Prepartation of soluble chromatin and specific chromatin fractions with restriction nucleases.

By digestion of rat liver nuclei with EndoR HaeIII, EndoR EcoRI, and EndoR Bam and subsequent lysis of the nuclei approx. 90%, 40%, and 45%, respectively, of the chromatin were solubilized. The plateau values of solubilization are in agreement with a model in which the chromatin strands are crosslinked and/or attached to a supporting structure. The distribution of DNA lengths in the soluble and insoluble chromatin fractions were determined. According to digestion experiments with restriction nucleases rat liver DNA contains highly repetitive sequences, some of which are arranged in tandem repeats of 95 and 380 nucleotide pairs, respectively. With EndoR EcoRI chromatin containing the repetitive RNA was preferentially solubilized and, by subsequent sucrose gradient centrifugation, purified to about 90%. The useful properties of chromatin prepared by the specific action of restriction nucleases are discussed.     

Engineered apoptotic nucleases for chromatin research

We have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal DNA cleavage, DNA fragmentation factor (DFF). Normally, in its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone inhibitor subunit (DFF45 or ICAD), and a 40-kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms active endonuclease homo-oligomers. Although Saccharomyces cerevisiae lacks DFF, expression of caspase-3 is lethal in this organism, but expression of the highly sequence-specific tobacco etch virus protease (TEVP) is harmless. Therefore, we inserted TEVP cleavage sites immediately downstream of the two caspase-3 cleavage sites within DFF45, generating a novel form of DFF (DFF-T) whose nuclease activity proved to be exclusively under the control of TEVP. We demonstrate that co-expression of TEVP and DFF-T under galactose control results in nucleosomal DNA laddering and cell death in S. cerevisiae. We also created synthetic DFF genes with optimized codons for high-level expression in Eschericia coli or S. cerevisiae. We further demonstrate the excellence of the synthetic gene products for in vitro mapping of the nucleosome positions and hypersensitive sites in specific genes such as the yeast PHO5.     

Specific cleavage of chromatin by restriction nucleases.

Digestion of mouse and rat liver nuclei with a restriction nuclease from Bacillus subtilis (Bsu) is examined in continuation of previous work from this laboratory (Pfeiffer et al., 1975, Nature 258, 450). The finding of more than 95% C in the 5'-termini of the DNA fragments generated during digestion with Bsu shows that the participation of endogenous nucleases in Bsu digestion is extremely small. The restriction nuclease Hae III, an isoschizomer of Bsu, yields identical degradation patterns. The patterns conform to what one expects from statistical calculations based on a nucleosome structure of chromatin with a region preferentially accessible to the nuclease of 40-50 nucleotide pairs per nucleosome. Integrity of the histones is maintained during digestion with restriction nucleases. Digestion of mouse liver nuclei with EcoRII shows that most if not all of the satellite DNA is organized in a nucleosome structure. Also in rat liver, much of the repetitive DNA appears to be present in nucleosomes.     

Stability of DNA-protein interactions in chromatin fractions with different sensitivity to nucleases

It was revealed by means of nucleoprotein-celite-chromatography that DNA-protein interactions in the chromatin fraction sensitive to micrococcal nuclease and DNase II are weaker that in the resistant one. The micrococcal nuclease destroys the DNA-matrix bond resistant to salt-urea, while DNase II does not change the DNA-matrix integrity. Tightness of the DNA-protein interactions is weakened by the increasing chromatin fragmentation, but does not depend on the size of chromatin particles.     

Organospecificity of chromatin fragmentation by endonuclease

The analysis of chromatin fragments from liver, kidney, spleen and brain by means of electrophoresis in polyacrylamide gel concentration gradient has revealed the organospecificity of the chromatin fragmentation with endonucleases. The highest endonucleolysis of chromatin was observed in spleen nuclei, it was moderate in kidney and liver and it was practically absent in brain. Differences in endonucleolysis of the organs studied demonstrate their correlation with the mitotic activity.     

