Sequence specific cleavage of African green monkey alpha-satellite DNA by micrococcal nuclease

The sequence specificity of micrococcal nuclease complicates its use in experiments addressed to the still controversial issue of nucleosome phasing. In the case of alpha-satellite DNA containing chromatin from African green monkey (AGM) cells cleavage by micrococcal nuclease in the nucleus was reported to occur predominantly at only one location around position 126 of the satellite repeat unit (Musich et al. (1982) Proc. Natl. Acad. Sci. USA 79, 118-122). DNA control experiments conducted in the same study indicated the presence of many preferential cleavage sites for micrococcal nuclease on the 172 bp long alpha-satellite repeat unit. This difference was taken as evidence for a direct and simple phase relationship between the alpha-satellite DNA sequence and the position of the nucleosomes on the DNA. We have quantitatively analyzed the digestion products of the protein-free satellite monomer with micrococcal nuclease and found that 50% of all cuts occur at positions 123 and 132, 5% at position 79, and to a level of 1-3% at about 20 other positions. We also digested high molecular weight alpha-satellite DNA from AGM nuclei with micrococcal nuclease. Again cleavage occurred mostly at positions 123 and 132 of the satellite repeat unit. Thus digestion of free DNA yields results very similar to those reported by Musich et al. for the digestion of chromatin. Therefore no conclusions on a possible phase relationship can be drawn from the chromatin digestion experiments.     

Nucleosome arrangement in alpha-satellite chromatin of African green monkey cells.

By analyzing the accessibility of restriction endonuclease sites in African green monkey alpha-satellite chromatin, we demonstrate the absence of a unique phase relationship between nucleosomes and alpha-satellite DNA. The data indicate a minimum of three different positions for nucleosome cores relative to the alpha-satellite sequence and suggest a random distribution in at least some regions. In addition, while we confirm published reports that staphylococcal nuclease cuts the alpha-satellite sequence in chromatin at a highly preferred site, two-dimensional gel electrophoresis of nuclear digests demonstrates that this site is preferentially cut by staphylococcal nuclease even when it is within the nucleosome core. These data indicate that staphylococcal nuclease is not useful for determining nucleosome positions on alpha-satellite DNA, and perhaps on other specific DNA sequences as well.     

Positioning of nucleosomes in satellite I-containing chromatin of rat liver

The location of nucleosomes on rat satellite I DNA has been investigated using a new approach. Nucleosome cores were prepared from rat liver nuclei with micrococcal nuclease, exonuclease III and nucleases S1. From the total population of core DNA fragments the satellite-containing fragments were isolated by molecular cloning and the complete sequence of 50 clones was determined. The location of nucleosomes along the satellite sequence was found to be non-random. Our results show that nucleosomes occupy a number of positions on satellite I DNA. About 35 to 50% of all nucleosomes are positioned in two corresponding major sites, the remainder in about 16 less preferred sites. The major nucleosome positions are apparently strictly defined with the precision of a single base-pair. These results were confirmed by other approaches, including restriction nuclease digestion experiments. There are good indications of a defined long-range organization of the satellite chromatin fiber in two or more oligonucleosomal arrays with distinct nucleosome configurations.     

Centromere protein B of African green monkey cells: gene structure, cellular expression, and centromeric localization.

Centromere protein B (CENP-B) is a centromeric DNA-binding protein which recognizes a 17-bp sequence (CENP-B box) in human and mouse centromeric satellite DNA. The African green monkey (AGM) is phylogenetically closer to humans than mice and is known to contain large amounts of alpha-satellite DNA, but there has been no report of CENP-B boxes or CENP-B in the centromere domains of its chromosomes. To elucidate the AGM CENP-B-CENP-B box interaction, we have analyzed the gene structure, expression, biochemical properties, and centromeric localization of its CENP-B. The amino acid sequence deduced from the cloned AGM CENP-B gene was established to be highly homologous to that of human and mouse CENP-B. In particular, the DNA binding and homodimer formation domains demonstrated 100% identity to their human and mouse counterparts. Immunoblotting and DNA mobility shift analyses revealed CENP-B to be expressed in AGM cell lines. As predicted from the gene structure, the AGM CENP-B in the cell extracts exhibited the same DNA binding specificity and homodimer forming activity as human CENP-B. By indirect immunofluorescent staining of AGM mitotic cells with anti-CENP-B antibodies, a centromere-specific localization of AGM CENP-B could be demonstrated. We also isolated AGM alpha-satellite DNA with a CENP-B box-like sequence with CENP-B affinity. These results not only prove that CENP-B functionally persists in AGM cells but also suggest that the AGM genome contains the recognition sequences for CENP-B (CENP-B boxes with the core recognition sequence or CENP-B box variants) in centromeric satellite DNA.     

