LIM Protein KyoT2 Negatively Regulates Transcription by Association with the RBP-J DNA-Binding Protein

The RBP-J/Su(H) DNA-binding protein plays a key role in transcriptional regulation by targeting Epstein-Barr virus nuclear antigen 2 (EBNA2) and the intracellular portions of Notch receptors to specific promoters. Using the yeast two-hybrid system, we isolated a LIM-only protein, KyoT, which physically interacts with RBP-J. Differential splicing gave rise to two transcripts of the KyoT gene, KyoT1 and KyoT2, that encoded proteins with four and two LIM domains, respectively. With differential splicing resulting in deletion of an exon, KyoT2 lacked two LIM domains from the C terminus and had a frameshift in the last exon, creating the RBP-J-binding region in the C terminus. KyoT1 had a negligible level of interaction with RBP-J. Strong expression of KyoT mRNAs was detected in skeletal muscle and lung, with a predominance of KyoT1 mRNA. When expressed in F9 embryonal carcinoma cells, KyoT1 and KyoT2 were localized in the cytoplasm and the nucleus, respectively. The binding site of KyoT2 on RBP-J overlaps those of EBNA2 and Notch1 but is distinct from that of Hairless, the negative regulator of RBP-J-mediated transcription in Drosophila. KyoT2 but not KyoT1 repressed the RBP-J-mediated transcriptional activation by EBNA2 and Notch1 by competing with them for binding to RBP-J and by dislocating RBP-J from DNA. KyoT2 is a novel negative regulatory molecule for RBP-J-mediated transcription in mammalian systems.

RBP-J/Su(H) DNA结合蛋白（RBP-J/Su(H) DNA-binding protein）通过将爱泼斯坦-巴尔病毒核抗原2（Epstein-Barr virus nuclear antigen 2，EBNA2）以及Notch受体的胞内结构域靶向至特定启动子，在转录调控中发挥关键作用。本研究借助酵母双杂交系统，分离得到一种仅含LIM结构域的蛋白质KyoT，其可与RBP-J发生物理相互作用。KyoT基因经可变剪接产生两种转录本：KyoT1与KyoT2，分别编码含4个LIM结构域和2个LIM结构域的蛋白质。因可变剪接导致某外显子缺失，KyoT2缺失了C端的两个LIM结构域，且在最后一个外显子中发生移码突变，于C端形成了RBP-J结合区域。KyoT1与RBP-J的相互作用水平可忽略不计。在骨骼肌与肺组织中可检测到KyoT mRNA的高表达，其中以KyoT1转录本为主。当在F9胚胎癌细胞中表达时，KyoT1与KyoT2分别定位于细胞质与细胞核内。KyoT2在RBP-J上的结合位点与EBNA2和Notch1的结合位点存在重叠，但与果蝇中RBP-J介导转录的负调控因子Hairless的结合位点不同。KyoT2（而非KyoT1）可通过竞争结合RBP-J以及将RBP-J从DNA上解离下来，抑制EBNA2与Notch1介导的RBP-J依赖型转录激活。KyoT2是哺乳动物系统中一类全新的RBP-J介导转录负调控分子。