Specific fragmentation of adenovirus heteroduplex DNA molecules with single-strand specific nucleases of Neurospora crassa

Heteroduplex DNA molecules, prepared by hybridizing DNA of adenovirus serotypes 1, 2 and 5 in all three combinations, have been shown by electron microscopy to contain two specifically located mismatched regions.Single-strand specific nucleases from Neurospora crassa cleaved the heteroduplexes into three fragments as demonstrated by sucrose gradient analysis, agarose gel electrophoresis and electron microscopy. These were produced in equimolar amounts, and were shown by alkaline sucrose gradient analysis to be free of internal single-strand interruptions. The size of each of the fragments was determined by sucrose gradient analysis and by electron microscopic measurements of the contour lengths. The three values obtained correspond to the lengths of the three double-stranded regions in the untreated heteroduplex molecules. The fragments, free of detectable single-stranded tails, have been isolated preparatively from neutral sucrose gradients. Renaturation kinetic analyses support the conclusion that each fragment represents a unique segment of the adenovirus genome.     

Effect of irradiation and endogenous nucleases on rat liver chromatin

These assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases. Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin. Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes. When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease, the levels of monosomes and oligosomes are identical to suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease. Histones isolated from monosomes of irradiated and unirradiated nuclei were intact, showing no fragmentation or loss of residues, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis.     

Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments

Extensive sonication of formaldehyde-crosslinked chromatin can generate DNA fragments averaging 200 bp in length (range 75–300 bp). Fragmentation is largely random with respect to genomic region and nucleosome position. ChIP experiments employing such extensively fragmented samples show 2- to 4-fold increased enrichment of protein binding sites over control genomic regions, when compared to samples sonicated to a more conventional size range (300–500 bp). The basis of improved fold enrichments is that immunoprecipitation of protein-bound regions is unaffected by fragment size, whereas immunoprecipitation of control genomic regions decreases progressively along with reduced fragment size due to fewer nonspecific binding sites. The use of extensively sonicated samples improves mapping of protein binding sites, and it extends the dynamic range for quantitative measurements of histone density. We show that many yeast promoter regions are virtually devoid of histones.     

The possible role of endogenous nucleases in the structural organization of chromatin]

Two fractions of rat liver nuclei with different buoyant density have been obtained. The electrophoretic analysis of the oligonucleosome patterns of DNA out of nuclei of these two fractions revealed different levels of activity in endonucleases. In case of inhibition during the extraction of activity in Ca, Mg-dependent endonucleases, the average size of high polymeric DNA is larger for nuclei with bigger buoyant density (fraction I) than for nuclei with smaller ones (fraction II). This finding is evidence of in situ existence of two pools of liver nuclei with different endogenic nuclease activities. In nuclear chromatin fraction I DNA is torsionally stressed; in fraction II it is relaxed that correlates with larger activity of endonucleases and smaller buoyant density of this fraction. A hypothesis on a possible role of endonucleases in chromatin structure organization has been put forward. According to this hypothesis a modulation of activity in nuclear endonucleases can determine different packaging and activity of chromatin from different pools of cellular nuclei.     

Computer analysis of chromatin arrangement and nuclear texture in follicular thyroid tumours

The differentiation of the thyroid glands follicular neoplasias into adenomas and carcinomas is currently done using the histological criteria recommended by WHO. This pilot study of 10 human follicular carcinomas and 10 folliculars adenomas demonstrates the possibility of a cytological classification using digital picture processing of high resolution cell images. Giemsa stained paraplast sections were scanned with a Colour-TV-camera, different channels were used with respect to staining and analyzing methods and computed with an image processing system. The computer aided cytophotometric methods detected significant differences in the chromatin arrangement and structure.     

Fragmentation of chromatin by endogenous nucleases, accumulation of the subnucleosomal fragments with high electrophoretic mobility]

Digestion of chromatin by endogenous nucleases to nucleosomes (140-160 base pairs of DNA) is accompanied by the accumulation of subnucleosomal DNP particles with high electrophoretic mobility (20-40 base pairs of DNA). All histones associate with the 140-160 base pairs fragment. The production of subnucleosomal DNP particles does not correlate with the degradation of histone H1 and the appearance of nucleosomes lacking histone H1. Degradation of the protein in this fragment is accompanied by the appearance of free DNA. The data obtained are in agreement with the hypothesis on the origin of subnucleosomes from the nucleosomal locus preferentially associated with the non-histone proteins and on the autonomy of these loci and of the loci associated with histone H1 in the nucleosome.     

Differences in chromatin from normal and leukemic human cells as shown by digestion with restriction nucleases

Sequence homology was found by computer analysis between potato spindle tuber viroid (PSTV) RNA and U3B snRNA of Novikoff hepatoma cells. This homology is colinear in arrangement, extends in length to 81% of the entire U3B snRNA molecule and is involved in the PSTV molecule unique sites which, if depicted in terms of the secondary structure of the circular PSTV molecule, reveal a conspicuous regularity in their location. A strong relation in primary structure between PSTV and U3B snRNA is demonstrated by statistical analysis.     

