North JA et al. (2012), Regulation of the nucleosome unwrapping rate co...

XB-ART-46609

Nucleic Acids Res 2012 Nov 01;4020:10215-27. doi: 10.1093/nar/gks747.

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Regulation of the nucleosome unwrapping rate controls DNA accessibility.

North JA , Shimko JC , Javaid S , Mooney AM , Shoffner MA , Rose SD , Bundschuh R , Fishel R , Ottesen JJ , Poirier MG .

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Eukaryotic genomes are repetitively wrapped into nucleosomes that then regulate access of transcription and DNA repair complexes to DNA. The mechanisms that regulate extrinsic protein interactions within nucleosomes are unresolved. We demonstrate that modulation of the nucleosome unwrapping rate regulates protein binding within nucleosomes. Histone H3 acetyl-lysine 56 [H3(K56ac)] and DNA sequence within the nucleosome entry-exit region additively influence nucleosomal DNA accessibility by increasing the unwrapping rate without impacting rewrapping. These combined epigenetic and genetic factors influence transcription factor (TF) occupancy within the nucleosome by at least one order of magnitude and enhance nucleosome disassembly by the DNA mismatch repair complex, hMSH2-hMSH6. Our results combined with the observation that ∼30% of Saccharomyces cerevisiae TF-binding sites reside in the nucleosome entry-exit region suggest that modulation of nucleosome unwrapping is a mechanism for regulating transcription and DNA repair.

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Species referenced: Xenopus laevis
Genes referenced: h2ac21 nps nucb1

