Structural diversity of nucleosomes detected by

single pair FRET

KatalinTóthBiophysics of Macromolecules,

DKFZ Heidelberg

nucleosome: smallest unit of DNA compaction

H3

H3

H4H4

H2BH2B

H2A

H2A

(Olins & Olins 1974)

(Arents,...Moudrianakis 1991)

(Luger,...Richmond 1997)

(Bussiek, Toth et al 2006)

(Bussiek, Toth et al 2005)

How is the accessibility of nucleosomal DNA regulated?

-unwrapping of DNA? -release - rebinding of histones?-continuous or stepwise?

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

Fluorescence resonance energy transfer

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

Fluorescence in bulk vs. single molecule conditions

In-house built single molecule spectrometer (A.Gansen)

Setup for spFRET in solution

--DNA sequence dependence--histone modification--salt concentration, --low particle concentration

Nucleosome structural variations are induced by:

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)

Histone acetylation

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

effect.

Nucleosome disassembly

In collaboration with Claus Seidel, Düsseldorf

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low FRET / donor only

medium FRET

high FRET

Increasing salt concentration destabilizes nucleosomes

Multiparameter analysis shows three major species

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

The compact nucleosome (HF) is more stable

than the partially opened form (MF)

salt-induced dissociation

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

Addition of unlabeled nucleosomes stabilizes the compact form

HF to MF transition includes dissociation of histones (probably H2A/H2B dimer)

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Structural interpretation of the FRET substates

(Gansen et al 2009)

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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

Thanks:

Jörg LangowskiAlex GansenFlorian HaugerNathalie Schwarz

Biophys. of MacromoleculesDKFZ, HeidelbergText

and Claus Seidel`s group: Molecular Physical Chemistry,University H. Heine Düsseldorf