Measurement of Dynamic Protein Binding to Chromatin In Vivo, Using Photobleaching Microscopy

doi:10.1016/S0076-6879(03)75025-3

Methods in Enzymology

Volume 375, 2003, Pages 393-414

https://doi.org/10.1016/S0076-6879(03)75025-3 Get rights and content

Publisher Summary

Chromatin-binding proteins play a crucial part in every aspect of chromatin structure and gene expression. An experimental approach to studying the binding of protein to chromatin in living cells is the use of photobleaching methods. 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, 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. Qualitative analysis of photobleaching data gives an impression of whether a protein binds stably or transiently to chromatin in vivo. 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. 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.

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

References (26)

P.H. Barrett
Metabolism
(1998)
D. Axelrod et al.
Bio Phys. J.
(1976)
G. Felsenfeld et al.
Nature
(2003)
R.D. Phair et al.
Nature
(2000)
T. Misteli et al.
Nature
(2000)
J.G. McNally et al.
Science
(2000)
M.A. Lever et al.
Nature
(2000)
D.L. Stenoien
Nat. Cell Biol.
(2001)
H. Kimura et al.
J. Cell Biol.
(2001)
J. Lippincott-Schwartz et al.
Nat. Rev. Mol. Cell. Biol.
(2001)

A.B. Houtsmuller et al.

Histochem. Cell. Biol.

(2001)

O. Seksek et al.

J. Cell Biol.

(1997)

R.D. Phair et al.

Nat. Rev. Mol. Cell. Biol.

(2001)

Cited by (280)

