1. Structural diversity of nucleosomes detected by single pair FRET KatalinTóth Biophysics of Macromolecules, DKFZ Heidelberg

2. nucleosome: smallest unit of DNA compaction H3 H4 H2B H2A (Olins & Olins 1974) (Arents,...Moudrianakis 1991) (Luger,...Richmond 1997) (Bussiek, Toth et al 2006) (Bussiek, Toth et al 2005)

3. How is the accessibility of nucleosomal DNA regulated? -unwrapping of DNA? -release - rebinding of histones? -continuous or stepwise?

4. Mononucleosome reconstitution from - DNA sequence: strong positioning (Selex 601) - recombinant histone octamer Can we see structural variations related to functionally different states? method: distance measurement between fluorescent labels 170 base pairs (length: ca.60 nm) ∅ ca. 10 nm Alexa 594 Alexa 488

5. Fluorescence resonance energy transfer Ensemble methods measure quantities averaged over time and populations. For observing subpopulations or dynamics, single particle methods can be used.

6.  Fluorescence in bulk vs. single molecule conditions

7. In-house built single molecule spectrometer (A.Gansen) Setup for spFRET in solution

8. --DNA sequence dependence --histone modification --salt concentration, --low particle concentration Nucleosome structural variations are induced by:

9. DNA sequence variations Nucleosomes reconstituted on the 601 sequence (an in vitro selected good positioner) have a more homogeneous population than on the 5S rDNA sequence (one of the best natural positioners). The sequence effect is more pronounced in the linker region than in the core. (Gansen, et al 2007, 2009)

10. Histone acetylation Histone acetylation strongly influences the linker DNA arm geometry, but not the core. H3 acetylation has the largest effect.

11. Nucleosome disassembly

12. In collaboration with Claus Seidel, Düsseldorf

13. 13 low FRET / donor only medium FRET high FRET Increasing salt concentration destabilizes nucleosomes Multiparameter analysis shows three major species

14. 25 pM nucleosome, 25mM NaCl Identification of the subpopulations The size of the most compact population HF corresponds to the intact nucleosome. The MF species may be a partially opened form

15. The compact nucleosome (HF) is more stable than the partially opened form (MF) salt-induced dissociation

16. Population-filtered FCS analysis As dissociation proceeds, diffusion coefficient of LF approaches that of free DNA. At low salt, LF is probably small-molecule contamination FCS ‘signature’ of LF is different from free DNA (large triplet component). This suggests that proteins are still associated LF form is open DNA associated with histones

17. Addition of unlabeled nucleosomes stabilizes the compact form HF to MF transition includes dissociation of histones (probably H2A/H2B dimer)

18. 18 Structural interpretation of the FRET substates (Gansen et al 2009)

19. 19 Summary: --spFRET revealed changes in the nucleosome structure induced by histone acetylation and DNA sequence variations. --multiparameter analysis of single molecule fluorescence signals enabled us to distinguish steps in the nucleosome disassembly Outlook: --spFRET study of enzymatic remodelling of nucleosomes --spFRET study in living cells

20. Thanks: Jörg Langowski Alex Gansen Florian Hauger Nathalie Schwarz Biophys. of Macromolecules DKFZ, Heidelberg Text and Claus Seidel`s group: Molecular Physical Chemistry,University H. Heine Düsseldorf