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J Biol Chem, Vol. 273, Issue 29, 18028-18039, July 17, 1998


In Vitro Reconstitution of Artemia Satellite Chromatin*

Maria Carla MottaDagger , Nicoletta Landsberger, Claudia Merli, and Gianfranco Badaracco§

From the Dipartimento di Biologia Stutterale e Funzionale III Facoltá di Scienze, Universitá di Milano, 21100 Varese, Italy

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We report the characterization of an in vitro chromatin assembly system derived from Artemia embryos and its application to the study of AluI-113 satellite DNA organization in nucleosomes. The system efficiently reconstitutes chromatin templates by associating DNA, core histones, and H1. The polynucleosomal complexes show physiological spacing of repeat length 190 ± 5 base pairs, and the internucleosomal distances are modulated by energy-using activities that contribute to the dynamics of chromatin conformation. The assembly extract was used to reconstitute tandemly repeated AluI-113 sequences. The establishment of preferred histone octamer/satellite DNA interactions was observed. In vitro, AluI-113 elements dictated the same nucleosome translational localizations as found in vivo. Specific rotational constraints seem to be the central structural requirement for nucleosome association. Satellite dinucleosomes showed decreased translational mobility compared with mononucleosomes. This could be the consequence of interactions between rotationally positioned nucleosomes separated by linker DNA of uniform length. AluI-113 DNA led to weak cooperativity of nucleosome association in the proximal flanking regions, which decreased with distance. Moreover, the structural properties of satellite chromatin can spread, thus leading to a specific organization of adjacent nucleosomes.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Most higher eukaryotic DNA is folded into a dynamic nucleoprotein structure, which is subject to progressive and reversible modifications of its condensation state during transitions between interphasic and metaphasic chromatin. However, there are chromosomal regions that maintain cytological properties comparable with those of the metaphase chromosome throughout the cell cycle (1). Termed heterochromatin, these highly condensed regions consist of simple DNA sequences repeated in long tandem arrays and are typically localized around centromeres and telomeres (reviewed in Refs. 2 and 3). Heterochromatic regions are replicated late during S phase (4, 5); they do not participate in meiotic recombination and are generally associated with the transcriptionally repressed state (reviewed in Ref. 6). These regions can influence the expression of juxtaposed genes in a manner dependent on their distance from the point of juxtaposition, a phenomenon called "position effect variegation" (7-9). Position effect variegation is thought to take place either by compartmentalization within transcriptionally inactive nuclear regions or by virtue of the spread of the heterochromatic structure (reviewed in Refs. 10 and 11).

Heterochromatin is generally defined as highly organized chromatin structures stabilized by multiprotein complexes and is functionally correlated with diffusible transcription repressing properties (11). Genetic and molecular studies have shown that the process of heterochromatinization involves the spread of particular chromatin structures in the cases of pericentric insertions of euchromatic genes in Drosophila (12), centromeric insertion of the ura4 gene in Schizosaccharomyces pombe (13), and the silencing of the HML and HMR loci in S. cerevisiae (14). Analysis of the silencing processes in the yeast mating type loci indicates a fundamental role for histones H3 and H4 in the stabilization of the repressed state of yeast telomeric heterochromatin (15, 16). Nucleosome arrangement therefore appears to be an important structural element that allows specific silencing proteins to assemble packed, repressive chromatin structures.

The study of the role of heterochromatic DNA on chromatin structure may help to clarify how specific heterochromatic structures are maintained. To this end, we reconstituted and characterized the chromatin features of chromosomal regions that are cytologically distinguishable as Artemia franciscana (Crustacea Phyllopoda) heterochromatin. These regions are mainly composed of satellite DNA with a repeat unit length of 113 bp1 (AluI-113). AluI-113 DNA has already been characterized by electrophoretic analysis and electron microscopy (17). AluI-113, like other satellite DNAs (18-23), shows an intrinsic curvature of the longitudinal axis of the double helix, the structural basis of which is determined by adenine blocks positioned in phase with the pitch of the double helix. The structural properties of satellite DNAs are widely considered to be fundamental for the organization of highly condensed nucleoprotein complexes. Extensive evidence of in vivo nucleosome positioning along various satellite DNAs (reviewed in Ref. 24) has suggested a role for specific chromatin structures in heterochromatin condensation (25). However, in vitro studies of interactions between histones and satellite DNAs are few. The only examples of in vitro reconstitution aimed at the analysis of nucleosome positioning on satellite DNAs are studies of histone octamer assembly on 200-250-bp-long fragments by dialysis (26, 27).

In order to determine the structural properties of polynucleosomal complexes on multimeric AluI-113 fragments, we developed and characterized a cell-free assembly system from Artemia at the nauplius embryo stage. We have used this to analyze: (i) the organization of bent AluI-113 DNA into nucleosomes; (ii) the effects of interactions between consecutive physiologically spaced AluI-113 nucleosomes; and (iii) how AluI-113 sequences could affect the chromatin structure of non-satellite flanking regions.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Artemia Strains-- A. franciscana dry cysts were provided by the Laboratory of Mariculture (Artemia Reference Center) of the University of Ghent (Ghent, Belgium).

Extract Preparation-- Cysts were rehydrated in synthetic sea water and developed at 24 °C for 20 h (nauplius stage). Embryos were then rinsed extensively in distilled water and collected. All subsequent manipulations were carried out at 4 °C. Embryos (30 g) were resuspended in 50 ml of extraction buffer (50 mM Tris/HCl, pH 8.0, 30 mM NaCl, 250 mM sucrose, 5 mM beta -mercaptoethanol, 1% dimethyl sulfoxide, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml pepstatin) and homogenized using Ultra-Turrax T 25 (Ika Labortechnik) until the integrity of the cells was disrupted as judged by optical microscopy. Nuclei were pelleted by centrifugation for 5 min at 8000 rpm in a JA20 rotor (Beckman) and resuspended in 12 ml of extraction buffer. Nuclei were disrupted by increasing the NaCl concentration to 2 M. The resulting lysate (20 ml) was clarified by centrifugation for 2 h at 60,000 rpm in a 70.1 Ti rotor (Beckman). Aliquots of the supernatant were frozen and stored at -80 °C. Protein concentrations were monitored by the Bradford assay (Bio-Rad) and were usually between 10 and 20 mg/ml.

DNA Templates-- Plasmids YEp-24 (7769 bp) (Biolabs; GenBankTM accession number L09156), pUC18m (2686 bp) (28), pU-He6-, and pU-He6+ were used in this study. pU-He6+ contains a heterochromatic AluI-113 hexameric (678 bp) DNA fragment inserted in the SmaI site of pUC18m (17), and pU-He6- contains a 680-bp A/T-rich fragment not homologous to the heterochromatic DNA (17). YEp-24 was used as relaxed plasmid. The relaxation reaction was carried out by adding 100 units of Artemia topoisomerase I (29) per µg of supercoiled DNA in 25 mM Tris/HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA at 30 °C for 2 h. The relaxed DNA was phenol/chloroform-extracted and ethanol-precipitated.

Chromatin Assembly Reaction and Supercoiling Analysis-- In a standard assembly reaction (25 µl), 1 µg of plasmid DNA was incubated at 30 °C for 2 h with 1.5 µl of extract (extract protein concentration of 10 µg/µl in 2 M NaCl), resulting in a final NaCl concentration of 120 mM, in 25 mM Tris/HCl, pH 8.0, 2 mM MgCl2, 1 mM ATP, 50 ng/µl poly-L-glutamic acid (Fluka), 20 mM disodium creatine phosphate (Sigma), 1 µg/ml creatine phosphokinase (Sigma).

