Analyzing G1-S transcriptional control

Methods Mol Biol. 2014:1170:463-76. doi: 10.1007/978-1-4939-0888-2_25.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques / methods
  • Chromatin Immunoprecipitation / methods
  • Flow Cytometry / methods
  • G1 Phase*
  • Gene Expression Regulation, Fungal
  • Reverse Transcriptase Polymerase Chain Reaction / methods
  • S Phase*
  • Saccharomycetales / cytology*
  • Saccharomycetales / genetics
  • Transcriptional Activation*