A NEW MYOCYTE-SPECIFIC ENHANCER-BINDING FACTOR THAT RECOGNIZES A CONSERVED ELEMENT ASSOCIATED WITH MULTIPLE MUSCLE-SPECIFIC GENES
A NEW MYOCYTE-SPECIFIC ENHANCER-BINDING FACTOR THAT RECOGNIZES A CONSERVED ELEMENT ASSOCIATED WITH MULTIPLE MUSCLE-SPECIFIC GENES
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DOI：
10.1128/mcb.9.11.5022
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发表时间：
1989-11-01
影响因子：
5.3
通讯作者：
OLSON, EN
中科院分区：
文献类型：
--
作者：
GOSSETT, LA;KELVIN, DJ;OLSON, EN
骨骼肌成肌细胞暴露于生长因子缺乏的培养基中导致肌肉特异性基因的转录激活，包括肌肉肌酸激酶基因（mck）。mck的组织特异性、发育调节和高水平表达主要由位于相对于转录起始位点的碱基对（bp）-1350和-1048之间的肌肉特异性增强子（E. A.斯滕贝格，G. Spizz，W. M.佩里，D.沃伊特河Weil和E. N.奥尔森，摩尔。Cell. 8：2896-2909，1988）。为了开始定义介导mck增强子在分化肌细胞中选择性激活的调节机制，我们进一步界定了这种增强子的边界，并分析了它与来自各种肌源性和非肌源性细胞类型的核因子的相互作用。缺失突变表明，mck上游1，204至1，095 bp之间的区域作为弱肌肉特异性增强子发挥作用，其依赖于邻近的增强子元件来获得强活性。该相邻的激活元件在单拷贝中不表现出增强子活性，但在多聚化时充当强增强子。结合DNA酶I足迹和焦碳酸二乙酯干扰的凝胶阻滞试验表明，核因子从分化的C2肌管和BC 3 H1肌细胞识别的保守的A+ T-丰富的序列内的外周激活区。这种肌细胞特异性增强子结合因子，命名为MEF-2，在C2或BC 3 H1成肌细胞或几种非肌细胞系的核提取物中检测不到。MEF-2在成肌细胞暴露于丝裂原缺陷培养基后2小时内首次检测到，此后24至48小时内丰度增加。MEF-2的出现需要持续的蛋白质合成，并被成纤维细胞生长因子和β型阻止。转化生长因子，其阻断肌肉特异性基因的诱导。一个成肌细胞特异性因子，在去除生长因子后4小时内下调，也被发现与MEF-2识别位点结合。在大鼠和人mck增强子、大鼠肌球蛋白轻链（mlc）-1/3增强子和鸡心脏mlc-2A启动子中鉴定了一个10 bp序列，该序列通过DNase I足迹法和焦碳酸二乙酯干扰显示与MEF-2直接相互作用。对应于mlc-1/3增强子区域的寡聚体，其包含该保守序列，结合MEF-2并竞争其与mck增强子的结合。因此，这些结果提供了一种新的肌细胞特异性增强子结合因子，MEF-2，这是在分化程序的早期表达，并抑制特定的多肽生长因子的证据。MEF-2识别与多个肌肉特异性基因相关的保守激活元件的能力表明，该因子可能参与肌肉发生过程中基因的协调调节。
Exposure of skeletal myoblasts to growth factor-deficient medium results in transcriptional activation of muscle-specific genes, including the muscle creatine kinase gene (mck). Tissue specificity, developmental regulation, and high-level expression of mck are conferred primarily by a muscle-specific enhancer located between base pairs (bp) -1350 and -1048 relative to the transcription initiation site (E. A. Sternberg, G. Spizz, W. M. Perry, D. Vizard, R. Weil, and E. N. Olson, Mol. Cell. Biol. 8:2896-2909, 1988). To begin to define the regulatory mechanisms that mediate the selective activation of the mck enhancer in differentiating muscle cells, we have further delimited the boundaries of this enhancer and analyzed its interactions with nuclear factors from a variety of myogenic and nonmyogenic cell types. Deletion mutagenesis showed that the region between 1,204 and 1,095 bp upstream of mck functions as a weak muscle-specific enhancer that is dependent on an adjacent enhancer element for strong activity. This adjacent activating element does not exhibit enhancer activity in single copy but acts as a strong enhancer when multimerized. Gel retardation assays combined with DNase I footprinting and diethyl pyrocarbonate interference showed that a nuclear factor from differentiated C2 myotubes and BC3H1 myocytes recognized a conserved A+T-rich sequence within the peripheral activating region. This myocyte-specific enhancer-binding factor, designated MEF-2, was undetectable in nuclear extracts from C2 or BC3H1 myoblasts or several nonmyogenic cell lines. MEF-2 was first detectable within 2 h after exposure of myoblasts to mitogen-deficient medium and increased in abundance for 24 to 48 h thereafter. The appearance of MEF-2 required ongoing protein synthesis and was prevented by fibroblast growth factor and type .beta. transforming growth factor, which block the induction of muscle-specific genes. A myoblast-specific factor that is down regulated within 4 h after removal of growth factors was also found to bind to the MEF-2 recognition site. A 10-bp sequence, which was shown by DNase I footprinting and diethyl pyrocarbonate interference to interact directly with MEF-2, was identified within the rat and human mck enhancers, the rat myosin light-chain (mlc)-1/3 enhancer, and the chicken cardiac mlc-2A promoter. Oligomers corresponding to the region of the mlc-1/3 enhancer, which encompasses this conserved sequence, bound MEF-2 and competed for its binding to the mck enhancer. These results thus provide evidence for a novel myocyte-specific enhancer-binding factor, MEF-2, that is expressed early in the differentiation program and is suppressed by specific polypeptide growth factors. The ability of MEF-2 to recognize conserved activating elements associated with multiple muscle-specific genes suggests that this factor may participate in the coordinate regulation of genes during myogenesis.