Comparative analysis of the nucleosomal structure of rye,wheat and their relatives

Analysis of the structure of chromatin in cereal species using micrococcal nuclease (MNase) cleavage showed nucleosomal organization and a ladder with typical nucleosomal spacing of 175–185 bp. Probing with a set of DNA probes localized in the authentic telomeres, subtelomeric regions and bulk chromatin revealed that these chromosomal regions have nucleosomal organization but differ in size of nucleosomes and rate of cleavage between both species and regions. Chromatin from Secale and Dasypyrum cleaved more quickly than that from wheat and barley, perhaps because of their higher content of repetitive sequences with hairpin structures accessible to MNase cleavage. In all species, the telomeric chromatin showed more rapid cleavage kinetics and a shorter nucleosome length (160 bp spacing) than bulk chromatin. Rye telomeric repeat arrays were shortest, ranging from 8 kb to 50 kb while those of wheat ranged from 15 kb up to 175 kb. A gradient of sensitivity to MNase was detected along rye chromosomes. The rye-specific subtelomeric sequences pSc200 and pSc250 have nucleosomes of two lengths, those of the telomeric and of bulk nucleosomes, indicating that the telomeric structure may extended into the chromosomes. More proximal sequences common to rye and wheat, the short tandem-repeat pSc119.2 and rDNA sequence pTa71, showed longer nucleosomal sizes characteristic of bulk chromatin in both species. A strictly defined spacing arrangement (phasing) of nucleosomes was demonstrated along arrays of tandem repeats with different monomer lengths (118, 350 and 550 bp) by combining MNase and restriction enzyme digestion.     

An evolutionary change in telomere sequence motif within the plant section Asparagales had significance for telomere nucleoprotein complexes

In association with a phylogenetic tree of Asparagales, our previous results showed that a distinct clade included plant species where the ancestral, Arabidopsis-type of telomeric repeats (TTTAGGG)n had been partially, or fully, replaced by the human-type telomeric sequence (TTAGGG)n. Telomerases of these species synthesize human repeats with a high error rate in vitro. Here we further characterize the structure of telomeres in these plants by analyzing the overall arrangement of major and minor variants of telomeric repeats using fluorescence in situ hybridization on extended DNA strand(s). Whilst the telomeric array is predominantly composed of the human variant of the repeat, the ancestral, Arabidopsis-type of telomeric repeats was ubiquitously observed at one of the ends and/or at intercalary positions of extended telomeric DNAs. Another variant of the repeat typical of Tetrahymena was observed interspersed in about 20% of telomerics. Micrococcal nuclease digestions indicated that Asparagales plants with a human-type of telomere have telomeric DNA organised into nucleosomes. However, unexpectedly, the periodicity of the nucleosomes is not significantly shorter than bulk chromatin as is typical of telomeric chromatin. Using electrophoretic mobility shift assays we detected in Asparagales plants with a human type of telomere a 40-kDa protein that forms complexes with both Arabidopsis- and human-type G-rich telomeric strands. However, the protein shows a higher affinity to the ancestral Arabidopsis-type sequence. Two further proteins were found, a 25-kDa protein that binds specifically to the ancestral sequence and a 15-kDa protein that binds to the human-type telomeric repeat. We discuss how the organisation of the telomere repeats in Asparagales may have arisen and stabilised the new telomere at the point of mutation.     

Differential association of linker histones H1 and H5 with telomeric nucleosomes in chicken erythrocytes.

Rat liver telomeric DNA is organised into nucleosomes characterised by a shorter and more homogeneous average nucleosomal repeat than bulk chromatin as shown by Makarov et al. (1). The latter authors were unable to detect the association of any linker histone with the telomeric DNA. We have confirmed these observations but show that in sharp contrast chicken erythrocyte telomeric DNA is organised into nucleosomes whose spacing length and heterogeneity are indistinguishable from those of bulk chromatin. We further show that chicken erythrocyte telomeric chromatin contains chromatosomes which are preferentially associated with histone H1 relative to histone H5. This contrasts with bulk chromatin where histone H5 is the more abundant species. This observation strongly suggests that telomeric DNA condensed into nucleosome core particles has a higher affinity for H1 than H5. We discuss the origin of the discrimination of the lysine rich histones in terms of DNA sequence preferences, telomere nucleosome preferences and particular constraints of the higher order chromatin structure of telomeres.     

Assembly kinetics of replicating chromatin: Isolation and characterization of prenucleosomal and nucleosomal DNA

Replicating chromatin is known to be more sensitive to micrococcal nuclease than bulk chromatin. We have used this property and a fractionation procedure based on the specific release of replicating material under mild micrococcal nuclease digestion, in order to analyse both the kinetics of maturation of newly replicated DNA into nucleosomes and the structure of the replicating material. As other authors, we initially observed that repetitive unit of newly replicated chromatin was shorter than that of bulk chromatin, however this result appears to be due to sliding of nucleosomes along the chromatin fibers close to the replicating fork. Replicative chromatin was fractionated and analysed. A prenucleosomal peak was observed and preliminary characterized.     

