Degradation of chromosomal proteins during dissociation and reconstitution of chromatin

Histones and nonhistone chromosomal proteins are degraded when chromatin is exposed to 2 M NaCl-5 M urea (pH 6–8) which has been most often used for disscciation and reconstitution of chromatin. Histones and nonhistone proteins are also degraded in 5 M urea (pH 6–8).     

Complete displacement of somatic histones during transformation of spermatid chromatin: a model experiment.

Displacement of histones from calf thymus chromatin has been studied in an attempt to postulate the mechanisms involved in the total removal of somatic-type histones during transformation of spermatid chromatin. When chromatin is saturated with protamine (protamine/DNA, 0.5), histone I becomes displaceable at 0.15-0.3 M NaCl, suggesting that direct replacement by highly basic sperm histone could be a mechanism for its removal. While histone I is the only histone which is extensively degraded upon incubation of chromatin and, therefore, proteolysis might provide an additional mechanism for the removal of this histone, acetylation of chromatin by acetic anhydride greatly increases suscpetibility of histones IIb1, IIb2, and III to the chromosomally associated protease. These histones are extensively degraded and displaced from the DNA upon incubation of the acetylated chromatin. Although histone IV is not appreciably degraded, the proteolytic removal of acetylated histone III from chromatin weakens the interaction of acetylated histone IV to the DNA, and this histone becomes dissociable at 0.3 M NaCl. A comparison of the extent of chemical acetylation of individual histones observed in this investigation with that of enzymatic acetylation which can be achieved in vivo suggests that acetylation and proteolysis could be a mechanism for the removal of histone IIb2 and III. The displacement of histones IIb1 and IV could be explained on the basis of decreased binding to DNA as a result of their acetylation together with the proteolytic removal of their respective partner histones, IIb2 and III.     

Chromatin proteins as a possible target for antitumour agents: alterations of chromatin proteins in dibromodulcitol treated Yoshida tumors

The effect of dibromodulcitol (DBD) on Yoshida sarcoma chromatin components has been investigated. Measurements on the radioactivity of nuclear components after in vivo treatment with [3H]DBD for 1 h indicated preferential drug binding to the high molecular weight component of the nuclear residual acidic protein (non-histones) and also to Histone 1 (H1) (very lysine rich, F1). Two-hour DBD treatment resulted in partial degradation and reduced [3H]leucine incorporation into the same fractions of chromatin. However, 6 h after DBD treatment, the synthesis of the degraded chromatin proteins began and by 24 h was completed. During the same treatment period the composition of chromatin showed a remarkable alteration; 2 h after DBD treatment the amount of the nuclear residual acidic proteins relative to DNA decreased by approx. 50%, but returned to control value 24 h after drug treatment. This in conjunction with the data on [3H]leucine incorporation suggests that certain chromatin proteins are degraded and subsequently newly synthesised after DND treatment resulting in an exchange of chromatin components. The formation of a nucleohistone complex between H1 and DNA was inhibited by pretreatment of H1 and DBD, dianhydrodulcitol (DAD) and bischloroethylnitrosourea (BCNU).     

DNA Methylation is Reduced in DNasel-Sensitive Regions of Plant Chromatin

Pea, barley, and corn chromatin was isolated and subjected to limited digestion with DNasel. The preferentially degraded, small molecular weight fraction, presumably consisting of sequences in an active state of expression, was isolated and its 5-methylcytosine content determined. The DNasel-sensitive chromatin fraction from all three plant species investigated contained a markedly reduced level of DNA methylation compared to total DNA.     

Properties of condensed chromatin in barley nuclei

A method for isolation and purification of intact nuclei from barley leaves was developed and several properties of the chromatin were studied. The dense structure of the main part of the chromatin does not alter the accessibility of the DNA to nucleases. 60% of the nuclear DNA can be degraded by micrococcal endonuclease. Nevertheless the solubility of the chromatin fragments depends on the extent of nuclease digestion; solubilisation occurring only when the major part of the internucleosomal DNA was degraded (30% of digestion). Electron microscopic observations suggest that this was due to particularly dense organization of the chromatin in situ. The possible physiological meaning of some of these properties are discussed.     