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	Figure 1. H3(K56ac) increases the rate of nucleosome unwrapping. (A) DNA constructs for FRET measurements of nucleosome unwrapping kinetics. Both higher affinity 601 NPS or lower affinity X. borealis contain a LexA protein binding site from bases 8–27 and a Cy3 molecule on the 5′-end. (B) Structure of FRET-labeled nucleosome (41) containing 601L or 5SL DNAs; the LexA-binding site in red, Cy3 in green, Cy5 on H2A(K119C) in magenta and H3(K56Ac) in orange. (C) A three-state model for LexA binding to its target site within a nucleosome. (D and E) Stopped Flow Cy5 emission versus time of 601L nucleosomes containing unmodified H3 or H3(K56Ac) nucleosomes, respectively, at 1 mM NaCl mixed with 0–900 nM LexA. (F) Normalized stopped flow Cy5 emission versus time at 900 nM LexA for nucleosomes containing unmodified H3 (blue circles), H3(K56Q) (red circles) and H3(K56ac) (orange circles) at 1 mM NaCl. (G) Relative unwrapping and calculated rewrapping rates of nucleosomes containing H3(K56Q) and H3(K56ac) versus unmodified at 1 mM NaCl. (H) Normalized stopped flow Cy5 emission versus time at 30 μM LexA for nucleosomes containing unmodified H3 (blue circles), H3(K56Q) (red circles) and H3(K56ac) (orange circles) at 130 mM NaCl. (I) Relative unwrapping and calculated rewrapping rates of nucleosomes containing H3(K56Q) and H3(K56ac) versus unmodified H3 at 130 mM NaCl (see also Supplementary Figure S1).
	Figure 2. DNA sequence does not influence H3(K56ac) enhancement of TF binding. (A) Cy3 fluorescence image of native PAGE analysis of purified FRET-labeled nucleosomes containing the 5SL NPS and unmodified H3 (lane 2), H3(K56Q) (lane 3) or H3(K56ac) (lane 4). (B and C) Fluoresence emission spectra of 5SL FRET-labeled nucleosomes containing unmodified H3 or H3(K56ac), respectively, excited at 510 nm (donor excitation) in the presence of 0.5× TE, 1 mM NaCl and LexA at 0 nM (black), 30 nM (red) or 1000 nM (blue), (D) Steady-state FRET efficiency, as determined by the (ratio)A method (36), versus LexA concentration for nucleosomes containing unmodified H3 (blue), H3(C110) (purple), H3(K56Q) (red), and H3(K56a) (orange) at 1 mM NaCl. Plots are the average of three LexA titrations and the error bars were determined from the SD of the three measurements. The data were fit to a non-cooperative binding curve, which determines S0.5-nuc, the LexA concentration at which 50% of the nucleosomes are bound by LexA. (E) Relative change in Keq for nucleosomes with the 601L (14) or 5SL NPS containing H3(K56Q) or H3(K56ac) versus unmodified H3 at 1 mM NaCl. This is inversely related to the relative change in S0.5.
	Figure 3. DNA sequence between the nucleosome entry–exit and the TF-binding site impacts nucleosome unwrapping. (A) DNA constructs with the 601 sequence (blue) iteratively replaced by the 5S sequence (lavender), where the parenthetical numbers represents the bases in 601 replaced by 5S; lexA site in red. (B) Steady-state FRET efficiency, as determined by the (ratio)A method, versus lexA concentration for nucleosomes containing 601L and unmodified H3 (blue), 601L and H3(K56Q) (red), 5SL(1–7) and unmodified H3 (green), and 5SL(1–7) and H3(K56Q) (olive) at 75 mM NaCl. Plots are the average of three LexA titrations and the error bars were determined from the SD of the three measurements. The data were fit to a non-cooperative binding curve. (C) Relative Keq for the indicated nucleosome versus nucleosomes containing 601L and unmodified H3 at 75 mM NaCl. (D) Normalized stopped flow Cy5 emission versus time at 15 μM LexA for nucleosomes containing 601L and unmodified H3 (blue), 601L and H3(K56Q) (red), 5SL(1–7) and unmodified H3 (green), and 5SL(1–7) and H3(K56Q) (olive) at 75 mM NaCl. (E and F) Relative unwrapping and calculated rewrapping rates, respectively, for the indicated nucleosomes versus nucleosome containing 601L and unmodified H3 at 75 mM NaCl (see also Supplementary Figure S4).
	Figure 4. Relation between DNA–histone binding and DNA unwrapping free energies. (A) Cy3 fluorescence image of native PAGE analysis of competitive reconstitutions performed in triplicate for unmodified HO with the DNA chimeras used for site exposure measures. (B) Change in free energy of nucleosome formation (ΔΔGnuc, light gray) relative to 601L as determined by competitive reconstitution from the gel in (A), and the change in nucleosome free energy for wrapping (ΔΔGwrap, dark gray) relative to 601L as determined from site accessibility measures (Figure 3B and C). The error bars for ΔΔGnuc are the SD of the three independent measurements for each nucleosome type. (C) Table of the change in free energy values in kcal/mol for nucleosome formation of each DNA species relative to Lumbriculus variegatus 5S NPS , for nucleosome formation of each DNA species relative to 601L DNA and for nucleosome wrapping for each DNA species relative to 601L DNA .
	Figure 5. TF-binding sites predominantly reside within nucleosomes. (A) Distribution of TF-binding site distances from the nearest nucleosome dyad (black circles), with distances of 0–36 bases considered within the dyad region, distances of 37–74 bases considered within the entry–exit region, and distance greater than 74 bases considered to be in naked DNA (see Experimental Procedures and Supplementary Figure S5 for details). The black line represents the expected TF-binding site distribution if they were distributed randomly throughout the genome. (B) Measured and expected percentages of TFs in each region calculated from the distribution in (A).
	Figure 6. H3(K56ac) facilitates nucleosome dissociation by the mismatch recognition complex hMSH2–hMSH6. (A) DNA constructs containing the X. borealis 5S NPS with (5S-GT) or without (5S-GC) a DNA mismatch and a 3′-biotin. (B and C) Electrophoretic mobility shift analysis of H3(C110) nucleosomes adjacent to a GT mismatch, and H3(K56ac) nucleosomes adjacent to a GT mismatch disassembled by hMSH2–hMSH6, respectively. Lane 1: sucrose gradient–purified nucleosomes, Lane 2: nucleosomes bound by streptavidin, Lane 3: nucleosomes bound by streptavidin and hMSH2–hMSH6, Lanes 4–9: kinetic analysis of streptavidin-bound nucleosome disassembly by hMSH2–hMSH6 in the presence of 1 mM ATP. (D) The fraction of unmodified 5S-GC NPS nucleosomes (black square), H3(C110) 5S-GT NPS nucleosomes (lavender diamond) and H3(K56ac) 5S-GT 5S-GT NPS nucleosomes (orange triangle) versus time in the presence of hMSH2–hMSH6 (250 nM) and ATP (1 mM). The error bars were determined from the SD of at least three separate experiments. The fraction of nucleosomes versus time were fit excluding the zero time point to a single exponential decay, A × e−t/τ (see also Supplementary Figure S6).
	Figure 7. Kinetic model of H3(K56Ac) and DNA sequence modulation of nucleosome unwrapping rate. (A and B) Nucleosomes containing TF-binding sites at two different loci within the genome (nucleosome 1 with entry–exit in blue, nucleosome 2 with entry–exit in magenta). The DNA sequence between the entry–exit and TF-binding site influences the inherent nucleosome unwrapping rate to regulate TF binding within each nucleosome. Acetylation/de-acetylation of H3 lysine 56 at nucleosome 1 enhances/suppresses the DNA unwrapping rate to adjust TF occupancy. This influences both the TF occupancy within nucleosome 1 but also TF occupancy relative to nucleosome 2.

References [+] :

Adams, Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative. 1995, Pubmed