Single-Molecule Fluorescence Microscopy Methods - American

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Downloaded by CENTRAL MICHIGAN UNIV on January 1, 2016 | http://pubs.acs.org Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch007

Single-Molecule Fluorescence Microscopy Methods in Chromatin Biology Roubina Tatavosian, Chao Yu Zhen, and Xiaojun Ren* Department of Chemistry, University of Colorado Denver, Campus Box 194, P.O. Box 173364, 1201 5th Street, Denver, Colorado 80217-3364 *E-mail: [email protected].

Classical biochemical and molecular genetic techniques have provided fundamental insights into genomic functions and chromatin organization. Recent advances in the development of fluorescence microscopy imaging methods as well as new labeling techniques enable visualizing chromatin structure and tracking dynamics of transcription factors within mammalian cells at a single-molecule level. Single particle tracking of transcription factors within cells provides insight into how transcription factors explore the complex nuclear environment and assemble on their target sites. Superresolution microscopy based on single-molecule centroid determination has been applied to map chromatin structure at a nucleosomal resolution. This chapter outlines recent advances in the application of single molecule-based fluorescence microscopy techniques in chromatin biology within mammalian cells.

Introduction In the nucleus, genome organization is shaped by nucleosomes―the basic building unit of chromatin. The nucleosome is formed by wrapping ~147 bp of DNA around a histone octamer core consisting of two copies of histones H2A, H2B, H3, and H4 (1). Chromatin organization has been studied at different levels within the last a few decades. The spatial organization of the genome is hierarchical and characterized by many local and long-range contacts among genes and other sequence elements (2–4). The repeating nucleosomal arrays create the first level of chromatin structures known as “beads on a string” with © 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


a diameter of about 11 nm (5). The association of linker histone H1 organizes nucleosomal arrays into 30 nm chromatin fibers. The studies of 30 nm chromatin fibers suggest two distinct structural models, a one-start helix (solenoid) and a two-start helix (zig-zag) (6). A variety of techniques have made it possible to characterize chromatin structure at high resolution (7–10). Transcription factors play an essential role in the control of gene transcription. The transcription processes are intrinsically highly dynamic, involving transcription factors and RNA polymerases assembly on chromatin during transcription initiation, transcription elongation, and transcription termination (11). The binding transcription factor specific to the DNA sequence encoded in the genome recruits RNA polymerases and other effectors that regulate the structure of chromatins. Classic biochemical and genetic methods have provided fundamental insights into these processes (9). Recent single particle/molecule tracking has provided additional layers of information about how transcription factors explore the complex nuclear environment, search for, and assemble on their target sites. In this chapter, we focus on recent advances in visualizing chromatin architecture and in tracking dynamics of transcription factors at the single-molecule level.

Labelling Strategies and Dyes Chromatin in living cells can be labeled by fusion of a variety of fluorescence proteins (FPs) at one of the histone core subunits (H2A, H2B, H3, and H4). Green FP (GFP) from jellyfish Aequorea Victoria has been widely used for genetically labelling chromatin within cells. For instance, H2B fused with GFP was used to image interphase chromosomes and mitotic chromosomes without perturbing cell-cycle progression or intercellular structure (12) and to assess the direct correlation between histone modifications and the structural stability of higher order chromatin assembly (13). Recently developed photoswitchable FPs provide a versatile toolkit for photoactivated localization microscopy (PALM) imaging of chromatin structure and cellular components (14). Besides for genetically labelling chromatin, GFP is also used to label transcription factors or other cellular proteins. GFP fusion proteins can be transiently or stably expressed within cells via delivery by recent developed lentiviral vectors (15). A specific gene locus can be labelled with genetically encoded FPs fused with gene editing proteins such as TALENs (transcription activator-like effector nuclease) (Figure 1A) or CRISPR/Cas9 (clustered regularly interspaced short palindrome repeats/CRISPR associated protein 9) (Figure 1B) in living cells (16–18). TALENs are sequence-specific DNA binding proteins and can bind to target DNA via 33-aa to 35-aa repeats arranged in tandem arrays (19). TALENs fused with different FPs can be used to tag specific genomic loci in living cells for visualizing chromatin structure and dynamics. TALENs have been used to visualize endogenous repetitive genomic sequence in cultured cells and in living organisms (17, 18). Thus, it can be a powerful technique to investigate heterochromatin formation and dynamics of chromatin segregation during the cell cycle in living cells. 130 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 1. (A) Schematic representation of TALEN-mediated genomic modification unit. TALEN consists of N-terminal domain for nuclear localization signal (NLS); a TAL-central DNA binding domain with 33-aa to 35-aa repeats arranged in tandem array that can bind to target DNA; and C-terminal domain that fused with functional endonucleas Fok I (a restriction enzyme). (B) Schematic of RNA-Guided Cas9 nuclease. Cas9 is guided to the 20 nt dsDNA target by single guided RNA (sgRNA) and it recognizes the specific protospacer associated motif (PAM) sequence and cut the DNA with two active nuclease domains: RuvC (an endonuclease domain named for an E. coli protein involved in DNA repair) and HNH (an endonuclease domain named for characteristic histidine and asparagine residues).

