Compact structure of ribosomal chromatin in Xenopus laevis.

Micrococcal nuclease digestion was used as a tool to study the organization of the ribosomal chromatin in liver, blood and embryo cells of X. laevis. It was found that in liver and blood cells, ribosomal DNA is efficiently protected from nuclease attack in comparison to bulk chromatin. Although ribosomal chromatin is fragmented in a typical nucleosomal pattern, a considerable portion of ribosomal DNA retains a high molecular weight even after extensive digestion. A greater accessibility of the coding region in comparison to the non-coding spacer was found. In embryos, when ribosomal DNA is fully transcribed, these genes are even more highly protected than in adult tissues: in fact, the nucleosomal ladder can hardly be detected and rDNA is preserved in high molecular weight. Treatment of chromatin with 0.8 M NaCl abolishes the specific resistance of the ribosomal chromatin to digestion. The ribosomal chromatin, particularly in its active state, seems to be therefore tightly complexed with chromosomal proteins which protect its DNA from nuclease degradation.     

A structure of potentially active and inactive genes of chicken erythrocyte chromatin upon decondensation.

In the presence of 3 mM MgCl2 DNase I cleavage of bulk, globin and ovalbumin gene chromatin in chicken erythrocyte nuclei generates fragments which are multiples of a double-nucleosome repeat. However, in addition to the dinucleosomal periodicity beta-globin gene chromatin was fragmented into multiples of a 100 b.p. interval which is characteristic for partially unfolded chromatin. This distinction correlates with higher sensitivity of beta-globin domain to DNase I and DNase II as compared to the inactive ovalbumin gene. At 0.7 mM MgCl2 where these DNases fragment bulk chromatin into series of fragments with a 100 b.p. interval, the difference in digestibility of the investigated genes is dramatically decreased. When chromatin has been decondensed by incubation of nuclei in 10 mM Tris-buffer, DNase II generates a typical nucleosomal repeat, and the differential nuclease sensitivity of the analyzed genes is not observed. The data suggest that higher nuclease sensitivity of potentially active genes is due to irregularities in higher order chromatin structure.     

The chromatin structure of specific genes: I. Evidence for higher order domains of defined DNA sequence.

When the chromatin of Drosophila is examined by digestion with DNAase I or micrococcal nuclease, no general structural organization above the level of the nucleosome is revealed by the cleavage pattern. In contrast, the DNAase I cleavage pattern of specific regions of the Drosophila chromosome shows discrete bands with sizes ranging from a few kilobase pairs (kb) to more than 20 kb. Visualization of such higher order bands was achieved by the use of the Southern blotting technique. The DNAase I-cleaved fragments were transferred onto a nitrocellulose sheet after size fractionation by gel electrophoresis. Hybridization was then carried out with radioactively labeled cloned fragments of DNA from D. melanogaster. For the five different chromosomal regions examined, each gives a unique pattern of higher order bands on the autoradiogram; the patterns are different for different regions. Restriction enzyme cleavage of the fragments generated indicates that the preferential DNAase I cleavage sites in chromatin are position-specific. The chromosomal regions bounded by preferential DNAase I cleavage sites are referred to as supranucleosomal or higher order domains for purposes of discussion and analysis. The micrococcal nuclease cleavage pattern of chromatin at specific loci was also examined. In the one case studied in detail, this nuclease also cleaves at position-specific sites.     

Adenovirus chromatin structure at different stages of infection.

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.     

The 87A7 chromomere. Identification of novel chromatin structures flanking the heat shock locus that may define the boundaries of higher order domains

The chromatin fiber of eukaryotic chromosomes is thought to be organized into a series of discrete domains or loops. To learn more about these large-scale structures, we have examined the sequence and chromatin organization of the DNA segments surrounding the two hsp 70 genes at the Drosophila melanogaster cytogenetic locus 87A7. These studies indicate that this heat shock locus is flanked on both the proximal and distal sides by novel chromatin structures, which we have called, respectively, scs and scs' (specialized chromatin structures). Each structure is defined by two sets of closely spaced nuclease-hypersensitive sites arranged around a central nuclease-resistant segment. Our findings suggest that these two structures define the proximal and distal boundaries of the 87A7 chromomere and, hence, may be one of the first examples of anchor points for the organization of eukaryotic chromosomes into a series of discrete higher order domains. Moreover, these structures may provide focal points both for the decondensation of the chromomere when the hsp 70 genes are induced by heat shock and for the subsequent rewinding and condensation of the chromomere during recovery from heat shock.     