Integration of human alpha-satellite DNA into simian chromosomes: centromere protein binding and disruption of normal chromosome segregation.

Centromeres of mammalian and other complex eukaryotic chromosomes are dominated by one or more classes of satellite DNA. To test the hypothesis that alpha-satellite DNA, the major centromeric satellite of primate chromosomes, is involved in centromere structure and/or function, human alpha-satellite DNA was introduced into African green monkey (AGM) cells. Centromere protein binding was apparent at the sites of integrated human alpha-satellite DNA. In the presence of an AGM centromere on the same chromosome, human alpha-satellite was associated with bridges between the separating sets of chromatids at anaphase and an increased number of lagging chromosomes at metaphase, both features consistent with the integrated alpha-satellite disrupting normal chromosome segregation. These experiments suggest that alpha-satellite DNA provides the primary sequence information for centromere protein binding and for at least some functional aspect(s) of a mammalian centromere, playing a role either in kinetochore formation or in sister chromatid apposition.     

Analysis of nucleosome arrangement on satellite DNA of rat liver chromatin

The arrangement of nucleosomes on the nucleotide sequence of satellite DNA of Oceanian rat (Rattus rattus) has been studied. Nucleosome cores were prepared from rat liver nuclei with micrococcal nuclease, exonucleaseIII and nuclease Sl. From the total population of core DNA fragments, the satellite-containing fragments were selected by molecular cloning and the complete nucleotide sequence of these clones was determined. The data show that nucleosomes occupy a number of preferred positions on satellite DNA. These positions are strictly defined. Thus location of nucleosomes along the satellite sequence is non-random. Such finding may have important biological significance.     

Surprising deficiency of CENP-B binding sites in African green monkey alpha-satellite DNA: implications for CENP-B function at centromeres.

Centromeres of mammalian chromosomes are rich in repetitive DNAs that are packaged into specialized nucleoprotein structures called heterochromatin. In humans, the major centromeric repetitive DNA, alpha-satellite DNA, has been extensively sequenced and shown to contain binding sites for CENP-B, an 80-kDa centromeric autoantigen. The present report reveals that African green monkey (AGM) cells, which contain extensive alpha-satellite arrays at centromeres, appear to lack the well-characterized CENP-B binding site (the CENP-B box). We show that AGM cells express a functional CENP-B homolog that binds to the CENP-B box and is recognized by several independent anti-CENP-B antibodies. However, three independent assays fail to reveal CENP-B binding sites in AGM DNA. Methods used include a gel mobility shift competition assay using purified AGM alpha-satellite, a novel kinetic electrophoretic mobility shift assay competition protocol using bulk genomic DNA, and bulk sequencing of 76 AGM alpha-satellite monomers. Immunofluorescence studies reveal the presence of significant levels of CENP-B antigen dispersed diffusely throughout the nuclei of interphase cells. These experiments reveal a paradox. CENP-B is highly conserved among mammals, yet its DNA binding site is conserved in human and mouse genomes but not in the AGM genome. One interpretation of these findings is that the role of CENP-B may be in the maintenance and/or organization of centromeric satellite DNA arrays rather than a more direct involvement in centromere structure.     

Non-random arrangement of nucleosomes in satellite I containing chromatin of rat liver.