Cas9 from the microbial adaptive immune system S. pyogenes is a RNA-guided DNA endonuclease enzyme that can be directed to a specific location in the genome of living cells using properly designed single guided RNA (sgRNA) (20). Three distinct types (I-III) of CRISPR/Cas9 system have been developed. Type II has been widely used in genomic editing and labeling for its simplicity, high efficiency, and multiplexing capability over the other two types (21). The endonuclease-defective Cas9 protein fused with GFP has been used to image specific genomic loci and to visualize repetitive elements of telomeres in living cells by construction of sgRNA targeting these DNA sequences (16). CRISPR/Cas9 and TALEN imaging technologies are revolutionizing the chromatin field since these techniques allow visualizing nuclear organization and its architecture in living cells. 131 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Other convenient methods to image chromatin and its associated proteins in living cells are the development of tag-mediated labeling proteins called SNAP (22), CLIP (23), and HaloTag (24), respectively (Figure 2). These labeling technologies are based on self-labeling proteins (tag) genetically fused with the protein of interest. Fluorescent dyes covalently bind to the tag proteins. The tag proteins have been widely used to label a variety of proteins in living cells due to their irreversible and photophysical features (25).

Figure 2. Schematic representation of tag-meditated labeling proteins. (A-B) Covalently labeling of hAGT fusion protein with benzylguanine (BG) derivatives (A) and benzylcytosine (BC) derivatives (B) in conjugation with fluorophores. (C) Halo-tag protein fused with protein of interest covalently interacts with chloroalkane linker conjugated with fluorophore.

The SNAP-tag (Figure 2A) is a self-labeling fusion protein that can be fused with the protein of interest and covalently tagged with an appropriate ligand such as a fluorescent dye. It is derived from the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) and can covalently react with O6-benzylguanine (BG) derivatives bearing a chemical probe in living cells (22). This method was used to label several subcellular compartments of zebrafish embryos including the nucleus in conjugation with a variety of fluorophore dyes (26). Furthermore, the same technique was also used for live-cell direct stochastic optical reconstruction microscopy (dSTORM) imaging of histone H2B protein fused with SNAP tag (27). An orthogonal tag, CLIP-tag (Figure 2B) was engineered via mutagenesis of eight amino acids of the SNAP-tag and used in conjugation with a SNAP-tag for simultaneous labeling of two different proteins in one cell (23).The CLIP self-labeling tag specifically and irreversibly interacts with O2-benzylcytosibe (BC) derivatives to create a covalent bond with an exogenously supplied substrate that is linked to a fluorescent dye (23). 132 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


The HaloTag self-labeling technology (Figure 2C) is another novel protein tagging method for imaging chromatin and its associated proteins within living cells. The HaloTag is a modified haloalkane dehalogenase that covalently binds to synthetic ligands called HaloTag ligands including fluorescent dyes. The covalent bond formation between the HaloTag and its ligand is very specific and essentially irreversible and occurs rapidly under physiological conditions (25). HaloTag was used to study the binding of transcriptional factors to chromatin within living cells at the single-molecule level (28).

Visualizing Chromatin Structure by Superresolution Microscopy Based Single-Molecule Centroid Determination Fluorescent microscopy has been used for many years to visualize cellular and subcellular components in living cells due to its high specificity and non-invasive visualization of biological structures. Its spatial resolution is typically limited to 200-300 nm due to diffraction of light; thus, only large-scale chromatin structure can be resolved (29, 30), such as heterochromatin and mitotic chromosomes. Chromatin compaction within different organizational stages occurs often in the range of 10-200 nm, which falls outside of the diffraction limit of the ordinary microscopy techniques. Recently developed superresolution fluorescent imaging techniques have revolutionized the spatial resolution of optic microscopy (31). Among the various approaches, stochastic optical reconstruction microscopy (STORM) (29) and PALM (30) take advantage of single fluorescent probes attached to biomolecules, which are detected while they transiently reside in their fluorescent “on” state, and a location map of their centroid positions are subsequently reconstructed. STORM utilizes photoswitchable dyes such as red Cyanin dye pairs (Cy3-Cy5) (29) with lateral spatial resolution up to 20 nm while PALM uses photoactivable proteins (PA) such as (PA- GFP) (30). STORM/PALM has been applied to study structures of chromosome regions or nucleosomal organization of chromatin. The de Lange and Zhuang laboratories have applied STORM to visualize the structure of telomeres and confirmed previous findings in which functional telomeres frequently exhibit a t-loop configuration (32). They also showed that TRF2, a component of shelterin, is required for the formation and/or maintenance of t-loops. The Darzacq lab has used PALM to visualize 3-D chromatin densities in human U2OS cells by tagging histone H2B with photoactivable GFP (PA-GFP). This showed that the H2B distribution in the nucleoplasm occurs heterogeneously and chromatin is organized in regions of enriched density that can be observed within 10 nm-1.0 µm (33). The Cosma laboratory has utilized STORM to visualize and count nucleosomes along the chromatin fiber in 10-20 nm of diffraction limit and reported that nucleosomes assembled in heterogeneous groups of different sizes, called “clutches” (34). Furthermore, they were able to conclude that the ground state of pluripotent stem cells has less dense clutches and fewer nucleosomes in comparison to the differentiated cells. 133 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Dynamics of Transcription Factors by Single Particle Tracking Transcription factors dynamically interact with chromatin. Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) have been broadly used to measure the binding dynamics of transcription factors on chromatin within cells and have provided profound insights into the dynamic processes of gene transcription (35–37). Single particle tracking of individual transcription factors has advantages over FRAP and FCS since it can resolve hidden kinetic populations and easily measures the residence time of transcription factors on chromatin (38, 39). Furthermore, analysis of individual trajectories of transcription factors can provide information on how transcription factors explore the nuclear environment and search for their cognate binding sites (40). Early studies of the dynamics of transcription factors by single particle tracking rely on the injection of fluorescently labelling transcription factors into living cells, which controls the protein concentration within cells (41). Recently developed HaloTag and SNAP tags can be genetically fused with transcription factors and the labelling density can be controlled by varying concentrations of fluorescent dye ligands. To increase signal-to-background ratio, highly inclined and laminated optical sheet (HILO) microscopy and light-sheet microscopy have been used to track dynamics of transcription factors (28, 39, 40, 42). A few groups have used fluorescence microscopy imaging methods and labeling techniques to dissect dynamics of transcription factors within the environment of the nucleus (28, 39, 40, 42). For example, single molecule tracking has revealed that transcription factors employ different searching strategies within living cells to measure their residence time on chromatin and to dissect their assembly on chromatin.

Summary In summary, chromatin organization and dynamics of transcriptional factors are essential for understanding of gene transcription. By utilizing genetic fluorescent protein with the aid of TALENS and CRISPER/Cas9, visualization of chromatin spatial organization and its dynamic in living cell can be achieved. Furthermore, spatiotemporal dynamics of chromatin associated proteins can be measured at a single-molecule level by using high resolution microscopy techniques. Although single molecule imaging technologies is still at its infancy, various techniques that have been discussed above have already provided a collectable new understanding of chromatin spatiotemporal organization and its associated proteins architecture in living cells.

Acknowledgments The authors would like to thank Huy Duc, Arthur Boo, and Jun Lee, Marko Kokotovic for constructive criticisms. This work was supported by the University of Colorado Denver and by the CU Denver Office of Research Services. 134 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, Chengshan, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Single-Molecule Fluorescence Microscopy Methods - American

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