Chromatin assembly in isolated mammalian nuclei.

Cellular DNA replication was stimulated in confluent monolayers of CV-1 monkey kidney cells following infection with SV40. Nuclei were isolated from CV-1 cells labeled with [3H]thymidine and then incubated in the presence of [alpha-32P]deoxyribonucleoside triphosphates under conditions that support DNA replication. To determine whether or not the cellular DNA synthesized in vitro was assembled into nucleosomes the DNA was digested in situ with either micrococcal nuclease or pancreatic DNase I, and the products were examined by electrophoretic and sedimentation analysis. The distribution of DNA fragment lengths on agarose gels following micrococcal nuclease digestion was more heterogeneous for newly replicated than for the bulk of the DNA. Nonetheless, the state of cellular DNA synthesized in vitro (32P-labeled) was found to be identical with that of the DNA in the bulk of the chromatin (3H-labeled) by the following criteria: (i) The extent of protection against digestion by micrococcal nuclease of DNase I. (ii) The size of the nucleosomes (180 base pairs) and core particles (145 base pairs). (iii) The number and sizes of DNA fragments produced by micrococcal nuclease in a limit digest. (iv) The sedimentation behavior on neutral sucrose gradients of nucleoprotein particles released by micrococcal nuclease. (v) The number and sizes of DNA fragments produced by DNase I digestion. These results demonstrate that cellular DNA replicated in isolated nuclei is organized into typical nucleosomes. Consequently, subcellular systems can be used to study the relationship between DNA replication and the assembly of chromatin under physiological conditions.     

DNA and protein components of nuclear acceptor sites for androgen receptors in the rat prostate

Androgen receptor-acceptor complexes in nuclei from rat ventral prostates were cross-linked in situ with formaldehyde and partially purified using affinity chromatography. To isolate acceptor DNA, the cross-linked receptor-acceptor complexes in formaldehyde-treated chromatin samples were adsorbed to dihydrotestosterone-17 beta-succinyl agarose, eluted with 75 microM dihydrotestosterone-1% SDS, digested with proteinase K and extracted with phenol-chloroform. After 32P end-labelling and PAGE, this DNA contained two distinct bands of DNA (about 300 and 400 base pairs respectively) which were unique relative to the total prostatic DNA. As an alternative approach for characterizing acceptor DNA, the DNA in prostatic nuclei and cross-linked chromatin was labelled with 32P by nick translation and analysed in glycerol density gradients for associations with cross-linked androgen receptors. A symmetrical 7s peak of 32P-DNA with a small amount of coincident receptor was observed in the gradients after mild trypsin treatment. In the absence of trypsin treatment, both the cross-linked receptors and the labelled DNA sedimented to the bottom of the gradients. Isolation of acceptor proteins involved iodination of cross-linked chromatin with 125I and androgen affinity chromatography. A comparison of the relative efficiency of retention and elution of 125I-proteins from different affinity columns revealed that testosterone-17 beta-succinyl agarose was potentially most suitable for purification of acceptor proteins. After electrophoresis on SDS-polyacrylamide gels, the eluates from this type of affinity matrix were found to contain two major peaks of 125I-labelled proteins--one corresponding to a protein with a similar molecular weight as the nuclear androgen receptor (33,000 Da); the other having a molecular weight of 20,000 Da. While the precise identity of this latter entity is unknown, its enrichment and retention by the affinity gel implies that it is closely associated with the androgen receptor and may be a component of the acceptor sites.     

Chromatin organization in nuclei of sea urchin embryos. Comparison with the chromatin organization of the sperm.

The chromatin in sea urchin embryo nuclei and that in sperm heads are both organized in nucleosomes but show marked differences when analyzed by endonuclease digestion. Sperm chromatin DNA appears to be totally organized in nucleosomes that are highly resistant to nuclease hydrolysis. The kinetics of formation of acid-soluble oligonucleotides is slow and concerns only about 50% of the total DNA. In contrast, the DNA of embryo chromatin does not appear to be totally organized in nucleosomes since 5 to 10% is rapidly and preferentially hydrolysed into acid-soluble oligonucleotides without any appreciable fragmentation of the remaining parts. Futher digestion causes the formation of the usual pattern of DNA bands, as detected by gel electrophoresis. The length of the DNA segment associated with the embryo nucleosomes appears to be shorter than that of the DNA segment associated with the sperm nucleosomes. The kinetics of formation of acid-soluble oligonucleotides upon digestion of embryo chromatin is much faster than that of sperm chromatin and concerns almost all the chromatin DNA.     