For supercoiling analysis, reactions were stopped by adding SDS and EDTA to final concentrations of 0.4% and 2.5 mM, respectively. Samples were deproteinized with 2 mg/ml proteinase K for 1 h at 37 °C. DNA was resolved on a 1% agarose gel in TAE buffer (30).

Core Histone Preparation from Chicken Erythrocytes and Nucleosome Reconstitution from Purified Components-- Core histones were prepared from adult chicken erythrocytes according to the procedure described by Simon and Felsenfeld (31). High molecular weight chromatin was sonicated and subsequently dialyzed against 0.6 M NaCl, 0.1 M potassium phosphate, pH 6.7, 0.25 mM phenylmethylsulfonyl fluoride and run on a hydroxyapatite column. After elution of histone H5 at 0.65 M NaCl, H2A and H2B were eluted with 0.93 M NaCl, and H3 and H4 were eluted with 2 M NaCl. Quantitation was estimated by measuring absorbance at 230 nm. The profile of the eluted histones was subsequently analyzed by SDS-polyacrylamide gel electrophoresis.

Relaxed YEp-24 plasmid (5 µg) and stoichiometric amounts of core histones (concentrations are indicated in the figure legends) were reconstituted by incubating at 30 °C for 4 h in 25 mM Tris/HCl, pH 8.0, 150 ng/µl poly-L-glutamic acid (Fluka), 2 mM MgCl2, 1 mM ATP, 20 mM disodium creatine phosphate (Sigma), and 1 µg/ml creatine phosphokinase (Sigma) in the presence of 10 units of Artemia purified topoisomerase I (29) per µg of plasmid DNA.

Plasmid Chromatin Purification and Histone Composition Analysis-- 12-ml linear 10-30% sucrose gradients in 25 mM Tris/HCl, pH 8.0, 160 mM NaCl, were made in Beckman SW40 centrifuge tubes. 600 µl of chromatin assembly reaction mixture or assembly reactions lacking DNA or extract were layered on top of the gradients and centrifuged at 38,000 rpm for 3 h at 4 °C. The gradients were fractionated, and aliquots from each fraction were proteinase K-treated and subjected to 1% agarose gel electrophoresis in TAE buffer to determine the position of the plasmid chromatin. Fractions from three gradients containing plasmid chromatin or corresponding fractions of the control gradients lacking DNA were pooled, trichloroacetic acid-precipitated, and electrophoresed in SDS 15% polyacrylamide gels. Protein gels were stained with Coomassie Blue.

Micrococcal Nuclease (MNase) Analysis-- MNase digestions were performed by adding 5 mM CaCl2 (final concentration) and MNase (1 unit/µg assembled DNA) (Sigma) to the reconstitution reactions. Reactions were incubated at 30 °C for the times indicated in the figure legends. Digestions were stopped by adding SDS and EDTA to final concentrations of 0.4% and 20 mM, respectively, and the mixtures were incubated for 1 h at 37 °C with 2 mg of proteinase K per ml. The DNA was phenol/chloroform-extracted and ethanol-precipitated. The digested DNA was resolved on a 1.6% agarose gel. Hybridization analyses were carried out by transfer to Hybond N membrane (Amersham Pharmacia Biotech) and probing with the end-labeled oligonucleotides specified in the figure legends.

MNase digestions of nuclei isolated from Artemia nauplii were performed as described above. Nuclei were resuspended in assembly buffer (25 mM Tris/HCl, pH 8.0, 120 mM NaCl, 2 mM MgCl2, 1 mM ATP, 50 ng/µl poly-L-glutamic acid, 20 mM disodium creatine phosphate, 1 µg/ml creatine phosphokinase), and the same assay was carried out as indicated for reconstituted chromatin except for RNase A treatment before electrophoretic resolution. End-labeled AluI-113 monomer or Artemia genomic DNA radioactively labeled by the random primer method (32) was used to probe Southern blots.

Restriction Nuclease Digestions of Nuclei-- Nuclei were resuspended in assembly buffer, and MgCl2 was added to a final concentration of 5 mM. The nuclei were then incubated at 37 °C in the presence of the restriction endonuclease AluI (0.001-0.6 units/µg of DNA) for 2 h. The reaction was stopped by adding EDTA and SDS to final concentrations of 20 mM and 0.4%, respectively. The DNA was phenol/chloroform-extracted and ethanol-precipitated. After RNase A treatment, the DNA was again phenol/chloroform-extracted, ethanol-precipitated, and resolved on a 1% agarose gel.

Nucleosome Border Analysis and High Resolution Mapping of Micrococcal Nuclease Cleavage Sites-- For nucleosome border analysis, 3 µg of pU-He6+ plasmid was reconstituted into chromatin. 5 mM CaCl2 (final concentration) and MNase (1 unit/µg of DNA) were added, and the reaction mixture was incubated at 30 °C for 10 or 20 min. The digestions were stopped, and the DNA was deproteinized with proteinase K and purified by phenol/chloroform extraction and ethanol precipitation as described previously. Digested DNA was resolved on a 1.6% agarose gel, and fragments of dinucleosomal and mononucleosomal sizes were then recovered (after 10 and 20 min of digestion, respectively) using silica gel membrane (Qiagen). After denaturation in formamide, the DNA was resolved on a denaturing (7 M urea) 6% polyacrylamide gel and then eluted in 50 mM Tris/HCl, pH 7.5, 0.5 mM EDTA. Approximately 40 fmol of DNA was used as a template for linear amplification in a mixture containing 1× Taq buffer, 100 µM deoxynucleoside triphosphates (each), 0.8 pmol of 32P-labeled primer (5'-CTACGTATGTTGGAAAAATG-3' or 5'-CTATTACCCTCGAAAACTAA-3') complementary to AluI-113, and 1 unit of Taq polymerase (Promega). Thermal cycling was performed at 95 °C for 30 s, 47 °C for 30 s, and 70 °C for 60 s. This process was repeated 30 times. The DNA samples were resolved on a denaturing (7 M urea) 6% polyacrylamide gel. DNAs from dinucleosomes and mononucleosomes obtained from MNase digestion of nuclei were identically processed.

For high resolution mapping of MNase cleavage sites, pUC18m and pU-He6+ plasmids (0.5 µg) were reconstituted and digested with MNase (0.8 unit/µg of DNA) for 2 and 4 min at 30 °C. Purified DNA (50 ng) was used as a template for linear amplification using the reaction conditions described above. The primer utilized for the extension was 5'-GAGTCGACCTGCAGGCATGCAAGC-3', complementary to pUC18m plasmid (424-401). Thermal cycling was done at 95 °C for 30 s, 50 °C for 30 s, and 70 °C for 60 s. This process was repeated three times.

DNase I Analyses-- To perform DNase I digestion of AluI-113 mononucleosomes, approximately 0.4 pmol of end-labeled AluI-113 dimer (226 bp) was reconstituted with 1 µg of assembly extract. 2 units of DNase I was added, and the reaction mixture was incubated for 60 s at 30 °C. The digestion was stopped by adding EDTA to a final concentration of 5 mM. Glycerol was then added to a final concentration of 3% (v/v), and the samples were resolved on a 0.7% agarose gel run in 0.5 × TBE buffer (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA). Mononucleosomes were recovered, and the DNA was deproteinized, phenol/chloroform-extracted, ethanol-precipitated, and analyzed by denaturing 6% polyacrylamide gel electrophoresis. Maxam and Gilbert sequencing reactions (reagent kit: Oligonucleotide Sequence Analysis, Merck) were used for sequence alignment.

