Buescher Jessica L, El-Hodiri Heithem M
Center for Molecular and Human Genetics, Nationwide Children's Research Institute, Columbus, OH.
Department of Pediatrics, College of Medicine, The Ohio State University, Columbus, OH.
Mol Vis. 2019 Feb 23;25:165-173. eCollection 2019.
The evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite Rax's biologic significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. The gene encodes a conserved octapeptide motif (OP) near the N-terminus and several conserved regions in the C-terminus of unknown function, including the orthopedia, aristaless, rax (OAR) domain and the RX domain. The purpose of this study is to investigate the contribution of these conserved domains in Rax function.
N-and C-terminal deletion and point mutations were generated in (previously known as Rx1A) using PCR-based methods. We examined the ability of mutated Rax to transactivate a reporter gene consisting of a portion of a target gene promoter (from the gene) fused to a firefly luciferase coding region and transfected into human embryonic kidney 293T (HEK293T) cells. Portions of the Rax C-terminal region were also assayed for transactivation activity in the context of a heterologous DNA binding domain with an appropriate reporter gene.
Full-length Rax weakly activated the reporter. Deletion of the Rax C-terminus increased Rax activity, suggesting that the C-terminus functions to repress Rax activity. Further deletion eventually resulted in a decrease in activity, suggesting that the C-terminal region also can function to enhance Rax activity. Deletion or mutation of the OP motif resulted in a slight decrease in Rax activity. Mutation or deletion of the N-terminal OP motif resulted in a mild decrease in activity and dampened the activity levels of the C-terminal deletions. Further, fusion of the C-terminus of Rax to a heterologous DNA binding domain enhanced transactivation.
The present data indicate that the C-terminus of Rax can function to repress or activate transcription in a context-dependent manner. These data support our hypothesis that the highly conserved OAR domain, in combination with other regulatory elements in the Rax C-terminus, coordinates Rax activity, perhaps through functional interaction with the N-terminal OP motif. Taken together, these data provide insight into the structural features that regulate Rax activity.
进化上保守的视网膜同源框（Rax）转录因子对所有脊椎动物的正常眼睛发育至关重要。尽管Rax具有生物学意义，但其作为转录调节因子的分子功能背后的分子机制仍不清楚。该基因在N端附近编码一个保守的八肽基序（OP），在C端有几个功能未知的保守区域，包括正位框、无触角、rax（OAR）结构域和RX结构域。本研究的目的是探讨这些保守结构域在Rax功能中的作用。
使用基于PCR的方法在（以前称为Rx1A）中产生N端和C端缺失及点突变。我们检测了突变型Rax激活由一部分靶基因启动子（来自基因）与萤火虫荧光素酶编码区融合而成的报告基因的能力，并将其转染到人胚肾293T（HEK293T）细胞中。Rax C端区域的部分片段也在具有适当报告基因的异源DNA结合结构域的背景下检测其反式激活活性。
全长Rax对报告基因的激活作用较弱。Rax C端的缺失增加了Rax的活性，表明C端起到抑制Rax活性的作用。进一步缺失最终导致活性降低，表明C端区域也可起到增强Rax活性的作用。OP基序的缺失或突变导致Rax活性略有下降。N端OP基序的突变或缺失导致活性轻度降低，并减弱了C端缺失的活性水平。此外，将Rax的C端与异源DNA结合结构域融合可增强反式激活作用。
目前的数据表明，Rax的C端可根据具体情况起到抑制或激活转录的作用。这些数据支持了我们的假设，即高度保守的OAR结构域与Rax C端的其他调节元件相结合，可能通过与N端OP基序的功能相互作用来协调Rax的活性。综上所述，这些数据为调节Rax活性的结构特征提供了见解。