The location of nucleosomes on the nucleotide sequence of rat satellite I DNA was investigated using micrococcal nuclease, exonuclease III, and restriction nucleases as tools. Hae III cleaved the satellite DNA containing chromatin very preferentially in the linker region. Nucleosomes were found predominantly in three defined positions on the 370 bp satellite I monomer unit. This type of arrangement occurs on not more than half of the satellite DNA containing chromatin while the rest of this chromatin is arranged differently. The arrangement of nucleosomes with high probability in preferred frames and with low probability in less preferred frames may be a general phenomenon which can be discussed as a possible mechanism to modulate sequence recognition.     

Subunit structure of alpha-satellite DNA containing chromatin from African green monkey cells.

alpha-Satellite DNA containing chromatin from African green monkey cells (CV-1 cells) has been used to study the question whether or not nucleosomes are arranged in phase with the 172 bp repeat unit of the satellite DNA. Digestion experiments with DNAase II led us to exclude a simple phase relationship between the nucleosomal and the satellite DNA repeats. Digestion of CV-1 nuclei with micrococcal nuclease and endogenous nuclease (s) produced a series of sharp bands in the satellite DNA register over a background of heterogeneous length fragments. This observation is explained by a preferential cleavage of certain nucleotide sequences by these nucleases and is not in contradiction to our conclusion that a simple phase relationship does not exist.     

CENP-A, -B, and -C chromatin complex that contains the I-type alpha-satellite array constitutes the prekinetochore in HeLa cells

CENP-A is a component of centromeric chromatin and defines active centromere regions by forming centromere-specific nucleosomes. We have isolated centromeric chromatin containing the CENP-A nucleosome, CENP-B, and CENP-C from HeLa cells using anti-CENP-A and/or anti-CENP-C antibodies and shown that the CENP-A/B/C complex is predominantly formed on alpha-satellite DNA that contains the CENP-B box (alphaI-type array). Mapping of hypersensitive sites for micrococcal nuclease (MNase) digestion indicated that CENP-A nucleosomes were phased on the alphaI-type array as a result of interactions between CENP-B and CENP-B boxes, implying a repetitive configuration for the CENP-B/CENP-A nucleosome complex. Molecular mass analysis by glycerol gradient sedimentation showed that MNase digestion released a CENP-A/B/C chromatin complex of three to four nucleosomes into the soluble fraction, suggesting that CENP-C is a component of the repetitive CENP-B/CENP-A nucleosome complex. Quantitative analysis by immunodepletion of CENP-A nucleosomes showed that most of the CENP-C and approximately half the CENP-B took part in formation of the CENP-A/B/C chromatin complex. A kinetic study of the solubilization of CENPs showed that MNase digestion first released the CENP-A/B/C chromatin complex into the soluble fraction, and later removed CENP-B and CENP-C from the complex. This result suggests that CENP-A nucleosomes form a complex with CENP-B and CENP-C through interaction with DNA. On the basis of these results, we propose that the CENP-A/B/C chromatin complex is selectively formed on the I-type alpha-satellite array and constitutes the prekinetochore in HeLa cells.     

DNA sequence directs placement of histone cores on restriction fragments during nucleosome formation.

Restriction fragments, 203 and 144 base pairs in length, bearing the Escherichia coli lac control region have been reconstituted with the core histones from calf thymus to form nucleosomes. By several criteria the reconstituted nucleosomes are similar to native nucleosomes obtained by micrococcal nuclease digestion of calf thymus nuclei. However, sensitive nuclease digestion studies reveal subtle and important differences between native monosomes and the lac reconstitutes. Each reconstitute consists mainly of nucleosomes containing histone cores placed nonrandomly with respect to the DNA sequence. The shorter reconstitute forms asymmetric nucleosomes as evidenced by the DNase I digestion pattern. Exonuclease III digestion followed by 5'-end analysis of the larger reconstitute suggests that, of the many possible arrangements of histone core with DNA sequence, only two are highly favored.     

