Foster D N, Min B, Foster L K, Stoflet E S, Sun S, Getz M J, Strauch A R
Ohio State Biochemistry Program, Ohio State University, Columbus 43210.
J Biol Chem. 1992 Jun 15;267(17):11995-2003.
Segments of the 5'-flanking region of the mouse vascular smooth muscle alpha-actin gene were assayed for promoter activity in transfected mouse BC3H1 myogenic cells and AKR-2B embryonic fibroblasts. The region between -150 and -191 that functions as a positive transcriptional element in myogenic and fibroblastic cells contains a mammalian-specific inverted CC(A/T)6GG-type consensus sequence. Expression was restricted to fully differentiated myogenic cells when an additional sequence spanning -191 to -224 was included in reporter gene constructs. This 33-base pair (bp) negative regulatory element is 70% conserved between the mouse and human genes and contains a 10-bp motif at its 3' end that only partially resembles a CC(A/T)6GG element. Retention of a GGGA motif at the 3' boundary of the 33-bp region is sufficient to maintain full transcriptional repression in fibroblasts and is partly responsible for repression in undifferentiated myoblasts. Complete muscle tissue-restrictive expression requires an additional 8 bp from the CC(A/T)6GG-like element immediately 5' to the GGGA motif, since replacement of this region with an unrelated 10-bp sequence completely eliminated restrictive transcriptional behavior in undifferentiated myoblasts. The distal portion of the 5'-flanking region between -224 and -1074 contains six E-box motifs (CANNTG) and mediates high level transcription only in postconfluent BC3H1 myoblasts. Analysis of reporter gene constructs including either the proximal E-box at -240 or all six E-boxes indicate that the five distal E-boxes are not required for high level transcription. A 724-bp segment of the 5'-flanking region consisting of the proximal E-box flanked upstream by a mammalian-specific 352-bp region was sufficient for maximal transcriptional activation in postconfluent BC3H1 myoblasts. Deletion of the 352-bp region restricts the early transcriptional response to high cell density in temporal studies of promoter activity during BC3H1 myogenic cell differentiation.
对小鼠血管平滑肌α-肌动蛋白基因5'-侧翼区的片段进行了分析，以检测其在转染的小鼠BC3H1成肌细胞和AKR-2B胚胎成纤维细胞中的启动子活性。在-150至-191之间的区域在成肌细胞和成纤维细胞中作为正转录元件起作用，该区域包含一个哺乳动物特异性的反向CC(A/T)6GG型共有序列。当报告基因构建体中包含跨越-191至-224的额外序列时，表达仅限于完全分化的成肌细胞。这个33个碱基对（bp）的负调控元件在小鼠和人类基因之间有70%的保守性，并且在其3'端包含一个10 bp的基序，该基序仅部分类似于CC(A/T)6GG元件。在33 bp区域的3'边界保留GGGA基序足以维持在成纤维细胞中的完全转录抑制，并且部分负责未分化成肌细胞中的抑制。完全的肌肉组织限制性表达需要来自紧邻GGGA基序5'端的CC(A/T)6GG样元件的另外8 bp，因为用不相关的10 bp序列替换该区域完全消除了未分化成肌细胞中的限制性转录行为。5'-侧翼区-224至-1074之间的远端部分包含六个E-盒基序（CANNTG），并且仅在汇合后的BC3H1成肌细胞中介导高水平转录。对包含-240处近端E-盒或所有六个E-盒的报告基因构建体的分析表明，高水平转录不需要五个远端E-盒。由近端E-盒及其上游侧翼的一个352 bp哺乳动物特异性区域组成的5'-侧翼区的一个724 bp片段足以在汇合后的BC3H1成肌细胞中实现最大转录激活。在BC3H1成肌细胞分化过程中启动子活性的时间研究中，删除352 bp区域会限制对高细胞密度的早期转录反应。
