The promoter activity of SUMO5 and conjugation ability on Pc2

• Main Authors: I-Pu Chen, 陳意樸
• Other Authors: 楊文明
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/84903034948376859237

碩士 === 國立中興大學 === 分子生物學研究所 === 103 === 1. The promoter activity of SUMO5 Small ubiquitin-related modifier (SUMO) plays critical roles in several cellular functions. Previously, our lab identified a novel SUMO variant, dubbed SUMO5, with functions of Promyelocytic Leukemia Protein (PML) nuclear bodies formation and Paired box protein 3 activity regulation. However, according to genome annotation by Human genome organization (HGNC), SUMO5 is regarded as a pseudogene of SUMO1. To address the issue whether SUMO5 is a genuine functional gene, I focused on the characterization of SUMO5 promoter activity. In this study, I identified that SUMO5 has instinct promoter activity, which locates 200 bp upstream of translation start site. Base on the transcription factor binding profiles, I found that liver-enriched transcription-activating protein 1 (LAP1) and LAP2 repress the limited SUMO5 promoter while GATA binding protein 1 (GATA 1) has no effect on SUMO5 promoter activity, suggesting that the regulation of SUMO5 may be tissue specific. In short, our results indicated SUMO5 is a genuine functional gene and it contains instinct promoter activity. 2. The conjugating function of SUMO5 on Pc2 Polycomb group protein 2 (Pc2) belongs to polycomb group family, which controls development. Pc2 has strong repressive activity and forms Pc2 bodies. However, little is known about how SUMO conjugation regulates function of Pc2. Previously, our lab found Pc2 can be conjugated by SUMO5. We hypothesize that the SUMO5 conjugation contributes to transcription regulation and bodies formation for Pc2. Here, we showed that SUMO5 conjugates on Pc2 by a mono-SUMOylation manner, which is not dependent on the SUMO E3 activity of Pc2 and identified the potential SUMOylation sites of Pc2. Furthermore, Gal4-based luciferase assays showed that SUMO5 enhance repressive activity of Pc2 through its conjugation ability. Finally, western results from glutaraldehyde treatment and non-reducing gel suggest that SUMO5 conjugation is not essential for Pc2 polymerization and body formation. Taken together, our results suggest that SUMO5 conjugates Pc2 on potential SUMOylation sites in a mono-SUMOylation manner to enhance transcriptional repression of Pc2.