Analysis of the chromatin assembled in germinal vesicles of Xenopus oocytes

We have injected circular DNA, labeled with 32P at a single restriction site, into germinal vesicles of Xenopus laevis oocytes in order to study the nucleosome arrangement on the assembled minichromosomes. Two types of genes were used in these studies, the somatic 5 S RNA gene unit of Xenopus borealis and the histone gene unit of Drosophila melanogaster. We find that injections of labeled DNA alone, at 1 ng DNA per oocyte, results in irregularly spaced nucleosomes and partially supercoiled DNA molecules. However, perfectly spaced nucleosomes are assembled and fully supercoiled DNA is recovered if 5 to 20 nanograms of cold vector DNA is coinjected with the labeled DNA. At the optimum chromatin assembly conditions, the nucleosomes are perfectly spaced with a 180 base-pair periodicity, but they are randomly positioned on the DNA. The assembly of a periodic chromatin structure is accompanied by a dramatic enhancement in the expression of the injected 5 S RNA gene.     

Distribution of H1 histone in chromatin digested by micrococcal nuclease.

The relative amount of H1 histone associated with isolated nucleosomes from calf thymus was determined as a function of the extent of DNA digestion by micrococcal nuclease. Generally the amount of H1 histone associated with mononucleosomes decreases with increasing digestion until 60% of the original H1 remains associated with DNA 150 base pirs or less in size. Coincidentally, H1 histone increases relative to the other histones in aggregated material that sediments through sucrose gradients to form a pellet. However, the level of H1 histone remains at control values for oligonucleosomes (dimer to hexamer) over the 30% digestion range studied. An increase in ionic strength to 0.3 M NaCl in the density gradient reveals a different pattern of H1 binding, whereby the amount of H1 reflects the average size of the DNA fragments with which it is associated. Although there is significant binding to nucleosomes per se, it appears that the major ionic involvement of H1 is with internucleosomal spacer DNA.     

Subunit structure of simian-virus-40 minichromosome.

Electron microscopic evidence indicates that Simian virus 40 (SV40) minichromosomes extracted from infected cells consist of 20 +/- 2 nucleosomes, each containing 190 -- 200 base pairs of DNA. About 50% of the nucleosomes are not close together, but connected by segments of DNA of irregular lengths which correspond to about 15% of the viral genome, irrespective of the ionic strength. Micrococcal nuclease digestion studies show that there is about 200 base pairs of DNA in the biochemical unit of SV40 chromatin. Therefore, the visible internucleosomal DNA of the SV40 minichromosome does not arise from an unfolding of a fraction of the 190 - 200 base pairs of DNA initially wound in the nucleosome. These results support the chromatin model which proposes that the same DNA length is contained in the nucleosome and the biochemical unit. Results from extensive micrococcal nuclease digestion suggest that an SV40 nucleosome consists of a 'core' containing a DNA segment of about 135 base pairs associated to a DNA fragment more susceptible to nuclease attack. The addition of histone H1 results in a striking condensation of the SV40 minichromosome, which supports the assumption that histone H1 is involved in the folding of chromatin fibers.     

Isolation of a chromatin fraction from calf endometrium highly enriched in estradiol binding sites.

The intranuclear distribution of [3H]-estradiol binding sites was studied in highly purified nuclei isolated from calf endometrial tissue pre-incubated with the labeled hormone. The major part (approximately 85%) of the receptor bound estradiol was found associated with the extranucleolar chromatin; only a negligible amount of [3H]-estradiol (approximately 8%) sedimented with the nucleolar fraction. [3H]-estradiol labeled chromatin was then fragmented by sonication and fractionated by sucrose density gradient sedimentation under different conditions of centrifugation. The vast majority of the [3H]-estradiol was invariably found to be associated with a fast sedimenting fraction which contained only 5 to 10% of the nuclear DNA. The concentration of estradiol receptors (per weight of DNA) in this fraction was 25- to 50-fold higher than that found in the slow sedimenting major chromatin component. Chemical analysis showed this fraction to have a high protein/DNA ratio but no phospholipids were detected.     

