1. Dynamics of nucleosomal DNA

2. Felsenfeld, G. & Groudine, M. (2003), Nature 421: 448-453

3. GCN4; Ellenberger et al., Cell 71: 1223, 1992
NF-KB; Chen et al., Nature 391: 410, 1998
Zif268; Pavletich & Pabo, Science 252: 809, 1991
Bam H1; Newman et al., Science 269: 656, 1995

4. A model for the spontaneous accessibility
of nucleosomal DNA target sites R +
k12
k21
k23
k32
Polach & Widom, 1995

5. Kd,nucleosome = Kd,naked DNA / Keqconf R +
k12
k21
k23
k32
Kd,nucleosome = Kd,naked DNA / Keqconf
Polach & Widom, 1995
Any protein can bind to DNA in a nucleosome,
and we can predict its affinity

6. Cooperative invasion of a nucleosome by
DG01
DG02
DG03 N
N-X
N-Y
N-XY X Y
dGXY = - DG01
Cooperative invasion of a nucleosome by
collaborative competition with histone octamer
Polach & Widom, 1996
Miller & Widom, 2003

7. Uncoiling vs sliding for a mononucleosome
Energetic costs are similar
Anderson et al., 2002

8. Test of uncoiling vs sliding for a mononucleosome
Extra DNA favors mobility but does not affect uncoiling
Anderson et al., 2002

9. Site exposure occurs without nucleosome sliding
Relative Keq
Location in nucleosome
Left
end
Right
middle 1
10–1
10–2
10–3
Anderson et al., 2002

10. Site exposure via stepwise unwrapping from one end of the nucleosomal DNA
Luger & Richmond, 1997

11. FRET systems for analysis of nucleosome dynamics
Li & Widom, 2004

12. Nucleosome conformational fluctuations in physiological ionic strength solution
Artificially stable wrapping in sub-physiological ionic strength
Completely dissociated at > 1.5 M NaCl
Titrate between these to measure Keq in physiological ionic strength
Wavelength (nm)
Intensity
Acceptor on
H2A K119C
Li & Widom, 2004

13. FRET efficiency vs monovalent cation concentration
Monovalent cation concentration (M) 1
0.5
1.5 2
E/E0
H2A-K119C
H3-V35C
FRET efficiency vs monovalent cation concentration
Li & Widom, 2004

14. Equilibrium constant for DNA unwrapping
Keq
Monovalent cation concentration (M)
0.001
0.01
0.1 1 10
100
H2A-K119C
H3-V35C
Equilibrium constant for DNA unwrapping
vs monovalent cation concentration
Li & Widom, 2004

15. Site exposure at internal nucleosome target sites: Cy3-labeled DNA constructs
601–147 LexA L Cy3 35
601–147 LexA L Cy3 57
601–147 Cy3 69
Cy3 fluorescence donor
Hannah Tims

16. Site exposure at internal nucleosome target sites: Cy5-labeled histone octamers and Cy3-labeled DNA
Cy5 H3V35C 1 69
Cy5 H2AK119C
Cy5 H4S47C 57
Cy5 H2BT112C
Cy5 H4L22C
Cy5 H2AA45C 35
Hannah Tims

17. Reduced equilibrium constant for site exposure near nucleosome dyad
V35C Dyad
Hannah Tims

18. R +
k12
k21
k23
k32
Polach & Widom, 1995

19. DNA constructs for LexA protein binding :
LexA147-left:
LexA147-right:
Li & Widom, 2004

20. No change in FRET when LexA binds to DNA end opposite fluorescence donor
Wavelength (nm)
Intensity
Li & Widom, 2004

21. DNA unwrapping detected by FRET when LexA binds near fluorescence donor
Wavelength (nm)
Intensity
Li & Widom, 2004

22. Nucleosome conformational change driven by LexA binding near fluorescence donor
[LexA] (nM)
0.2
0.4
0.6
0.8 1
100
101
102
103
104
105
FRET Efficiency
Li & Widom, 2004

23. Site exposure at internal nucleosome target sites
Cy3 601–147
Cy3 601–147 LexA L
Cy3 601–147 LexA 17
Cy3 601–147 LexA 27
Cy3 fluorescence donor
LexA binding site
Hannah Tims

24. Protein binding to internal DNA target sites
LexA 7
LexA 17
LexA27
Cy5 acceptor
Cy3 donor
Hannah Tims

25. Sites further inside the nucleosome are less accessible (more costly) for protein binding
Hannah Tims

26. Two assays for the rates of site exposure and
re-wrapping in nucleosomes
Stopped-flow FRET
k12
k21
k23
k32 +
LexA
FRET-FCS
k12
k21
Li, Levitus, Bustamante, & Widom, 2005

