Organization of telomeric and subtelomeric chromatin in the higher plant Nicotiana tabacum

We have examined the structure and chromatin organization of telomeres in Nicotiana tabacum. In tobacco the blocks of simple telomeric repeats (TTTAGGG)_n are many times larger than in other plants, e.g., Arabidopsis thatiana or tomato. They are resolved as multiple fragments 60–160 kb in size (in most cases 90–130 kb) on pulsed-field gel electrophoresis (PFGE) of restriction endonuclease-digested DNA. The major subtelomeric repeat of the HRS60 family forms large homogeneous blocks of a basic 180 by motif having comparable lengths. Micrococcal nuclease (MNase) cleaves tobacco telomeric chromatin into subunits with a short repeat length of 157±5 bp; the subtelomeric heterochromatin characterized by tandemly repeated sequences of the HRS60 family is cut by MNase with a 180 by periodicity. The monomeric and dimeric particles of telomeric and subtelomeric chromatin differ in sensitivity to MNase treatment: the telomeric particles are readily digested, producing ladders with a periodicity of 7 bp, while the subtelomeric particles appear to be rather resistant to intranucleosomal cleavage. The results presented show apparent similarities in the organization of telomeric chromatin in higher plants and mammals.     

Organization of telomeric and subtelomeric chromatin in the higher plant Nicotiana tabacum

We have examined the structure and chromatin organization of telomeres in Nicotiana tabacum. In tobacco the blocks of simple telomeric repeats (TTTAGGG)_n are many times larger than in other plants, e.g., Arabidopsis thatiana or tomato. They are resolved as multiple fragments 60–160 kb in size (in most cases 90–130 kb) on pulsed-field gel electrophoresis (PFGE) of restriction endonuclease-digested DNA. The major subtelomeric repeat of the HRS60 family forms large homogeneous blocks of a basic 180 by motif having comparable lengths. Micrococcal nuclease (MNase) cleaves tobacco telomeric chromatin into subunits with a short repeat length of 157±5 bp; the subtelomeric heterochromatin characterized by tandemly repeated sequences of the HRS60 family is cut by MNase with a 180 by periodicity. The monomeric and dimeric particles of telomeric and subtelomeric chromatin differ in sensitivity to MNase treatment: the telomeric particles are readily digested, producing ladders with a periodicity of 7 bp, while the subtelomeric particles appear to be rather resistant to intranucleosomal cleavage. The results presented show apparent similarities in the organization of telomeric chromatin in higher plants and mammals.     

Identification and chromosomal organization of two rye genome-specific RAPD products useful as introgression markers in wheat.

Two rye genome-specific random amplified polymorphic DNA (RAPD) markers were identified for detection of rye introgression in wheat. Both markers were amplified in all of the tested materials that contained rye chromatin such as rye, hexaploid triticale, wheat-rye addition lines, and wheat varieties with 1BL.1RS translocation. Two cloned markers, designated pSc10C and pSc20H, were 1012 bp and 1494 bp, respectively. Sequence analysis showed that both pSc10C and pSc20H fragments were related to retrotransposons, ubiquitously distributed in plant genomes. Using fluorescence in situ hybridization (FISH), probe pSc10C was shown to hybridize predominantly to the pericentromeric regions of all rye chromosomes, whereas probe pSc20H was dispersed throughout the rye genome except at telomeric regions and nucleolar organizing regions. The FISH patterns showed that the two markers should be useful to select or track all wheat-rye translocation lines derived from the whole arms of rye chromosomes, as well as to characterize the positions of the translocation breakpoints generated in the proximal and distal regions of rye arms.     

The large-scale genomic organization of repetitive DNA families at the telomeres of rye chromosomes.

Repetitive DNA sequences in the terminal heterochromatin of rye (Secale cereale) chromosomes have consequences for the structural and functional organization of chromosomes. The large-scale genomic organization of these regions was studied using the telomeric repeat from Arabidopsis and clones of three nonhomologous, tandemly repeated, subtelomeric DNA families with complex but contrasting higher order structural organizations. Polymerase chain reaction analysis with a single primer showed a fraction of the repeat units of one family organized in a "head-to-head" orientation. Such structures suggest evolution of chromosomes by chromatid-type breakage-fusion-bridge cycles. In situ hybridization and pulse field gel electrophoresis showed the order of the repeats and the heterogeneity in the lengths of individual arrays. After Xbal digestion and pulse field gel electrophoresis, the telomeric and two subtelomeric clones showed strong hybridization signals from 40 to 100 kb, with a maximum at 50 to 60 kb. We suggest that these fragments define a basic higher order structure and DNA loop domains of regions of rye chromosomes consisting of arrays of tandemly organized sequences.     

