| Summary: | 博士 === 國立中興大學 === 分子生物學研究所 === 97 === KAP1 (KRAB-association protein 1) is a transcriptional intermediary factor, which acts as a scaffold in many transcriptional regulation complexes. The recruitment of KAP1 by a large family of Kruppel-like zinc finger proteins (KRAB-ZFPs) to repress transcription is indispensable for KRAB-ZFPs function. However, the basis of KAP1 in transcription repression is not fully understood. In this dissertation, we will focus on what controls the repressional ability of KAP1 and further investigate other cellular functions of KAP1.
First, KAP1 was identified to stably associate with members of the heterochromatin protein 1 (HP1) by affinity purification. Using glycerol gradient and immunostaining analysis, we found that the dynamic movement of HP1 is controlled by its association with KAP1. KAP1 relocates HP1 from heterochromatin to euchromatin; HP1 also increases the heterochromatin localization of KAP1. Furthermore, the physical localization of HP1 to heterochromatin not only is required for HP1 to repress transcription but also affect cellular physiology.
Second, we found that the repressional ability of KAP1 is regulated by histone modifiers. Coimmunoprecipitation experiment results show that KAP1 interacts with histone modifiers, including histone methylases, demethylases and deacetylases. H3K9 methyltransferases but not H3K27 methyltransferases enhance the repressional activity of KAP1. JMJD2A (demethlase for H3K9/36) but not LSD1 (demethylase for H3K4/K9) increases the repressional activity of KAP1. KAP1 interacts with HDACs and only HDAC9 enhances the repressional activity of KAP1. These results suggest that different histone modifiers have different effects on KAP1- mediated repression.
Furthermore, we also found that post-translational modifications affect subcellular localization and the repressional activity of KAP1. KAP1 is sumoylated by SUMO1 and SUMO3. SUMO1-conjugation enhances the repressional activity of KAP1; however, SUMO3-conjugation decreases the repressional activity of KAP1. We also identified that KAP1 is subjected to acetylation and ubiquitination. By using MALDI-TOF-MS and site-directed mutagenesis, we determined that Lys-238 is the acetylation site of KAP1. Mimicking the acetylation on K238 (K238Q) decreases the repressional activity of KAP1. We also found that Lys-779 is the ubiquitination site of KAP1. Ubiquitinated-
KAP1 translocates from the nucleus to the cytoplasm and losses the repressional activity. In summary, KAP1 exhibits multiple post-translational modifications, suggesting that KAP1 might play different roles in cells regulated by various post-translational modifications.
Finally, we found that KAP1 not only interacts with p53 but also restores nuclear import of p53 and further enhances the transcriptional repression activity of p53. Specifically, KAP1 facilitates nuclear import of p53 by recruiting p53 through its KRAB domain and interacting with importin α1 through its HP1-binding domain. HP1β competes with importin α1 for binding to KAP1, releasing KAP1 and p53 inside the nucleus. On p53 target promoters such as the survivin promoter, HDAC9 and SETDB1 interact with KAP1 and enhance the repressional activity of p53. The results highlight the significance of KAP1 in the regulation of nuclear import and repressional ability of p53.
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