Wavelet-Vermuse Cindy, Odnokoz Olena, Xue Yifan, Lu Xinghua, Cristofanilli Massimo, Wan Yong
Department of Pharmacology and Chemical Biology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA.
Department of Biomedical Informatics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15206, USA.
Cancers (Basel). 2022 Jul 31;14(15):3732. doi: 10.3390/cancers14153732.
Cell division cycle 20 (CDC20) functions as a critical cell cycle regulator. It plays an important role in cancer development and drug resistance. However, the molecular mechanisms by which CDC20 regulates cellular drug response remain poorly understood. Chromatin-associated CDC20 interactome in breast cancer cells was analyzed by using affinity purification coupled with mass spectrometry. hnRNPU as a CDC20 binding partner was validated by co-immunoprecipitation and immunostaining. The molecular domain, comprising amino acid residues 461-653, on hnRNPU required for its interaction with CDC20 was identified by mapping of interactions. Co-immunoprecipitation showed that CDC20-mediated hnRNPU ubiquitination promotes its interaction with the CTCF and cohesin complex. The effects of CDC20-hnRNPU on nuclear size and chromatin condensation were investigated by analyzing DAPI and H2B-mCherry staining, respectively. The role of CDC20-hnRNPU in tumor progression and drug resistance was examined by CCK-8 cell survival and clonogenic assays. Our study indicates that CDC20-mediated ubiquitination of hnRNPU modulates chromatin condensation by regulating the interaction between hnRNPU and the CTCF-cohesin complex. Dysregulation of the CDC20-hnRNPU axis contributes to tumor progression and drug resistance.
细胞分裂周期20（CDC20）作为一种关键的细胞周期调节因子发挥作用。它在癌症发展和耐药性中起着重要作用。然而，CDC20调节细胞药物反应的分子机制仍知之甚少。通过使用亲和纯化结合质谱分析乳腺癌细胞中与染色质相关的CDC20相互作用组。通过共免疫沉淀和免疫染色验证了hnRNPU作为CDC20结合伴侣。通过相互作用图谱鉴定了hnRNPU与CDC20相互作用所需的包含氨基酸残基461 - 653的分子结构域。共免疫沉淀表明，CDC20介导的hnRNPU泛素化促进其与CTCF和黏连蛋白复合物的相互作用。分别通过分析DAPI和H2B - mCherry染色研究了CDC20 - hnRNPU对核大小和染色质凝聚的影响。通过CCK - 8细胞存活和克隆形成试验检测了CDC20 - hnRNPU在肿瘤进展和耐药性中的作用。我们的研究表明，CDC20介导的hnRNPU泛素化通过调节hnRNPU与CTCF - 黏连蛋白复合物之间的相互作用来调节染色质凝聚。CDC20 - hnRNPU轴的失调导致肿瘤进展和耐药性。