Structure of the extrachromosomal ribosomal RNA chromatin of Physarum polycephalum

Isolated nucleoli from exponentially growing microplasmodia of Physarum polycephalum were digested with micrococcal nuclease or DNAase I, or were photoreacted with trimethyl psoralen. In the coding region for the precursor of the ribosomal RNA, micrococcal nuclease and DNAase I digestions show predominantly a smear, and treatment with psoralen leads to a fairly continuous crosslinking of the DNA. All three assays are compatible with the absence of a typical nucleosomal array in most of the gene copies. In contrast, in the central non-transcribed spacer, except in the immediate 5'-flanking region, micrococcal nuclease and DNAase I digestions yield fragments that are multiples of a basic repeat, compatible with a nucleosomal packing of this region. The crosslinking pattern with psoralen confirms this conclusion. In addition, there are three sites over 400 base-pairs long that are inaccessible for psoralen crosslinking. Two of these sites have been mapped to the putative origins of replication. In the terminal non-transcribed spacer, except in the immediate 3'-flanking region, digestions with micrococcal nuclease and DNAase I give a smeared repeat. The crosslinking pattern after treatment with psoralen suggests that this region is packed in nucleosomes, except for about 900 base-pairs constituting the telomere regions of the linear extrachromosomal palindromic rDNA. Micrococcal nuclease digestion of the immediate 5'-flanking region shows a complete absence of any nucleosomal repeat, but digestion with DNAase I leads to a faint ten base-pair repeat. In contrast, in the 3'-flanking regions both nuclease assays indicate a chromatin structure similar to the coding region. Both flanking regions are unusual with respect to psoralen crosslinking, in that crosslinking is reduced both in chromatin and deproteinized DNA. On the basis of the known sequence-dependent psoralen crosslinking and the established sequences in these regions, crosslinking should be expected to occur. However, it does not and we therefore propose the presence of an unusual DNA conformation in these regions.     

TRF2 controls telomeric nucleosome organization in a cell cycle phase-dependent manner

Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization.     

Organization of 5S genes in chromatin of Xenopus laevis.

The chromatin organization of the genes coding for 5S RNA in Xenopus laevis has been investigated with restriction endonucleases and micrococcal nuclease. Digestion of nuclei from liver, kidney, blood and kidney cells maintained in culture with micrococcal nuclease reveals that these Xenopus cells and tissues have shorter nucleosome repeat lengths than the corresponding cells and tissues from other higher organisms. 5S genes are organized in nucleosomes with repeat lengths similar to those of the bulk chromatin in liver (178 bp) and cultured cells (165 bp); however, 5S gene chromatin in blood cells has a shorter nucleosome repeat (176 bp) than the bulk of the genome in these cells (184 bp). From an analysis of the 5S DNA fragments produced by extensive restriction endonuclease cleavage of chromatin in situ, no special arrangement of the nucleosomes with respect to the sequence of 5S DNA can be detected. The relative abundance of 5S gene multimers follows a Kuhn distribution, with about 57% of all HindIII sites cleaved. This suggests that HindIII sites can be cleaved both in the nucleosome core and linker regions.     

Human cancer cells harbor T-stumps, a distinct class of extremely short telomeres

Using a modified single telomere length analysis protocol (STELA) to clone and examine the sequence composition of individual human XpYp telomeres, we discovered a distinct class of extremely short telomeres in human cancer cells with active telomerase. We name them "t-stumps," to distinguish them from the well-regulated longer bulk telomeres. T-stumps contained arrangements of telomeric repeat variants and a minimal run of seven canonical telomeric TTAGGG repeats, but all could bind at least one TRF1 or TRF2 in vitro. The abundance of t-stumps was unaffected by ATM alteration but could be changed by manipulating telomerase catalytic subunit (hTERT) levels in cancer cells. We propose that in the setting of active telomerase and compromised checkpoints characteristic of human cancer cells, t-stumps define the minimal telomeric unit that can still be protected by a TRF1/TRF2-capping complex and, further, that hTERT (or telomerase) may have a role in protecting t-stumps.     

