Gupta Madhu, Sueblinvong Viranuj, Raman Jai, Jeevanandam Valluvan, Gupta Mahesh P
Hope Children's Hospital, University of Illinois, Chicago, Illinois 60612, USA.
J Biol Chem. 2003 Nov 7;278(45):44935-48. doi: 10.1074/jbc.M307696200. Epub 2003 Aug 21.
The alpha-myosin heavy chain is a principal molecule of the thick filament of the sarcomere, expressed primarily in cardiac myocytes. The mechanism for its cardiac-restricted expression is not yet fully understood. We previously identified a purine-rich negative regulatory (PNR) element in the first intron of the gene, which is essential for its cardiac-specific expression (Gupta, M., Zak, R., Libermann, T. A., and Gupta, M. P. (1998) Mol. Cell. Biol. 18, 7243-7258). In this study we cloned and characterized muscle and non-muscle factors that bind to this element. We show that two single-stranded DNA-binding proteins of the PUR family, PURalpha and PURbeta, which are derived from cardiac myocytes, bind to the plus strand of the PNR element. In functional assays, PURalpha and PURbeta repressed alpha-myosin heavy chain (alpha-MHC) gene expression in the presence of upstream regulatory sequences of the gene. However, from HeLa cells an Ets family of protein, Ets-related protein (ERP), binds to double-stranded PNR element. The ERP.PNR complex inhibited the activity of the basal transcription complex from homologous as well as heterologous promoters in a PNR position-independent manner, suggesting that ERP acts as a silencer of alpha-MHC gene expression in non-muscle cells. We also show that PUR proteins are capable of binding to alpha-MHC mRNA and attenuate its translational efficiency. Furthermore, we show robust expression of PUR proteins in failing hearts where alpha-MHC mRNA levels are suppressed. Together, these results reveal that (i) PUR proteins participate in transcriptional as well as translational regulation of alpha-MHC expression in cardiac myocytes and (ii) ERP may be involved in cardiac-restricted expression of the alpha-MHC gene by preventing its expression in non-muscle cells.
α-肌球蛋白重链是肌节粗肌丝的主要分子，主要在心肌细胞中表达。其心脏特异性表达的机制尚未完全阐明。我们先前在该基因的第一个内含子中鉴定出一个富含嘌呤的负调控（PNR）元件，它对于其心脏特异性表达至关重要（古普塔，M.，扎克，R.，利伯曼，T. A.，以及古普塔，M. P.（1998年）《分子与细胞生物学》18卷，7243 - 7258页）。在本研究中，我们克隆并鉴定了与该元件结合的肌肉和非肌肉因子。我们发现，源自心肌细胞的PUR家族的两种单链DNA结合蛋白PURα和PURβ与PNR元件的正链结合。在功能试验中，PURα和PURβ在该基因上游调控序列存在的情况下抑制α-肌球蛋白重链（α-MHC）基因的表达。然而，在HeLa细胞中，Ets家族的一种蛋白，即Ets相关蛋白（ERP），与双链PNR元件结合。ERP-PNR复合物以与PNR位置无关的方式抑制同源及异源启动子的基础转录复合物的活性，这表明ERP在非肌肉细胞中作为α-MHC基因表达的沉默子发挥作用。我们还表明，PUR蛋白能够与α-MHC mRNA结合并降低其翻译效率。此外，我们发现在α-MHC mRNA水平受到抑制的衰竭心脏中PUR蛋白有强烈表达。总之，这些结果表明：（i）PUR蛋白参与心肌细胞中α-MHC表达的转录及翻译调控；（ii）ERP可能通过阻止α-MHC基因在非肌肉细胞中的表达而参与其心脏特异性表达。
