Nucleosomes are responsible for packaging the genome into the nucleus and regulating access to genetic information during various cellular processes. We study the nucleosome structure and dynamics using a variety of approaches.
1. Unzipping through a nucleosome
A typical experimental configuration for unzipping. An optical trap is used to apply a force necessary to unzip through the DNA as the coverslip is moved laterally so that the end of the DNA bound to the coverslip is moved away from the trapped microsphere.
Representative force unzipping signatures of naked DNA (black), DNA containing a nucleosome (red), and DNA containing a tetrasome (green).
2. High resolution histone-DNA interaction map in a nucleosome
We applied the unzipping technique to generate a high resolution map of the strengths of histone-DNA interactions in a nucleosome (Hall et al., NSMB, 2009). These interactions represent a fundamental level of gene regulation through which nucleosome stability, DNA accessibility, transcription, and chromatin higher order structures are controlled. Our map revealed highly non-uniform strengths of histone-DNA interactions along the DNA, highlighted by three broad regions: a region of strong interactions at the dyad and two regions of relatively weaker interactions ± 40 bp on either side of the dyad. According to the nucleosome structure, histone core domains are expected to make strong contacts with the DNA minor groove every 10 bp (Luger et al., Nature, 1997). However, we identified a distinctive 5-bp periodicity throughout all three regions, indicating that two distinct interactions at each minor groove contact (one from each strand) could be disrupted sequentially, rather than simultaneously. Furthermore, we demonstrated that, as histone-DNA interactions were disrupted beyond the dyad region, the entire histone octamer dissociated from the DNA. This study has significant implications for the mechanism of motor protein movement through nucleosomal DNA.
A high-resolution map of histone-DNA interactions within a nucleosome. (A) An optical trap was used to unzip through DNA containing a single nucleosome held under constant force. (B) The resulting dwell time histogram of the DNA fork along the DNA clearly identified the locations and strengths of histone-DNA interactions. There are three regions of strong interactions and a finer 5-bp periodicity.
3. Unzipping to study nucleosome remodeling
DNA unzipping can accurately locate a nucleosome on a long DNA template such that undesired effects of close proximity to DNA ends may be minimized. We have used the unzipping technique to probe the structure of individual nucleosomes after SWI/SNF remodeling (Shundrovsky et al., NSMB, 2006). We observed that, under our experimental conditions, SWI/SNF remodeling does not alter the overall nucleosome structure. The histone octamer remains intact and the overall strength and position of histone-DNA interactions within the nucleosome are essentially unchanged. However, nucleosomes were moved bidirectionally along the DNA with a characteristic spreading of 28 bp per remodeling event. Taken together, these results on SWI/SNF mediated nucleosome remodeling generated by unzipping provide direct measurements of the structure and location of remodeled nucleosomes.
Unzipping through nucleosomes before (top panels) and after (bottom panels) remodeling. Left panels show unzipping signatures for 30 unremodeled and remodeled data curves. Right panels show histograms of unremodeled and remodeled nucleosome positions on the DNA.
4. The behavior of chromatin under torsion
It is by now well established that DNA in both prokaryotic and eukaryotic cells experiences torsional stress that can modify its structure and influence genetic transactions. While the topological alterations of the DNA itself are relatively well studied, much less is known about the effect of torsion on chromatin. It has been hypothesized that supercoiling waves generated by elongating RNA polymerase play a role in nucleosome disruption and regeneration. Enabled by our angular optical trap, we are using a single nucleosome as a model system to understand the implications of force and torsion in this system.