DNA engineering shows that nucleosome phasing on the African green monkey alpha-satellite is the result of multiple additive histone-DNA interactions

The mechanism underlying sequence-specific positioning of nucleosomes on DNA was investigated. African green monkey alpha-satellite DNA was reconstituted in vitro with histones. Histone octamers were found to adopt one major and several minor positions on the satellite repeat unit, very similar to those positions found previously in vitro, demonstrating that sequence-specific histone-DNA interactions are responsible for nucleosome positioning on this DNA. In order to understand the nature of these interactions in more detail, we have constructed a variant satellite fragment containing an insertion of half a helical DNA turn. The parent fragment directs histones to one major and two overlapping minor positions that are all affected by the insertion. All three frames respond in a unique fashion to the additional five base-pairs. From a quantitative analysis of the nucleosome positions on the engineered fragment, consensus "phasing boxes" as the basis for nucleosome positioning can be ruled out. Instead, our results argue very strongly that nucleosome positioning is due to the independent contribution of many different DNA-histone contacts along the entire core particle, in an apparently additive fashion.     

Nucleosomal structure of two Drosophila melanogaster simple satellites

Nucleosomes have been fractionated on nondenaturing polyacrylamide gels, and nucleosome subtypes containing the Drosophila melanogaster specific protein D1 and ubiquitinated core histone H2A were identified by solubility in 0.1 M NaCl before nucleoprotein gel electrophoresis. Nucleosomes which contain DNA complementary to the 1.672 density simple satellite (sequence-AATAT-) bind protein D1, as demonstrated by two-dimensional hybridization mapping. This hybridization pattern allows the identification of D1 dinucleosomes, which, like D1 mononucleosomes, are reduced in mobility on the first dimension (nucleoprotein) gel by the addition of D1, an AT sequence-specific DNA-binding protein. The 1.705 density simple satellite (sequence-AAGAG-) is also found in nucleosomes, in a radically different subset from those of the -AATAT- DNA sequence. -AAGAG- nucleosomes do not contain D1 protein, but appear to be enriched in ubiquitinated core histone H2A. One-dimensional hybridization patterns suggest that -AAGAG- nucleosomal DNA is rapidly trimmed to a shorter DNA length than either bulk or -AATAT- nucleosomes.     

A functional role for nucleosomes in the repression of a yeast promoter.

Induction of the PHO5 gene in S. cerevisiae was previously shown to be accompanied by the removal of four positioned nucleosomes from the promoter. In order to assess the role of nucleosomes in the cascade of gene activation, DNA corresponding to one of these nucleosomes was excised. In its place two foreign DNA segments of the same length were inserted: a fragment from the African green monkey alpha-satellite DNA which is known to associate with histones in a highly specific fashion to give a uniquely positioned nucleosome or, alternatively, a fragment derived from pBR322 DNA. The promoter constructs were fused to the lacZ gene on centromere plasmids and transformed into yeast cells. The satellite fragment formed a nucleosome which persisted under inducing conditions. At the same time the inducibility of the PHO5 promoter was virtually abolished. When various subfragments containing between 35 and 100 bp of the satellite segment were tested, they were all found to decrease the inducibility of the promoter, full repression required the full length molecule, however. In contrast, the pBR fragment made the promoter weakly constitutive, and induction proceeded to levels even higher than with a promoter lacking an insert. Analysis of the chromatin structure reveals a nucleosome on the pBR segment at noninducing conditions which is removed upon induction. It is concluded that the quality of the histone-DNA interactions at the promoter makes an intrinsic contribution to the regulation of the gene.     

Fractionation of nucleosomes by salt elution from micrococcal nuclease- digested nuclei

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-- 17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.     

Organisation of nucleosomal arrays reconstituted with repetitive African green monkey α-satellite DNA as analysed by atomic force microscopy

Alpha-satellite DNA (AS) is part of centromeric DNA and could be relevant for centromeric chromatin structure: its repetitive character may generate a specifically ordered nucleosomal arrangement and thereby facilitate kinetochore protein binding and chromatin condensation. Although nucleosomal positioning on some satellite sequences had been shown, including AS from African green monkey (AGM), the sequence-dependent nucleosomal organisation of repetitive AS of this species has so far not been analysed. We therefore studied the positioning of reconstituted nucleosomes on AGM AS tandemly repeated DNA. Enzymatic analysis of nucleosome arrays formed on an AS heptamer as well as the localisation of mononucleosomes on an AS dimer by atomic force microscopy (AFM) showed one major positioning frame, in agreement with earlier results. The occupancy of this site was in the range of 45–50%, in quite good agreement with published in vivo observations. AFM measurements of internucleosomal distances formed on the heptamer indicated that the nucleosomal arrangement is governed by sequence-specific DNA-histone interactions yielding defined internucleosomal distances, which, nevertheless, are not compatible with a uniform phasing of the nucleosomes with the AGM AS repeats.     