To perform DNase I digestion of reconstituted AluI-113 hexamers cloned into pU-He6+, 250 ng of pU-He6+ plasmids was assembled. 10 units of DNase I was added, and the reaction mixture (12.5 µl) was incubated for 60 s at 30 °C. The digestion was stopped by adding SDS and EDTA to final concentrations of 0.4% and 5 mM, respectively. DNA was purified by proteinase K treatment, phenol/chloroform extraction, and ethanol precipitation. For linear amplification, P-labeled primers complementary to AluI-113 (5'-CTACGTATGTTGGAAAAATG-3' or 5'-CTATTACCCTCGAAAACTAA-3') were extended for 30 cycles with Taq polymerase using the reaction conditions previously described. The DNA thermal cycling used was as follows: 95 °C for 30 s, 47 °C for 30 s, 70 °C for 60 s.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

General Nucleosomal Organization of the Native AluI-113 Heterochromatic Structure-- Previous analyses of the chromatin structures of different satellite DNAs in vivo (33-36) have revealed the prevalence of organization into multiple, defined nucleosome frames. To investigate the chromatin organization of A. franciscana AluI-113 DNA in vivo, we performed MNase and restriction enzyme digestions on nuclei obtained by homogenization of embryos at the nauplius stage. In order to avoid disrupting the physiological DNA, histone, and non-histone protein interactions, extraction of chromatin was not undertaken, since any rearrangement of nucleosomes would complicate the interpretation of the results (24).

Satellite and bulk chromatin organization was examined by MNase digestion and hybridization with an AluI-specific probe (Fig. 1A, panel I) and labeled genomic DNA (panel II), respectively. No significant differences were revealed except for a slightly clearer banding pattern in the case of the satellite DNA, possibly indicative of more uniform nucleosome arrangements on the AluI-113 elements.


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  		Fig. 1.   General nucleosome organization of AluI-113 DNA in vivo. A, nuclei from nauplii embryos were isolated and incubated with MNase at 30 °C for 1 min using 0, 0.4, 0.8, and 1.6 units/µg of DNA (indicated by open triangles). DNA was purified, separated by size on a 1.6% agarose gel, blotted, and and hybridized to probes: AluI-113 monomer (panel I) or Artemia genomic DNA (panel II). The 123-bp ladder (Life Technologies, Inc.) is indicated as a marker. B, left, A. franciscana DNA digested with AluI at 0.001 (lane 1) and 0.06 units/µg of DNA (lane 2) for 1 h; right, nuclei (lanes 4-6) and control DNA (lanes 1-3) were incubated with AluI at 37 °C for 2 h using 0.001 (lanes 1 and 4), 0.3 (lanes 2 and 5) and 0.6 units/µg of DNA (lanes 3 and 6). Purified DNA samples were electrophoresed on a 1% agarose gel and stained with ethidium bromide.

The accessibility of the satellite chromatin to the restriction endonuclease AluI was also examined. Restriction endonucleases are frequently used to study nucleosomal arrays, because they generally cut in linker regions, since their activity is severely sterically obstructed by nucleosomes (37, 38). Naked DNA and nuclei were incubated with increasing amounts of AluI. Ethidium bromide staining showed less enzyme accessibility of satellite DNA folded into chromatin compared with naked DNA (Fig. 1B, right; compare lanes 2 and 5 and lanes 3 and 6). Monomeric AluI-113 DNA fragments were not detectable in the nuclei samples, while trimeric and pentameric AluI-113 fragments (339 and 565 bp, respectively) accumulated (lanes 5 and 6). This result was confirmed by blotting and hybridizing with an AluI-specific probe (data not shown). The 339- and 565-bp fragments, defined by AluI cuts, are likely to correspond to di- and trinucleosomes and may reflect a specific nucleosomal organization.

These data suggest that AluI-113 sequences fold into highly ordered polynucleosomal arrays.

Chromatin Assembly in Vitro-- In order to develop an in vitro chromatin reconstitution system capable of the assembly of physiological nucleosomal arrays, we prepared a protein extract from Artemia by homogenizing embryos (nauplius stage) and lysing pelleted nuclei in a high salt extraction buffer (2 M NaCl) (see "Materials and Methods"). Western blot analysis and immunostaining were used to verify the presence of the core histones, H1 and topoisomerase I (not shown). A topological variation assay was performed using relaxed plasmid YEp-24 in order to evaluate the assembly activity of the extract. The activity was optimized by testing parameters that are known to affect chromatin assembly in other reconstitution systems, such as protein/DNA ratio, ionic conditions, and the quantity of polyglutamic acid added to the reaction (39-48). A high efficiency of assembly was obtained using a protein/DNA ratio of approximately 15:1, a NaCl concentration in the range 120-160 mM, and 50 µg/ml polyglutamic acid. All covalently closed plasmid molecules (YEp-24) migrated as negative supercoils after 2 h (Fig. 2A, lane 4). Two-dimensional gel analysis demonstrated that only nicked molecules migrated to the relaxed position (data not shown). DNA supercoiling analysis revealed that nucleosome assembly could be detected within 15 min (lane 2). No substantial variations in assembly efficiency were detected when polyglutamic acid was added in the range 50-250 µg/ml (data not shown).


