Dynamics of nucleosomal DNA

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

R + k12 k21 k23 k32 Polach & Widom, 1995

DNA constructs for LexA protein binding 1 147 601-147: LexA147-left: 1 8 27 147 LexA147-right: 1 121 140 147 Li & Widom, 2004

No change in FRET when LexA binds to DNA end opposite fluorescence donor Wavelength (nm) Intensity 1 121 140 147 Li & Widom, 2004

DNA unwrapping detected by FRET when LexA binds near fluorescence donor Wavelength (nm) Intensity 1 8 27 147 Li & Widom, 2004

Nucleosome conformational change driven by LexA binding near fluorescence donor 1 8 27 147 [LexA] (nM) 0.2 0.4 0.6 0.8 1 100 101 102 103 104 105 FRET Efficiency Li & Widom, 2004

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

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

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

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

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

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

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

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

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

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

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

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

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.

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.

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

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

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

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

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