bp）区域（N2 d）以细胞类型和分化状态依赖性方式激活转录，最大活性在NIH 3 T3成纤维细胞中观察到活性（类似于20倍），在增殖和未分化的C2 C12成肌细胞中观察到中等活性（7倍）。该区域在终末分化的C2 C12肌管中几乎是无活性的，其中内源性非肌肉MHC-A表达被下调。使用NIH 3 T3核提取物进行凝胶迁移率变动分析和甲基化干扰分析以确定转录因子的蛋白质结合元件。在N2 d区域中已经鉴定了三个结合元件，抗体超移位实验，以及使用特异性转录因子的共有结合序列的竞争实验，揭示了大多数5 '元件，C（GGGAGGGGCC）被Sp1和Sp3转录因子特异性地和排他地识别，元件C紧接着一个新元件A（GTGACCC），第三个元件F（GTGTCAGGTG），其含有E盒，位于元件A的3'端50 bp处。元件F可以部分地被上游刺激因子USF 1和/或USF 2识别。用荧光素酶报告基因构建体的转染研究表明，在NIH 3 T3细胞中，A和C结合因子协同激活转录活性。
In an attempt to identify cis-acting elements for transcriptional regulation of the human nonmuscle myosin II heavy chain (MHC)-A gene, the region extending 20 kilobases (kb) upstream and 40 kb downstream from the transcription start sites, which includes the entire 37-kb intron 1, was examined. Using transient transfection analysis of luciferase reporter constructs, a 100-base pair (bp) region (N2d) in intron 1, located 23 kb downstream from the transcriptional start sites, has been found to activate transcription in a cell type-and differentiation state-dependent manner, Maximum activity (similar to 20-fold) is seen in NIH 3T3 fibroblasts and intermediate activity (7-fold) in proliferating and undifferentiated C2C12 myoblasts, In contrast, this region is almost inactive in terminally differentiated C2C12 myotubes, in which endogenous nonmuscle MHC-A expression is down-regulated,Gel mobility shift assays and methylation interference analyses were performed using NIH 3T3 nuclear extracts to determine the protein-binding elements for transcription factors, Three binding elements have been identified within the N2d region, Antibody-supershift experiments, as well as competition experiments using consensus binding sequences for specific transcription factors, revealed that the most 5'-element, C (GGGAGGGGCC) is recognized specifically and exclusively by Sp1 and Sp3 transcriptional factors, Element C is immediately followed by a novel element, A (GTGACCC), A third element, F (GTGTCAGGTG), which contains an E-box, is located 50 bp 3' to element A. Element F can be recognized partially by upstream stimulatory factors, USF1 and/or USF2, Transfection studies with luciferase reporter constructs which include mutations in all three elements in various combinations demonstrate that the A and C binding factors cooperatively activate transcriptional activity in NIH 3T3 cells, The F binding factor shows an additive effect on transcription.