27. Stopped-flow analysis of LexA binding
to buried nucleosomal target site
Fluorescence (a.u.)
Time (s) A B
0.7
0.8
0.9
1.0
0.5
1.5
2.0
-0.1
0.1
Residuals
Mock reaction (no LexA)
200 nM LexA
Li, Levitus, Bustamante, & Widom, 2005

28. Nucleosome dynamics analyzed by fluorescence correlation spectroscopy
acceptor
donor
Li, Levitus, Bustamante, & Widom, 2005

29. FCS analysis of nucleosomes labeled with donor-only
Diffusion-only model
G (t)
Li et al., 2005

30. Nucleosome dynamics analyzed by fluorescence correlation spectroscopy
lag time (ms) -5 5 10 15
0.001
0.01
0.1 1
100
1000
Donor Autocorrelation Function
0.0
0.2
0.4
0.3
0.5 A B
GDA () / GD ()
Donor-only
Donor-acceptor
(D–A / D) Ratio function
Li, Levitus, Bustamante, & Widom, 2005

31. Rapid spontaneous site exposure in nucleosomes
~4 sec–1
~20–90 sec–1
> 5x108 M–1 sec–1 +
(slow)
Rapid spontaneous site exposure in nucleosomes
Explains how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale
Suggests that the major impediment to polymerase elongation is re-wrapping of the nucleosomal DNA
Sets tight limits to kinetic efficiency in regulatory protein binding
Li, Levitus, Bustamante, & Widom, 2005

32. Site exposure in long chains of nucleosomes
k12
k21
k23
k32 + k4
Single Nucleosomes + k4
k23
k32
k12*
k21*
Nucleosome Array
Purpose: Describe the experimental approach of how we determine DNA accessibility within nucleosome arrays.
Michael Poirier

33. Site exposure in long chains of nucleosomes
Heptadecamer
mp2
mp1
Dimer
BamH1
NsiI
SacI
RsrII
PmlI
DraI
HaeIII
PstI
HindII
StyI
Monomer
Purpose: Show the DNA template used to reconstitute mono-, di- and heptadecanucleosomes.
601 to mp1, 8 bp changes, bp 11: C->T, bp12: C->T, bp 21: G->A, bp 50: ->C, bp 70: ->A, bp 72: ->G, bp 100: ->A, bp 112: ->C
601.2 to mp2, 2 base changes, bp16: C->G, bp92: C->A
Michael Poirier

34. Cation-dependent folding of 17-mers analyzed by AFM
Extended
(low [NaCl]) c d
Compact
(+ Mg2)
1 
250 nm
Poirier, Bussiek, Langowski, & Widom

35. Nucleosomal site accessibility in a chromatin fiber
mp2
mp1
Heptadecamer
Dimer
BamH1
PmlI
DraI
HaeIII
PstI
HindII
StyI
Basepair (in nucleosome)
Nucleosomal site accessibility in a chromatin fiber
Michael Poirier
Purpose: Summarize the restriction enzyme kinetic method results for Kequ of heptadecanucleosomes and dinucleosomes relative to mononucleosomes.

36. Site accessibility in linker DNA
mp2
mp1
17mer
Dimer
Basepair (in linker)
NsiI
SacI
RsrII
Keq
Michael Poirier
Site accessibility in linker DNA
Purpose: Summarize the restriction enzyme kinetic method results for Kequ of heptadecanucleosomes and dinucleosomes in the linker region.

37. Site exposure equilibrium constants depend on
the affinity of histone-DNA interactions R +
k12
k21
k23
k32
Anderson & Widom, 2000
Widom, 2001

38. Site exposure equilibrium constants depend on
the affinity of histone-DNA interactions
5S rRNA gene DNA
SELEX Non-natural
high affinity DNA
Anderson & Widom, 2000
Widom, 2001

39. Site exposure equilibrium constants depend on
the affinity of histone-DNA interactions R +
k12
k21
k23
k32
Therefore, the equilibrium locations of nucleosomes along DNA depend on the local affinities of histone-DNA interactions
Anderson & Widom, 2000
Widom, 2001
Segal et al., 2006

40. Felsenfeld, G. & Groudine, M. (2003), Nature 421: 448-453

41. Acknowledgements Nucleosome dynamics Peggy Lowary Kevin Polach
Jeff Anderson
Gu Li
Marcia Levitus (Berkeley, Arizona State)
Carlos Bustamante (Berkeley)
Michael Poirier
Karissa Fortney
Hannah Tims
Georgette Moyle