Nucleosome positioning and periodicity of satellite DNA in the liver of aging rats – Nucleosome positioning and periodicity of satellite DNA

The positioning of nucleosomes has been analysed by comparing the pattern of cutting sites of a probing reagent on chromatin and naked DNA. For this purpose, high molecular weight DNA and nuclei from the liver of young (18±2 weeks) and old (100±5 weeks) Wistar male rats were digested with micrococcal nuclease (MNase) and hybridized with ³²P-labelled rat satellite DNA probe. A comparison of the ladder generated by MNase with chromatin and nuclei indicates long range organization of the satellite chromatin fiber with distinct non-random positioning of nucleosomes. However, the positioning of nucleosomes on satellite DNA does not vary with age. For studying the periodicity and subunit structure of satellite DNA, high molecular weight DNA from the liver of young and old rats were digested with different restriction enzymes. Surprisingly, no noteworthy age-related change is visible in the periodicity and subunit structural organization of the satellite DNA. These results suggest that the nucleosome positioning and the periodicity of liver satellite DNA do not vary with age.     

Nucleosomes are positioned on mouse satellite DNA in multiple highly specific frames that are correlated with a diverged subrepeat of nine base-pairs

Nucleosome phasing on highly repetitive DNA was investigated using a novel strategy. Nucleosome cores were prepared from mouse liver nuclei with micrococcal nuclease, exonuclease III and nuclease S1. The core DNA population that contains satellite sequences was then purified from total core DNA by denaturation of the DNA, reassociation to a low Cot value and hydroxyapatite chromatography to separate the renatured satellite fraction. After end-labeling, the termini of the satellite core DNA fragments were mapped with an accuracy of +/- 1 base-pair relative to known restriction sites on the satellite DNA. Sixteen dominant nucleosome positions were detected. There is a striking correlation between these nucleosome frames and an internal highly diverged 9 base-pair subrepeat of the satellite DNA. The results are consistent with a sequence-dependent association of histone octamers with the satellite DNA. Our finding that histone octamers can interact with a given DNA in a number of different defined frames has important implications for the possible biological significance of nucleosome phasing.     

Structure of chromatin containing extensively acetylated H3 and H4

I have grown HeLa cells in 5 mM sodium n-butyrate leading to extensive in vivo histone acetylation, and have characterized the structure of chromatin containing the modified histones. Nuclear DNA in butyrate-treated cells is digested 5-10 fold more rapidly by DNAase I than the DNA of control cells. Staphylococcal nuclease degrades the two nuclear samples to acid-soluble material with identical rates; this nuclease, however, does excise nucleosomes with extensively acetylated histones from the nucleoprotein chain preferentially. The physical properties of unsheared chromatin and isolated core particles from control and butyrate-treated cells are closely similar, as are the rates of digestion of core particles from the two cell preparations by DNAase I. Determination of the relative susceptibilities of cleavage sites for DNAase I demonstrates that the site 60 bases from the ends of the DNA resistant in control cells, becomes susceptible to the nuclease in core particles containing acetylated histones. Similarly, the 5' terminal phosphate at the end of DNA in core prticles is removed by staphylococcal nuclease 2-3 fold faster in particles containing acetylated histones than in particles from control cells.     

Eukaryotic DNA does not form nucleosomes as readily as some prokaryotic DNA.

Nucleosomal-length DNA was prepared from the genomic DNA of various prokaryotic and eukaryotic organisms by limited nuclease digestion after reconstitution with core histones. The DNAs ranged in base composition from 26.5% to 72% guanosine-plus-cytosine (%GC). The nucleosomal-length DNAs were then used in a competitive reconstitution assay in order to quantitatively determine their relative abilities to form nucleosomes. The results of the assay indicate a linear dependence of the free energy of nucleosome formation on base composition and, surprisingly, show that several prokaryotic DNAs form nucleosomes as well as or better than eukaryotic DNAs.