The structure of inactive interphase macronuclear chromatin of the ciliate Bursaria truncatella. Radial loops in the structure of chromatin clumps

The organization of chromatin in macronuclei of Bursaria truncatella cells that completed their growth and differentiation was electron microscopically studied. The data obtained showed that (1) inactive macronuclear chromatin was organized in compact chromatin clumps 120 to 180 nm in diameter linked by one or several chromatin fibres, and (2) in low salt buffer the chromatin clumps gradually unraveled, radial loops of supranucleosomal or, more often, nucleosomal structure appearing around chromatin clumps. Upon prolonged incubation in low salt buffer chromatin clumps were completely transformed into nucleosomal fibres. The data obtained evidenced in favour of a loop-packed structure of chromatin clumps.     

Chromatin structure of histone genes in sea urchin sperms and embryos.

The nucleosomal organization of active and repressed alpha subtype histone genes has been investigated by micrococcal nuclease digestion of P. lividus sperm, 32-64 cell embryo and mesenchyme blastula nuclei, followed by hybridization with 32P-labeled specific DNA probes. In sperms, fully repressed histone genes are regularly folded in nucleosomes, and exhibit a greater resistance to micrococcal nuclease cleavage than bulk chromatin. In contrast, both coding and spacer alpha subtype histone DNA sequences acquire an altered conformation in nuclei from early cleavage stage embryos, i.e., when these genes are maximally expressed. Switching off of the alpha subtype histone genes, in mesenchyme blastulae, restores the typical nucleosomal organization on this chromatin region. As probed by hybridization to D.melanogaster actin cDNA, actin genes retain a regular nucleosomal structure in all the investigated stages.     

The effect of various conditions of chromatin isolation on the nucleosomal structure of the isolated chromatin

Chromatin from calf thymus isolated under hypotonic conditions in the presence of various agents was investigated by methods of electron microscopy prior to and after EDTA treatment. It is shown that the presence of chelating agents and, especially, the application of considerable mechanical forces in the course of isolation may cause damage to the nucleosomal structure of the chromatin. Moreover, sufficiently great mechanical forces are liable to destroy the structure of the chromatin nucleosomal fibres even when they are packed in structures of a higher order of organization of the chromatin.     

Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status.

There are now many mammalian examples in which single cell assays of transgene activity have revealed variegated patterns of expression. We have previously reported that transgenes in which globin regulatory elements drive the lacZ reporter gene exhibit variegated expression patterns in mouse erythrocytes, with transgene activity detectable in only a sub-population of circulating erythroid cells. In order to elucidate the molecular mechanism responsible for variegated expression in this system, we have compared the chromatin structure and methylation status of the transgene locus in expressing and non-expressing populations of erythrocytes. We find that there is a difference in the chromatin conformation of the transgene locus between the two states. Relative to active transgenes, transgene loci which have been silenced exhibit a reduced sensitivity to general digestion by DNase I, as well as a failure to establish a transgene-specific DNase I hypersensitive site, suggesting that silenced transgenes are situated within less accessible chromatin structures. Surprisingly, the restrictive chromatin structure observed at silenced transgene loci did not correlate with increased methylation, with transgenes from both active and inactive loci appearing largely unmethylated following analysis with methylation-sensitive restriction enzymes and by sequencing PCR products derived from bisulphite-converted genomic DNA.     

Current insights into chromatin structure organization

This review summarizes current insights into organization of chromatin structure at different levels of DNA compaction. Analysis of available experimental data allowed concluding that only nucleosomal level of structural organization was sufficiently investigated, whereas structure of a 30-nm chromatin fiber remains an open issue. The data on the chromatin structure obtained at the level of the nucleus speak in favor of a biphasic fractal organization of chromatin.