Heterogeneous organization of a tandem repeat family in subtelomeric heterochromatin of rye

The presence of tandem repeat multicopy families in subtelomeric regions of all chromosomes is a characteristic feature of the rye karyotype, in contrast to the organization of these regions in chromosomes of extensively studied species, such as human, rice, and Arabidopsis. To study the molecular structure of these regions, we analyzed BAC clones from a library constructed from the genetic material of rye chromosome 1 short arm (1RS). Screening of the library detected numerous clones that contained copies of multicopy tandem families of DNA sequences pSc200, pSc250, and pSc119.2. An examination of the molecular organization of tandem arrays of the pSc200 family, which is the most common in the rye genome, showed that the subtelomeric 1RS region includes several such arrays, each of which contains characteristic blocks of multimers of various periodicity. Such pattern of heterogeneous organization of tandem repeat arrays differs from the view of the tandem arrays as monotonous sequence of identical monomers, which was generally accepted in recent past.     

Long-range nucleosome ordering is associated with gene silencing in Drosophila melanogaster pericentric heterochromatin

We have used line HS-2 of Drosophila melanogaster, carrying a silenced transgene in the pericentric heterochromatin, to investigate in detail the chromatin structure imposed by this environment. Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region. The repeating unit is ca. 10 bp larger than that observed for bulk Drosophila chromatin. The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged. The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin. Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin. However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole. The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing.     

Unusual chromatin in human telomeres.

We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere.     

Highly repetitive sequences in B chromosomes of Secale cereale revealed by fluorescence in situ hybridization.

An analysis of the presence and distribution of the rye and wheat repeated sequences in rye B chromosomes was carried out by fluorescent in situ hybridization. Probes used consisted of three highly repetitive sequences from rye (pSc119.2, pSc74, and pSc34) and the multigene families for the 25S-5.8S-18S and 5S rDNA from wheat (pTa71 and pTa794, respectively). pSc74 and pSc119.2 showed hybridization signals in the telomeric regions of rye B chromosomes. The remaining DNA clones did not hybridize to the B chromosomes.     

Painting the rye genome with genome-specific sequences

We used rye-specific repetitive DNA sequences in fluorescence in situ hybridization (FISH) to paint the rye genome and to identify rye DNA in a wheat background. A 592 bp fragment from the rye-specific dispersed repetitive family R173 (named UCM600) was cloned and used as a FISH probe. UCM600 is dispersed over the seven rye chromosomes, being absent from the pericentromeric and subtelomeric regions. A similar pattern of distribution was also observed on the rye B chromosomes, but with weaker signals. The FISH hybridization patterns using UCM600 as probe were comparable with those obtained with the genomic in situ hybridization (GISH) procedure. There were, however, sharper signals and less background with FISH. UCM600 was combined with the rye-specific sequences Bilby and pSc200 to obtain a more complete painting. With these probes, the rye chromosomes were labeled with distinctive patterns; thus, allowing the rye cultivar 'Imperial' to be karyotyped. It was also possible to distinguish rye chromosomes in triticale and alien rye chromatin in wheat-rye addition and translocation lines. The distribution of UCM600 was similar in cultivated rye and in the wild Secale species Secale vavilovii Grossh., Secale sylvestre Host, and Secale africanum Stapf. Thus, UCM600 can be used to detect Secale DNA introgressed from wild species in a wheat background.     

Inter- and intra-genomic transfer of small chromosomal segments in wheat-rye allopolyploids

Newly synthesized wheat-rye allopolyploids, derived from Triticum aestivum Mianyang11 × S. cereale Kustro, were investigated by sequential fluorescent in situ hybridization (FISH) and genomic in situ hybridization (GISH) using rye tandem repeat pSc200 and rye genomic DNA as probes, respectively, over the first, second and third allopolyploid generations. FISH signals of pSc200 could be observed at both telomeres/subtelomeres of all 14 chromosomes of the parental rye. In the first allopolyploid generation, there were ten rye chromosomes bearing FISH signals at both telomeres/subtelomeres and four rye chromosomes bearing FISH signals at only one telomere/subtelomere. However, in the second and the third allopolyploid generations, there were 12 rye chromosomes bearing FISH signals at both telomeres/subtelomeres and 2 rye chromosomes bearing FISH signals at only one telomere/subtelomere. Rye telomeric segments were transferred to the centromeric region of wheat chromosomes in some cells and small segments derived from non-telomeric regions of rye chromosome were transferred to the telomeric region of wheat chromosomes in some other cells. These observations indicated that the rye telomeric/subtelomeric region was unstable in newly synthesized wheat-rye allopolyploids and allopolyploidization was accompanied by rapid inter/intra-genomic exchange. The inter-genomic exchange may have occurred in somatic cells.     