Effect of cycloheximide on the morphological and biochemical changes in chromatin in non-irradiated and irradiated rat thymocytes

The level of chromatin degradation was studied and the method of electron-microscopy was used to estimate the changes in the ultrastructure of irradiated and nonirradiated thymocytes of rats treated with cycloheximide. The latter was found to decrease the degree of derangement of nuclear ultrastructure and the level of chromatin degradation in exposed animals and to increase the yield of these damages in thymocytes of nonirradiated animals. The electronmicroscopic determinations showed that the percentage of thymocytes with the impaired nucleus structure is twice as high as that of degraded chromatin. The causes of the quantitative disagreement between the morphological and biochemical indices of the interphase thymocyte death are discussed.     

Structure of chromatin subunits: an endonuclease Serratia marcescens study.

Electrophoretic properties of chromatin subunits--nucleosomes--obtained by treatment of chromatin with the Serratia marcescens endonuclease have been studied. Double-stranded breaks of DNA between adjacent nucleosomes do not necessarily lead to their disjunction. Fragmentation of the DNA within the nucleosomes may proceed simultaneously with the breakdown of the DNA between the nucleosomes at early stages of the endonuclease digestion. Electrophoretic mobility and chromatographic properties of mononucleosomes, their dimers and trimers with internally degraded DNA was not changed. It has been deduced that the integrity of chromatin particles with internally fragmented DNA is supported by histone interaction inside and between the nucleosomes.     

Template active chromatin structures: Degradation by deoxyribonuclease I

Chicken embryos were pulse-labelled in vivo with [³H]uridine (10 min), the chromatin isolated and treated with DNAse I. The residual chromatin was separated from the degradation products by centrifugation. The nascent pulse-labelled RNA is completely recovered in the residual chromatin even after prolonged incubation with DNAase I, whereas the DNA is completely degraded to 80 base polynucleotide fragments and smaller fragments.Abbreviations cDNA DNA complementary to mRNA - DOC deoxycholate - EDTA ethylendiamintetraacetic acid - SDS sodium-dodecylsulfate     

Integrity of proteins in reconstituted chromatin.

Rat liver chromatin reconstituted from fractionated histones, chromosomal non-histone proteins, and DNA is extensively degraded by chromatin-bound protease.     

Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

 Apoptotic cell nuclei are known to stain hyperchromatically with absorption dyes and dimly with many DNA fluorochromes. We hypothesised that both optical phenomena have the same cause - the ability of apoptotic chromatin to aggregate cationic dyes. This hypothesis was tested using prednisolone-primed rat thymus, which is known to contain apoptotic cells. The apoptotic cells were classified as early and late, based on their morphology, in thin and semithin sections and in thymus imprints on slides. Direct reaction for DNA strand breaks (TUNEL) indicated the presence of breaks in both categories of cells, with more intense labelling in late apoptosis. The chromatin ultrastructure of early apoptotic cells initially retained the supranucleosomal order of packaging which characterises control cells, whereas the dense chromatin of late apoptotic cells possessed the degraded structure. Absorption spectra of the toluidine blue-stained early apoptotic cell chromatin revealed a metachromatic shift, indicating a change of DNA conformation and polymerisation of the dye. When the staining was performed by acridine orange (preceded by a short acid treatment), a paradoxical several-fold increase of fluorescence intensity at a several-fold dilution of the dye was found. The simultaneous reduction of the ratio of red to green components of fluorescence confirmed that the concentration-dependent fluorescence quenching was due to aggregation of the dye. The results suggest that the enhanced affinity of the chromatin of early apoptotic cells for cationic dyes is associated with conformational relaxation rather than degradation of DNA. In late apoptotic cells, the very dense packaging of degraded DNA promotes further aggregation of dyes. The results suggest alternative methods for detection and discrimination of early and late apoptotic cells. Accepted: 12 February 1997     

A lysine-rich protein functions as an H1 histone in Dictyostelium discoideum chromatin.

