Digestion of insect chromatin with micrococcal nuclease, DNase I and DNase I combined with single-strand specific nuclease S1.

The chromatin of the lepidopteran Ephestia kuehniella was digested by micrococcal nuclease, DNase I and S1-nuclease combined with DNase I pretreatment. The resulting DNA fragments were analyzed by gel electrophoresis and compared with the DNA fragments of rat liver nuclei obtained by the same process. Extensive homology was revealed between insect and mammalian chromatin structure. The combined DNase I- S1-nuclease digestion yields double-stranded DNA fragments of lengths from 30 to 110 base-pairs. These DNA fragments are not obtained from nuclei predigested extensively with micrococcal nuclease. The results are discussed with respect to the internal structure of the chromatin subunit.     

Physical-chemical properties of the estrogen receptor released by deoxyribonuclease I

The physical-chemical properties of the nuclear estrogen receptor released by DNase I were characterized. Nuclei were isolated from MCF-7 cells previously exposed to 10-nM-[3H]estradiol. The parameters determined were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. The properties of the receptor released by DNase I obtained from Worthington were compared to the properties of the receptor released by DNase I obtained from Sigma. Digestion with DNase I (Worthington) excised a receptor form which could be solubilized from nuclei by EDTA. This form sedimented at 5.2S with a Rs = 7.08 nm and a calculated Mr = 152.000. About 40% of this receptor form bound to a DNA-cellulose column. 0.4 M KCl dissociated this receptor form into a smaller form sedimenting at 4.2S with Rs = 4.64 nm and a calculated Mr = 80.000. The properties of the receptor solubilized by micrococcal nuclease followed by DNase I (Worthington) digestion were identical to the properties of the DNase I (Worthington) released receptor. Digestion with DNase I (Sigma) released a 3.2S receptor form, which diffused through the nuclear membrane and a 4-5S form which could be extracted from nuclei by EDTA. The 3.2S receptor had a Rs = 2.41 nm, a calculated Mr = 32.000 and less than 5% of it bound to a DNA-cellulose column. Digestion with micrococcal nuclease followed by DNase I (Sigma) solubilized a receptor form with identical properties to the 3.2S receptor. These results suggest that DNase I (Worthington) released a receptor form still associated with some molecules, probably chromatin proteins, which complexed it to DNA, while DNase I (Sigma) released the estradiol binding fragment of the receptor (meroreceptor) as a result of a proteolytic activity present in this preparation.     

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.     

Subunit organization of Euglena chromatin

Digestion of $\text{Euglena}$ nuclei or extracted chromatin with micrococcal nuclease results in the identification of a repeating structure. The DNA repeat size, analyzed on agarose and polyacrylamide gels, is found to be 225±13 base pairs. DNase I digestion produces a serie of fragments multiples of roughly 10 bases. Eventhough pressure shearing is necessary to disrupt the though pellicule of the phytoflagellate, we confirm that, in $\text{Euglena}$, chromatin organization is similar to that of other eukaryotes.     

DNA-histone interaction in the vicinity of replication points.

Chromatin replication was studied in isolated nuclei from Concanavalin A activated lymphocytes. Digestion with micrococcal nuclease revealed that the resistant fraction of in vitro replicated DNA is associated with nucleosomes. Earlier experiments had shown that the nuclease resistant fraction of nascent DNA is composed of fragments which are shorter than the nuclease resistant fragments of bulk DNA. In this communication we demonstrate that the short fragments of nascent DNA are differently bound to nucleosome like structures compared to bulk DNA. At 0.5 M NaCl a fraction of pulse labeled labeled DNA is released from these structures and appears as free double stranded DNA of about 140 base pair length (5S DNA) while the 185 pair fragments of mature replicated DNA remain attached to nucleosomes under these conditions. The experiments may indicate that the interaction of a fraction of replicating DNA with histones differs from that of bulk DNA.     

Analysis of the 4-hydroxytamoxifen (4-OHTAM) bound nuclear estrogen receptor from MCF-7 cells by limited proteolysis

The assumption that a different conformational form was induced in the nuclear estrogen receptor following binding by antiestrogens compared to estrogens was studied by analysing the proteolytic fragments of the receptor following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from MCF-7 cells previously exposed to [3H] 4-OHTAM. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients (S) determined on a sucrose gradient and from the Stokes radii (Rs) estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 155,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 63,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. A similar receptor molecule was released by chymotrypsin from intact nuclei. Digestion of the micrococcal nuclease hydrolysate with trypsin degraded the receptor to a form of a Mr = 67,000 which could not be further dissociated by 0.4 M KCl and 3 M urea. Digestion of intact nuclei with trypsin followed by micrococcal nuclease, solubilized a receptor form of Mr = 80,000 which could be further dissociated with 0.4 M KCl and 3 M urea to a receptor form of Mr = 67,000. This trypsin degraded receptor form seems to be similar in Mr to the chymotrypsin degraded form. On the other hand different receptor fragments of Mr = 33,000 and Mr = 60,000 were excised by chymotrypsin and trypsin respectively from the estradiol ligated estrogen receptor. (Geier et al., J. steroid Biochem. 26 [1987] 35-40.) These results support the assumption of a different conformational form for the antiestrogen ligated receptor, compared to the estrogen ligated receptor since they were differentially susceptible to proteolytic degradation by chymotrypsin.     