Distribution of estradiol receptor and vitellogenin gene in chick liver chromatin fractions.

The distribution of estradiol receptor and vitellogenin gene was studied in estradiol stimulated chick liver chromatin fractions prepared by limited DNAse II digestion and MgCl2 precipitation. The receptor was found in all fractions, undigested chromatin (P1), Mg2+ insoluble chromatin (P2) and Mg2+ soluble chromatin (S2). This last fraction was rich in acidic proteins, had a high protein:DNA ratio (7.0 w/w), contained 28% of rapidly labelled RNA, 20% of the receptor, 3-5% of chromatin DNA and showed a 2 fold enrichment of vitellogenin DNA sequences over unfractionated chromatin as well as P1 and P2 DNA. On isopycnic metrizamide gradients, all chromatin fractions showed a receptor peak banding at 1.23 g/cm3, the density of nucleoproteins. Hybridization experiments showed that the DNA banding at this density in fraction S2 was enriched 4 fold in vitellogenin DNA sequences over unfractionated chromatin as well as P1 and P2 DNA. These results suggest an association of hormone receptor complex with nucleoprotein structures of an apparently active chromatin fraction.     

Copper-rich nucleoprotein generated by micrococcal nuclease

Nuclei from calf thymus tissue digested with micrococcal nuclease under nonchelating conditions yielded soluble nucleoprotein enriched in copper. Following limited digestion, the ratio of μg Cu:mg DNA was inversely related either to percent solubility of chromatin or to levels of enzyme maintaining an enzyme:A ₂₆₀ ratio of 0.059. The enzyme appeared to cleave preferentially regions of chromatin where copper is localized, releasing no additional metal upon further digestion. Moreover, the highest copper: DNA ratio was always associated with the least-digested sample. The distribution between copper and angiotensin II (AII) in chromatin fragments following slight nuclease digestion suggests a possible link between copper and nuclear AII binding. When nuclei are incubated with AII prior to digestion and dialysis, solubilized chromatin contained about three times more copper than buffer control. Metal profiles generated from gel (A-5 M) chromatography for these samples were distinctive: copper peaks appeared near or adjacent to linker DNA regions, and in the case of AII, coincided with fragments containing specific AII receptors; thus, there appears to be an enrichment of copper in these active nucleoprotein fragments.     

Distribution of estrogen receptors in subfractions of hen oviduct chromatin.

Hen oviduct chromatin was digested with DNase II and separated into two fractions. The MgCl2 insoluble chromatin fraction (43% of the total DNA) was enriched in nucleosome-like particles, which sedimented at 11 S and contained 185 base pairs of DNA. The MgCl2 soluble chromatin fraction (5% of the total DNA) was characterized by 5 S and 14 S peaks in sucrose gradients; Estrogen receptors in the chromatin fractions were labelled with [3H] estradiol using the steroid exchange assay. The concentration of receptors in the MgCl2 soluble chromatin was 4;5 times higher than that in the MgCl2 insoluble chromatinmin sucrose gradient analysis the 11 S particles displayed a negligible specific radioactivity suggesting that estrogen receptors mainly bind to extranucleosomal chromatin.     

Effects of ionic strength on endogenous nuclease activity in chelated and nonchelated chromatin

Calf thymus chromatin, isolated using a standard (low ionic strength, but nonchelating) isolation protocol, dialyzed against either Tris-PMSF or Tris-EDTA, was reconstituted in a high salt compacting buffer (COM) or a low salt dispersing buffer (DIS) prior to digestion with endogenous nucleases. A greater level of enzyme activity occurred when chromatin was in a condensed state (COM buffer) and not chelated prior to digestion. In contrast, chromatin chelated by dialysis against Tris-EDTA prior to digestion showed higher levels of enzyme activity in the dispersed state (DIS buffer). Nonchelated undigested chromatin contained 0.280 +/- 0.16 ug copper/mg DNA and and 0.305 +/+- 0.09 ug zinc/mg DNA. Chelation removed about 78% of copper per mg DNA and approximately 65% of zinc per mg DNA. In COM buffer after a 20 min digestion, the solubilized fraction was enriched in copper showing about 20 X more metal per mg DNA than nonchelated chromatin. Approximately the same amount of zinc was found in both chelated and nonchelated chromatin while there was less zinc in chelated chromatin solubilized in DIS buffer. Thus, chelation has important effects on the digestibility of chromatin and on the type of ionic environment that provides the most favorable conditions for endogenous nuclease activity.     