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  		Fig. 2.   In vitro chromatin assembly. A, time course of DNA linking number reduction. Relaxed YEp-24 plasmid (7 µg) was incubated with 10.5 µl of 10 mg/ml assembly extract (120 mM NaCl final concentration) in the presence of 25 mM Tris/HCl, pH 8.0, 1 mM ATP, 0.5 mM EDTA, 50 ng/µl poly-L-glutamic acid in a final reaction volume of 175 µl. Aliquots (25 µl) were taken at the times indicated. DNA samples were isolated as described under "Materials and Methods" and resolved on a 1% agarose gel. The positions of relaxed and supercoiled DNA are indicated by R and Sc, respectively. B, Mg2+ and ATP-dependent modifications of nucleosome arrays. Relaxed YEp-24 DNA (5 µg) was incubated for the times indicated with 7.5 µl of 10 mg/ml assembly extract (120 mM NaCl final concentration) in the presence (lanes 4-7) and absence (lanes 16-19) of 1 mM ATP, 2 mM MgCl2, 20 mM disodium creatine phosphate, 1 µg/ml creatine phosphokinase. Chromatin assembled for 2 h in the absence of ATP, MgCl2, disodium creatine phosphate, and creatine phosphokinase (lanes 12-15) was further incubated for 2 h after the addition of ATP, MgCl2, and the ATP regeneration system (lanes 8-11). Reconstituted chromatin was digested with MNase (1 unit/µg of DNA) for 1, 2, 4, and 8 min (indicated by open triangles). Purified DNA samples were resolved by agarose gel electrophoresis and stained with ethidium bromide. The MNase digestion pattern of nuclei isolated from Artemia nauplii is also shown (lanes 2 and 3). Lane 1 shows the 123-bp ladder (Life Technologies, Inc.). Densitometer scans of lanes 2, 5, 9, and 13 are shown (right panel). Peaks of mono- (mon), di- (din), and trinucleosomes (tri) for samples 2, 5, and 9 are indicated. Peaks of di- and trinucleosomes for sample 13 are marked by asterisks. C, requirement of both Mg2+ and ATP to achieve the physiological nucleosome repeat length. Relaxed YEp-24 DNA (15 µg) was incubated for 4 h with 18 µl of 10 mg/ml assembly extract (120 mM NaCl final concentration) in the presence of 2 mM MgCl2, 25 mM Tris/HCl, pH 8.0, 20 mM disodium creatine phosphate, 1 µg/ml creatine phosphokinase (lanes 1-6); 1 mM ATP, 5 mM EDTA, 25 mM Tris/HCl, pH 8.0, 20 mM disodium creatine phosphate, 1 µg/ml creatine phosphokinase (lanes 7-12); or 1 mM ATP, 2 mM MgCl2, 25 mM Tris/HCl, pH 8.0, 20 mM disodium creatine phosphate, 1 µg/ml creatine phosphokinase (lanes 13-18). Reconstituted chromatin was then digested with MNase (0.4 units/µg of DNA) for 1, 2, 5, 10, 15, and 30 min (indicated by open triangles). Purified DNA samples were resolved by agarose gel electrophoresis and stained with ethidium bromide. The MNase digestion pattern of nuclei isolated from Artemia nauplii is also shown (lanes 19 and 20). DNA fragments corresponding to mononucleosomes (mon), dinucleosomes (din), and trinucleosomes (tri) are indicated. D, histone composition of reconstituted chromatin. Panel I shows the protein composition of the assembly extract; the positions of protein size standards are indicated to the left. Proteins that co-sedimented with assembled plasmids on a sucrose gradient were resolved by SDS-polyacrylamide gel electrophoresis and stained with Coomassie Blue (panel II). E, nucleosome reconstitution from purified core histones under Artemia assembly reaction conditions. Relaxed YEp-24 DNA (5 µg) was incubated with 1, 4, or 6 µg of core histones (histone/DNA ratios of 0.2, 0.8, and 1.2, respectively, as indicated) purified from chicken erythrocytes (see "Materials and Methods"). Reconstitution reaction conditions were 30 °C for 4 h in 25 mM Tris/HCl, pH 8.0, 2 mM MgCl2, 1 mM ATP, 150 ng/µl poly-L-glutamic acid, 20 mM disodium creatine phosphate, 1 µg/ml creatine phosphokinase, 10 units of Artemia purified topoisomerase I per µg of plasmid DNA. Reconstituted templates were then digested with MNase (1 unit/µg of DNA) for 1 and 2 min (indicated by open triangles). Purified DNA samples were electrophoresed on an agarose gel and stained with ethidium bromide. The 123-bp ladder (Life Technologies, Inc.) is shown as a marker.

Since ATP and Mg2+ are necessary for physiologically spaced nucleosomal arrays in all assembly systems characterized to date (41, 47, 49, 50), MNase digestion was used to verify whether these two cofactors affect the spacing of the reconstituted chromatin templates. Both in the presence (Fig. 2B, lanes 4-7) and absence (lanes 16-19) of ATP and Mg2+, partial digestions produced a ladder of fragments corresponding to oligonucleosomal DNAs. In the absence of ATP and Mg2+, chromatin templates were linker-deficient. However, when ATP and Mg2+ were included in the reaction, the nucleosome arrays were characterized by a repeat length of 190 ± 5 bp, equal to that found in vivo (lanes 2 and 3).

The dependence of chromatin dynamics on ATP was also examined. MNase digestions revealed that closely packed nucleosomal arrays, assembled in the absence of ATP and Mg2+ (Fig. 2B, lanes 12-15), were involved in general remodeling after the addition of ATP and Mg2+ (lanes 8-11), thus resulting in an increase in the internucleosomal distance to the physiological distance. In order to verify whether physiological nucleosome spacing was acquired by virtue of the presence of the divalent cation or the presence of ATP, chromatin was reconstituted in the presence of Mg2+ and the absence of ATP (Fig. 2C, lanes 1-6) and in the presence of ATP and the absence of Mg2+ (lanes 7-12). Physiological spacing between nucleosomes was not acquired in either case, thus confirming that nucleosome arrangements are not solely dependent on the ionic conditions of the reaction (determined by the presence of Mg2+) but also on energy-dependent mechanisms.

The protein composition of chromatin templates reconstituted with the Artemia extract under standard assembly conditions and purified by sucrose gradient centrifugation was examined. SDS-polyacrylamide gel electrophoresis analysis and Coomassie Blue staining (Fig. 2D, panel II) revealed the presence of the four core histones and histone H1. This result was confirmed by Western blot analysis and immunostaining with monoclonal anti-histone antibodies (data not shown). H1 levels were lower than the physiological stoichiometry among the histones (51). This might be due either to the purification procedure used to obtain the chromatin templates or to competition by polyglutamic acid for H1 binding locations (52).

In order to evaluate the assembly properties of the Artemia extract, nucleosome reconstitution in the absence of the extract and in the presence of purified chicken erythrocyte core histones was performed under assembly reaction conditions similar to those employed for chromatin reconstitution with the Artemia extract. Increased quantities of purified core histones were used to reconstitute nucleosomes on YEp-24 DNA in the presence of ATP, Mg2+, the energy regeneration system (disodium creatine phosphate and creatine phosphokinase), and polyglutamic acid (150 ng/µl). Reconstituted YEp-24 templates were then analyzed by MNase digestion (Fig. 2E). A histone/DNA ratio of 1.2 (lanes 6 and 7), equal to that used for nucleosome assembly with the Artemia extract (Fig. 2B, lanes 4-19), resulted in the occurrence of DNA and histone aggregation phenomena and the consequent absence of a distinguishable MNase pattern. When a histone/DNA ratio of 0.8 was used (Fig. 2E, lanes 4 and 5), a ladder of oligonucleosomal DNA fragments was obtained, indicating the reconstitution of closely packed nucleosomes onto YEp-24. At this ratio, the reconstitution efficiency is slightly diminished compared with that achieved using the Artemia extract at a histone/DNA ratio of 1.2 (Fig. 2, compare E, lanes 5 and 4, and B, lanes 7 and 6; also confirmed (data not shown) by topology analysis). No differences in either assembly efficiency or nucleosome spacing were detected when nucleosome reconstitution using purified chicken histones and plasmid DNA was carried out in the absence of ATP and Mg2+ (data not shown). The presence in the Artemia extract of additional proteins other than the presumed chaperones and histone-binding factors seems to prevent the occurrence of aggregates at a histone/DNA ratio of 1.2, since even bovine serum albumin could partially relieve the aggregation phenomenon at this ratio when purified core histones were used. However, the presence of bovine serum albumin did not result in the production of completely assembled plasmid chromatin (data not shown) such as that obtained with the Artemia assembly system.

Satellite Chromatin Reconstitution in Vitro-- A tandemly repeated set of AluI-113 sequences constrained in a topological unit was reconstituted in order to study the behavior of Artemia satellite DNA during the process of nucleosome organization. pU-He6+, a 3364-bp plasmid obtained by cloning six AluI-113 (678-bp) monomeric units into the SmaI site of pUC18m (17) (Fig. 3A) was used for this analysis.


