Spontaneous access to DNA target sites in folded chromatin fibers - PubMed (original) (raw)

Spontaneous access to DNA target sites in folded chromatin fibers

Michael G Poirier et al. J Mol Biol. 2008.

Abstract

DNA wrapped in nucleosomes is sterically occluded from many protein complexes that must act on it; how such complexes gain access to nucleosomal DNA is not known. In vitro studies on isolated nucleosomes show that they undergo spontaneous partial unwrapping conformational transitions, which make the wrapped nucleosomal DNA transiently accessible. Thus, site exposure might provide a general mechanism allowing access of protein complexes to nucleosomal DNA. However, existing quantitative analyses of site exposure focused on single nucleosomes, while the presence of neighbor nucleosomes and concomitant chromatin folding might significantly influence site exposure. In this work, we carried out quantitative studies on the accessibility of nucleosomal DNA in homogeneous nucleosome arrays. Two striking findings emerged. Organization into chromatin fibers changes the accessibility of nucleosomal DNA only modestly, from approximately 3-fold decreases to approximately 8-fold increases in accessibility. This means that nucleosome arrays are intrinsically dynamic and accessible even when they are visibly condensed. In contrast, chromatin folding decreases the accessibility of linker DNA by as much as approximately 50-fold. Thus, nucleosome positioning dramatically influences the accessibility of target sites located inside nucleosomes, while chromatin folding dramatically regulates access to target sites in linker DNA.

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Figures

Figure 1

Restriction enzyme digestion assay for site exposure. k12 and k21 are the forward and reverse rates of site exposure of a unique DNA site in a single nucleosome; k12* and k21* are the corresponding rates for exposure of a unique site in a nucleosome array; k23 and k32 are the rates for restriction enzyme binding and unbinding; and k4 is the rate of restriction enzyme catalysis. The restriction enzyme kinetics method determines the equilibrium constant for site exposure, Kequ = k12 / k21 or = k12* / k21*.

Figure 2

DNA templates used. The nucleosome positioning sequence mp1 is a variant of positioning sequence 601, while mp2 is a variant of 601.2. mp2 contains many restriction enzyme recognition sites that are not present in mp1, allowing analysis of accessibility at unique sites within the dimer and 17-mer nucleosome arrays.

Figure 3

Reconstitution, purification, and characterization of mono- and di-nucleosomes. (a) Native 5% polyacrylamide gel of dinucleosome template DNA reconstituted with increasing concentrations of histone octamer. The shifts in DNA mobility are due to the formation of one and then two nucleosomes on each DNA. Numbers at the top (0–0.8) indicate the mass ratio (w/w) of histone octamer to total DNA used in the reconstitution reaction in that lane. Total DNA includes both the specific (high affinity) dinucleosome template DNA plus low affinity cpDNA competitor present as a histone buffer (see Methods). The specific template DNA saturates with histone octamer even though the amount of octamer is substoichiometric for the total amount of DNA. (b) Mononucleosome (lanes 1, 2) and dinucleosome (lanes 3, 4) reconstitutions before (lanes 1, 3) and after (lanes 2, 4) purification on sucrose gradients. Lane 5, purified dinucleosomes after digestion with 20 units ml−1 Sac I for 1 hour. Sac I cuts in the linker region, resulting in two bands having gel mobilities similar to that of mononucleosomes (lane 2), and two faint bands from digested naked template DNA (e.g., any template DNA that was not incorporated into nucleosomes). Quantification of these bands shows that greater than 95% of the dinucleosome’s positioning sequences are wrapped into nucleosomes.

Figure 4

Reconstitution, purification, and characterization of nucleosome 17-mers. (a) Native gel analysis of 17-mer template DNA reconstituted with increasing concentrations of histone octamer. The mass ratio (w/w) of histone octamer to total DNA used in the reconstitution reaction in each lane is indicated. The decrease in mobility is due to nucleosome formation along the DNA template. The mass ratios 0.1 and 0.2 have wide range of gel mobilities owing to the inherent heterogeneity of DNA templates that are partially filled with nucleosomes. In contrast, the mass ratios of 0.4 and 0.8 yield a well-defined shift in gel mobility, suggesting that the DNA template is saturated with nucleosomes. (b) Ethidium-stained native gel analysis of the 17-mer DNA template (lane 1), and reconstituted nucleosome array (0.8 histone to DNA mass ratio) before (lane 2) and after purification (lane 3). The sucrose gradient removes the short low affinity competitor DNA and any aggregates. (c) Native 5% polyacrylamide gel analysis of DNA products from a Mlu I digestion of purified 17-mer nucleosomes mixed with naked DNA. The 17-mer contains 16 Mlu I sites and the naked DNA contains a single MluI site. (d) Quantification of results from (c) showing the fraction of naked DNA and the nucleosome 17-mer remaining uncut, as a function of time. The curves show best fits to the appropriate exponential decays (see Methods). These results show that 0.03 of the positioning sequences are not incorporated into nucleosomes, i.e., that 97 % of all positioning sequences in these nucleosome 17-mers are wrapped into nucleosomes.

Figure 4

Figure 5

Atomic Force Microscopy images of nucleosome 17-mers. (a, b) purified nucleosome 17-mers adsorbed onto mica in 0.2 × TE (which causes them to adopt extended conformations) and imaged in air; (b) 4× zoom of the upper array in (a). These and additional images confirm the biochemical results showing that the 17-mers are greater than 95% saturated with nucleosomes. (c, d) 17-mers adsorbed in 0.5× TE plus 1 mM MgCl2 (which allows them to compact) and imaged in that buffer. (d) is a 4× zoom of the lower array in (c). As expected, the arrays fold into more compact structures in the presence of MgCl2.

Figure 6

Site exposure in dinucleosomes and nucleosome 17-mers measured relative to mononucleosomes. (a) Native gel analysis of DNA products from Hae III digestions of a mixture of purified dinucleosomes and mononucleosomes; times of digestion (minutes) are indicated. (b) Quantification of the fraction of uncut DNA from (a), fit with an exponential decay (see Methods). (c, d) As in (a, b) except purified nucleosome 17-mers are used in place of dinucleosomes. (e) Summary of site accessibilities in dinucleosomes and nucleosome 17-mers, measured relative to accessibilities in mononucleosomes, for sites spanning the test nucleosome. Averages and standard deviations (n=2 or 3) are shown.

Figure 6

Figure 7

Site exposure in linker DNA within dinucleosomes and nucleosome 17-mers measured relative to naked DNA. (a) Native gel analysis of DNA products from Sac I digestions of a mixture of purified dinucleosomes and naked DNA; times of digestion (minutes) are indicated. (b) Quantification of the fraction of uncut DNA from (a), fit with an exponential decay. (c, d) As in (a, b) except purified nucleosome 17-mers are used in place of dinucleosomes. (e) Summary of site accessibilities in dinucleosome and nucleosome 17-mer linker DNA, measured relative to accessibilities in naked DNA, for sites spanning one linker DNA region. Averages and standard deviations (n=2–5) are shown.

Figure 7

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Spontaneous access to DNA target sites in folded chromatin fibers - PubMed (original) (raw)