Molecular biologists have come to realize that chromatin structure and dynamics is of major importance in regulating transcription, replication, and repair. Our interests are to understand at the single nucleosome and single chromatin fiber level the interrelationship between biological processes using DNA as a template and the structure and dynamics of chromatin. 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. Single-molecule techniques provide the sensitivity to detect and to elucidate small, yet physiologically relevant, changes in chromatin structure and dynamics. For example, recently, we have been able to:
- detect conformational changes in fiber structure due to the presence of 24 methyl groups per nucleosome (AFM);
- investigate the role of histone variants in chromatin fiber structure (AFM);
- determine the piconewton forces to unravel individual nucleosomes in a fiber context (optical tweezers);
- follow in real time the assembly and disassembly of nucleosomes on a single DNA molecule (magnetic tweezers);
- demonstrate fast, long-range, reversible conformational fluctuations in nucleosomes between two states: fully folded (closed) with the DNA wrapped around the histone core, or open, with the DNA significantly unraveled from the histone octamer (spFRET).
- In collaboration with Saleem Khan (Microbiology and Molecular Genetics), we have used spFRET to demonstrate that PcrA DNA helicase displaces RecA from both ssDNA as well as dsDNA as a model for regulation of homologous recombination.
- In collaboration with Pedro Rodriguez-Collazo (Cell Biology and Physiology), we have developed a method to isolate in one-step histones containing their native post-translational modifications (Rodriguez-Collazo et al., 2009). This method has also been patented and licensed.
The kinds of experiments we perform and the expertise in our laboratory range from molecular biology/biochemistry to engineering and physics. We believe that our considerable knowledge and experience in the chromatin field, combined with our ever-increasing capabilities in using single-molecule approaches put us in a unique position to approach a number of unresolved structural issues and to seek the elusive link between chromatin structure and function.
- Rodriguez-Collazo P, Leuba SH, Zlatanova J. Robust methods for purification of histones from cultured mammalian cells with the preservation of their native modifications.Nucleic Acids Res. 2009 37:e81. Epub 2009 May 13.PMID:
- Anand, S. P., Zheng, H., Bianco, P., Leuba, S. H. and Khan, S. A. DNA helicase activity of PcrA is not required for the displacement of RecA from DNA and inhibition of RecA-mediated strand exchange. J. Bacteriology 189, 4502-4509, 2007.
- P. Rodriguez-Collazo, S. K. Snyder, R. C. Chiffer, J. Zlatanova, S. H. Leuba and C. L. Smith. cAMP Signaling Induces Rapid Loss of Histone H3 Phosphorylation in Mammary Adenocarcinoma-Derived Cell Lines. Experimental Cell Research. Epub ahead of print, 2007.
- Zheng, H., Goldner, L. S. and Leuba, S. H. Homebuilt scanning confocal fluorescence microscope for the study of DNA/protein interactions, one-at-a-time. Methods 41, 342-352, 2007.
- Zlatanova, J., William T. McAllister, W. T., Borukhov, S. and Leuba, S. H. Single-molecule approaches reveal the idiosyncrasies of RNA polymerases. Structure 14, 953-966, 2006.
- Tomschik, M., Zheng, H., van Holde, K., Zlatanova, J. and Leuba, S. H. (2005) Fast, long-range, reversible conformational fluctuations in nucleosomes revealed by spFRET. Proc. Natl. Acad. Sci. USA 102, 3278-3283.
- Zlatanova, J. & Leuba, S. H., Eds. (2004) Chromatin Structure & Dynamics: State-of-the-Art. Elsevier New Comprehensive Biochemistry, vol. 39, Amsterdam, 507 pg. ISBN: 0-444-515941.
- Leuba, S. H., Karymov, M. A., Tomschik, M., Ramjit, R., Smith, P. and Zlatanova, J. (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.
- An, W., Palhan, V. B., Karymov, M. A., Leuba, S. H. and Roeder, R. G. (2002) Selective requirements for histone H3 and H4 N termini in p300-dependent transcriptional activation from chromatin. Mol. Cell 9, 811-821.
- Leuba, S. H. and Zlatanova, J., Eds. (2001) Biology at the single-molecule level. Pergamon, Amsterdam. ISBN: 0-08-044031-2 (Book reviewed by Ishii, Y. and Yanagida, T. Toward single molecule biochemistry. Cell 109, 686-688, 2002).
- Bennink, M. L., Leuba, S. H., Leno, G. H., Zlatanova, J., de Grooth, B. G. and Greve J. (2001) Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers. Nat. Struct. Biol. 8, 606-610. Leuba, S. H. and Bustamante, C. (1999) Analysis of chromatin by scanning force microscopy. Methods Mol. Biol. 119, 143-160.
- Leuba, S. H., Bustamante, C., van Holde, K. and Zlatanova, J. (1998) Linker histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers. Biophys J. 74, 2830-2839.
- Zlatanova, J., Leuba, S. H. and van Holde, K. (1998) Chromatin fiber structure: morphology, molecular determinants, structural transitions. Biophys J. 74, 2554-2566.
- Leuba, S. H., Yang, G., Robert, C., Samori, B., van Holde, K., Zlatanova, J. and Bustamante, C. (1994) Three-dimensional structure of extended chromatin fibers as revealed by tapping-mode scanning force microscopy. Proc. Natl. Acad. Sci. USA 91, 11621-11625.
University of Pittsburgh
Department of Cell Biology
2.26a Hillman Cancer Center