YG001: A Usp7-Tip60 pathway regulates early adipogenesis

USP7, a dominant DUB activity in 3T3-L1 adipocytes and in mouse adipose tissue, increases Tip60 protein levels, and deubiquitinates Tip60 both in intact cells and in vitro. Treatment with a pan deubiquitinase (DUB) inhibitor, or knockdown of USP7, decreases adipogenesis. Transcriptome analysis reveals a common set of cell cycle genes to be co-regulated by both Tip60 and USP7. Knock down of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results therefore reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis Mature 3T3-L1 adipocytes were subjected to RNAi-mediated knock down with control(C), USP7(U)- or Tip60(T)-specific oligonucleotides. For this, 4 replicates of differentiated 3T3-L1 cells were transfected with Amaxa technology. Two days after transfection cells were washed twice with PBS twice and lysed in 0.5ml Trizol (Invitrogen). mRNA expression of Tip60 and USP7 was assessed by qRT-PCR. Amplified cRNA samples were labeled with either cy3 or cy5 and put on microarray together with and oppositely labeled common reference sample consisting of cRNA derived from undifferentiated 3T3-L1 cells.

USP7是3T3-L1脂肪细胞与小鼠脂肪组织中主要的去泛素化酶（deubiquitinase, DUB）活性分子，其可提升Tip60蛋白水平，并在完整细胞与体外环境中对Tip60进行去泛素化修饰。采用广谱去泛素化酶（pan deubiquitinase, DUB）抑制剂处理，或敲低USP7基因，均会抑制脂肪生成过程。转录组分析显示，存在一组共有的细胞周期基因同时受Tip60与USP7共同调控；单独敲低二者任一因子，均会损伤成脂早期关键步骤——有丝分裂克隆扩增。本研究结果揭示，对转录共调控因子的去泛素化修饰，是调控早期成脂过程的核心机制。成熟3T3-L1脂肪细胞通过RNA干扰（RNA interference, RNAi）介导的方式进行基因敲低，所用寡核苷酸分别为对照序列（C）、USP7特异性序列（U）以及Tip60特异性序列（T）。本实验共设置4个生物学重复，将分化成熟的3T3-L1细胞通过Amaxa技术完成转染。转染两天后，细胞先用磷酸盐缓冲液（phosphate buffered saline, PBS）洗涤两次，随后加入0.5mL TRIzol（Invitrogen）进行裂解。通过实时定量反转录聚合酶链反应（quantitative real-time reverse transcription PCR, qRT-PCR）检测Tip60与USP7的mRNA表达水平。扩增得到的互补RNA（complementary RNA, cRNA）样本分别以Cy3或Cy5标记，并与由未分化3T3-L1细胞提取的cRNA制备的通用参考样本进行反向标记后，共同点样于基因芯片（microarray）进行杂交分析。