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.     

Preparation and physical characterization of a homogeneous population of monomeric nucleosomes from HeLa cells.

We describe a method of isolating a homogeneous population of "trimmed" monomeric nucleosomes from Hela cells. These nucleoprotein particles contain a 140 +/- 5 base pair length of DNA and have a histone/DNA ratio of 1.2. They lack H1 and contain equal amounts of the four smaller histones. The DNA contains no single strand nicks. The particles sediment with an S20,w of 11S in D2O density gradients. After formaldehyde fixation, they band at a density of 1.4370 in neutral CsCl. Digestion of nucleosomes with either micrococcal nuclease or DNase I generates the same pattern of DNA fragments observed when intact nuclei are digested. Circular dichroism spectra indicate that the 280 nm positive ellipticity maximum of nucleosomes is about one-half that of chromatin. In the presence of 6 M urea, nucleosomes sediment with an S20,w of 6S, have a multiphasic thermal denaturation profile, and exhibit a circular dichroic spectrum nearly identical to that of B-form DNA. Our yield of purified nucleosomes (10-15% of the input DNA) is similar to the yields of other methods; our nucleosome population is substantially more homogeneous than those previously reported.     

Changes in chromatin structure at the replication fork. II The DNPs containing nascent DNA and a transient chromatin modification detected by DNAase I.

Discrete deoxyribonucleoproteins (DNPs) containing nascent and/or bulk DNA, were obtained by fractionating micrococcal nuclease digests of nuclei form 3H-thymidine pulse (15-20 sec) and 14C-thymidine long (16 h) labeled sea urchin embryos in polyacrylamide gels. One of these DNPs was shown to contain the micrococcal nuclease resistant 300 bp "large nascent DNA" described in Cell 14, 259-267, 1978. The bulk and nascent mononucleosome fractions provided evidence for the preferential digestion by micrococcal nuclease of nascent over bulk linker regions to yield mononucleosome cores with nascent DNA. DNAase I was used to probe whether any nascent DNA is in nucleosomes. Nascent as well as bulk single-stranded DNA fragments occurred in multiples of 10.4 bases with higher than random frequencies of certain fragment sizes (for instance 83 bases) as expected from a nucleosome structure. However, a striking background of nascent DNA between nascent DNA peaks was observed. This was eliminated by a pulse-chase treatment or by digestion of pulse-labeled nuclei with micrococcal nuclease together with DNAase I. One of several possible interpretations of these results suggests that a transient change in nucleosome structure may have created additional sites for the nicking of nascent DNA by DNAase I; the micrococcal nuclease sensitivity of the interpeak radioactivity suggest its origin from the linker region. Endogenous nuclease of sea urchin embryos cleaves chromatin DNA in a manner similar to that of DNAase I.     

The higher order repeat structure of chromatin is built up of globular particles containing eight nucleosomes

Mild nuclease digestion of rat liver chromatin generates particles with sedimentation coefficients of about 33S, 60S, and 90S (in 50 mM NaCl). The kinetics of appearance and disappearance of these particles with progressive digestion suggest that they are produced by cleavage from a higher order repeat structure, the 33S particle representing the monomer. At an intermediate stage of digestion, about 75 % of the nuclear chromatin can be recovered as monomers to trimers of this higher order structure. Sedimentation profiles indicate that monomer particles containing 7–8 nucleosomes occur at the highest frequency. The DNA fragments in monomers have a size corresponding to hepta- and octanucleosomes, and those in dimers have a size corresponding to chains of sixteen nucleosomes. The higher order repeat structure is only stable between 30 and 200 mM NaCl; the particles unfold below 30 and above 200 mM NaCl. When examined by electron microscopy, monomers and dimers appear as compact globular structures. Relaxation by lowering the salt concentration results in the appearance of polynucleosomes with a chain length of eight beads in the monomer and sixteen in the dimer particle. These results indicate that the unit particle of the higher order repeat structure of rat liver chromatin contains eight nucleosomes.