Stable Set8 effect on gene expression in U2OS cells

Ubiquitin-mediated proteolysis play a significant role in various biological processes including transcription, DNA repair and cell cycle progression. The identification of Set8 and Set8b (a splice isoform) histone H4K20 methyl transferase as a substrate for the cullin-based ubiquitin ligase (CRL4-Cdt2) demonstrate that this pathway plays a significant role in promoting cell cycle progression, specifically promoting G2 progression. This study investigate the effect of failure to degrade Set8 in S-phase of the cell cycle via CRL4-Cdt2 on gene expression. We used microarrays to detail the effect of the expression of stable form of Set8b H4K20 mono-methyl transferase (Set8b_deltaPIP2) on gene expression Human osteosarcoma-derived human cells were transduced with retroviruses encoding either wt-Set8b or a mutant of Setb8 which is resistant to degradation via CRL4-Cdt2 ubiquitin ligase complex (Set8b_deltaPIP2) or with an a control pMSCV empty virus. 5 days after transduction, cells were harvested and the RNA was extracted by Trizol (Invitrogen) and hybridized to the affymetrix chips array.

泛素介导的蛋白水解（ubiquitin-mediated proteolysis）在转录、DNA修复及细胞周期进程等多种生物学过程中发挥重要调控作用。将Set8与剪接异构体Set8b（组蛋白H4K20甲基转移酶，histone H4K20 methyl transferase）鉴定为基于cullin的泛素连接酶（CRL4-Cdt2，cullin-based ubiquitin ligase）的底物，这一发现表明该通路在促进细胞周期进程，尤其是G2期进展中发挥关键作用。本研究旨在探究细胞周期S期内无法通过CRL4-Cdt2降解Set8对基因表达产生的影响。我们采用微阵列（microarrays）技术，详细解析稳定型Set8b H4K20单甲基转移酶（Set8b_deltaPIP2）的表达对基因表达的调控效应。实验中将人骨肉瘤来源的细胞分别用编码野生型Set8b、抗CRL4-Cdt2泛素连接酶复合物降解的Set8b突变体（Set8b_deltaPIP2）的逆转录病毒（retroviruses），以及空载对照pMSCV病毒进行转导。转导5天后收集细胞，采用Trizol（Invitrogen）提取总RNA，并将其与Affymetrix基因芯片进行杂交。