Structure of rat liver nuclei after depletion and reassociation of histone H1

Chromatin in isolated rat liver nuclei was compared with chromatin in (i) nuclei depleted of H1 by acid extraction; (ii) nuclei treated at pH 3.2 (without removal of H1), and (iii) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination.     

Responses of mammalian metaphase chromosomes to endonuclease digestion

Digestion of fixed metaphase chromosomes by endonucleases (micrococcal nuclease and DNase II) under optimal digestion conditions followed by Giemsa staining produces sharp banding patterns identical to G-bands. In ³H-thymidine labeled, synchronized metaphase cells of the chinese hamster (CHO line), the band induction is accompanied by the removal of DNA. The single strand specific nuclease S1 and DNase I do not produce such banding patterns.     

Nuclease sensitivity of active chromatin.

The active regions of chicken erythrocyte nuclei were labeled using the standard DNase I directed nick translation reaction. These nuclei were then used to study the characteristics and, in particular, the nuclease sensitivity of active genes. Although DNase I specifically attacks active genes, micrococcal nuclease solubilizes these regions to about the same degree as the total DNA. On the other hand micrococcal nuclease does selectively cut the internucleosomal regions of active genes resulting in the appearance of mononucleosomal fraction which is enriched in active gene DNA. A small percentage of the active chromatin is also released from the nucleus by low speed centrifugation following micrococcal nuclease treatment. The factors which make active genes sensitive to DNase I were shown to reside on individual nucleosomes from these regions. This was established by showing that isolated active mononucleosomes were preferentially sensitive to DNase I digestion. Although the high mobility group proteins are essential for the maintenance of DNase I sensitivity in active regions, these proteins are not necessary for the formation of the conformation which makes these genes preferentially accessible to micrococcal nuclease. The techniques employed in this paper enable one to study the chromatin structure of the entire population of actively expressed genes. Previous studies have elucidated the structure of a few special highly prevalent genes such as ovalbumin and hemoglobin. The results of this paper show that this special conformation is a general feature of all active genes irregardless of the extent of expression.     

Chromatin structure of a 3-methylcholanthrene-induced cytochrome P-450 gene

Plasmids carrying fragments of a cytochrome P-450 gene, inducible by 3-methylcholanthrene, were used to study the chromatin structure of this gene in the liver of normal and carcinogen-treated rats. Digestion with micrococcal nuclease revealed that the gene is not present in the typical 200 base pair nucleosomal structure. By use of indirect end-label hybridization, four DNase I hypersensitive sites were mapped in the 5'-terminal region of the gene. An S1 nuclease sensitive site is located close to a DNase I site. Gene induction by treatment with 3-methylcholanthrene does not result in detectable changes in the DNase I hypersensitive sites. Rat thymus chromatin does not contain DNase I hypersensitive sites in the P-450 gene, suggesting that in the liver the chromatin structure is altered so as to allow tissue-specific expression of the gene. This paper is the first study on the chromatin structure of a gene coding for a member of the cytochrome P-450 family of enzymes. The implications of our results to the understanding of gene regulation of the P-450 genes are discussed.     

Methylation of nucleosomal and nuclease sensitive DNA.

The proportion of cytosines methylated in the DNA of nucleosome oligomers and of core particles appears indistinguishable from that of total nuclear DNA from CHO cells. However the DNA in nucleoprotein which is initially released from nuclei by treatment with very low levels of micrococcal nuclease and the first 10% of material rendered acid soluble by treatment of nuclei with DNase I are enriched 2 fold in their content of 5 methylcytosine. (Cessation of hydrolysis by nuclease occurs concomitantly with precipitation of nucleosomal core particles).     

A site of discontinuity in the interaction between DNA and histones in nucleosomes of sea urchin embryo chromatin.

Digestion of chromatin DNA in nuclei of sea urchin embryos with pancreatic nuclease and with micrococcal nuclease give additional details concerning the interaction between DNA and histones. A specific site of hydrolysis appears to be located on the nucleosome in such a position as to split the DNA unit length in two equivalent fragments of about 60–70 base pairs in length. The complete digestion of chromatin DNA appears to depend on the low stability of the nucleosome containing the split DNA fragments.     

