Klier Steffi, Farmer Sarah, de Bruin Robertus A M
MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, UK.
Methods Mol Biol. 2014;1170:463-76. doi: 10.1007/978-1-4939-0888-2_25.
G1-S transcriptional control involves the coordination of the expression of a large set of co-regulated genes as a function of cell cycle progression (Bertoli et al., Nat Rev Mol Cell Biol 14:518-528, 2013). Confining transcription to the G1 phase of the cell cycle requires the regulation of specific transcription factor activity through either co-factors or regulation of promoter DNA binding. Therefore, the analysis of G1-S transcriptional control involves cell cycle synchronization and monitoring cell cycle synchrony, in order to establish DNA binding of G1-S transcription factors to G1-S promoters and to investigate changes in gene expression during the different phases of the cell cycle. Here, we describe a cell cycle synchrony method and ways to monitor synchrony. We also describe a chromatin immunoprecipitation (ChIP) method to locate G1-S transcription factor components to promoters and a quantitative PCR (qPCR) protocol to determine gene expression. Defining the binding dynamics of G1-S transcription factors and changes in gene expression during the cell cycle should provide new insights into the mechanism that control G1-S transcription and will allow for investigation of the biological relevance of confining gene expression to G1.
G1-S转录调控涉及大量共同调控基因的表达协调，这是细胞周期进程的一个函数（Bertoli等人，《自然综述：分子细胞生物学》14:518 - 528，2013年）。将转录限制在细胞周期的G1期需要通过辅助因子或启动子DNA结合的调控来调节特定转录因子的活性。因此，对G1-S转录调控的分析涉及细胞周期同步化以及监测细胞周期同步性，以便确定G1-S转录因子与G1-S启动子的DNA结合，并研究细胞周期不同阶段的基因表达变化。在此，我们描述一种细胞周期同步化方法以及监测同步性的方法。我们还描述一种染色质免疫沉淀（ChIP）方法，用于将G1-S转录因子成分定位到启动子上，以及一种定量PCR（qPCR）方案，用于确定基因表达。确定G1-S转录因子的结合动态以及细胞周期中基因表达的变化，应该能为控制G1-S转录的机制提供新的见解，并将有助于研究将基因表达限制在G1期的生物学相关性。
