Introduction
DNA and Histone Protein
Histone protein and DNA are the basic repeating units of eukaryotic chromatin, which forms a secondary structure called nucleosome. Because histone protein has positive charges, it allows negatively charged DNA to associate with histone protein and form a nucleosome. Each nucleosome is composed of eight histone proteins that coat DNA and are called histone octamer.
The successive nucleosomes then form a 30nm spiral resembling a solenoid, where additional histone proteins called H1 are associated with nucleosomes to form solenoid structure1,7.
Histone Tail and its Epigenetic Modification Site
Magnetic Tweezers for Nucleosome Dynamics
For Z-position calibration, we used piezo stages (FT-2150, ASI), which enable us to precisely control the stage and track the z-position of magnetic bead accurately. When the histone protein was injected, 3pN magnetic force was applied to the magnetic bead to prevent the bead from being bound to slide nonspecifically. After nucleosome reconstitution, with 0.1 pN/s force increment, magnetic bead position was observed in real time.
The center position of the magnetic bead was tracked and the rotation diameter was calculated by measuring the camera pixels. If the diameter exceeds 1um, a bead was excluded from observation due to off-center attachment, which will interfere with the real-time observation of magnetic bead. After assembly of nucleosome on NRL sequence, we increased the magnetic force on the magnetic bead at a rate of 0.1 pN/s, and the position of magnetic bead was simultaneously tracked in real time.
Similar to tracking wild-type nucleosome, the H4-tailed nucleosome showed discrete changes in the position of the magnetic bead, signifying the disassembly of histone proteins from DNA. By tracking the H4-tailed nucleosome we were able to recognize that the average number of discrete changes in magnetic bead position is significantly reduced compared to wild-type histone. Real-time observation of disassembly event of H2A/H2B tail removed (top) and H2A/H2B, H4 tail removed (bottom) nucleosome.
H2A/H2B tail deleted nucleosome showed different dynamics compared to wild type or H4 tail deleted nucleosome by its separation step size and the number of discrete changes of magnetic bead position. First, the number of sudden jumps in magnetic bead position is less than that of the wild-type and H4 tail-deleted nucleosome. Wild-type nucleosomes also show a lot of discrete changes of magnetic bead position, indicating that it has the most efficient ability for nucleosome reconstitution.
Our self-built magnetic tweezers worked successfully for picking up the beads that do not show eccentric attachment and tracking the 3D position of the magnetic bead, which shows sudden changes in the position of the magnetic bead in real time, which can be considered as the disassembly of the nucleosome.
Materials and Method
Handle Sequence preparation
To prevent free rotation of the magnetic beads, the NRL 16x197 sequence is ligated to handle the sequence which is 500 base pairs in length and multiple modified nucleotides. Each sequence was constructed by PCR from the pet28a backbone with Phusion PCR MM (NEB), biotin-dUTP (Jena Bioscience), and dig-dUTP (Jena Bioscience). The BamHI site of pet28a is amplified for the biotin handle and the XhoI site for the dig handle.
With a ratio of modified dUTP to normal dTTP of 1:3, the final handle product can be expected to have about thirty biotin or ground sites that allow the handle to bind to multiple neutravidin sites on a slide or an anti-ground site on an M280 magnetic bead. After constructing each handle sequence, the 16x 197NRL sequence (approximately 3200 bp) and each handle sequence (500 bp) were ligated in 16C overnight. The ratio of insert to handle sequence was 10:1 to prevent self-ligation of the 16x 197NRL sequence.
Magnetic Bead Coating
Magnetic Tweezers
Tweezers Sample Preparation
For nucleosome assembly, 6 nM histone octamer and 2 nM NAP1L1 were injected into the channel and incubated for 15 min. As the magnetic force increases, histone compaction or nucleosome disassembly will be shown as a change in the position of the magnetic bead5.
Code for preventing to pick the off-center attachment of bead
To investigate the role of the histone H4 tail on the physical property of the nucleosome, we also observe the disassembly of the H4 tail-deleted nucleosome in real time. It suggests that when the H4 tail deletion is given, nucleosome reconstitution is not as complete as in wild type and it also reduces the efficiency of nucleosome reconstitution. It suggests that reconstitution efficiency decreases further and that the H2A/H2B tails are related to NAP1 work efficiency.
Also, the disassembly step size was reduced compared to wild type, which is similar to H4 tail-deleted nucleosome compared to wild type. When we measure the segregation step of the H4 tail-deleted nucleosome, the number of populations that have step size larger than 22nm is lowered, whereas every other population was similar to the wild-type segregation population. It suggests that the H4 tail has a role in creating a higher structure of the nucleosome, such as the outer turn of the nucleosome or part of the inner turn of the nucleosome.
Also, disassembly of H2A/H2B tail-deleted nucleosome was measured by measuring the length of discrete changes of tracks, H2A/H2B tail-deleted nucleosome shows more loosened structure by showing its separation population of 16~18nm mostly. It suggests that H2A and H2B tails are related to the stability of the inner turn of the nucleosome by showing that its step size and reconstitution efficiency are much lower than WT and H4 tail deleted. When we introduced H2A/H2B/H4 tail deletion to nucleosome, the disassembly step size is significantly reduced compared to wild type, H4 deletion and H2A/H2B deletion.