Nascent DNA in nucleosome like structures from chromatin

We have used chromatin sensitivity to cleavage by micrococcal nuclease as a probe for differences between chromatin containing nascent DNA and that containing bulk DNA. Micrococcal nuclease digested the nascent DNA in chromatin of swimming blastulae of sea urchins more rapidly to acid-soluble nucleotides than the DNA of bulk chromatin. A part of the nascent DNA occurred in micrococcal nuclease-resistant structures which were either different from, or temporary modifications of, the bulk nucleosomes. This was inferred from the size differences between bulk and nascent DNA fragments in 10% polyacrylamide gels after micrococcal nuclease digestion of nuclei from a mixture of ¹⁴C-thymidine long- and ³H-thymidine pulse-labeled embryos. Bulk monomer and dimer DNA fragments contained about 170 and 410 base pairs (bp), respectively, when 18% of the bulk DNA had been rendered acid-soluble. At this level of digestion, “nascent monomer DNA” fragments of about 150 bp as well as 305 bp “large nascent DNA fragments” were observed. Increasing levels of digestion indicated that the large nascent DNA fragment was derived from a chromatin structure which was more resistant to micrococcal nuclease cleavage than bulk dimer chromatin subunits. Peaks of ³H-thymidine-labeled DNA fragments from embryos which had been pulse-labeled and then chased or labeled for several minutes overlapped those of ¹⁴C-thymidine long-labeled monomer, dimer and trimer fragments. This indicated that the chromatin organization at or near the replication fork which had temporarily changed during replication had returned to the organization of its nonreplicating state.     

The telomere binding protein TRF2 induces chromatin compaction

Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.     

TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection.     

Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo

Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.     

Variation in chromatin structure in two cell types from the same tissue: a short DNA repeat length in cerebral cortex neurons

We have used micrococcal nuclease as a probe of the repeating structure of chromatin in four nuclear populations from three tissues of the rabbit. Neuronal nuclei isolated from the cerebral cortex contain about 160 base pairs of DNA in the chromatin repeat unit, as compared with about 200 base pairs for nonastrocytic glial cell nuclei from the same tissue, neuronal nuclei from the cerebellum and liver nuclei. All four types of nuclei show the same features of nucleosomal organization as other eucaryotic nuclei so far studied: nucleosomes liberated by digestion with micrococcal nuclease give a “core particle” containing 140 base pairs as a metastable intermediate on further digestion and a series of single-strand DNA fragments which are mutiples of 10 bases after digestion with DNAase I. Nuclei from cerebral cortex neurons, which have a short repeat, are distinct from the others in being larger, in having a higher proportion of euchromatin (dispersed chromatin) as judged by microscopy and in being more active in RNA synthesis in vitro.     

Protection of expressed immunoglobulin genes against nuclease cleavage.

Fragmentation of the actively transcribed kappa immunoglobulin gene in mouse myeloma nuclei with micrococcal nuclease and the restriction nuclease Bsp RI reveals a chromatin structure without the regularity of repeating nucleosomes found in bulk chromatin. Such regularity is restored about 2.2 kb 3' of the coding region. An only moderately increased micrococcal nuclease sensitivity and a 65% average protection of the Bsp RI sites indicates a DNA-protein interaction in the transcribed region which is not very different from that of an inactive gene. As determined by indirect endlabeling the frequency of Bsp RI cleavage both, after very mild and exhaustive digestion, varied moderately from site to site along the gene. In addition, it was not in each case the same at analogous sites on both alleles which are both transcribed. Thus, the experiments demonstrate differences between the chromatin structures of the genes which may be related to regulatory phenomena and thereby corroborate earlier findings made with DNAase I.     

Methidiumpropyl-EDTA-iron(II) cleavage of ribosomal DNA chromatin from Dictyostelium discoideum.

We have used methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium. Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.     

Chromatin structure of the chicken lysozyme gene domain as determined by chromatin fractionation and micrococcal nuclease digestion

The chromatin structure encompassing the lysozyme gene domain in hen oviduct nuclei was studied by measuring the partitioning of coding and flanking sequences during chromatin fractionation and by analyzing the nucleosome repeat in response to micrococcal nuclease digestion. Following micrococcal nuclease digestion, nuclei were sedimented to obtain a chromatin fraction released during digestion (S1) and then lysed in tris(hydroxymethyl)aminomethane-(ethylenedinitrilo)tetraacetic acid-[ethylenebis(oxyethylenenitrilo)]tetraacetic acid and centrifuged again to yield a second solubilized chromatin fraction (S2) and a pelleted fraction (P2). By dot-blot hybridization with 14 specific probes, it is found that the fractionation procedure defines three classes of sequences within the lysozyme gene domain. The coding sequences, which partition with fraction P2, are flanked by class I flanking sequences, which partition with fractions S1 and P2 and which extend over 11 kilobases (kb) on the 5'side and probably over about 4 kb on the 3' side. The partitioning of class II flanking sequences, which are located distal of class I flanking sequences, is different from that of class I flanking sequences. Coding sequences lack a canonical nucleosome repeat, class I flanking sequences possess a disturbed nucleosome repeat, and class II flanking sequences generate an extended nucleosomal ladder. Coding and class I flanking sequences are more readily digested by micrococcal nuclease than class II flanking sequences and the inactive beta A-globin gene. In hen liver, where the lysozyme gene is inactive, coding and class I flanking sequences fractionate into fractions S2 and P2.(ABSTRACT TRUNCATED AT 250 WORDS)