Since the discovery of the nucleosome in the early 1970's, scientists have sought to correlate chromatin structure and dynamics with biological function. More recently, we have learned that nucleosomes and chromatin play a critical role in the regulation of transcription, replication, recombination, and repair (Zlatanova and Leuba, 2004). Our laboratory uses an interdisciplinary approach combining the disciplines of molecular biology, biochemistry, engineering, and physics to try to understand at the single nucleosome and single chromatin fiber level how chromatin structure and dynamics regulate biological processes that use DNA as a template. To this end, we are applying several single-molecule approaches such as atomic force microscopy (AFM), magnetic tweezers, optical tweezers and single-pair fluorescence resonance energy transfer (spFRET) to native or reconstituted chromatin fibers of different protein compositions with the latter three methods using homebuilt instrumentation. Single-molecule techniques provide the sensitivity to detect and to elucidate small, yet physiologically relevant, changes in chromatin structure and dynamics. Recent examples of what we have been able to discover include the following:

• We have been able to use AFM to detect conformational changes in chromatin fiber structure due to the presence of 24 methyl groups per nucleosome (Karymov et al., 2001) implying that the combined action of the DNA methylation and linker histone binding required to compact chromatin may affect the transcription of large chromatin domains.
• We also used AFM to investigate the role of histone variants in chromatin fiber structure (Tomschik et al., 2001). Eukaryl and archaeal organisms have similar fiber structure with differences likely related to the more complex needs of eukaryl organisms to regulate transcription.
• We have used optical tweezers to determine the piconewton forces necessary to unravel individual nucleosomes in a fiber context (Bennink et al., 2001) and found that the measured forces for individual nucleosome disruptions are in the same range of forces reported to be exerted by RNA- and DNA-polymerases.
• We have used magnetic tweezers to observe a dynamic equilibrium between force dependent nucleosomal assembly and disassembly on a single DNA molecule in real time (Leuba et al., 2003) as a model of what happens to nucleosomes when a transcribing polymerase passes through the region where they are located.
• We have used spFRET to demonstrate fast, long-range, reversible conformational fluctuations in nucleosomes between two states: fully folded (closed) with the DNA wrapped around the histone core, and open, with the DNA significantly unraveled from the histone octamer (Tomschik et al., 2005), implying that most of the DNA on the nucleosome can be sporadically accessible to regulatory proteins and proteins that track the DNA double helix.
  

Our future goals are to build combination single-molecule instruments to image and manipulate intramolecular nanometer movements in submillisecond real-time with piconewton force sensitivity (e.g., we want to observe directly what happens to the histones in a nucleosome in the path of a transcribing polymerase). We want to observe what changes in superhelicity occur upon nucleosome formation, nucleosome by nucleosome. We hope to resolve whether the positive supercoils generated by a transcribing polymerase are sufficient to displace histone octamers. In addition to chromatin, we are studying the mechanism of action of individual helicases unwinding DNA. We are also working on the capability to observe in real time single nucleosome dynamics in living cells.

Atomic Force Microscope (AFM)

Evanescent Field Fluorescent Microscope (EFFM)

Magnetic Tweezers

Optical Tweezers

Scanning Confocal Fluorescence Microscope (SCFM)

• Bennink, ML, SH Leuba, GH Leno, J Zlatanova, BG de Grooth & J Greve (2001) Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers. Nature Struct. Biol. 8, 606-610.
• Karymov, MA, M Tomschik, SH Leuba, P Caiafa & J Zlatanova (2001) DNA Methylation-dependent chromatin fiber compaction in vivo and in vitro: Requirement for linker histone. FASEB J. 15, 2631-2641.
• Leuba, SH, MA Karymov, M Tomschik, R Ramjit, P Smith & J Zlatanova (2003) Assembly of single chromatin fibers depends on the tension in the DNA molecule: magnetic tweezers study. Proc. Natl. Acad. Sci. USA 100, 495-500.
• Tomschik, M, MA Karymov, J Zlatanova & SH Leuba (2001) The archaeal histone-fold protein HMf organizes DNA into bona fide chromatin fibers. Struct. Fold. Des. 9, 1201-1211.
• Tomschik, M, H Zheng, K van Holde, J Zlatanova & SH Leuba (2005) Fast, long-range, reversible conformational fluctuations in nucleosomes revealed by spFRET. Proc. Natl. Acad. Sci. USA 102, 3278-3283.
• Zlatanova, J & SH Leuba, Eds. (2004) Chromatin Structure and Dynamics: State-of-the-Art. New Comprehensive Biochemistry Vol. 39, Elsevier, Amsterdam, 507 pg. ISBN: 0-444-515941.