Also, the number of changes in the footprint also becomes smaller than that of deleted H2A/H2B tail nucleosomes. In this dissertation, we constructed magnetic tweezers for tracking DNA and histone protein dynamics, and compared the stability of wild-type and tail-deleted nucleosomes in order to test the hypothesis that histone tails may have role in nucleosome stability and chromatin compaction. . Corresponding to our hypothesis of changes in nucleosome stability, our MNase assay data and magnetic tweezer data indicate that histone tails have several roles in nucleosome stability, the efficiency of nucleosome remodeling in the Widom 601 sequence, which can binds to the nucleosome assembly protein (NAP1), which is supported by the known interaction of the H2A/H2B tail with NAP1.
In conclusion, single-molecule experiments showed that the H2A/H2B tail and the H4 tail have roles in maintaining nucleosome structure and, furthermore, also interfere with nucleosome remodeling when using NAP1 for histone protein reassembly.
Results
Construction of plasmid having 16x 197NRL sequence
To confirm the binding affinity of the histone protein to the Widom sequence, each combination of histone subunits (from left; DNA ladder, no histone, AB4D, ABD, 4D) was incubated with the 2x197NRL sequence. As we observed the Coomassie blue band to fall, we concluded that the histone protein wraps the DNA. Since MNase provides more direct information about nucleosome reconstitution, we reconfirmed nucleosome reconstitution by MNase treatment.
The reconstituted WT nucleosome after MNase treatment shows that the WT nucleosome is well formed by showing the intense band around the 170 bp region, while the other modified nucleosome after MNase treatment shows its band smaller than 100 bp which means that the nucleosome is reconstituted only with the tetramer and has low efficiency. It suggests that the histone tail has an interaction with the nucleosome assembly protein (NAP1L1) which helps the histone protein form nucleosomes. To study the force-dependent stability of wild-type nucleosomes, we measure the WT histone disassembly event.
While this magnetic force change resulted in WLC adaptation to bare DNA extension, in the case of WT histone aggregated DNA, additional discrete changes of bead position were observed due to the disassembly of the individual nucleosome, which is the release of The Widom sequence that wraps around the nucleosome. Through this real-time observation of nucleosome disassembly, we could observe wild-type histone protein has high efficiency for nucleosome reconstitution, and on average, wild-type nucleosome disassembly has a step size of about 24nm, which coincides with the length of the nucleosome internal turn. To investigate the structural difference between wild-type and modified nucleosomes, we measured the separation step length of each type of modifications on the nucleosome from tracks.
First, our MNase gel data showed that the nucleosome reconstitution ability of each modified histones and wild-type histone is different. Wild-type histone protein showed that it works well with a high-intensity band around 170 bp, which corresponds to the 1xWidom sequence, while the other modified histone showed its band only at less than 100 bp, which may mean that histone protein is only tetrameric or less forms. And our magnetic tweezers data show that the histone tail plays a number of roles in reconstructing and maintaining the inner or outer turn of the nucleosome by showing the differences in step size of nucleosome disassembly.
These data provide insights into each histone tail's role on nucleosome stability and basis for future works that will work to reveal specific epigenetic modifications on histone tails. Effect of specific modifications in histone tail on nucleosome stability: this research can be repeated in the same way with another epigenetic modification to reveal the role of that modification's role on nucleosome stability. Effect of polyamine on nucleosome stability: as the ion concentration becomes high, nucleosome is known to become unstable.
Confirmation Nucleosome Formation through agarose gel
Real-time observation of nucleosome disassembly events with magnetic tweezers
Stepsize of nucleosome disassembly
First, the wild-type disassembly length histogram has most of its population at 22~26 nm, which is known as the inner turn of the normal nucleosome. It also shows the populations at 42~48nm and 60~66nm, which represent the disassembly length of dinucleosomes and trinucleosomes. Most of its steps are less than 10 nm, suggesting that it almost loses its ability to independently turn nucleosomes inside and out.
As we delete more histone tails, the step size of disassembly and the number of disassembly were lowered, which means that the binding affinity and wrapping length are lowered. Dialysis to reconstitute the nucleosomes with modified histones: because modified histone has low efficiency for nucleosome reconstitution with NAP1, it was difficult to find out and collect its physical properties. If it induces nucleosome to be unstable, we can do further research to reveal the relationship between polyamine and transcription efficiency.
Quantitative analysis of single-molecule force spectroscopy on coiled chromatin fibers, Nucleic Acids Research, 2015; vol. Timothee L, Jean A, Andrey R, Terence S, Omar S, David B and Vincent C, Single-Molecule Studies Using Magnetic Traps, Cold Spring Harbor Protoc; 2012; doi:10.1101/pdb. Daniel K and Ralf S, Torsional stiffness of individual superparamagnetic microspheres in an external magnetic field, PRL.
Discussion& Conclusion
