Quantifying chromatin-associated interactions: the HI-FI system.
Chromatin plays a vital role in regulating cellular processes that occur on the DNA. Modulation of chromatin structure is conducted through interactions with binding factors that direct critical actions such as posttranslational modifications, nucleosome remodeling, and incorporation of histone variants. Specific factors recognize and act upon the various physical states of chromatin to modulate DNA accessibility. The ability to quantitatively characterize these interactions in vitro can provide valuable insight into the mechanisms that dictate chromatin architecture. Here, we describe in detail fluorescence methodologies for quantifying the thermodynamic principles that guide interactions between nucleosomal arrays, mononucleosomes, or nucleosome components and chromatin-associated factors through application of the HI-FI (High-throughput Interactions by Fluorescence Intensity) system. These measurements utilize fluorescence (de)quenching and FRET assays performed in 384-well microplates, making the assays suitable for high-throughput characterization of interactions at low concentrations. Further, this system can be used to determine the stoichiometric composition of complexes and specific sites of interaction. After quantification on a plate reader or similar instrument, the solution-based assays can be directly transferred to native gels for visualization of interaction(s). We also highlight procedural details on the efficient attachment of fluorescent dyes to histones and DNA. In all, the HI-FI system of assays can be used to elucidate mechanistic details of how specific chromatin-associated factors function at the molecular level.
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado, USA.
Duane D
DD
Winkler
Karolin
K
Luger
Aaron R
AR
Hieb
D000818:Animals; D002021:Buffers; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006657:Histones / Q000737:chemistry; D006801:Humans; D009707:Nucleosomes / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D058849:Protein Refolding; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D013816:Thermodynamics; D029867:Xenopus Proteins / Q000737:chemistry
D002021:Buffers; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D006657:Histones; D009707:Nucleosomes; D012996:Solutions; D029867:Xenopus Proteins