Measuring the dynamics of chromatin proteins during differentiation

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1042; p. 173
• Main Authors: Harikumar, Arigela, Meshorer, Eran
• Format: Journal Article
• Language: English
• Published: United States 2013
• Subjects: Animals; Cell Differentiation - genetics; Cells, Cultured; Chromatin - metabolism; Chromosomal Proteins, Non-Histone - analysis; Chromosomal Proteins, Non-Histone - metabolism; Cloning, Molecular; Embryonic Stem Cells - cytology; Fluorescence Recovery After Photobleaching - methods; Green Fluorescent Proteins - genetics; Histones - analysis; Histones - metabolism; Mice; Photobleaching; Single-Cell Analysis - methods
• ISSN: 1940-6029
• DOI: 10.1007/978-1-62703-526-2_12

Chromatin-protein interactions are important in determining chromosome structure and function, thereby regulating gene expression patterns. Most chromatin associated proteins bind chromatin in a transient manner, with residence times on the order of a few seconds to minutes. This is especially pertinent in mouse embryonic stem cells (ESCs), where hyperdynamic binding of chromatin associated proteins to chromatin is thought to regulate genome plasticity. In order to quantitatively measure binding dynamics of such chromatin proteins in living cells, a combination of GFP-fusion proteins and photobleaching-based assays such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are advantageous over other existing biochemical assays, because they are applied in living cells at a single cell level. In this chapter we describe a detailed protocol for performing FRAP and FLIP assays for measuring structural chromatin protein dynamics such as Heterochromatin Protein 1 (HP1) and linker histone H1 in mouse ESCs and during ESC differentiation.