Mononucleosomes released from Dictyostelium discoideum chromatin by micrococcal nuclease contained two distinctive DNA sizes (166-180 and 146 bp). Two dimensional gel electrophoresis suggested a lysine-rich protein protected the larger mononucleosomes from nuclease digestion. This was confirmed by stripping the protein from chromatin with Dowex resin. Subsequently, only the 146 bp mononucleosome was produced by nuclease digestion. Reconstitution of the stripped chromatin with the purified lysine-rich protein resulted in the reappearance of the larger mononucleosomes. Two-dimensional gel electrophoresis showed the protein was associated with mononucleosomes. Hence, the protein functions as an H1 histone in bringing the two DNA strands together at their exit point from the nucleosome. Trypsin digestion of the lysine-rich protein in nuclei resulted in a limiting peptide of approx. 10 kilodaltons. Trypsin concentrations which degraded the protein to peptides of 12-14 kilodaltons and partially degraded the core histones did not change the DNA digestion patterns obtained with micrococcal nuclease. Thus, the trypsin-resistant domain of the lysine-rich protein is able to maintain chromatosome structure.     

Digestion of chromatin with deoxyribonuclease II

The action of DNAse II on DNA in chromatin was studied. The formation of acid-soluble products followed a two-phase kinetic curve. At the end of the first more rapid phase about 25% of DNA was degraded. Early in the degradation process DNA was converted into double stranded fragments, whose sizes were multiples of about 180 base pairs. As the degradation proceeded these fragments were reduced in size. After denaturation DNA from digested chromatin was resolved into discrete single stranded fractions, exact multiples of a ten-nucleotide length, forming a pattern very similar to that observed with DNAse I.     

Proliferating cell nuclear antigen-dependent rapid recruitment of Cdt1 and CRL4Cdt2 at DNA-damaged sites after UV irradiation in HeLa cells

The licensing factor Cdt1 is degraded by CRL4(Cdt2) ubiquitin ligase dependent on proliferating cell nuclear antigen (PCNA) during S phase and when DNA damage is induced in G(1) phase. Association of both Cdt2 and PCNA with chromatin was observed in S phase and after UV irradiation. Here we used a micropore UV irradiation assay to examine Cdt2 accumulation at cyclobutane pyrimidine dimer-containing DNA-damaged sites in the process of Cdt1 degradation in HeLa cells. Cdt2, present in the nucleus throughout the cell cycle, accumulated rapidly at damaged DNA sites during G(1) phase. The recruitment of Cdt2 is dependent on prior PCNA chromatin binding because Cdt2 association was prevented when PCNA was silenced. Cdt1 was also recruited to damaged sites soon after UV irradiation through its PIP-box. As Cdt1 was degraded, the Cdt2 signal at damaged sites was reduced, but PCNA, cyclobutane pyrimidine dimer, and XPA (xeroderma pigmentosum, complementation group A) signals remained at the same levels. These findings suggest that Cdt1 degradation following UV irradiation occurs rapidly at damaged sites due to PCNA chromatin loading and the recruitment of Cdt1 and CRL4(Cdt2), before DNA damage repair is completed.     

Bleomycin-induced aggregation of presolubilized and nuclear chromatin from L1210 cells

These studies have identified a new activity of bleomycin (in addition to the well-known DNA cleavage): drug-induced chromatin aggregation. Bleomycin treatment of presolubilized chromatin from L1210 nuclei resulted in two types of effect as shown by sedimentation analysis of intact nucleoprotein. The first effect was a dose-dependent fragmentation of chromatin to mononucleosomes (12 S) and subnucleosomal fragments (under 5 S). The second effect was aggregation manifested by the generation of large chromatin particles (over 120 S) that sedimented faster than the original material (20-40 S). Bleomycin treatment of nuclei from L1210 cells resulted in a similar, almost bimodal, size distribution of drug-released chromatin fragments. Increasing levels of bleomycin produced a gradual enhancement of the amount of small fragments (under 5 S) accompanied by generation of large, aggregated particles. Aggregation caused by high drug concentrations significantly reduced the overall extent of chromatin solubilization and allowed only the release of the most degraded fragments from nuclei. The aggregation required intact nucleoprotein, since it was not detected after high-salt deproteinization of bleomycin-treated presolubilized or nuclear chromatin. The aggregation phenomenon reflects a novel activity of bleomycin which may contribute to the drug's antiproliferative properties.     

Barriers to nuclease Bal31 digestion across specific sites in simian virus 40 chromatin.

