Identification of a rapidly formed nonnucleosomal histone-DNA intermediate that is converted into chromatin by ACF

Abstract

Chromatin assembly involves the combined action of histone chaperones and ATP-dependent motor proteins. Here, we investigate the mechanism of nucleosome assembly with a purified chromatin assembly system containing the histone chaperone NAP1 and the ATP-dependent motor protein ACF. These studies revealed the rapid formation of a stable nonnucleosomal histone-DNA intermediate that is converted into canonical nucleosomes by ACF. The histone-DNA intermediate does not supercoil DNA like a canonical nucleosome, but has a nucleosome-like appearance by atomic force microscopy. This intermediate contains all four core histones, lacks NAP1, and is formed by the initial deposition of histones H3-H4. Conversion of the intermediate into histone H1-containing chromatin results in increased resistance to micrococcal nuclease digestion. These findings suggest that the histone-DNA intermediate corresponds to nascent nucleosome-like structures, such as those observed at DNA replication forks. Related complexes might be formed during other chromatin-directed processes such as transcription, DNA repair, and histone exchange.

Figure 1. Core histones associate with the DNA template prior to ACF-catalyzed nucleosome assembly

(A) Three models for the mechanism of chromatin assembly. In model A, core histone deposition and nucleosome formation occur in a single step, whereas in models B and C, the histones are initially deposited onto the DNA to give a non-nucleosomal intermediate that is subsequently converted into canonical (“mature”) chromatin by ACF. (B) Core histones stably associate with DNA prior to chromatin assembly by ACF. Template association experiments were performed with equivalent masses of two different circular DNA templates of approximately 3 kbp (3K) and 8kbp (8K). Core histones (amount sufficient for complete assembly of one DNA template) were initially incubated with Template 1 (either 3K or 8K). After 5 min, Template 2 (either 8K or 3K) was added to the mixture, which was then allowed to incubate for another 5 min (to allow potential exchange of histones from Template 1 to Template 2) prior to the addition of ACF. Purified topoisomerase I was included in the reactions, and the formation of nucleosomes was monitored by the DNA supercoiling assay. The 3K and 8K templates were resolved by agarose gel electrophoresis. For each reaction, the % supercoiling of Template 1 DNA [(supercoiled DNA/total DNA) × 100] is indicated. Due to the higher resolution of the different supercoiled species with the 3K plasmid than the 8K plasmid, the quantitation of % supercoiling is likely to be more accurate with the 3K plasmid than the 8K plasmid. As references, supercoiled and relaxed DNAs were also included. The positions of relaxed, supercoiled, and nicked DNA species are shown. The asterisk indicates a contaminant in the preparation of the 3K plasmid.

Figure 2. Dynamics of the formation of the non-nucleosomal histone-DNA intermediate and its conversion to canonical nucleosomes by ACF

(A) The non-nucleosomal histone-DNA complexes remain associated with the DNA template for at least 120 min. Template association experiments were performed as in Figure 1B, except that the time of incubation of the Template 1-histone complex with Template 2 (to allow potential exchange of histones from Template 1 to Template 2 prior to the addition of ACF) was varied as indicated. (B) The non-nucleosomal histone-DNA complexes are formed within 15 seconds. Template association experiments were performed as in Figure 1B, except that the time of addition of Template 2 to the Template 1 + NAP1-histone mixture was varied as specified. (C) The non-nucleosomal histone-DNA complexes can be converted into chromatin by ACF at a rate that is comparable to that of the overall ACF-catalyzed chromatin assembly reaction. Standard (one-template) chromatin assembly reactions were performed in parallel under conditions that compared the rate of overall chromatin assembly with the rate of conversion of the non-nucleosomal histone-DNA complexes into chromatin by ACF. Purified topoisomerase I was included in the reactions, and the formation of nucleosomes was monitored by the DNA supercoiling assay.

Figure 3. The non-nucleosomal histone-DNA intermediate does not contain NAP1

(A) NAP1 is not associated with the non-nucleosomal histone-DNA complexes. Non-nucleosomal histone-DNA complexes were formed by incubation of NAP1-histones and relaxed template DNA, and then incubated with Ni-NTA agarose beads (Qiagen) to which the His₆-tagged NAP1 can bind (lanes 4 and 5). The beads were pelleted, and the resulting supernatant (S) and pellet (P) fractions were analyzed by western blot with antibodies against NAP1 and histones H2A-H2B. In addition, the DNA content of the supernatant and pellet was analyzed by agarose gel electrophoresis and staining with ethidium bromide. As a reference, NAP1-histone complexes in the absence of DNA were analyzed in parallel (lanes 2 and 3). Samples of the input (I) NAP1, histones, and DNA were also included (lane 1). (B) The NAP1-depleted intermediate can be converted into chromatin by ACF at a rate that is comparable to that of the overall chromatin assembly reaction. Upon depletion of NAP1 in (A), the non-nucleosomal histone-DNA complexes in the supernatant (S) fraction were incubated with ACF for the indicated reaction times. Chromatin assembly was monitored by DNA supercoiling analysis in the presence of purified topoisomerase I. Assembly reactions were performed in parallel with NAP1-depleted samples (middle panel), NAP1-depleted samples that were subsequently supplemented with purified NAP1 (equivalent amount of NAP1 as depleted; lower panel), and mock-depleted samples (upper panel). The % supercoiling [(supercoiled DNA/total DNA) × 100] versus time for each series of reactions is shown in Figure S3. The positions of relaxed (Rel), supercoiled (SC), and nicked (N) DNA are indicated. (C) The NAP1-depleted histone-DNA complexes can be converted into periodic nucleosome arrays by ACF. NAP1-depleted histone-DNA complexes were incubated with ACF and ATP under standard chromatin assembly conditions. Samples that were obtained either before or after incubation with ACF were subjected to partial micrococcal nuclease digestion analysis. As a reference, mock-depleted histone-DNA complexes that were incubated with ACF were analyzed in parallel.

