Transcriptional repression by the orphan steroid receptor RVR/Rev-erb beta is dependent on the signature motif and helix 5 in the E region: Functional evidence for a biological role of RVR in myogenesis
Transcriptional repression by the orphan steroid receptor RVR/Rev-erb beta is dependent on the signature motif and helix 5 in the E region: Functional evidence for a biological role of RVR in myogenesis
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DOI：
10.1093/nar/24.18.3481
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发表时间：
1996-09-15
影响因子：
14.9
通讯作者：
Muscat, GEO
中科院分区：
文献类型：
--
作者：
Burke, L;Downes, M;Muscat, GEO
RVR/Rev-erb beta/BD 73是一种孤儿类固醇受体，在“经典”意义上没有已知的配体。RVR作为单体与由共有六聚体半位点上游的富含A/T序列组成的元件结合。然而，RVR不激活转录，并通过ROR/RZR阻断该元件的反式激活。RVR的作用机制仍然不清楚，因此我们使用GAL 4杂交系统来鉴定和表征RVR的配体结合结构域（LED）中的活性转录沉默子。严格的缺失和突变分析表明，该阻遏物结构域由RVR的氨基酸416-449编码。此外，我们证明了有效的抑制依赖于所谓的LED特异性特征基序，（F/W）AKxxxxFxxLxxxDQxxLL（其跨越环3 -4和螺旋4）和螺旋5（H5;在类固醇受体LBD的晶体结构中鉴定）。尽管RVR在许多成体组织（包括骨骼肌）和胚胎发育过程中表达，但其在分化和哺乳动物发育中的生理功能仍不清楚。由于其他的“转录因子”，如COUP-TF II和Rev-erbA α，已被证明可以调节肌肉和脂肪细胞的分化，我们研究了RVR在小鼠肌发生过程中的表达和功能作用。在C2 C12肌源性细胞中，RVR mRNA在增殖的成肌细胞中被检测到，并且当细胞被诱导分化成有丝分裂后的多核肌管时，通过血清提取被抑制。RVR mRNA的减少与肌肉特异性标志物（例如肌生成素mRNA）的出现相关。通过显性阴性RVR Delta E的组成性过度表达进行的RVR“功能丧失”研究导致血清戒断后p21（Cip 1/Waf 1）和肌细胞生成素mRNA水平升高。时程研究表明，RVR Delta E mRNA的表达导致血清戒断后肌生成素和p21 mRNA的过早诱导和积累。此外，我们证明了COUP-TF II和Rev-erbA α受体在C2 C12细胞中的过表达完全阻断了血清戒断后p21 mRNA的诱导。总之，我们的研究确定了一个有效的转录抑制结构域RVR，其特征在于沉默区域内的关键氨基酸，并提供证据的生理作用，RVR在肌发生。
RVR/Rev-erb beta/BD73 is an orphan steroid receptor that has no known ligand in the 'classical' sense. RVR binds as a monomer to an element which consists of an A/T-rich sequence upstream of the consensus hexameric half-site. However, RVR does not activate transcription and blocks transactivation of this element by ROR/RZR. The mechanism of RVR action remains obscure, hence we used the GAL4 hybrid system to identify and characterize an active transcriptional silencer in the ligand binding domain (LED) of RVR. Rigorous deletion and mutational analysis demonstrated that this repressor domain is encoded by amino acids 416-449 of RVR. Furthermore, we demonstrated that efficient repression is dependent on the so-called LED-specific signature motif, (F/W)AKxxxxFxxLxxxDQxxLL (which spans loop3-4 and helix 4) and helix 5 (H5; identified in the crystal structures of the steroid receptor LBDs). Although RVR is expressed in many adult tissues, including skeletal muscle, and during embryogenesis, its physiological function in differentiation and mammalian development remains unknown. Since other 'orphans', e.g. COUP-TF II and Rev-erbA alpha, have been demonstrated to regulate muscle and adipocyte differentiation, we investigated the expression and functional role of RVR during mouse myogenesis. In C2C12 myogenic cells, RVR mRNA was detected in proliferating myoblasts and was suppressed when the cells were induced to differentiate into post-mitotic, multinucleated myotubes by serum withdrawal. This decrease in RVR mRNA correlated with the appearance of muscle-specific markers (e.g. myogenin mRNA). RVR 'loss of function' studies by constitutive over-expression of a dominant negative RVR Delta E resulted in increased levels of p21(Cip1/Waf1) and myogenin mRNAs after serum withdrawal. Time course studies indicated that expression of RVR Delta E mRNA results in the precocious induction and accumulation of myogenin and p21 mRNAs after serum withdrawal. In addition, we demonstrated that over-expression of the COUP-TF II and Rev-erbA alpha receptors in C2C12 cells completely blocked induction of p21 mRNA after serum withdrawal. In conclusion, our studies identified a potent transcriptional repression domain in RVR, characterized critical amino acids within the silencing region and provide evidence for the physiological role of RVR during myogenesis.