A portion of the nucleoprotein containing viral DNA extracted from cells infected by simian virus (SV40) is preferentially cleaved by endonucleases in a region of the genome encompassing the origin of replication and early and late promoters. To explore this nuclease-sensitive structure, we cleaved SV40 chromatin molecules with restriction enzymes and digested the exposed termini with nuclease Bal31. Digestion proceeded only a short distance in the late direction from the MspI site, but some molecules were degraded 400 to 500 base pairs in the early direction. By comparison, BglI-cleaved chromatin was digested for only a short distance in the early direction, but some molecules were degraded 400 to 450 base pairs in the late direction. These barriers to Bal31 digestion (bracketing the BglI and the MspI sites) define the borders of the same open region in SV40 chromatin that is preferentially digested by DNase I and other endonucleases. In a portion of the SV40 chromatin, Bal31 could not digest through the nuclease-sensitive region and reached barriers after digesting only 50 to 100 base pairs from one end or the other. Chromatin molecules that contain barriers in the BglI to MspI region are physically distinct from molecules that are open in this region as evidenced by partial separation of the two populations on sucrose density gradients.     

DNaseI sensitivity of the rat albumin and alpha-fetoprotein genes.

We have analyzed the DNaseI sensitivity of chromatin from the rat albumin and alpha-fetoprotein genes in the fetal liver (which synthesizes albumin and alpha-fetoprotein), adult liver (which synthesizes albumin), fetal yolk sac (which synthesizes alpha-fetoprotein), and adult kidney (which synthesizes neither). Active genes were much more sensitive than their kidney counterparts, and the adult liver alpha-fetoprotein and fetal yolk sac albumin genes showed intermediate levels of sensitivity. Sensitivity was analyzed as a function of the extent of DNaseI digestion. Rate constants were calculated for the degradation of individual DNA hybridization bands and normalized to the intrinsic rate constants of the same bands degraded in purified DNA. This enabled us to eliminate the inconsistencies that otherwise result from comparing chromatin sensitivity of different DNA sequences, or chromatin sensitivity in different nuclear environments.     

Sperm nucleosomes disassembly is a requirement for histones proteolysis during male pronucleus formation

We had previously reported that a cysteine-protease catalyzes the sperm histones (SpH) degradation associated to male chromatin remodeling in sea urchins. We found that this protease selectively degraded the SpH leaving maternal cleavage stage (CS) histone variants unaffected, therefore we named it SpH-protease. It is yet unknown if the SpH-protease catalyzes the SpH degradation while these histones are organized as nucleosomes or if alternatively these histones should be released from DNA before their proteolysis. To investigate this issue we had performed an in vitro assay in which polynucleosomes were exposed to the active purified protease. As shown in this report, we found that sperm histones organized as nucleosomes remains unaffected after their incubation with the protease. In contrast the SpH unbound and free from DNA were readily degraded. Interestingly, we also found that free DNA inhibits SpH proteolysis in a dose-dependent manner, further strengthening the requirement of SpH release from DNA before in order to be degraded by the SpH-protease. In this context, we have also investigated the presence of a sperm-nucleosome disassembly activity (SNDA) after fertilization. We found a SNDA associated to the nuclear extracts from zygotes that were harvested during the time of male chromatin remodeling. This SNDA was undetectable in the nuclear extracts from unfertilized eggs and in zygotes harvested after the fusion of both pronuclei. We postulate that this SNDA is responsible for the SpH release from DNA which is required for their degradation by the cysteine-protease associated to male chromatin remodeling after fertilization.     

A high molecular weight RNA fraction in chromatin.

Utilizing a new chromatin isolation and fractionation technique we have obtained a high molecular weight RNA fraction from L-929 cell chromatin. The synthesis of this RNA is not greatly inhibited by concentrations of 0.04 mug/ml actinomycin D in the medium. Its synthesis appears to be strongly inhibited by 2 mug/ml of alpha-amanitin. The RNA appears to be quickly degraded (or removed from the chromatin) and does not contain a poly(A) sequence at its 3'-OH terminal end. Our working hypothesis is that this RNA is "nascent" heterogenous nuclear RNA partially transcribed from regions of the chromatin.