title: Analyzing G1-S transcriptional control.
abstract: 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.
authors.affiliation: MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London, WC1E 6BT, UK.
authors.forename: Steffi
authors.initials: S
authors.lastname: Klier
authors.forename: Sarah
authors.initials: S
authors.lastname: Farmer
authors.forename: Robertus A M
authors.initials: RA
authors.lastname: de Bruin
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D047369:Chromatin Immunoprecipitation / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D016193:G1 Phase; D015966:Gene Expression Regulation, Fungal; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D016196:S Phase; D004718:Saccharomycetales / Q000166:cytology* / Q000235:genetics; D015533:Transcriptional Activation