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  		Fig. 3.   Satellite chromatin reconstitution in vitro. A, diagrammatic representation of pU-He6+ plasmid used for satellite chromatin assembly. Six AluI-113 (678-bp) monomeric units were cloned into the SmaI site of pUC18m. The restriction sites BamHI and KpnI (2 and 1 bp, respectively, from the hexameric insert) are indicated. AluI-113 and 1-4 denote oligonucleotides (22 bp) used for the hybridizations shown in Figs. 3B, 5 (A and B), and 6A. AluI-113 is complementary to AluI-113 DNA (5'-CATACGTAGCTATTACCCTCGA-3') (positions 9-101) (see Fig. 4C); regions 1-4 are complementary to pUC18m plasmid: 1 (5'-GTCACGACGTTGTAAAACGACG-3') (positions 368-389), 2 (5'-TGGTGCACTCTCAGTACAATCT-3') (positions 184-163), 3 (5'-TCTTACGGATGGCATGACAGTA-3') (positions 2157-2136), 4 (5'-GCCGGTGAGCGTGGGTCTCGCG-3') (positions 1784-1763). Distances (bp) of probes from the BamHI site are also indicated. B, preferential histone-AluI-113 DNA interactions. Supercoiled pU-He6+ DNA (8 µg) was incubated with 3, 6, 12, 24, 60, or 120 µg of assembly extract under standard reconstitution conditions. Micrococcal nuclease (0.5 units/µg of DNA) was then added to the samples, and aliquots were taken at 2, 4, and 8 min (indicated by open triangles). Samples were processed as described under "Materials and Methods." DNA blots were hybridized sequentially to oligonucleotides AluI-113, 1, 3, and 4. Uncut plasmid is shown as a control of exposure of each autoradiograph to the right of panels AluI-113, 1, 3, and 4. MNase digestion of nuclei (N) is shown as a marker. DNA fragments corresponding to mononucleosomes (mon) and dinucleosomes (din) are indicated.

Early experiments on in vitro reconstitution of purified core histones with DNA by salt dilution or dialysis established that highly curved DNA assembles histone octamers with greater affinity than noncurved DNA (53-56). The physiological relevance of the data obtained by this approach has been questioned (57), since the reconstitution process starts at a salt concentration (0.5 M NaCl) that is close to dissociating conditions, thus possibly altering the affinity of sequences for histone octamers. We therefore examined whether the unidirectional curvature of the AluI-113 DNA helix resulted in a particular affinity for histone octamers under physiological assembly conditions. Increased quantities of extract were used to reconstitute nucleosomal templates, which were then analyzed by MNase digestion (Fig. 3B). Sequential hybridizations were performed with oligonucleotides complementary to AluI-113 or other plasmid regions (regions 1, 3, and 4; see Fig. 3A). With small amounts of extract (3 and 6 µg), 3-4 times more satellite DNA fragments of mononucleosomal size accumulated compared with non-satellite DNA fragments (compare panels AluI-113, 1, 3, and 4). Greater quantities of extract (24 and 60 µg) generated greater amounts of dinucleosome-sized fragments in the heterochromatic regions (compare panels AluI-113, 1, 3, and 4). These observations indicate a high affinity of histone octamers for AluI-113 DNA, suggesting that in the presence of a limited concentration of extract the assembly process is not random but stabilizes selected histone/DNA interactions. Moreover, this confirms previous data obtained by salt dilution or dialysis reconstitution onto bent DNA sequences (53-56).

We then investigated whether the heterochromatic DNA causes specific nucleosome positioning and whether the dynamics of the positioning phenomenon were reproducible in vitro. The translational positions of nucleosomes on both native and reconstituted satellite chromatins were examined by high resolution mapping of nucleosome borders using MNase (58). Nuclei and completely assembled pU-He6+ chromatin templates were extensively digested with MNase to obtain only mononucleosome-sized fragments (146 bp). Nucleosome borders were then detected by primer extension/linear amplification analysis using purified single strand DNA fragments (see "Materials and Methods") as substrates for the extensions. Identical start points (mapped by primer P1) and end points (mapped by primer P2) were found both in vivo and in vitro (Fig. 4A, panel I, compare lanes N and M). MNase digestion of naked DNA was also performed to exclude the possibility that only sequence preferences of the enzyme were visualized (panel II). All possible nucleosome locations (14 locations) on AluI-113 DNA (Fig. 4C, borders marked by triangles) were determined by integrating the data derived from extensions with different primers (data not shown). From these results, we deduced that (i) in vitro the six AluI-113 monomeric elements direct the histone octamers to the same translational positions as found in vivo and (ii) nucleosomes exhibit mobility with respect to the AluI-113 sequence as suggested by the large number of translational positions found (59, 60). While six consecutive AluI-113 elements allowed us to reproduce the native nucleosome locations, it was not possible to recover identical positioning if plasmids carrying AluI-113 dimers instead of hexamers were used (data not shown).


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  		Fig. 4.   Nucleosome positioning by AluI-113 DNA in chromatin. A, MNase borders detected over AluI-113 mononucleosomes. pU-He6+ plasmid chromatin reconstituted under standard assembly conditions and A. franciscana nuclei were digested with MNase (1 unit/µg of DNA) for 20 min. Fragments of mononucleosomal size obtained from digestions of nuclei (N) or minichromosomes (M) were recovered and analyzed by primer extension linear amplification analysis (see "Materials and Methods"). After denaturation in formamide, DNA samples were resolved on a denaturing 6% polyacrylamide gel (panel I). P1 and P2 indicate the 32P-labeled primers complementary to the AluI-113 sequence (1-20 and 113-94, respectively; see C) utilized for the extension. High resolution mapping of MNase preferential cleavage sites on naked AluI-113 DNA is shown (panel II). Naked DNA was digested with MNase for 1 min using 0.01 and 0.1 unit/µg of DNA (indicated by open triangles). The DNA was purified and analyzed by primer extension linear amplification analysis with P1 primer. Sequencing reactions are shown as markers (lanes A, C, G, and T). B, MNase borders detected over AluI-113 dinucleosomes. pU-He6+ plasmid chromatin and A. franciscana nuclei were digested with MNase (1 unit/µg of DNA) for 10 min. Fragments of dinucleosomal size were recovered and analyzed as described for A. P1 and P2 extensions are shown (din). P1 and P2 extensions of DNA fragments of mononucleosomal size, obtained by MNase digestion of minichromosomes, were used as controls (mon). C, translational locations of nucleosomes. Start point (open triangles) and end point (filled triangles) nucleosome borders detected by extension of P1 and P2 primers, respectively, over AluI-113 fragments of mononucleosome size are indicated. Start point (open circle) and end point (filled circle) nucleosome borders over AluI-113 fragments of dinucleosome size are also indicated. Oligonucleotides P1 and P2 are shown (arrows). D, DNase I cleavage of AluI-113 DNA in a nucleosome. End-labeled AluI-113 dimer (226 bp) was reconstituted and digested with DNase I as described under "Materials and Methods." Mononucleosomes were isolated by agarose (0.7%) gel electrophoresis. DNA was recovered, purified (see "Materials and Methods"), and analyzed by denaturing polyacrylamide (6%) gel electrophoresis. The locations of cleavage sites on the 3'-5' strand are indicated. E, DNase I cleavage of nucleosomes reconstituted on the AluI-113 hexamer inserted in the plasmid pU-He6+. pU-He6+ chromatin reconstituted under standard conditions was digested for 1 min at 30 °C with DNase I (10 units). DNA was purified and used as a template for primer extension linear amplification analysis (see "Materials and Methods"). P1 and P2 indicate the primers used for the extensions. DNase I cleavage sites on naked DNA and on 5'-3' (P1) and 3'-5' (P2) strands are shown. Sequencing reactions are also shown as markers (lanes T, G, C, and A (left) and lanes A, C, G, and T (right)). F, DNase I cleavage site positions on the 3'-5' strand are indicated. Short runs of poly(dA-dT) are underlined.

