鸡AChRγ亚基基因远端E盒子与myogenin的相互作用

业已证明,鸡ｎＡｃｈＲγ亚基基因５＇连接区,－５０９／－３０２,－３２４／＋３６片段可独立激活异源启动子ＳＶ４０的转录活性;γ基因近端两个Ｅ盒子（ＣＡＮＮＴＧ）与生肌调节因子（ｍｙｏｇｅｎｉｎ）的相互作用及转录激活分析表明,近端两个Ｅ盒于是γ启动子活性必不可少的,但却是不充分的。利用胶阻滞和ＤＮａｓｅⅠ足纹试验观察了鸡ｎＡｃｂＲγ基因远端Ｅ盒子与ＧＳＴ－ｍｙｏｇｅｎｉｎ融合蛋白的相互作用。胶阻滞试验证明,－５３８／－２８８片段可与ｍｙｏｇｅｎｉｎ相互结合,引起（３２）￣ｐ标记的ＤＮＡ片段在ＰＡＧＥ中迁移率减慢,这种胶阻滞现象可被含Ｅ盒子的未标记片段消除,并被抗鸡ｍｙｏｇｅｎｉｎ单克隆抗体或抗血清所改变。ＤＮａｓｅⅠ足纹试验证明,ｍｙｏｇｅｎｉｎ系通过γ基因转录起始点上游－４２９／－４２４及－４６３／－４５８两个Ｅ盒子与γ基因５＇连接区－５３８／－２８８相互作用。     

Localization of mRNAs coding for CMD1, myogenin and the alpha-subunit of the acetylcholine receptor during skeletal muscle development in the chicken.

Myogenin and CMD1, the chicken homologue of MyoD, transactivate the promoter of the alpha-subunit of the acetylcholine receptor (AChR) in chicken fibroblasts. The expression of these three genes was followed by in situ hybridization. In two-day-old embryos the CMD1 gene is expressed shortly before the AChR alpha-subunit and the myogenin genes. At day 19 extrajunctional AChR mRNA clusters have disappeared and myogenin mRNAs are no longer detected in PLD muscle. Moreover, both myogenin and CMD1 mRNA levels increase after muscle denervation in chicks. These data are compatible with a role for myogenic factors in the induction and maintenance of extra-junctional expression of the AChR genes during early muscle development. Using digoxygenin labelled RNA probes, we also show that the mRNAs for the AChR alpha-subunit display a punctated, probably perinuclear distribution, whereas mRNAs for myogenic genes accumulate in the sarcoplasm around subsets of nuclei in the muscle fiber.     

鸡AChRγ亚基基因远上游增强子样作用依赖生肌因子

先前曾报告鸡ＡＣｈＲγ亚基基因５＇连接区－５３８／－２８８片段（含一对Ｅ盒子）与生肌调节因子（ｍｙｏｇｅｎｉｎ）的相互作用,为进一步观察ＤＮＡ－ｍｙｏｇｅｎｉｎ相互作用对γ基因转录激活功能的影响,利用瞬时表达体系研究了鸡ｎＡＣｈＲγ亚基基因５＇端－５３８／－２８８片段调控机能。ＣＡＴ分析表明：γ基因－５３８／－２８８片段可显著激活ｔｋ启动子在分化的Ｃ２Ｃ１２肌细胞中的转录活性,但不能激活在未分化Ｃ２Ｃ１２肌细胞中的转录活性。这种组织特异性转录激活作用与距离无关,因而是一个典型的增强子;－５３８／－２８８片段的增强子样作用既依赖于两毗邻Ｅ盒子的完整性,又依赖于ｍｙｏｇｅｎｉｎ等生肌调节因子的存在。强化表达ｍｙｏｇｅｎｉｎ可激活ｐＢＬＣＡＴ２－γ（－５３８／－４３３）和ｐＢＬＣＡＴ２－γ（－４３２／－２８８）（各含一个Ｅ盒子）在分化肌细胞中的表达,究竟是克隆片段中的独立Ｅ盒子对这种表达起贡献还是出现在克隆位点附近的反向Ｅ盒子（ＧＴＣＧＡＣ）或ｔｋ启动子中的另一个Ｅ盆子起作用？尚不十分清楚。     

Alternative multimeric structures affect myogenin DNA binding activity.

The native molecular weight of the basic helix-loop-helix (bHLH) proteins myogenin, MyoD, and E12 was calculated from their mobilities on sucrose gradients and molecular sieve chromatography. The muscle bHLH proteins associate to form a variety of higher order complexes, most of which are larger than dimers. Homodimers bind to DNA sequences such as the MEF-1 site in the creatine kinase enhancer whereas homotetramers and larger forms do not recognize this DNA sequence. The ubiquitous bHLH protein E12 forms monomers or homodimers with little evidence for higher order complexes. Mixtures of myogenin and E12 show some heterodimeric structures, but most of the myogenin remains in large complexes. This result using purified proteins is also obtained in nuclear extracts from differentiated myotubes, in which most of the myogenin is present in large complexes that do not bind to the creatine kinase enhancer. A fusion protein containing only the myogenin HLH region forms large homomeric complexes. A model is presented in which each helix associates with a different subunit to form chains or ring structures to explain these observations. The partition of myogenin in nuclear extracts into dimers that recognize known DNA sequences and higher order complexes that do not raises important new issues concerning the regulation of skeletal muscle bHLH protein activity during myogenesis.     

Activation of fgf4 gene expression in the myotomes is regulated by myogenic bHLH factors and by sonic hedgehog

The Fgf4 gene encodes an important signaling molecule which is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, we previously identified overlapping but distinct enhancer elements in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. Here we have investigated the hypothesis that Fgf4 is a target of myogenic bHLH factors. We show by mutational analysis that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. We show here that expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner.