Single-epitope recognition imaging of native chromatin
- Hongda Wang†1, 2Email author,
- Yamini Dalal†3, 4Email author,
- Steven Henikoff3, 5 and
- Stuart Lindsay1
© Wang et al; licensee BioMed Central Ltd. 2008
Received: 11 September 2008
Accepted: 17 December 2008
Published: 17 December 2008
Direct visualization of chromatin has the potential to provide important insights into epigenetic processes. In particular, atomic force microscopy (AFM) can visualize single nucleosomes under physiological ionic conditions. However, AFM has mostly been applied to chromatin that has been reconstituted in vitro, and its potential as a tool for the dissection of native nucleosomes has not been explored. Recently we applied AFM to native Drosophila chromatin containing the centromere-specific histone 3 (CenH3), showing that it is greatly enriched in smaller particles. Taken together with biochemical analyses of CenH3 nucleosomes, we propose that centromeric nucleosomes are hemisomes, with one turn of DNA wrapped around a particle consisting of one molecule each of centromere-specific CenH3, H4, H2A and H2B.
Here we apply a recognition mode of AFM imaging to directly identify CenH3 within histone core particles released from native centromeric chromatin. More than 90% of these particles were found to be tetrameric in height. The specificity of recognition was confirmed by blocking with a CenH3 peptide, and the strength of the interaction was quantified by force measurements. These results imply that the particles imaged by AFM are indeed mature CenH3-containing hemisomes.
Efficient and highly specific recognition of CenH3 in histone core particles isolated from native centromeric chromatin demonstrates that tetramers are the predominant form of centromeric nucleosomes in mature tetramers. Our findings provide proof of principle that this approach can yield insights into chromatin biology using direct and rapid detection of native nucleosomes in physiological salt concentrations.
Eukaryotic genomes are packaged with octameric protein particles, consisting of two copies each of histones H2A, H2B, H3 and H4, which wrap nearly two turns of DNA to form nucleosomes . Since the discovery of nucleosomes in the early 1970s, a variety of techniques have been applied to their study. However, both ultrastructural technologies (e.g. crystallography and electron microscopy) and biochemical analyses (e.g. nuclease assays and sedimentation) have been limited in their scope, because they cannot simultaneously assay structure and dynamics. In recent years, progress has been made in applying new technologies that have the potential to bridge the gap between static ultrastructural features and dynamic physiological processes in the study of chromatin. These technologies, which include scanning confocal fluorescence microscopy , molecular tweezers [3, 4] and atomic force microscopy (AFM) [5, 6], have provided remarkable insights into the behavior of individual nucleosomes. The combination of single-molecule resolution, solution biochemistry and observation of native macromolecular complexes has made AFM especially attractive for studying nucleosomes.
A fundamental level of distinction between nucleosome types is provided by the incorporation of alternative variants of histones H2A and H3 . At chromosomal sites for spindle fiber attachment at mitosis, histone H3 is replaced with a centromere-specific variant (CenH3, CENP-A in humans) to form the specialized nucleosomes that comprise centromeric chromatin . CenH3s in a variety of eukaryotes are found to be absolutely essential for the organization of the kinetochore, which is the only chromosomal structure required for mitosis and meiosis . Therefore, CenH3 nucleosomes are thought to provide the molecular foundation for assembly of the kinetochore at mitosis .
Until recently, the structure of CenH3 nucleosomes was presumed to be the same as that for canonical H3 nucleosomes. However, our recent study showed that CenH3 nucleosomes consist of half the DNA and protein of octameric nucleosomes, with one molecule each of CenH3, H4, H2A and H2B . In that study, we applied AFM to complement detailed biochemical characterization, and found that CenH3 chromatin consists of particles that are half the height of bulk octameric nucleosomes. However, it has been argued that the tetrameric particles that we observed might be non-nucleosomal intermediates in the assembly of CenH3 chromatin . Given the novelty of tetrameric nucleosomes in eukaryotic biology , and the potential for controversy , it is important to directly test alternative interpretations of our observations.
To address the possibility that we have identified an immature intermediate in CenH3 nucleosome assembly as opposed to a mature form, we adapted the purification of the CenH3 nucleosomes to include a DNA-binding step, so that only stable nucleosomes are purified rather than soluble assembly intermediates. In addition, we have applied recognition imaging, wherein an anti-CenH3 antibody is covalently coupled to the AFM tip. Recognition imaging provides highly efficient and specific identification of epitopes within nucleosomes . This strategy has allowed us to show directly that by far the most predominant form of CenH3 particles isolated from chromatin is tetrameric in height, which is inconsistent with them being assembly intermediates. Our application of single-epitope recognition imaging to centromeric nucleosomes also illustrates the potential of recognition imaging by AFM for analyzing minute amounts of specialized chromatin fractions isolated from the nucleus.
Results and discussion
Recognition imaging of centromeric nucleosomes
Force analysis of tip-sample dissociation
Our application of recognition imaging to CenH3 nucleosomes also serves to highlight the exquisite sensitivity of this technique, wherein each particle detected represents the signal from a single CenH3 protein epitope that is present at the end of the 125-aa N-terminal tail. Single-epitope recognition also suggests broader applications of AFM to native chromatin. There has been an explosion of interest in the distribution of histone modifications and variants in chromatin to understand epigenetic regulation , and as a result, there are now dozens of excellent commercially available antibodies against a wide variety of histone modifications. The methods that we have described here for CenH3 nucleosomes should be broadly applicable to any histone variant or modification, especially in cases where only small amounts of native material are available. Therefore, AFM recognition imaging has the potential to provide single-molecule maps of a wide variety of epigenetic features for any chromatin fraction that can be isolated.
Direct measurement of single CenH3 epitopes in the tetramer-height particles released from native chromatin reveals that more than 90% contain CenH3. Insofar as an assembly intermediate would, by definition, comprise only a minor subset of the entire population, we conclude that CenH3 hemisomes represent the mature form of centromeric chromatin. Thus, the high sensitivity and specificity of single-epitope recognition imaging has made it possible to address a key issue in chromatin biology. With efficient single-epitope detection of native macromolecular complexes, AFM with recognition imaging should be generally applicable to the detection of epitopes in biological samples that are available in only limited amounts.
Biochemical purification of native histone core particles
A desiccator was purged with argon for 2 minutes and 30 μl of APTES (99% 3-aminopropyl triethoxysilane, Sigma-Aldrich, St. Louis, MO) placed into a small container at the bottom of the desiccator. Ten microliters of N,N-diisopropylethylamine (99%, distilled, Sigma-Aldrich) was placed into another small container, and the desiccator purged with argon for a further 2 minutes. Mica sheets were stripped on one side until smooth and immediately placed into the desiccator. The desiccator was purged for another 3 minutes and then sealed off, leaving the mica exposed to APTES vapor for 1 hour. After this exposure, the APTES was removed, the desiccator purged, and the APTES-mica stored in the sealed desiccator until needed.
Preparing samples for AFM imaging
Two hundred microliters of a 2 μM glutaradehyde (grade I, Sigma-Aldrich) solution in water was added via a pipette onto APTES-mica immediately upon removal from the storage desiccator and incubated for 10 minutes . The surface was rinsed with water from a Nanopure ultrapure water system, and 60 μl core particle solution (about 0.2 μg core particles per milliliter in PBS buffer) was added via a pipette onto the treated surface and allowed to incubate for 30 minutes. The surface was then rinsed again with PBS buffer (100 mM NaCl, 50 mM Na-phosphate, pH 7.5). The prepared sample was mounted into the scanning probing microscopy (SPM) liquid flow cell and imaged immediately.
Functionalizing AFM tips
Silicon-nitride cantilever tips (Microlever, Veeco, Santa Barbara, CA, coated for MacMode AFM by Agilent Technologies, Chandler, AZ) for recognition imaging were used as described . Briefly, anti-CenH3 antibody was reacted with N-Succinimidyl 3-(acetylthio)propionate (SATP, Sigma inc.) and purified in a PD-10 column (Amersham Pharmacia Biotech). The cantilevers were cleaned in a ultraviolet (UV) cleaner, vapor-treated with APTES and reacted with polyethylene glycol (PEG) crosslinker using triethylamine and CHCl3. The SATP-labeled antibodies were then bound to the PEG crosslinkers with NH2OH (Sigma) in NaCl/Phosphate buffer. The tips were then rinsed in PBS buffer and stored at 4°C until use.
Imaging of native core particles
Conventional imaging was performed on a Pico I MacMode AFM (Agilent Technologies, Chandler, AZ) with an amplitude setting between 2.0 and 2.5 V. Recognition imaging was performed on a Pico I AFM with a Picotrec recognition imaging attachment (Agilent Technologies) with an amplitude setting of about 14–16 nm. AFM engagement was performed at 30% amplitude reduction. The images were taken in PBS buffer. Peptide blocking experiments (Figure 2D) were performed by adding 200 μl CenH3 peptide (30 μg ml-1) into the liquid cell (diameter 1.2 cm, height 0.5 cm) during imaging of samples resulting in an effective peptide concentration of 10 μg cm-2.
Recognition imaging was analyzed using custom software [21, 22]. This program compiles histograms of the pixel intensity distribution from background regions containing no features, and compares them with regions containing visible recognition spots. Recognition events give rise to a second peak in the intensity distribution and these were clearly separated from the background by selecting a cut-off of 75% of the background intensity (recognition spots correspond to a decrease in the signal). Particle heights were measured using FemtoScan (Advanced Technologies Center). A maximum height was taken as the peak height relative to the local background. Only particles with an apparent diameter of more than 12 nm were counted. The true diameter of histone core particle ranges from 8 to 10 nm in the absence of DNA , which produces features from 15 to 25 nm in diameter in the AFM image owing to the limited resolution of the probe.
We thank members of our laboratories for helpful discussions and technical assistance.
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