Sartorelli V, Webster K A, Kedes L
Program in Molecular Biology and Genetics, University of Southern California School of Medicine, Los Angeles 90033.
Genes Dev. 1990 Oct;4(10):1811-22. doi: 10.1101/gad.4.10.1811.
Expression of the human cardiac alpha-actin gene (HCA) depends on the interactions of multiple transcriptional regulators with promoter elements. We report here that the tissue-specific expression of this promoter is determined by the simultaneous interaction of at least three specific protein-DNA complexes. The myogenic determinant gene MyoD1 activated the transcription of transfected HCA-CAT promoter constructs in nonmuscle cells, including CV-1 and HeLa cells. Gel mobility-shift and footprinting assays revealed that MyoD1 specifically interacted with a single consensus core sequence, CANNTG, at -50. Previously characterized sites interact with a protein identical with or related to the serum response factor (SRF) at -100 and Sp1 at -70. All three elements must be intact to support transcription in muscle cells: site-specific mutation within any one of these three elements eliminated transcriptional expression by the promoter. Furthermore, expression of the promoter in embryonic Drosophila melanogaster cells that lack MyoD1 and Sp1 is strictly dependent on all three sites remaining intact and on the presence of exogenously supplied Sp1 and MyoD1. These experiments suggest that the presence of three sequence-specific binding proteins, including MyoD1, and their intact target DNA sequences are minimal requirements for muscle-specific expression of the HCA gene.
人类心脏α-肌动蛋白基因（HCA）的表达取决于多种转录调节因子与启动子元件的相互作用。我们在此报告，该启动子的组织特异性表达是由至少三种特定蛋白质-DNA复合物的同时相互作用所决定的。生肌决定基因MyoD1在非肌肉细胞（包括CV-1和HeLa细胞）中激活了转染的HCA-CAT启动子构建体的转录。凝胶迁移率变动分析和足迹分析表明，MyoD1在-50处与单个共有核心序列CANNTG特异性相互作用。先前鉴定的位点在-100处与与血清反应因子（SRF）相同或相关的蛋白质相互作用，在-70处与Sp1相互作用。所有这三个元件必须完整才能支持肌肉细胞中的转录：这三个元件中任何一个的位点特异性突变都会消除启动子的转录表达。此外，在缺乏MyoD1和Sp1的胚胎果蝇细胞中，启动子的表达严格依赖于所有三个位点保持完整以及外源提供的Sp1和MyoD1的存在。这些实验表明，包括MyoD1在内的三种序列特异性结合蛋白的存在及其完整的靶DNA序列是HCA基因肌肉特异性表达的最低要求。
