The dual functions of FKBP25 in transcriptional regulation

• Main Authors: Huai-Huei Huang, 黃懷慧
• Other Authors: Wen-Ming Yang
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/84722677790980066703

碩士 === 國立中興大學 === 分子生物學研究所 === 99 === FK-506 binding protein family is one of protein family that has the binding capacity with immunosuppressant drug-FK-506. Previous studies of FKBP family proteins focus on their functions in the cytoplasm. In recent years, it has been identified that a FKBP protein homologue Fpr4, in yeast nucleus has the ability to regulate RNA polymerase I and RNA polymerase II gene expression. However, whether the Fpr4 homologue in mammalian, FKBP25, also has the capacity to regulate RNA polymerase I and RNA polymerase II gene expression remains unknown. Therefore, the study of FKBP25 in this thesis will be separated into two parts, one on the role of FKBP25 in RNA polymerase II, the other in RNA polymerase I. First, through florescent microscopy and transcriptional assay, we made sure that FKBP25 was a nuclear protein and contained transcriptional repression activity. To understand if FKBP25 is directly involved in transcription by the RNA polymerase II system, co-immunoprecipitation detect that FKBP25 interacted with histone demethylase-JMJD2A and enhanced FKBP25 repression activity in luciferase assay. Chip assay detected that FKBP25 had the ability to recruite JMJD2A on target promoter. Moreover, we identify c-myc as a target gene of FKBP25. In luciferase assay, FKBP25 repressed c-myc promoter expression. Chip assay showed that FKBP25 could target c-myc promoter, suggesting that c-myc was the target gene of FKBP25. Through florescent microscopy observation and co-immunoprecipitation result, we found that FKBP25 recruited nucleoplasmic Mdm2 into the nucleolus and interacted with Mdm2. Furthermore, by western blot and in vivo ubiquitination assay, we found that FKBP25 stabilized p53 protein expression and inhibited Mdm2-triggered p53 poly-ubiquitination. Also, we detected that overexpressed FKBP25 could repress the expression of p21, a p53 target gene, in normal cell by luciferase assay. We observed that FKBP25 with the possible nucleolus localization signal (NoLS)- (R/K)(R/K)X(R/K) motif deleted caused a drop in FKBP25 nucleolus localization from 19 % to 7 %. Transcriptional assay and Chip assay revealed that FKBP25 repressed rDNA gene expression and targeted the rDNA promoter. And co-immunoprecipitation result detected that FKBP25 interacted with a histone modifier JHDM1B that is related to rDNA repression. In summary, we suggest that FKBP25 interacts and recruits JMJD2A to further repress the expression of c-myc that is the down stream gene of transcription factor YY1. And also FKBP25 recruits Mdm2 into nucleolus, which activateing transcription factor p53 activity, to represses p53 target gene p21 expression in normal cell, FKBP25 also interacts with histone demethylase JHDM1B to represse rDNA gene expression, slowing down cell growth and causing p53 to properly perform its function in RNA polymerase II system, such as transcribing down stream gene, repairing DNA or leads cell cycle arrest etc.