We also analyzed the nucleosome borders determined by primer extension of MNase-digested fragments of dinucleosomal size (Fig. 4, B and C (borders marked by circles)). As previously observed, no difference was found between the in vitro and in vivo positions (Fig. 4B, din lanes, compare M and N). A decrease in the total number of borders (from 14 to eight) was revealed, mapping to positions identical to those of mononucleosome core particles (compare din and mon lanes). We conclude that the summation of the structural properties arising from the interactions of consecutive positioned nucleosomes results in uniformity of linker DNA lengths and a decrease in the mobility of the nucleoprotein complexes.

The multiple translational locations found for mononucleosomes suggested that the primary structural requirement of AluI-113 DNA is the stabilization of nucleosome cores having preferred rotational positions. This agrees with earlier work indicating that DNA bending tends to cause the binding of histone octamers in a specific orientation (54, 56, 61). Rotational positioning was examined by DNase I cleavage of an AluI-113 dimer (226 bp) assembled as a mononucleosome. Strong preferential cutting at approximately 10-bp intervals was observed (Fig. 4, D and F (cleavage sites marked by AluI-113 map positions)), implying that the A/T repeats face inward toward the histone surface. The same result was obtained by analyzing the DNase I cleavage sites of the reconstituted heterochromatic hexamer in plasmid pU-He6+ (Fig. 4, E and F). These findings support the hypothesis that AluI-113 dictates specific rotational positioning. The requirement of structural deformability of the double helix in a nucleosome is reminiscent of studies on chicken nucleosomal core DNA (62).

Chromatin Arrays of the Heterochromatic DNA Flanking Regions-- By analyzing the nucleosomal organization of different pU-He6+ regions located at increasing distances from the heterochromatic insert, we investigated (i) whether nucleosome arrangements on the satellite DNA could influence the chromatin organization of the plasmid DNA and (ii) whether this was dependent on the presence of physiologically spaced nucleosome arrays. Chromatin templates were assembled with a subsaturating amount of extract for increasing lengths of time, both in the presence and absence of ATP and Mg2+, and were subsequently digested with MNase. Sequential probing of satellite DNA and the regions progressively further away demonstrated that, with or without ATP, the amount of DNA fragments smaller than mononucleosomal size increased with the distance from the heterochromatic DNA (Fig. 5, compare panels AluI-113, 2, 3, and 4). This indicated a progressive reduction in nucleosome assembly in the specific regions tested. These experiments also showed that (i) the association of nucleosomes with DNA is rapid, only 5 min being required for a typical nucleosomal ladder to appear; (ii) within the same period remodeling activities occur, which, in the presence of ATP and Mg2+, influence nucleosome spacing although the physiological nucleosome repeat lengths are gradually acquired over a period of 2 h; (iii) MNase accessibility generally depends on ATP and Mg2+ (the appearance of larger amounts of subnucleosomal fragments in the presence of the two cofactors was revealed); and (iv) strong MNase hypersensitivity occurred in a region located approximately 300 bp from the insert in the presence of ATP but not in its absence (panel 2), thus indicative of different levels of structural constraint on nucleosomal arrangements that are dependent on energy requiring processes.


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  		Fig. 5.   Nucleosome density on different regions of pU-He6+ and pU-He6- assembled with a subsaturating quantity of assembly extract. A, nucleosome density on pU-He6+ template. pU-He6+ supercoiled plasmid (12 µg) was incubated in the presence and absence of ATP, Mg2+, and the ATP regeneration system with a subsaturating quantity of assembly extract (90 µg). Aliquots were taken at the times indicated, and MNase (1 unit/µg of DNA) was added. The reaction mixture was incubated for a further 1 min at 30 °C. Samples were processed as described under "Materials and Methods." Sequential hybridizations of the same filter were made with the following 32P-labeled oligonucleotides: AluI-113, 2, 3, and 4 (map positions are specified in Fig. 3A). MNase digestion of nuclei (N) is shown as a marker. DNA fragments corresponding to mononucleosomes (mon) and dinucleosomes (din) are indicated. B, nucleosome density on pU-He6- template. pU-He6- supercoiled plasmid (4 µg) was incubated at 30 °C for 240 min with a subsaturating quantity of assembly extract (30 µg) in the presence of ATP, Mg2+, and the ATP regeneration system. MNase (1 unit/µg of DNA) was then added, and aliquots were taken at 1 and 2 min (indicated by open triangles). Samples were processed as described under "Materials and Methods." Sequential hybridizations of the same filter were made with 32P-labeled oligonucleotides 2, 3, and 4 (map positions specified in Fig. 3A). DNA fragments corresponding to mononucleosomes (mon) and dinucleosomes (din) are indicated.

Since it has recently been shown, using randomly selected chicken genomic DNA sequences, that both the regularity of nucleosomal arrays and the value of the nucleosome repeat length are highly DNA sequence-dependent (63) and since the different plasmid regions tested might have different affinities for core histones, we wished to exclude the possibility that these phenomena were responsible for the MNase pattern obtained in the regions proximal to the heterochromatic insert, which indicate a higher nucleosome density in these regions compared with the more distant ones. A control was thus performed to confirm the effect of satellite DNA on the process of nucleosome formation in the flanking regions. pU-He6-, which contains an A/T-rich sequence of the same length as the heterochromatic hexamer inserted in pU-He6+ (17) but unrelated to it, was reconstituted under standard assembly conditions with a subsaturating amount of extract. The MNase pattern of the previously tested regions (regions 1-3) analyzed by sequential probing (Fig. 5B), even if exhibiting slight differences in the amount of subnucleosomal DNA fragments, did not reveal the same phenomenon of reduced nucleosome assembly with increased distance from the insert as described previously.

Potential nucleosome positioning mechanisms mediated by heterochromatic DNA and the extension of their influence to flanking regions were analyzed by carrying out MNase digestions followed by BamHI and KpnI digests on purified DNA fragments. Plasmid regions located at various distances from the satellite DNA were sequentially probed (Fig. 6A). Hybridization with probe 1 (panel 1) at 48 bp from the hexamer revealed (i) the disappearance of the mononucleosome-sized band, thereby indicating the presence of nucleosomes at the satellite DNA/plasmid junction, and (ii) a series of bands indicative of nucleosome positioning effects occurring over the flanking plasmid regions. From the lengths of the bands obtained by hybridization with probe 2 (panel 2) it was possible to determine that the positioning effect extends for at least three nucleosomes outside the heterochromatic insert.