A kinesin-1 adaptor complex controls bimodal slow axonal transport of spectrin in Caenorhabditis elegans
2023, Developmental Cell
An actin-spectrin lattice, the membrane periodic skeleton (MPS), protects axons from breakage. MPS integrity relies on spectrin delivery via slow axonal transport, a process that remains poorly understood. We designed a probe to visualize endogenous spectrin dynamics at single-axon resolution in vivo. Surprisingly, spectrin transport is bimodal, comprising fast runs and movements that are 100-fold slower than previously reported. Modeling and genetic analysis suggest that the two rates are independent, yet both require kinesin-1 and the coiled-coil proteins UNC-76/FEZ1 and UNC-69/SCOC, which we identify as spectrin-kinesin adaptors. Knockdown of either protein led to disrupted spectrin motility and reduced distal MPS, and UNC-76 overexpression instructed excessive transport of spectrin. Artificially linking spectrin to kinesin-1 drove robust motility but inefficient MPS assembly, whereas impairing MPS assembly led to excessive spectrin transport, suggesting a balance between transport and assembly. These results provide insight into slow axonal transport and MPS integrity.
Beyond analytic solution: Analysis of FRAP experiments by spatial simulation of the forward problem
2023, Biophysical Journal
Fluorescence redistribution after photobleaching is a commonly used method to understand the dynamic behavior of molecules within cells. Analytic solutions have been developed for specific, well-defined models of dynamic behavior in idealized geometries, but these solutions are inaccurate in complex geometries or when complex binding and diffusion behaviors exist. We demonstrate the use of numerical reaction-diffusion simulations using the Virtual Cell software platform to model fluorescence redistribution after photobleaching experiments. Multiple simulations employing parameter scans and varying bleaching locations and sizes can help to bracket diffusion coefficients and kinetic rate constants in complex image-based geometries. This approach is applied to problems in membrane surface diffusion as well as diffusion and binding in cytosolic volumes in complex cell geometries. In addition, we model diffusion and binding within phase-separated biomolecular condensates (liquid droplets). These are modeled as spherical low-affinity binding domains that also define a high viscosity medium for exchange of the free fluorescently labeled ligand with the external cytosol.
The analysis of protein recruitment to laser microirradiation-induced DNA damage in live cells: Best practices for data analysis
2023, DNA Repair
Laser microirradiation coupled with live-cell fluorescence microscopy is a powerful technique that has been used widely in studying the recruitment and retention of proteins at sites of DNA damage. Results obtained from this technique can be found in published works by both seasoned and infrequent users of microscopy. However, like many other microscopy-based techniques, the presentation of data from laser microirradiation experiments is inconsistent; papers report a wide assortment of analytic techniques, not all of which result in accurate and/or appropriate representation of the data. In addition to the varied methods of analysis, experimental and analytical details are commonly under-reported. Consequently, publications reporting data from laser microirradiation coupled with fluorescence microscopy experiments need to be carefully and critically assessed by readers. Here, we undertake a systematic investigation of commonly reported corrections used in the analysis of laser microirradiation data. We validate the critical need to correct data for photobleaching and we identify key experimental parameters that must be accounted for when presenting data from laser microirradiation experiments. Furthermore, we propose a straightforward, four-step analytical protocol that can readily be applied across platforms and that aims to improve the quality of data reporting in the DNA damage field.
Nanog organizes transcription bodies
2023, Current Biology
The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells.¹^–³ How proteins of the transcriptional machinery come together to form such bodies, however, is unclear. Here, we take advantage of two large, isolated, and long-lived transcription bodies that reproducibly form during early zebrafish embryogenesis to characterize the dynamics of transcription body formation. Once formed, these transcription bodies are enriched for initiating and elongating RNA polymerase II, as well as the transcription factors Nanog and Sox19b. Analyzing the events leading up to transcription, we find that Nanog and Sox19b cluster prior to transcription. The clustering of transcription factors is sequential; Nanog clusters first, and this is required for the clustering of Sox19b and the initiation of transcription. Mutant analysis revealed that both the DNA-binding domain as well as one of the two intrinsically disordered regions of Nanog are required to organize the two bodies of transcriptional activity. Taken together, our data suggest that the clustering of transcription factors dictates the formation of transcription bodies.
Mediator subunit MED1 differentially modulates mutant thyroid hormone receptor intracellular dynamics in Resistance to Thyroid Hormone syndrome
2023, Molecular and Cellular Endocrinology
Thyroid hormone receptor (TR) controls the expression of thyroid hormone (T3)-responsive genes, while undergoing rapid nucleocytoplasmic shuttling. In Resistance to Thyroid Hormone syndrome (RTH), mutant TR fails to activate T3-dependent transcription. Previously, we showed that Mediator subunit 1 (MED1) plays a role in TR nuclear retention. Here, we investigated MED1's effect on RTH mutants using nucleocytoplasmic scoring and fluorescence recovery after photobleaching in transfected cells. MED1 overexpression and knockout did not change the nucleocytoplasmic distribution or intranuclear mobility of C392X and P398R TRα1 at physiological T3 levels. At elevated T3 levels, however, overexpression increased P398R's nuclear retention and MED1 knockout decreased P398R's and A263V's intranuclear mobility, while not impacting C392X. Although A263V TRα1-transfected cells had a high percentage of aggregates, MED1 rescued A263V's impaired intranuclear mobility, suggesting that MED1 ameliorates nonfunctional aggregates. Results correlate with clinical severity, suggesting that altered interaction between MED1 and TRα1 mutants contributes to RTH pathology.
Directed cell invasion and asymmetric adhesion drive tissue elongation and turning in C. elegans gonad morphogenesis
2022, Developmental Cell
Development of the C. elegans gonad has long been studied as a model of organogenesis driven by collective cell migration. A somatic cell named the distal tip cell (DTC) is thought to serve as the leader of following germ cells; yet, the mechanism for DTC propulsion and maneuvering remains elusive. Here, we demonstrate that the DTC is not self-propelled but rather is pushed by the proliferating germ cells. Proliferative pressure pushes the DTC forward, against the resistance of the basement membrane in front. The DTC locally secretes metalloproteases that degrade the impeding membrane, resulting in gonad elongation. Turning of the gonad is achieved by polarized DTC-matrix adhesions. The asymmetrical traction results in a bending moment on the DTC. Src and Cdc42 regulate integrin adhesion polarity, whereas an external netrin signal determines DTC orientation. Our findings challenge the current view of DTC migration and offer a distinct framework to understand organogenesis.

View all citing articles on Scopus

View full text

Measurement of Dynamic Protein Binding to Chromatin In Vivo, Using Photobleaching Microscopy

Publisher Summary

Introduction

Section snippets

Photobleaching Microscopy

FRAP to Study Protein Binding

Collecting FRAP Data

FRAP Data Processing

Qualitative Analysis of FRAP Data

Quantitative FRAP Analysis

Approximation of Well-Mixed Nucleoplasm

Estimation of Number of Binding Site Classes

Summary

Metabolism

Bio Phys. J.

Nature

Nature

Nature

Science

Nature

Nat. Cell Biol.

J. Cell Biol.

Nat. Rev. Mol. Cell. Biol.

Histochem. Cell. Biol.

J. Cell Biol.

Nat. Rev. Mol. Cell. Biol.