Measurement of Dynamic Protein Binding to Chromatin In Vivo, Using Photobleaching Microscopy
Introduction
Chromatin-binding proteins play a crucial part in every aspect of chromatin structure and gene expression.1 Direct binding of proteins to chromatin maintains and regulates higher order chromatin structure, and leads to histone modifications and transcriptional activation. Once a gene is activated, components of the RNA polymerase machinery directly contact DNA and mediate transcription. Despite the crucial importance of chromatin proteins, most of what we know about the interaction of these proteins with DNA comes from in vitro experiments. Regardless of whether the DNA used in in vitro assays consists of naked DNA or reconstituted chromatin, it is unlikely that these templates reflect the physiological binding substrates that are found in a cell nucleus or that the buffer conditions accurately reproduce the ionic environment in a cell. Methods are required to probe the binding of proteins to native, unperturbed chromatin in intact cells.
An experimental approach to studying the binding of protein to chromatin in living cells is the use of photobleaching methods.2, 3, 4, 5, 6, 7, 8, 9 In these experiments a fluorescently tagged protein of interest is introduced into cells and its apparent mobility is measured as an indicator of its dynamic properties. Because nuclear proteins move passively by rapid diffusion through the nuclear space,2, 10 binding of a protein dramatically affects its overall mobility and therefore the measured mobility contains information about the in vivo binding properties of a protein.2, 8, 9, 11 Qualitative analysis of photobleaching data gives an impression of whether a protein binds stably or transiently to chromatin in vivo.2, 3, 4, 5, 6, 7, 8, 9
In addition to the standard qualitative analysis of photobleaching experiments, kinetic modeling methods can be applied for data analysis to permit extraction of quantitative information about simple biophysical properties of chromatin proteins.11 This method can be used to determine the residence time of a protein on chromatin, the size of the bound and free pools, and the number of kinetically distinct fractions of a protein. These parameters are important in quantitatively describing the in vivo behavior and function of a protein, and they are crucial as constraints in models of large-scale biological systems. In this chapter, we describe the basics of photobleaching microscopy, the rationale for using photobleaching methods to obtain information about binding properties, and we describe methods for the qualitative and quantitative analysis of the binding properties of proteins to chromatin in vivo by photobleaching methods.
Section snippets
Photobleaching Microscopy
Photobleaching methods were first applied to biological samples in the 1970s.12, 13 The early studies were limited to the investigation of lipids and proteins in the plasma membrane because these types of molecules could easily be fluorescently labeled. Subsequently, fluorescently labeled components of the cytoskeleton were microinjected into cells to study cytoskeleton dynamics by photobleaching methods.14 With the development of the green fluorescent protein, photobleaching of expressed
FRAP to Study Protein Binding
FRAP experiments primarily measure the apparent overall mobility of a protein. In an aqueous environment, the recovery kinetics are a direct reflection of diffusional mobility (Fig. 2A). The case of chromatin proteins is different. Chromatin proteins bind periodically to chromatin and reside on the chromatin fiber. The association of a protein with chromatin slows down its overall mobility (Fig. 2B). This complicates the assessment of the absolute mobility of a protein. However, because the
Collecting FRAP Data
For an FRAP experiment, adherent cultured cells stably or transiently expressing a green fluorescent protein (GFP) fusion protein of interest are plated at ∼50% density into glass-bottom dishes [MatTek (Ashland, MA) or LabTek II chambers (Nalge Nunc, Rochester, NY)] and are grown in their regular growth medium for at least 1 day before the experiment. For imaging, the incubation chamber is placed onto the stage of an inverted confocal microscope capable of executing bleaching routines. Optimal
FRAP Data Processing
Even qualitative analysis of FRAP data requires computation of normalized recovery curves. To generate a recovery curve, the average intensity in several regions of the cell is measured. Data sets can generally be directly exported from the microscope software and can be manipulated in general spreadsheet software packages (Excel, SigmaPlot, etc.). There are various methods to normalize the recovery curves. We describe two of the commonly used methods here.
Qualitative Analysis of FRAP Data
Inspection of the recovery curves allows qualitative conclusions regarding the binding of proteins to chromatin. Using this type of approach, it has been demonstrated that core histones H2B, H3, and H4 are virtually immobile and are thus largely statically bound to chromatin.7 In contrast, the linker histone H1 exchanges dynamically and recovery is on the order of minutes.3, 5 Several chromatin proteins including steroid receptors, transcriptional coactivators, and components of the
Quantitative FRAP Analysis
The goal in the following sections is to detail the process of FRAP data analysis in the context of a hypothesized kinetic model of chromatin protein binding in the nucleus.11 A kinetic model is a quantitative statement, based on biochemical and biophysical principles, of a working mechanistic hypothesis about a biological system. The process of extracting quantitative parameters from imaging data, using kinetic models, consists of four steps. First, the mechanistic hypothesis is translated
Approximation of Well-Mixed Nucleoplasm
The apparent mobility of a protein as measured by FRAP is affected by two major components: diffusional mobility between binding events and the binding events themselves. The simplest kinetic data analysis approach involves the assumption that the chromatin protein of interest is kinetically well mixed in the nucleoplasm. This simply means that if we were able to measure the free pool of the protein, we would obtain the same numerical value no matter where in the nucleoplasm we chose to sample.
Estimation of Number of Binding Site Classes
A first step in generating a kinetic model of chromatin protein binding is to determine how many classes of binding sites can be resolved from the data. This can be done with software tools capable of fitting data to sums of exponentials and providing statistical information about the parameter estimates. We recommend SAAM II20, 21 (SAAM Institute, Seattle WA; www.saam.com), but other tools, such as ADAPT (USC Biomedical Simulations Resource; www.bmsr.usc.edu/Software/Adapt/overview.html), have
Summary
We have described procedures for collecting, processing, and analyzing kinetic data obtained by photobleaching microscopy of GFP-tagged chromatin proteins in nuclei of cultured living cells. These procedures are useful for characterizing the in vivo binding of chromatin proteins to their natural template—unperturbed, native chromatin in an intact cell nucleus. These techniques have revealed several generalizations that significantly change our view of the nucleus. At the qualitative level, it
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