Gupta M P, Gupta M, Zak R
Department of Medicine (Section of Cardiology, University of Chicago, Illinois 60637.
J Biol Chem. 1994 Nov 25;269(47):29677-87.
Expression of the cardiac myosin heavy chain (MHC) genes is regulated developmentally and by numerous epigenetic factors. Here we report the identification of a cis-regulatory element and cognate nuclear binding protein(s) responsible for cAMP-induced expression of the rat cardiac alpha-MHC gene. By Northern blot analysis, we found that, in primary cultures of fetal rat heart myocytes, the elevation of intracellular levels of cAMP results in up-regulation of alpha-MHC and down-regulation of beta-MHC mRNA expression. This effect of cAMP was dependent upon the basal level of expression of both MHC transcripts and was sensitive to cycloheximide. In transient expression analysis employing a series of alpha-MHC/CAT constructs, we identified a 31-base pair fragment located in the immediate upstream region (-71 to -40), which confers both muscle-specific and cAMP-inducible expression of the gene. Within this 31-base pair fragment there are two regions, an AT-rich portion and a hybrid motif which contains overlapping sequences of E-box and M-CAT binding sites (GGCACGTGGAATG). By substitution mutation analysis, both elements were found important for the basal muscle-specific expression; however, the cAMP-inducible expression of the gene is conferred only by the E-box/M-CAT hybrid motif (EM element). Using mobility gel shift competition assay, immunoblotting, and UV-cross-linking analyses, we found that a protein binding to the EM element is indistinguishable from the transcription enhancer factor-1 (TEF-1) in terms of sequence recognition, molecular mass, and immunoreactivity. Methylation interference and point mutation analyses indicate that, besides M-CAT sequences, center CG dinucleotides of the E-box motif CACGTG are essential for protein binding to the EM element and for its functional activity. Furthermore, our data also show that, in addition to TEF-1, another HF-1a-related factor may be recognized by the alpha-MHC gene EM element. These results are first to demonstrate transcriptional activation of a sarcomeric gene by cAMP and support the role of TEF-1 and HF-1a-like factors in the regulation of alpha-MHC gene expression in cardiac myocytes.
心肌肌球蛋白重链（MHC）基因的表达受发育过程及众多表观遗传因素调控。在此，我们报告鉴定出一种顺式调控元件及相关核结合蛋白，它们负责cAMP诱导的大鼠心脏α-MHC基因表达。通过Northern印迹分析，我们发现，在原代培养的胎鼠心肌细胞中，细胞内cAMP水平升高导致α-MHC上调，β-MHC mRNA表达下调。cAMP的这种作用依赖于两种MHC转录本的基础表达水平，且对放线菌酮敏感。在使用一系列α-MHC/CAT构建体的瞬时表达分析中，我们鉴定出一个位于紧邻上游区域（-71至-40）的31个碱基对的片段，它赋予该基因肌肉特异性和cAMP诱导性表达。在这个31个碱基对的片段中有两个区域，一个富含AT的部分和一个杂交基序，该杂交基序包含E盒和M-CAT结合位点（GGCACGTGGAATG）的重叠序列。通过替代突变分析，发现这两个元件对基础肌肉特异性表达都很重要；然而，该基因的cAMP诱导性表达仅由E盒/M-CAT杂交基序（EM元件）赋予。使用迁移凝胶迁移竞争分析、免疫印迹和紫外线交联分析，我们发现与EM元件结合的一种蛋白质在序列识别、分子量和免疫反应性方面与转录增强因子-1（TEF-1）无法区分。甲基化干扰和点突变分析表明，除了M-CAT序列外，E盒基序CACGTG的中心CG二核苷酸对于蛋白质与EM元件的结合及其功能活性至关重要。此外，我们的数据还表明，除了TEF-1外，α-MHC基因EM元件可能还能识别另一种与HF-1a相关的因子。这些结果首次证明了cAMP对肌节基因的转录激活作用，并支持了TEF-1和HF-1a样因子在心肌细胞中α-MHC基因表达调控中的作用。
