Bouvagnet P F, Strehler E E, White G E, Strehler-Page M A, Nadal-Ginard B, Mahdavi V
Howard Hughes Medical Institute, Department of Cardiology, Children's Hospital, Boston, Massachusetts.
Mol Cell Biol. 1987 Dec;7(12):4377-89. doi: 10.1128/mcb.7.12.4377-4389.1987.
To identify the DNA sequences that regulate the expression of the sarcomeric myosin heavy-chain (MHC) genes in muscle cells, a series of deletion constructs of the rat embryonic MHC gene was assayed for transient expression after introduction into myogenic and nonmyogenic cells. The sequences in 1.4 kilobases of 5'-flanking DNA were found to be sufficient to direct expression of the MHC gene constructs in a tissue-specific manner (i.e., in differentiated muscle cells but not in undifferentiated muscle and nonmuscle cells). Three main distinct regulatory domains have been identified: (i) the upstream sequences from positions -1413 to -174, which determine the level of expression of the MHC gene and are constituted of three positive regulatory elements and two negative ones; (ii) a muscle-specific regulatory element from positions -173 to -142, which restricts the expression of the MHC gene to muscle cells; and (iii) the promoter region, downstream from position -102, which directs transcription initiation. Introduction of the simian virus 40 enhancer into constructs where subportions of or all of the upstream sequences are deleted (up to position -173) strongly increases the level of expression of such truncated constructs but without changing their muscle specificity. These upstream sequences, which can be substituted for by the simian virus 40 enhancer, function in an orientation-, position-, and promoter-dependent fashion. The muscle-specific element is also promoter specific but does not support efficient expression of the MHC gene. The MHC promoter in itself is not muscle specific. These results underline the importance of the concerted action of multiple regulatory elements that are likely to represent targets for DNA-binding-regulatory proteins.
为了鉴定调控肌肉细胞中肌节肌球蛋白重链（MHC）基因表达的DNA序列，将一系列大鼠胚胎MHC基因的缺失构建体导入成肌细胞和非成肌细胞后，检测其瞬时表达情况。发现5'侧翼DNA的1.4千碱基中的序列足以以组织特异性方式（即在分化的肌肉细胞中，而非未分化的肌肉细胞和非肌肉细胞中）指导MHC基因构建体的表达。已鉴定出三个主要的不同调控结构域：（i）从-1413至-174位的上游序列，其决定MHC基因的表达水平，由三个正调控元件和两个负调控元件组成；（ii）从-173至-142位的肌肉特异性调控元件，其将MHC基因的表达限制在肌肉细胞中；（iii）从-102位下游的启动子区域，其指导转录起始。将猿猴病毒40增强子导入上游序列部分或全部缺失（直至-173位）的构建体中，可强烈提高此类截短构建体的表达水平，但不改变其肌肉特异性。这些可被猿猴病毒40增强子替代的上游序列，以依赖于方向、位置和启动子的方式发挥作用。肌肉特异性元件也是启动子特异性的，但不支持MHC基因的高效表达。MHC启动子本身并非肌肉特异性的。这些结果强调了多个调控元件协同作用的重要性，这些元件可能是DNA结合调控蛋白的作用靶点。
