Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer

Abstract

Chromatin remodelers are sophisticated nano-machines that are able to alter histone-DNA interactions and to mobilize nucleosomes. Neither the mechanism of their action nor the conformation of the remodeled nucleosomes are, however, yet well understood. We have studied the mechanism of Remodels Structure of Chromatin (RSC)-nucleosome mobilization by using high-resolution microscopy and biochemical techniques. Atomic force microscopy and electron cryomicroscopy (EC-M) analyses show that two types of products are generated during the RSC remodeling: (i) stable non-mobilized particles, termed remosomes that contain about 180 bp of DNA associated with the histone octamer and, (ii) mobilized particles located at the end of DNA. EC-M reveals that individual remosomes exhibit a distinct, variable, highly-irregular DNA trajectory. The use of the unique "one pot assays" for studying the accessibility of nucleosomal DNA towards restriction enzymes, DNase I footprinting and ExoIII mapping demonstrate that the histone-DNA interactions within the remosomes are strongly perturbed, particularly in the vicinity of the nucleosome dyad. The data suggest a two-step mechanism of RSC-nucleosome remodeling consisting of an initial formation of a remosome followed by mobilization. In agreement with this model, we show experimentally that the remosomes are intermediate products generated during the first step of the remodeling reaction that are further efficiently mobilized by RSC.

Fig. 1.

AFM topography images of centrally positioned nucleosomes reconstituted on 255 bp 601 positioning sequence and incubated for 30 min with ATP in the absence of RSC (1st Row) or in the presence of RSC (2nd, 3rd, and 4th Rows).

Fig. 2.

The initial step of the RSC-nucleosome mobilization reaction is the generation of remosomes. (A) Schematics of the experiment. (B) Two-dimensional histogram L_c/ΔL representing the DNA complexed length L_c along with the nucleosome position ΔL (number of nucleosomes analyzed N = 1254 nucleosomes) for nucleosomes incubated in absence of RSC (Control) under the conditions described in (A) and gel eluted [Fraction I, see (A)]. (C) and (D), two-dimensional histograms for the upper gel band eluted nucleosomes incubated with 30 fmol [Fraction II, see (A] (N = 635) and 60 fmol [Fraction III, see (A)], (N = 255 nucleosomes) with RSC. (E) two-dimensional histogram for the nucleosomes eluted from the excised lower gel band after incubation for 30 mins with RSC (N = 538 nucleosomes). The inserts show the distinct nucleosome species corresponding to the different regions of the two-dimensional histograms. The color indicates the probability to find a nucleosome with the DNA complexed length L_c and the position ΔL. Blue corresponds to a low probability and Red to a high probability.

Fig. 3.

EC-M visualization of remosomes. (A) Centrally positioned nucleosomes reconstituted on a 255 bp 601 DNA were treated with RSC, immediately vitrified, and visualized by EC-M. Each micrograph is accompanied by schematic drawing illustrating the shape of the DNA observed in the micrographs; bar, 25 nm. (B) Same as (A), but for nucleosomes reconstituted with a 255 bp DNA fragment containing the 5S somatic gene of Xenopus borealis; bar, 25 nm. (C) Same as (A), but for trinucleosomes reconstituted on a DNA fragment consisting of three 601 repeats; bar, 25 nm. The First Row illustrates the structure of a trinucleosome unaffected by RSC. The Second and the Third Rows show a typical structure of trinucleosome, containing a remosome (the Black Arrowhead indicates the centrally located remosome within the trinucleosome). Note the altered structure of the remosome compared to the end-positioned nucleosome in the trinucleosome. The Fourth Row shows a trinucleosome in which the centrally positioned nucleosome has been mobilized.

Fig. 4.

In-gel and in-solution-one-pot restriction-accessibility assay of the RSC generated remosomes. (A) Schematics of the in-gel -one-pot assay. (B) HaeIII DNA digestion pattern of the non-slid nucleosomes incubated with RSC in the absence (Left ) or presence (right ) of ATP. The excised gel slices, containing the Control (incubated in the absence of ATP) or the non-mobilized (but treated with RSC in the presence of ATP) nucleosomes were incubated with the indicated units of HaeIII, DNA was then isolated and run on denaturing PAGE. Lane 11, HaeIII-digested naked DNA. The # indicates a fragment that corresponds to a HaeIII site present only in “dyad 7” 601.2 fragment and located at 4 bp from the dyad 7 (d₇) site (C) Quantification of the data presented in (B). (D) HaeIII accessibility of gel isolated remosomes in solution. An equimolar mixture of 11 centrally positioned nucleosomes (d₀ to d₁₀) were treated with RSC as in (B) and then run on a 5% PAGE. The bands corresponding to the Control (incubated with RSC in the absence of ATP) and the remosome fractions were excised from the gel and eluted. They were then digested with HaeIII (2 U/μl) in solution and the cleaved DNA was run on an 8% denaturing PAGE. Quantification was carried out as in (C).

Fig. 5.

The remosomes are intermediate structures generated during the RSC mobilization reaction. (A) Schematic presentation of the experiment. (B) 8% sequencing PAGE of the isolated DNA from the RSC remodeled and DNase I digested particles shown in (A). At the bottom of the gel, the numbers of the different fractions presented in (A) are indicated. At the top of the gel, the times of incubation with RSC are indicated. The last Two Lanes (48 and 64 min) show the DNase I digestion pattern of the gel purified mobilized particles (A). DNA, DNase I digestion profile of free 601 DNA. The position of the nucleosome is shown schematically on the Right; the Arrow shows the location of the nucleosome dyad. The bands, which change in intensity, are indicated by Asterisks and were used for calculation of the fraction of intact nucleosomes remaining in each remodeling reaction. (C) Normalized fractions of intact nucleosomes, remosomes, and slid nucleosomes (relative yields) determined from A and B versus the reaction time.

Fig. 6.

The remosomes are bona fide substrates for RSC. (A) Description of the remosome mobilization experiment. (B) Mobilization of the gel eluted remosome fractions R and R+, the control nucleosomes (N) and the slid-end-positioned nucleosomes (S). Note that both remosome fractions, R and R+, in contrast to the end-positioned nucleosomes (S), are mobilized by RSC. (C) Schematic representation of the two steps, RSC-induced, nucleosome mobilization.