Qasba P, Lin E, Zhou M D, Kumar A, Siddiqui M A
Department of Anatomy and Cell Biology, State University of New York Health Science Center, Brooklyn 11203.
Mol Cell Biol. 1992 Mar;12(3):1107-16. doi: 10.1128/mcb.12.3.1107-1116.1992.
The cardiac myosin light chain-2 (MLC-2) gene promoter contains several positive and negative cis-acting sequences that are involved in the regulation of its expression. We describe here the properties of two activator sequences, elements A and P, and their DNA-binding factors (ABFs). Element A (CCAAAAGTGG), located at -61, has homology with the evolutionarily conserved sequence CC(A/T)6GG, present in the genes of many contractile proteins. Element P (TAACCTTGAAAGC), located 114 bp upstream of element A, is conserved in both chicken and rat cardiac MLC-2 gene promoters. Deletion mutagenesis demonstrated that these two elements are involved in the positive regulation of MLC-2 gene transcription. At least two sequence-specific element A-binding proteins, ABF-1 and ABF-2, were identified by gel shift analysis of the fractionated cardiac nuclear proteins. ABF-1 binds to element A with strict dependence on the internal element A sequence AAAAGT. In contrast, ABF-2 exhibits a relaxed sequence requirement, as it recognizes the consensus CArG and CCAAT box sequences as well. ABF-2 also recognizes the distal element P despite the fact that the sequences of elements A and P are divergent. DNase I footprinting, methylation interference, and gel shift analyses demonstrated unequivocally that the element A-DNA affinity-purified protein ABF-2 binds to element P with sequence specificity. Since both elements A and P play a positive regulatory role in MLC-2 gene transcription and bind to a single protein (ABF-2), it would appear that ABF-2 is a key transcription factor with the ability to recognize divergent sequence elements involved in a common regulatory pathway during myogenesis.
心肌肌球蛋白轻链2（MLC - 2）基因启动子包含几个正向和负向顺式作用序列，这些序列参与其表达的调控。我们在此描述两个激活序列元件A和元件P及其DNA结合因子（ABFs）的特性。位于 - 61处的元件A（CCAAAAGTGG）与许多收缩蛋白基因中存在的进化保守序列CC(A/T)6GG具有同源性。元件P（TAACCTTGAAAGC）位于元件A上游114 bp处，在鸡和大鼠的心肌MLC - 2基因启动子中均保守。缺失诱变表明这两个元件参与MLC - 2基因转录的正向调控。通过对分级分离的心肌核蛋白进行凝胶迁移分析，鉴定出至少两种序列特异性元件A结合蛋白ABF - 1和ABF - 2。ABF - 1严格依赖元件A内部序列AAAAGT与元件A结合。相比之下，ABF - 2对序列的要求较为宽松，因为它也能识别共有CArG和CCAAT框序列。尽管元件A和元件P的序列不同，但ABF - 2也能识别远端元件P。DNase I足迹分析、甲基化干扰分析和凝胶迁移分析明确表明，经元件A - DNA亲和纯化的蛋白ABF - 2以序列特异性方式结合元件P。由于元件A和元件P在MLC - 2基因转录中均起正向调控作用，且都与单一蛋白（ABF - 2）结合，因此ABF - 2似乎是一种关键转录因子，能够识别在肌发生过程中参与共同调控途径的不同序列元件。
