Transcriptional repression by Rev-erbA alpha is dependent on the signature motif and helix 5 in the ligand binding domain: Silencing does not involve an interaction with N-CoR
Transcriptional repression by Rev-erbA alpha is dependent on the signature motif and helix 5 in the ligand binding domain: Silencing does not involve an interaction with N-CoR
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DOI：
10.1093/nar/24.18.3490
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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：
Downes, M;Burke, LJ;Muscat, GEO
Rev-erbA α是一种孤儿核受体，其功能是显性转录抑制因子。组织培养和原位杂交研究表明，Rev-erbA α在哺乳动物的分化和发育中起着重要作用。先前的研究已经将Rev-erbA α的沉默域定位在孤儿受体的D/E区域。本研究利用GAL4杂交系统证明了有效的抑制是由受体E区aa 455和488之间的34个氨基酸（aa）介导的。该结构域包含配体结合域（LBD）特征基序[(F/W)AKxxxxFxxLxxxDQxxLL]和一个区域，根据最近发表的类固醇受体晶体结构，该区域将形成典型LBD结构的螺旋5。精细缺失和位点特异性突变表明LBD特征基序和螺旋5都是有效沉默所必需的。利用哺乳动物双杂交技术，我们还证明了Rev-erbA α不与核受体协同抑制因子N-CoR的相互作用结构域（aa 2218-2451）结合，而已知N-CoR与甲状腺激素和视黄酸受体相互作用。这表明Rev-erbA α的转录抑制不是通过与N-CoR的相互作用介导的。总之，我们已经确定并表征了Rev-erbA α的最小结构域，该结构域介导这种孤儿受体的转录抑制。
Rev-erbA alpha is an orphan nuclear receptor that functions as a dominant transcriptional repressor. Tissue culture and in situ hybridisation studies indicated that Rev-erbA alpha plays an important role in mammalian differentiation and development. Previous studies have localised the silencing domain of Rev-erbA alpha to the D/E region of the orphan receptor. This study utilised the GAL4 hybrid system to demonstrate that efficient repression is mediated by 34 amino acids (aa) between aa 455 and 488 in the E region of the receptor. This domain contains the ligand binding domain (LBD)-signature motif [(F/W)AKxxxxFxxLxxxDQxxLL] and a region that, according to the recently published crystal structures of steroid receptors, would be predicted to form helix 5 of the canonical LBD structure. Fine deletions and site-specific mutagenesis indicated that both the LBD signature motif and helix 5 were necessary for efficient silencing. Utilising mammalian two hybrid technology, we have also demonstrated that Rev-erbA alpha does not associate with the interaction domain (aa 2218-2451) of the nuclear receptor corepressor, N-CoR, that is known to interact with the thyroid hormone and retinoic acid receptors. This suggested that transcriptional repression by Rev-erbA alpha is not mediated through an interaction with N-CoR. In conclusion, we have identified and characterised the minimal domain of Rev-erbA alpha, that mediates transcriptional repression by this orphan receptor.