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  		Fig. 6.   Nucleosome positioning effect is mediated by AluI-113 DNA. A, pU-He6+ supercoiled plasmid (10 µg) was reconstituted under standard conditions. MNase (1 unit/µg) was added to the reaction mixture, and aliquots were taken after 1, 2, and 4 min. DNA was purified, and half of each sample was digested to completion with BamHI and KpnI. DNA was resolved on a 1% agarose gel and blotted. MNase-treated DNA (2 and 4 min of digestion) prior to restriction enzyme digestion is shown. The blot was sequentially probed with end-labeled oligonucleotides: AluI-113, 1 (368-389), 2 (184-163), and 3 (2157-2136) oligonucleotides. B, high resolution mapping of MNase cleavage sites in AluI-113 flanking regions. pUC18m and pU-He6+ plasmids reconstituted in the presence and absence of ATP, Mg2+, and the ATP regeneration system were digested with MNase (0.8 unit/µg of DNA) for 2 and 4 min (indicated by open triangles). Primer extension linear amplification analysis of MNase cleavages was then performed as detailed under "Materials and Methods." A primer complementary to pUC18m (vertical arrow; map position is indicated) was used, extending from the insert (hex) toward the plasmid flanking regions. After denaturation in formamide, the DNA samples were resolved on a denaturing 6% polyacrylamide gel. Solid bars indicate regions where more frequent cleavages are detected (map positions are also indicated). Mapping of cleavage sites on naked pU-He6+ DNA is also shown. The approximate 10-bp recurrence of MNase cleavages (samples 1-4) is indicated by horizontal arrows. Sequencing markers (A, C, and G) are also shown.

We also analyzed the ATP-dependent influence of heterochromatic DNA on flanking regions by determining the MNase cleavage sites to single nucleotide resolution (Fig. 6B). Mapping of cleavage sites was performed by primer extension/linear amplification analysis using a primer extending away from the insert toward the plasmid. In the absence of ATP and Mg2+, MNase cleavage of closely packed pUC18m and pU-He6+ chromatin templates showed identical patterns of approximately 10-bp recurrence (compare lanes 1 and 2 and lanes 3 and 4) indicating the arrays of nucleosome boundaries. In the presence of ATP and Mg2+, MNase mapping revealed generally greater accessibility in both fully assembled templates (compare lanes 1-4 with lanes 7-10). This indicates that the nucleoprotein structures reconstituted on the flanking regions maintain a dynamic potential in the presence of the two cofactors. Comparison of pU-He6+ and pUC18m templates assembled in the presence of ATP (compare lanes 7 and 8 with lanes 9 and 10) revealed at least four regions (indicated as solid bars) showing greater frequencies of MNase cleavage as reflected in the multiplicity of DNA bands seen by gel electrophoresis and similar to that found for naked DNA (lanes 5 and 6). Since MNase preferentially cleaves DNA between nucleosome core particles, these regions, whose positions are consistent with the experiments noted above (Fig. 6A), must correspond to internucleosomal DNA. The influence of the heterochromatic DNA on the flanking regions in dynamic reconstituted chromatin is therefore exerted by constraining spaced nucleosomes in ordered arrays. The structural basis for this appears to be uniformity of linker DNA lengths rather than sequence-specific nucleosome binding (compare lanes 7 and 8 with lanes 9 and 10).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Artemia Chromatin Assembly System-- We have developed and characterized an in vitro chromatin assembly system from A. franciscana at the nauplius stage. This cell-free preparation is derived from late embryos unlike those of Drosophila (derived from preblastoderm embryos) or Xenopus (derived from eggs and oocytes) (47, 64). Although our assembly system cannot use large stores of histones as occurs in the two previously mentioned systems, we nevertheless obtained efficient chromatin reconstitution. Significant amounts (micrograms) of plasmids were totally assembled without the addition of purified histones. The reconstitution process relies completely on the endogenous core histones, H1, topoisomerase I, and chromatin remodeling proteins present at that particular larval stage. Characterization of the assembly system was achieved by analyses following three main criteria: (i) the efficiency of the reconstitution reaction; (ii) the spacing and conformational dynamics of the polynucleosomal complexes; and (iii) the protein composition of the chromatin templates formed. The Artemia extract was shown to be an appropriate system for proper reconstitution of chromatin in vitro. The assembly process is replication-independent (data not shown) and leads to a high nucleosome density. The reaction efficiency depends on (i) the specific DNA/extract ratio; (ii) ionic strength, the optimum being in the range 120-160 mM NaCl (data not shown); and (iii) the presence of polyglutamic acid, which minimizes aspecific aggregation (data not shown). The necessity of the polyanion to mask the electrostatic interactions among histones and between histones and DNA indicates a lack of those molecular chaperones that have been found to assist nucleosome formation in other systems (reviewed in Ref. 50). The resulting chromatin arrays are physiologically spaced (i.e. one nucleosome every 185-195 bp) in the presence of ATP, Mg2+, an ATP regeneration system, and a NaCl concentration in the range of 120-160 mM. This suggests that the molecular processes that lead to physiological spacing depend on energy-using activities (65, 66) and specific ionic conditions (48), as is the case in other characterized systems (see Refs. 41, 47, and 49; reviewed in Ref. 50). In light of recent results concerning the influence of cations on linker DNA lengths (48), the modification of spacing in the Artemia system is not governed merely by Mg2+ but also depends on the presence of ATP and an ATP regeneration system. Neither in the presence of added Mg2+ and the absence of exogenous ATP nor in the presence of exogenous ATP and the absence of added Mg2+ is physiological spacing achieved.

Chromatin dynamics, which allow nucleosome movement, have been proposed to be fundamental mechanisms for nuclear metabolic events (67). We thus tested whether our assembly system could reproduce dynamic chromatin properties in vitro. Energy-dependent activities, not yet characterized in Artemia, are able to progressively and continuously modulate the interactions between nucleosomes during the assembly process. A whole preconstituted closely packed chromatin plasmid can be remodeled after the addition of ATP (1 mM) and Mg2+ (2 mM) yielding physiologically spaced nucleosomal arrays.

Nucleosome assembly carried out under the same reaction conditions as used for chromatin reconstitution in the presence of the Artemia extract, but using only plasmid DNA and purified adult chicken erythrocyte histones (histone/DNA ratio 0.2-1.2), demonstrated that the conditions used are themselves sufficient for core histone assembly and lead to the formation of polynucleosomal chains (histone/DNA ratio 0.8 for optimum assembly efficiency). However, (i) complete assembly of DNA templates is obstructed due to the aggregation and precipitation of DNA and histones (this problem is not encountered in the Artemia assembly system nor in systems designed for highly efficient assembly of extended nucleosome arrays from purified histones (43, 46)); and (ii) physiological spacing is not achieved, nor is there any increase in nucleosome repeat length even in the presence of ATP and Mg2+.

The Artemia extract assembled the full complement of core histones. Moreover, endogenous histone H1 was incorporated into physiologically spaced chromatin, although there was partial loss of the physiological stoichiometry (51). We suggest that this might be due to the process of chromatin purification and/or possibly the presence of polyglutamic acid in the assembly reaction. It has been shown that the concentration of this polymer is a determining factor for assembly and extraction of H1 (52). The addition of exogenous H1 has been observed to increase the nucleosomal repeat lengths of chromatin reconstituted in Drosophila and Xenopus extracts, which are deficient in the normal somatic histone H1 (47, 48, 68). It has also been suggested that H1 influences nucleosome positions (69-71). H1 association would therefore appear to be relevant to the in vitro reconstitution of specific chromatin features on AluI-113 sequences.

