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 transcriptiona l regulation by targeting Epstein-Barr virus nuclear antigen 2 (EBNA2) and the intracellular portions of Notch receptors to specific promote rs, Using the yeast two-hybrid system, we isolated a LIM-only protein, KyoT, which physically interacts with RBP-J, Differential splicing ga ve rise to two transcripts of the KyoT gene, KyoT1 and KyoT2, that enc oded proteins with four and two LIM domains, respectively, With differ ential splicing resulting in deletion of an exon, KyoT2 lacked two LIM domains from the C terminus and had a frameshift in the last exon, cr eating the RBP-J-binding region in the C terminus, KyoT1 had a negligi ble level of interaction with RBP-J, Strong expression of KyoT mRNAs w as 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 bin ding 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-mediat ed 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, Kyo T2 is a novel negative regulatory molecule for RBP-J-mediated transcri ption in mammalian systems.