Department of Biological Engineering, Center for Environmental Health Sciences, Laboratory for Computational Biology and Biophysics, Department of Biology and The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Nucleic Acids Res. 2013 Nov;41(20):9310-24. doi: 10.1093/nar/gkt715. Epub 2013 Aug 9.
An effective response to DNA damaging agents involves modulating numerous facets of cellular homeostasis in addition to DNA repair and cell-cycle checkpoint pathways. Fluorescence microscopy-based imaging offers the opportunity to simultaneously interrogate changes in both protein level and subcellular localization in response to DNA damaging agents at the single-cell level. We report here results from screening the yeast Green Fluorescent Protein (GFP)-fusion library to investigate global cellular protein reorganization on exposure to the alkylating agent methyl methanesulfonate (MMS). Broad groups of induced, repressed, nucleus- and cytoplasm-enriched proteins were identified. Gene Ontology and interactome analyses revealed the underlying cellular processes. Transcription factor (TF) analysis identified principal regulators of the response, and targets of all major stress-responsive TFs were enriched amongst the induced proteins. An unexpected partitioning of biological function according to the number of TFs targeting individual genes was revealed. Finally, differential modulation of ribosomal proteins depending on methyl methanesulfonate dose was shown to correlate with cell growth and with the translocation of the Sfp1 TF. We conclude that cellular responses can navigate different routes according to the extent of damage, relying on both expression and localization changes of specific proteins.
除了 DNA 修复和细胞周期检查点途径外，有效应对 DNA 损伤剂还需要调节细胞内稳态的许多方面。基于荧光显微镜的成像技术提供了在单细胞水平上同时检测 DNA 损伤剂作用下蛋白质水平和亚细胞定位变化的机会。我们在此报告了筛选酵母绿色荧光蛋白 (GFP) 融合文库的结果，以研究暴露于烷化剂甲磺酸甲酯 (MMS) 时全局细胞蛋白重排。鉴定出诱导、抑制、富含核和细胞质的广泛蛋白群。基因本体论和相互作用组分析揭示了潜在的细胞过程。转录因子 (TF) 分析确定了反应的主要调控因子，所有主要应激响应 TF 的靶基因都富集在诱导蛋白中。根据针对单个基因的 TF 数量对生物功能进行的意外分区被揭示出来。最后，根据甲磺酸甲酯剂量的不同，核糖体蛋白的差异调节与细胞生长和 Sfp1 TF 的易位相关。我们得出结论，细胞反应可以根据损伤程度选择不同的途径，依赖于特定蛋白质的表达和定位变化。