The structure of sub-nucleosomal particles. The octameric (H3/H4)4--125-base-pair-DNA complex

Chicken erythrocyte chromatin was depleted of histones H1, H5, H2A and H2B. The resulting (H3/H4)-containing chromatin was digested with micrococcal nuclease to yield monomer, dimer, trimer etc. units, irregularly spaced on the DNA, with even-number multimers being more prominent. Sucrose density gradient centrifugation separated monomers and dimers (7.7 S and 10.5 S). Sodium dodecyl sulphate gel electrophoresis and cross-linking indicated: the monomer contains 50-base-pair (bp), 60-bp and 70-bp DNA and the dimer 125-bp DNA; the monomer contains a tetramer and the dimer an octamer of H3 and H4. Partial association of monomer units to dimers inhibits structural studies of monomers. The internal structure of the dimer, i.e. and (H3/H4)4-125-bp-DNA particle, was studied using circular dichroism, thermal denaturation and nuclease digestion. Both micrococcal nuclease and DNase I digestion indicate that, unlike core particles, accessible sites occur in the centre of the particle and it is concluded that the (H3/H4)4-125-bp-DNA particle is not a 'pseudo-core particle' in which the 'extra' H3 and H4 replace H2A and H2B. It is proposed that the octamer particle is formed by the sliding together of two 'monomer' units, each containing the (H3/H4)2 tetramer and 70 bp of DNA. Excision of this dimer unit with micrococcal nuclease results in the loss of 10 readily digestible base pairs at each end, leaving 125 bp.     

Analysis of the nuclear estrogen receptor from MCF-7 cells by limited proteolysis

The proteolytic fragments of the nuclear estrogen receptor in the MCF-7 cell line were characterized following limited digestion with chymotrypsin and trypsin. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The proteolytic digestion was performed either on the micrococcal nuclease hydrolysate or on intact nuclei. The molecular weights (Mr) were calculated from the sedimentation coefficients determined on a sucrose gradient and from the Stokes radii estimated by gel filtration. Digestion of the nuclei with micrococcal nuclease solubilized a receptor form of Mr = 151,000. This receptor form was degraded by chymotrypsin to a receptor of Mr = 33,000 and by trypsin to a receptor of Mr = 60,000. Digestion of intact nuclei with chymotrypsin solubilized a receptor form of Mr = 62,000 which dissociated in 0.4 M KCl to a receptor of Mr = 32,000. Digestion of intact nuclei with trypsin followed by micrococcal nuclease solubilized a receptor form of Mr = 75,000 which was further dissociated by 0.4 M KCl to a receptor form of Mr = 60,000. The ability of the receptor forms to bind DNA was tested using DNA-cellulose column chromatography. About 40% of the micrococcal nuclease solubilized receptor form, compared to about 7% of the chymotrypsin degraded receptor and to about 13% of the trypsin degraded receptor forms, all bound to the column and could be eluted by high salt concentrated buffer. We conclude that the nuclear estrogen receptor in the MCF-7 cell line can be partially degraded either in the micrococcal nuclease hydrolysate or in intact nuclei by chymotrypsin or trypsin generating protein moieties, probably receptor fragments of Mr = 33,000 and 60,000 respectively. Both fragments retain their estradiol binding domain and it may be hypothesized that the heavier fragment retains its chromatin binding domain.     

Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases

Summary The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and nondenaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosommc DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50–75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multinucleosomic chromatin segments containing a full complement of histones.     

Comparison of non histone proteins selectively associated with nucleosomes with proteins released during limited DNase digestions.

Cultured mammary cells from GR mouse were used to analyse proteins associated with the mononucleosomes and released by a short micrococcal DNase treatment of nuclei. On metrizamide density gradients, mononucleosomes appear to be heterogeneous according to their content of associated non-histone proteins. Proteins associated with the denser fraction (1.22 - 1.24 g/ml) were analysed by two dimensional electrophoresis and compared to the proteins released by DNase I treatment. All the proteins associated with mononucleosomes were also released by DNase I treatment. It could then be assumed that these proteins are associated with the active part of the genome. Additional proteins were released by micrococcal DNase treatment of the nuclei. They could be involved in a higher order organization of chromatin.     

Analysis of chromatin of skeletal muscle of developing rats using micrococcal nuclease and DNase I

Conformational changes in the chromatin of skeletal muscle of 3-, 14-and 30 day-old developing rats have been studied using DNase I and micrococcal nuclease (MCN). Purified nuclei were digested separately by MCN and DNase I. The rate and extent of digestion by MCN decreases gradually as development proceeds. The electrophoretic pattern of MCN digested DNA, however, shows no change. The kinetics of digestion of nuclei by DNase I show no change with development. However, the electrophoretic pattern of DNase I digested DNA shows a gradual decrease in the amount of 10–30 bp fragments with progressive development. These studies show that the chromatin of the skeletal muscle undergoes certain conformational changes during postnatal development, and such changes in chromatin may be necessary for terminal differentiation of this tissue.