Nucleosome Interactions with AluI-113 Heterochromatic DNA-- In vivo studies of satellite chromatin domains as well as in vitro analyses carried out using the Artemia assembly system allowed us to determine some of the fundamental structural properties of AluI-113 DNA within nucleosomes. The AluI-113 hexamer (678 bp) cloned in pUC18m (2686 bp) was used as a template (pU-He6+). Nucleosome reconstitution on multimeric satellite fragments allowed us to reproduce histone core/AluI-113 DNA interactions as well as the more complex structures arising from interactions between several nucleosomes. Various experimental approaches have shown that adjacent nucleosomes exert reciprocal influences. This phenomenon might be fundamental for the folding geometry of satellite polynucleosomal complexes in which the satellite element alignments relate to nucleosome recurrence (reviewed in Ref. 24). The efficiency of nucleosome association with heterochromatic DNA compared with other sequences belonging to the same topological unit was investigated. The analysis not only gave indications of histone core affinity for the sequences tested but also indicated the thermodynamic probability that octamers would assemble in a given topological unit. The heterochromatic DNA revealed a high dynamic propensity for histone octamer winding, leading to a nonrandom assembly process and the stabilization of preferred AluI-113 DNA/octamer interactions. This is reminiscent of results obtained by assembling nucleosomal core particles under stringent conditions (by salt dilution or dialysis) on natural, bent DNA sequences: (i) a segment of kinetoplast DNA from Crithidia fasciculata (53, 56); (ii) the terminus of replication and termini of transcription of SV40 DNA (53); and (iii) a bent cloned fragment of 223 bp from chicken erythrocytes (54). Furthermore, our results are in agreement with studies on a large number of satellite sequences, which suggest that the pattern of bending conserved in satellite DNAs consists of a modular structure of two bending elements separated by a low curvature region resembling the bending of DNA in the nucleosome (25).

DNA bending has been shown to be a strong energetic determinant of selective histone octamer associations (55, 61) and also one of the major determinants in directing translational and rotational positioning of nucleosomes (56, 72). In the case of satellite sequences, it has been widely assumed that the intrinsic structural features of DNA play a role in directing translational positioning (24). Recently, it has been suggested that the specific satellite patterns of DNA bending might be a general signal for nucleosome positioning (25). We established the translational nucleosome positioning of AluI-113 DNA at the nucleotide level by analyzing the nucleosome borders defined by MNase both in vivo and in vitro. Reconstituted chromatin yielded the same histone octamer localizations as found in the nucleus. The number of borders specified for mononucleosomes was 14. The multiplicity of nucleosome localizations found for AluI-113 DNA is atypical compared with those established for other satellite DNAs (see Refs. 26, 27, and 33-36; reviewed in Ref. 24). The approach used showed that individual nucleosomes maintain dynamic interactions with the satellite DNA sequence, leading to alternative translational states. This supports the assumption that short range nucleosome sliding is a general phenomenon that assures potential nucleosome mobility in relation to required nuclear functions (59).

The DNase I cleavage pattern of reconstituted AluI-113 DNA (it exhibits an approximately 10-bp recurrence) indicates that the primary structural requirement of the satellite sequence is the definition of a specific rotational phase around the histone core (24, 72, 73). We therefore suggest that the observed translational locations, although not precisely spaced by one helical repeat as expected (see Refs. 54 and 74; reviewed in Ref. 24), reflect the staggering of each single nucleosome maintaining specific rotational phases. Translational positioning of the histone octamers, as well as their rotational phase, remained unaltered in the presence or absence of ATP and Mg2+ (data not shown). Thus, the dynamics of the interactions of each histone octamer with the satellite DNA sequence are energy-independent, and the short range mobility of the nucleosomes is also typical of closely packed chromatin. The translational positioning of dinucleosomes showed a reduction in the number of the MNase borders (from 14 to eight). This indicates that the dinucleosomal complex, while maintaining the specific nucleosomal rotational positioning, is subject to a decrease in the extent of mobility compared with single nucleosomes. Adjacent nucleosomes constrained by preferential positioning along the satellite DNA interact, reciprocally limiting histone core mobility and establishing uniform linker DNA lengths. At present, we are unable to specify whether H1 plays a role in inhibiting nucleosome mobility on AluI-113 DNA as has been observed for mononucleosomes assembled on sea urchin 5 S rDNA (60) and dinucleosomes assembled on Xenopus 5 S RNA genes (75). The regular chromatin structure of heterochromatic AluI-113 domains seems to result from the constraints imposed by (i) nucleosome positioning as dictated by the anisotropic properties of the DNA sequence; (ii) nucleosome-nucleosome interactions; and (iii) the uniformity of internucleosomal distances.

The preferential quantitation of internucleosomal DNA (76-80) has led to the suggestion that linker DNA is one of the determinants of nucleosome positioning (81). Linker DNA has also been correlated with different states of chromatin folding. Models of symmetrical ribbon-like structures have been predicted for ordered nucleosomes spaced by linker DNA of uniform length (82). The uniformity of the distance between rotationally positioned nucleosomes would cause periodic identity of linker sequences and therefore periodic identity of DNA linker conformations. This may be important for the configuration of crystal-like higher order folding of AluI-113 chromatin.

Nucleosome associations on AluI-113 DNA seem to influence the energetic interactions that occur during the process of assembly on flanking regions. On subsaturated chromatin templates, we observed that heterochromatic DNA restricted the randomness of histone octamer binding. The proximal flanking regions exhibited greater nucleosome density compared with distal regions. Thus, the hexameric insert appears to lead to weak cooperativity of nucleosome association. However, our experimental data do not allow us to elucidate how the bend intrinsic to the AluI-113 sequence, which promotes the rotational orientation and translational positioning of DNA on nuclesomes, would affect the deformational anisotropy of flanking chromatin DNA and determine the observed effect of cooperativity on core histone assembly. Further analysis will therefore be required to address this issue, which might be implicated in the cooperative generation of higher order chromatin structures.

In dynamic, reconstituted chromatin, we observed the spread of satellite chromatin features and the imposition of a specific organization on adjacent nucleosomes, possibly mediated by the uniformity of the internucleosomal distances. Preliminary analyses of the extent of satellite chromatin spread revealed an influence on at least 60% of the entire plasmid chromatin. The observation that the satellite nucleosome arrays influence the patterns of nucleosome formation over flanking regions supports the hypothesis that nucleosome organization may play a role in repressing gene expression in position effect variegation phenomena (12). Synergic effects between ordered nucleosomes and silencing proteins might also be an explanation for local heterochromatinization induced by non-satellite DNA sequences, provided that they are tandemly arranged (see Ref. 83; reviewed in Ref. 3).

    ACKNOWLEDGEMENTS

We are grateful to Ida Ruberti for helpful suggestions in setting up the Artemia chromatin assembly system; G. Camilloni and E. Di Mauro for the protocols to map translational nucleosome positioning; and A. P. MacCabe and R. Mantovani for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by a grant from Consiglio Nazionale delle Ricerche (Project 97.04423.CT04) and in part by Ministers per l'Université e le Ricerce Scientifice, Project "Protein-Nucleic Acid Interactions."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

§ To whom correspondence should be addressed: III Facoltá di Scienze, Universitá di Milano, Via Ravasi 2, 21100 Varese, Italy. Tel.: 39-332-250206; Fax: 39-332-281308; E-mail: hetero{at}imiucca.csi.unimi.it.

1 The abbreviation used is: bp, base pair(s).

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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M. C. Motta, G. Caretti, G. F. Badaracco, and R. Mantovani
Interactions of the CCAAT-binding Trimer NF-Y with Nucleosomes
J. Biol. Chem., January 15, 1999; 274(3): 1326 - 1333.
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