AFM imaging and theoretical modeling studies of sequence-dependent nucleosome positioning

Telomeric chromatin has different features with respect to bulk chromatin, since nucleosomal repeat along the chain is unusually short. We studied the role of telomeric DNA sequences on nucleosomal spacing in a model system. Nucleosomal arrays, assembled on a 1500-bp-long human telomeric DNA and on a DNA fragment containing 8 copies of the 601 strong nucleosome positioning sequence, have been studied at the single molecule level, by atomic force microscopy imaging. Random nucleosome positioning was found in the case of human telomeric DNA. On the contrary, nucleosome positioning on 601 DNA is characterized by preferential positions of nucleosome dyad axis each 200 bp. The AFM-derived nucleosome organization is in satisfactory agreement with that predicted by theoretical modeling, based on sequence-dependent DNA curvature and flexibility. The reported results show that DNA sequence has a main role, not only in mononucleosome thermodynamic stability, but also in the organization of nucleosomal arrays.     

New Secale cereale (rye) DNA derivatives for the detection of rye chromosome segments in wheat

Subcloning of a clone of the 120-bp family of rye, pSc119, has produced two extremely useful probes. pSc119.1 assays rye-specific dispersed repetitive sequence families. It is present on all seven rye chromosomes and hybridizes to the entire length of each chromosome, with the exception of some telomeres and the nucleolar organiser region. pSc119.2, in contrast, hybridizes predominantly to the telomeric regions of rye chromosomes, with some interstitial sites. Unlike pSc119.1, it assays similar repetitive sequence families in both wheat and rye chromosomes.     

Diversified chromosomal distribution of tandemly repeated sequences revealed evolutionary trends in Secale (Poaceae)

Genomic in situ hybridization (GISH) with Secale cereale cv. ‘Jingzhou rye’ DNA as a probe to chromosomes of hexaploid triticale line Fenzhi-1 revealed that not only were all chromosomes of rye strongly hybridized along the entire chromosome length, but there were also stronger signals in terminal or subtelomeric regions. This pattern of hybridization signals is referred to as GISH banding. After GISH banding, sequential fluorescene in situ hybridizaion (FISH) with tandem repeated sequence pSc200 and pSc250 as probes showed that the chromosomal distribution of pSc200 is highly coincident with the GISH banding pattern, suggesting that GISH banding revealed chromosomal distribution of pSc200 in rye. In addition, FISH using pSc200 and pSc250 as probes to chromosomes of 11 species of the genus Secale and two artificial amphiploids (Triticum aestivum-S. strictum subsp. africanum amphiploid and Aegilops tauschii-S. silvestre amphiploid) showed that (1) the chromosomal distribution of pSc200 and pSc250 differed greatly in Secale species, and the trend towards an increase in pSc200 and pSc250 binding sites from wild species to cultivated rye suggested that pSc200 and pSc250 sequences gradually accumulated during Secale evolution; (2) the chromosomal distribution of pSc200 and pSc250 presented polymorphism on homologous chromosomes, suggesting that the same species has two heterogeneous homologous chromosomes; (3) the intensity and number of hybridization signals varied differently on chromosomes between pSc200 and pSc250, suggesting that each repetitive family evolved independently.     

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.     

The telomeric region is excluded from nucleosomal fragmentation during apoptosis, but the bulk nuclear chromatin is randomly degraded

Characteristic steps during cellular apoptosis are the induction of chromatin condensation and subsequent DNA fragmentation, finally leading to the formation of oligomers of nucleosomes. We have examined the kinetics and local distribution of this nucleosomal fragmentation within different genomic regions. For the induction of apoptosis, HL60 cells were treated with the water-soluble camptothecin derivative topotecan (a topoisomerase I inhibitor). The genomic origin of the fragments was analysed by Southern blot hybridisation of the cleaved DNA. In these experiments we observed similar hybridisation patterns of the fragmented DNA, indicating a random and synchronous cleavage of the nuclear chromatin. However, hybridisation with a telomeric probe revealed that, in contrast to the other analysed genomic regions, the telomeric chromatin was not cleaved into nucleosomal fragments despite our observation that the telomeric DNA in HL60 cells is organised in nucleosomes. We determined just a minor shortening of the telomeric repeats early during apoptosis. These observations suggest that telomeric chromatin is excluded from internucleosomal cleavage during apoptosis.     

Organization of telomeric nucleosomes: atomic force microscopy imaging and theoretical modeling

Telomeric chromatin has peculiar features with respect to bulk chromatin, which are not fully clarified to date. Nucleosomal arrays, reconstituted on fragments of human telomeric DNA and on tandemly repeated tetramers of 5S rDNA, have been investigated at single-molecule level by atomic force microscopy and Monte Carlo simulations. A satisfactory correlation emerges between experimental and theoretical internucleosomal distance distributions. However, in the case of telomeric nucleosomal arrays containing two nucleosomes, we found significant differences. Our results show that sequence features of DNA are significant in the basic chromatin organization, but are not the only determinant.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.