Karasseva Natalia, Tsika Gretchen, Ji Juan, Zhang Aijing, Mao Xiaoqing, Tsika Richard
Department of Biochemistry, School of Medicine, University of Missouri-Columbia, 1600 Rollins Road, Columbia, MO 65211, USA.
Mol Cell Biol. 2003 Aug;23(15):5143-64. doi: 10.1128/MCB.23.15.5143-5164.2003.
In adult mouse skeletal muscle, beta-myosin heavy chain (betaMyHC) gene expression is primarily restricted to slow type I fibers; however, its expression can be induced in fast type II fibers in response to a sustained increase in load-bearing work (mechanical overload [MOV]). Our previous betaMyHC transgenic and protein-DNA interaction studies have identified an A/T-rich element (betaA/T-rich -269/-258) that is required for slow muscle expression and which potentiates MOV responsiveness of a 293-bp betaMyHC promoter (beta293wt). Despite the GATA/MEF2-like homology of this element, we found binding of two unknown proteins that were antigenically distinct from GATA and MEF2 isoforms. By using the betaA/T-rich element as bait in a yeast one-hybrid screen of an MOV-plantaris cDNA library, we identified nominal transcription enhancer factor 1 (NTEF-1) as the specific betaA/T-rich binding factor. Electrophoretic mobility shift assay analysis confirmed that NTEF-1 represents the enriched binding activity obtained only when the betaA/T-rich element is reacted with MOV-plantaris nuclear extract. Moreover, we show that TEF proteins bind MEF2 elements located in the control region of a select set of muscle genes. In transient-coexpression assays using mouse C2C12 myotubes, TEF proteins transcriptionally activated a 293-bp betaMyHC promoter devoid of any muscle CAT (MCAT) sites, as well as a minimal thymidine kinase promoter-luciferase reporter gene driven by three tandem copies of the desmin MEF2 or palindromic Mt elements or four tandem betaA/T-rich elements. These novel findings suggest that in addition to exerting a regulatory effect by binding MCAT elements, TEF proteins likely contribute to regulation of skeletal, cardiac, and smooth muscle gene networks by binding select A/T-rich and MEF2 elements under basal and hypertrophic conditions.
在成年小鼠骨骼肌中，β-肌球蛋白重链（βMyHC）基因表达主要局限于慢肌I型纤维；然而，在承受负荷的工作持续增加（机械过载[MOV]）时，其表达可在快肌II型纤维中被诱导。我们之前的βMyHC转基因和蛋白质-DNA相互作用研究已鉴定出一个富含A/T的元件（β富含A/T的-269/-258），它是慢肌表达所必需的，并且能增强293 bp βMyHC启动子（β293wt）对MOV的反应性。尽管该元件具有GATA/MEF2样同源性，但我们发现有两种未知蛋白质与之结合，它们在抗原性上与GATA和MEF2亚型不同。通过在MOV-比目鱼肌cDNA文库的酵母单杂交筛选中使用富含βA/T的元件作为诱饵，我们鉴定出名义转录增强因子1（NTEF-1）为特定的富含βA/T的结合因子。电泳迁移率变动分析证实，NTEF-1代表仅在富含βA/T的元件与MOV-比目鱼肌核提取物反应时获得的富集结合活性。此外，我们表明TEF蛋白结合位于一组特定肌肉基因控制区域的MEF2元件。在使用小鼠C2C12肌管的瞬时共表达分析中，TEF蛋白转录激活了一个不含任何肌肉CAT（MCAT）位点的293 bp βMyHC启动子，以及由结蛋白MEF2或回文Mt元件的三个串联拷贝或四个串联富含βA/T的元件驱动的最小胸苷激酶启动子-荧光素酶报告基因。这些新发现表明，除了通过结合MCAT元件发挥调节作用外，TEF蛋白可能在基础和肥大条件下通过结合特定的富含A/T和MEF2元件，对骨骼肌、心肌和平滑肌基因网络的调节做出贡献。