Androgen treatment protects mouse liver chromatin from cleavage by endogenous nucleases during aging

We have examined the endogenous nuclease activity of the liver of intact, castrated and testosterone-treated mice of different ages. Both Mg²⁺- and Ca²⁺-dependent endogenous nuclease activities decline in old age. Withdrawal of the hormone increases nuclease activity in the immature and young. However, testosterone administration prevents the digestion of nuclei to different extents in all ages. These findings suggest a possible protective role of testosterone in the cleavage of liver chromatin by endogenous nucleases during the aging of mice.     

Restoration of the primer activity of gamma-irradiated DNA by nucleases from rat liver chromatin

gamma-Irradiation of DNA results in a several-fold decrease of its primer activity measured as one substrate synthesis catalyzed by DNA polymerase beta. However, the combined treatment of injured DNA with 3'----5' exonuclease and endonuclease I from rat liver chromatin almost normalizes primer activity of DNA. Therefore the above-mentioned nucleases are capable of excising the gamma-injured nucleotides from 3'-OH ends of DNA.     

Utilization of an in vitro assay to evaluate chromatin degradation by candidate apoptotic nucleases

Apoptosis is commonly associated with the catabolism of the genome in the dying cell. The chromatin degradation occurs in essentially two forms: (1) internucleosomal DNA cleavage to generate oligonucleosomal-length fragments (180-200 bp and multiples thereof), and (2) cleavage of higher order chromatin structures to generate approximately 30-50 Kb fragments. To investigate this component of apoptosis and identify the nuclease(s) responsible, we have developed and utilized an in vitro assay that recapitulates the genomic destruction seen during apoptosis in vivo and allows the simultaneous analysis of both forms of DNA degradation from the same sample. Using this assay we evaluated the digestion patterns of several candidate apoptotic nucleases: DNase I, DNase II, and cyclophilin (NUC18) as well as the bacterial enzyme micrococcal nuclease (not thought to be involved in apoptosis). Chromatin degraded by DNase I formed a smear of DNA on conventional static-field agarose gels and approximately amp;30 - 50 Kb DNA fragments on pulsed field gels. In contrast, DNase II, at a physiologically relevant pH, had no effect on the integrity of HeLa chromatin in either analysis. Similar to DNase I, cyclophilin C produced only approximately 30-50 Kb DNA fragments but did not generate internucleosomal fragments. In contrast, micrococcal nuclease generated both oligonucleosomal and approximately 30-50 Kb DNA fragments. Nuclear extracts from glucocorticoid-treated apoptotic thymocytes generated oligonucleosomal DNA fragments and the larger approximately 30-50 Kb DNA fragments, fully recapitulating both types of apoptotic DNA degradation. Previously, differential sensitivity of nucleases to inhibition by Zn2+ was used to argue that two distinct enzymes mediate approximately 30-50 Kb DNA cleavage and internucleosomal DNA degradation. While, the nuclease activity present in thymocyte nuclear extracts was differentially sensitive to inhibition by Zn2+ during short term incubations it was not during prolonged digestions, suggesting that differences in DNA detection are likely to account for previous results. Together our studies show that none of the nucleases commonly associated with apoptosis could fully recapitulate the DNA degradation seen in vivo.     

Higher order chromatin structure determines double-nucleosome periodicity of DNA fragmentation

Double-nucleosome periodicity of DNA fragmentation with DNAse I in the nuclei of cells differing in size of the linker DNA length and lysine-rich histone composition was analyzed by means of nondenaturing agarose gel electrophoresis. DNAse I revealed this type of periodicity in rat thymus and CHO cell nuclei as well as in erythrocyte nuclei. It has been deduced that the so-called nucleodisome structure is also typical of cells possessing a usual DNA repeat length (200 bp or less) and lysine-rich histone H1. Two probably related events are important for establishing a clear double-nucleosome periodicity of DNA fragmentation: the replacement of H1 histone by a specific arginine-rich histone fraction (H5 histone in the case of erythrocyte) and the increase of the linker DNA length. The results are interpreted in terms of supranucleosomal organization of chromatin which may determine the dinucleosome periodicity of DNA fragmentation due to a specific packing of nucleosomes.