Figure 4. Analysis of chromatin assembled with histone H1

(A) Template association during chromatin assembly occurs in the presence of histone H1. Chromatin assembly reactions were performed as in Figure 1B, except that histone H1 was included at approximately one molecule of H1 per core histone octamer. The asterisk denotes a contaminant in the preparation of the 3K plasmid. (B) Partial micrococcal nuclease digestion analysis of chromatin at different stages of assembly. Naked DNA (DNA only), Core Histone-DNA Complexes (DNA, NAP1, core histones), Chromatin lacking H1 (DNA, NAP1, core histones, ACF, ATP), and Histone H1-containing Chromatin (DNA, NAP1, core histones, H1, ACF, ATP) were subjected to micrococcal nuclease digestion analysis. Each sample was treated with three different concentrations (increasing from left to right) of micrococcal nuclease. The three concentrations of micrococcal nuclease used with the different samples were identical. The DNA size markers are the 123 bp ladder (Invitrogen). The white dots denote positions of DNA fragments derived from mono-, di-, and trinucleosomes. (C) Extensive micrococcal nuclease digestion analysis of chromatin at different stages of assembly. Samples that are similar to those in (B) were extensively digested with micrococcal nuclease for 1 min. The relative concentrations of micrococcal nuclease are indicated. The DNA size markers are the 123 bp ladder. The positions of DNA fragments derived from core particles (“Core”) and chromatosomes (“Chr”) are denoted. The relative amounts of total mononucleosome species (core particles and chromatosomes) are indicated.

Figure 5. The deposition of histones H3-H4, but not H2A-H2B, is sufficient for template association

(A) Chromatin assembly reactions with H2A-H2B alone, H3-H4 alone, or all four core histones. Chromatin assembly reactions were performed, as outlined in the scheme at the bottom of the figure, in the absence or presence of ACF with equimolar amounts of the indicated histones. The reaction products were analyzed by using the DNA supercoiling assay. (B) Order-of-addition reactions reveal that H3-H4, but not H2A-H2B, is sufficient for template association. Template association reactions were carried out, as outlined in the scheme at the bottom of the figure, by modification of the basic procedure used in Figure 1B. Histones (H2A-H2B, H3-H4) were added either at 0 min (prior to the addition of Template 2) or at 2 min (after the addition of Template 2). For each reaction, the order of addition of H3-H4 and H2A-H2B is indicated. The % supercoiling (%SC) is given for both the 3K and 8K templates.

Figure 6. Visualization of the non-nucleosomal histone-DNA complexes by atomic force microscopy

(A) Template association experiments were performed, as depicted in the diagram, in the absence or presence of ACF. The resulting samples were immobilized on mica and then analyzed by atomic force microscopy. Representative images are shown. The sample field for each image is 1 μm × 1 μm, and the height scale is from 0 to 4 nm, as shown on the right. The scale bars represent 200 nm. (B) Magnified images of histone-DNA complexes (−ACF) and nucleosomes (+ACF). (C) Distribution of apparent areas of individual histone-DNA intermediates (−ACF; N = 338) and nucleosomes (+ACF; N = 558).

Figure 7. A new working model for the steps in chromatin assembly

In the assembly of chromatin, a non-nucleosomal histone-DNA intermediate is rapidly formed in a process that does not require ACF. Unlike a canonical nucleosome, this intermediate does not significantly supercoil DNA. The intermediate is also more sensitive to micrococcal nuclease digestion than a canonical nucleosome. The formation of the intermediate appears to involve the initial deposition of histones H3-H4 followed by H2A-H2B. ACF is able to convert the intermediate into a canonical nucleosome in an ATP-dependent process at a rate that is comparable to that of the overall chromatin assembly reaction. By atomic force microscopy, the non-nucleosomal intermediate resembles a nucleosome. Thus, the intermediate may correspond to “nascent” chromatin that is formed immediately behind a DNA replication fork. This nascent chromatin is then converted by ACF into “mature” chromatin containing histone H1.