Functional study of C-terminal domain on KAP1

• Main Authors: Shiao-Ru Chang, 張筱茹
• Other Authors: Wen-Ming Yang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/46251599936660592030

碩士 === 國立中興大學 === 分子生物學研究所 === 98 === Transcription regulation plays a critical role in eukaryotic gene expression. KAP1 (KRAB-associated protein) is a transcriptional intermediary factor for repression, which asts as scaffold in many transcriptional regulation complexes. KAP1 serves as a co-repressor for KRAB-containing zinc finger proteins (KRAB-ZFPs) in the regulation of gene expression and KAP1 is required for KRAB-ZFPs-mediated transcription repression. However, the molecular mechanism of KAP1 functions as a co-repressor to connect transcription factors and regulatory proteins is unclear. Previous studies proved that N-terminal RBCC domain of KAP1 is responsible for oligomerization and KAP1 exists as both oligomers and monomers in cells, but how KAP1 oligomerization regulates transcriptional repression remains unknown. First, we want to understand wether KAP1 oligomerization affects its function on transcriptional repression. In addition, it has been previously demonstrated that C-terminal bromodomain (BD) of KAP1 regulates subcellular localization and transcriptional activity by interacting with its N-terminal RBCC domain. Above studies suggest that intramolecular and intermolecular interaction of KAP1 might cooperative regulate subcellular localization and transcriptional activity of KAP1. In this study, we want to address the molecular mechanism of intermolecular and intramolecular interaction of KAP1 regulates its subcellular localization and transcriptional activity. To investigate the role of KAP1 oligomerization in transcriptional repression, we blocked the self-interaction of KAP1 to mimic monomeric KAP1 by generating point mutations on RBCC domain. Here we demonstrated that monomeric KAP1 exhibited a decreased level of euchromatin localization but stronger repressional activity. The results suggest that monomeric KAP1 is an active form for transcriptional repression. Go further to explore the mechanism of KAP1 oligomerization in subcellular localization and transcriptional activity, we approach it from C-terminal domain of KAP1. Previously we found that missing of BD in KAP1 results in spots formation in euchromatin region and derepressional activity, but these phenomena could be reverted to wild type by addition of BD which through intramolecular interaction with RBCC domain. Thus, we proposed that intramolecular interaction mediated by BD of KAP1 contribute to subcellular localiztion and transcriptional activity by promoting KAP1 monomers formation to repress transcription, which blocks the RBCC domain for KAP1 oligomerization. To prove this idea, first, we perform co-immunoprecipitation assay to prove C-terminal BD interacts with N-terminal RBCC domain. To validate that BD of KAP1 regulates its oligomerization by intramolecular interaction, we demonstrated that missing BD of KAP1 leads to oligomers formationin glycerol gradient analysis, which reverted to monomers by addition of BD. Furthermore, we demonstrated that BD interacts with RBCC domain of KAP1, suggesting that intramolecular interaction mediated by BD regulates KAP1 oligomerization. Taken together, the results demonstrate that intramolecular interaction between C-terminal BD and N-terminal RBCC domain regulates KAP1 monomers formation, which is capable of heterochromatin localization and transcriptional repression.