Tissue-restricted expression of the cardiac α-myosin heavy chain gene is controlled by a downstream repressor element containing a palindrome of two Ets-binding sites
Tissue-restricted expression of the cardiac α-myosin heavy chain gene is controlled by a downstream repressor element containing a palindrome of two Ets-binding sites
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DOI：
10.1128/mcb.18.12.7243
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发表时间：
1998-12-01
影响因子：
5.3
通讯作者：
Gupta, MP
中科院分区：
文献类型：
--
作者：
Gupta, M;Zak, R;Gupta, MP
α-肌球蛋白重链（MHC）基因的表达主要限于心肌细胞。到目前为止，已经确定了几个积极的调控元件和它们的结合因子参与α-MHC基因的调控，然而，限制该基因的表达心肌细胞的机制尚未阐明。在这项研究中，我们已经确定了通过使用大鼠心脏α-MHC基因的连续缺失突变体的30 bp的嘌呤丰富的负调控（PNR）元件位于第一内含子区域，似乎是必不可少的组织特异性表达的α-MHC基因。单独去除该元件可使心肌细胞培养物和直接注射质粒DNA的心肌中α-MHC基因的表达升高（20- 30倍）。令人惊讶的是，这种缺失也允许α-MHC基因在HeLa和其他非肌肉细胞中显着表达，在那里它通常是无活性的。PNR元件需要α-MHC基因的上游序列进行负基因调控。通过对PNR元件的DNA酶I足迹分析，鉴定了两个高亲和力Ets结合位点（CTTCCCTGGAAG）的回文序列。此外，通过位点特异性碱基对突变，迁移率凝胶移位竞争和UV交联分析，发现两种不同的Ets样蛋白从心脏和HeLa细胞核提取物结合到PNR基序。此外，PNR结合因子的活性被发现在压力超负荷肥大的成年大鼠心脏中增加了2 - 3倍，其中α-MHC基因通常被抑制。这些数据表明，PNR元件起着双重作用，既下调心肌细胞中的α-MHC基因的表达，又沉默非肌肉细胞中的肌肉基因活性。在不同物种的α-MHC基因和其他心肌细胞限制性基因中发现了类似的回文Ets结合基序。这些结果首次揭示了Ets类蛋白在控制心肌基因的组织特异性表达中的作用。
The expression of the alpha-myosin heavy chain (MHC) gene is restricted primarily to cardiac myocytes. To date, several positive regulatory elements and their binding factors involved in alpha-MHC gene regulation have been identified; however, the mechanism restricting the expression of this gene to cardiac myocytes has yet to be elucidated. In this study, we have identified by using sequential deletion mutants of the rat cardiac alpha-MHC gene a 30-bp purine-rich negative regulatory (PNR) element located in the first intronic region that appeared to be essential for the tissue-specific expression of the alpha-MHC gene. Removal of this element alone elevated (20- to 30-fold) the expression of the alpha-MHC gene in cardiac myocyte cultures and in heart muscle directly injected with plasmid DNA. Surprisingly, this deletion also allowed a significant expression of the alpha-MHC gene in HeLa and other nonmuscle cells, where it is normally inactive. The PNR element required upstream sequences of the alpha-MHC gene for negative gene regulation. By DNase I footprint analysis of the PNR element, a palindrome of two high-affinity Ets-binding sites (CTTCCCTGGAAG) was identified. Furthermore, by analyses of site-specific base-pair mutation, mobility gel shift competition, and UV cross-linking, two different Ets-like proteins from cardiac and HeLa cell nuclear extracts were found to bind to the PNR motif. Moreover, the activity of the PNR-binding factor was found to be increased two- to threefold in adult rat hearts subjected to pressure overload hypertrophy, where the alpha-MHC gene is usually suppressed. These data demonstrate that the PNR element plays a dual role, both downregulating the expression of the alpha-MHC gene in cardiac myocytes and silencing the muscle gene activity in nonmuscle cells. Similar palindromic Ets-binding motifs are found conserved in the alpha-MHC genes from different species and in other cardiac myocyte-restricted genes. These results are the first to reveal a role of the Ets class of proteins in controlling the tissue-specific expression of a cardiac muscle gene.