From the Dipartimento di Biologia Stutterale e Funzionale III Facoltá di Scienze, Universitá di Milano, 21100 Varese, Italy
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 -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 |
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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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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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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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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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DISCUSSION |
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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 |
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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.
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FOOTNOTES |
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* 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.
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).
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REFERENCES |
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