Expansion of chicken erythrocyte nuclei upon limited micrococcal nuclease digestion. Correlation with higher order chromatin structure

A sensitive method for measuring nuclear volumes with a Coulter counter is described. It has been applied to the digestion of chicken erythrocyte nuclei by micrococcal nuclease and DNase I. Early in digestion, micrococcal nuclease induced a 20% increase in the effective spherical volume of the nuclei, followed by a gradual reduction. At the peak of nuclear swelling, about 17% of the chromatin was soluble after lysis and its average chain length was about 18 kilobase pairs (kb). DNase I digestion did not give rise to a corresponding expansion of the nuclei. Several preparation conditions, including the treatment of nuclei with 0.2% Triton X-100, led to a loss of the expansion effect upon subsequent micrococcal nuclease digestion. The results support the domain theory of higher order chromatin structure. In the context of this model, the observed maximum nuclear expansion correlates with an average of one nuclease scission per domain.     

Enrichment for the globin coding region in a chromatin fraction from chick reticulocytes by endonuclease digestion.

A nuclease-sensitive fraction was obtained from chick reticulocyte chromatin by brief digestion with an endonuclease (DNAase II, deoxyribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.6). The nuclease-sensitive fraction typically contained less than 1% of the chromatin-DNA but about 50% or more of the nascent chromatin-bound RNA. Hybridization of chick globin complementary DNA to the DNA component of the nuclease-sensitive fraction of reticulocyte chromatin indicated a 3--5 fold enrichment for the globin coding region of the chromatin. The control experiment utilizing DNA from a nuclease-sensitive fraction of chick liver chromatin did not show a comparable enrichment for the globin coding region. This suggests that the endonuclease-effected enrichment for the globin coding region in the nuclease-sensitive fraction of reticulocyte chromatin is to some degree specific for structural genes transcribed in reticulocytes.     

Polylysine titration of rat liver chromatin fractions after DNase II digestion.

The concentration of free phosphate groups is measured in rat liver chromatin after DNase II digestion using polylysine titration. The unsheared chromatin completely precipitates at lysine/DNA phosphate ratios of 0.5 to 0.6. Digestion of the chromatin reduces the lysine/DNA phosphate ratio of complete precipitation by about 0.2 units suggesting the removal of free phosphate groups. The two chromatin fractions: MgC12 insoluble (template-inactive) and Mg12 soluble (template-active) chromatins precipitate at about the same lysine/DNA phosphate ratio. Some 15% of the MgC12 soluble chromatin remains in solution at any polylysine concentration. The removal of histone H 1 FROM THE MgC12 insoluble chromatin increases the lysine/DNA phosphate ratio by about 0.2 units suggesting that 20% of the DNA phosphate groups in nucleosomes are masked by histone H 1.     

Identification of a rosette-enriched chromatin fraction from mouse fibroblast nuclei

We have characterized two components of DNA isolated from mouse L-M cell nuclei. These components, designated as HMW (high molecular weight) and VHMW (very high molecular weight) DNA, were characterized by rate zonal sedimentation, agarose gel electrophoresis, and for protein content. Our electron micrographs revealed that HMW-DNA contained mainly linear molecules with few single rosette structures, while the VHMW-DNA was enriched in rosettes, many of which were significantly larger and linked together in multimeric structural forms. The VHMW-DNA component was also enriched for residual protein, which we believe represents the core of the rosette. The characteristics of this residual protein are consistent with reported findings of the most tightly bound proteins. The rosette conformation does not appear to be an artifact of microscopy or of an aggregate nature for several reasons: (i) rosettes are preferentially found in the VHMW-DNA component; (ii) further manipulation or purification of the DNA disrupts the rosette structure and produces linear fragments; (iii) the amount of proteinaceous material at the core of the rosette is diminished when the DNA is further purified; and (iv) treatment of intact nuclei with a novel bisamine reagent putatively crosslinks DNA in vivo and minimizes the disruption of rosettes by shear. We believe this separation of chromatin is critical to establish the architectural forms of euchromatin and heterochromatin of interphase DNA in the eucaryotic system. Once established, fractionated chromatin can be used to identify specifically expressed or repressed genes with linear form DNA and rosette form DNA. We discuss rosettes as derivatives of chromosomal domains that retain structural features because of residual peptide elements.     

Biochemical and physiochemical characterization of chromatin fractions with different degrees of solubility isolated from chicken erythrocyte nuclei

Chicken erythrocyte chromatin was prepared according to two different methods [Fulmer, A. W., & Bloomfield, V. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 5968-5972; Ausio, J., Borochov, N., Seger, D., & Eisenberg, H. (1984) J. Mol. Biol. 177, 373-398] to give three main common fractions, according to its solubility (S) or insolubility (I) in 0.15 M NaCl buffers or to its further solubility in 0.25 mM ethylenediaminetetraacetic acid (E). From the biochemical point of view, all of them have been found to be undistinguishable. Analytical ultracentrifugation shows that all of these fractions can reversibly undergo the transition from the low to the higher order structure, through a nearly identical way of folding. Thermal denaturation profiles yielded three transitions having the same Tm's for the three fractions. The percentage of DNA melting in the first transition decreased in the order S greater than I greater than E, and the amount in the second transition increased in the same order. Together with the different solubility of these fractions in the presence of divalent ions, these results indicate that in the three fractions of chromatin studied, the amount of linker DNA bound to the nucleosome varied.     

Protein distribution amongst chromatin particles following DNAse II digestion

When active chromatin is released as a Mg-soluble fraction following digestion of nuclei with DNAse II, as concomitant release of HMG proteins, and hnRNP particles occurs. Release of HMG 14 and 17 is dependent on active chromatin release, whereas HMG 1 and 2, and hnRNP particles are released in an independent process. The Mg-soluble fraction comprises a heterogenous mixture of particles of less compact conformation than normal nucleosomes, and prone to protein-induced aggregation. Histone H1, and HMG 14 and 17 appear to be associated with these particles in a reversible manner, whereas HMG 1 and 2 are unbound.     

Kinetics of DNase I digestion of interphase chromatin in differentiated cell nuclei of the mouse: a flow cytometric study

The process of DNA digestion with DNase I was monitored in interphase chromatin of differentiated cells by flow cytometry after DNA staining with either the intercalating dye propidium iodide (PI) or the AT specific dye Hoechst 33258 (HO). Nuclei from the liver, kidney and spleen of the mouse were studied after different digestion times (0 to 120 min). During the first 30 min of treatment, a tissue specific digestion pattern was found after PI staining; from 60 min onward, the digestion curves ran parallel, with minor quantitative differences among the cell types. After HO staining, the digestion kinetics appeared to be similar for all the cell types; this is likely due to the peculiar base composition of the mouse genome, where inactive c-heterochromatin is exceptionally AT-rich. No quantitative correlation was found between interphase "heterochromatin" and chromatin DNA which is resistant to DNase I cleavage, while the amount of DNase-I-sensitive DNA does not correspond to the interphase "euchromatic" component. It was confirmed that the flow cytometric approach is a tool for quantifying relative changes in the functional state of chromatin in differentiated cell systems.     

The DNase I sensitive state of "active" globin gene chromatin resists trypsin treatments which disrupt chromatin higher order structure

Active genes in higher eukaryotes reside in chromosomal domains which are more sensitive to digestion by DNase I than the surrounding inactive chromatin. Although it is widely assumed that some modification of higher order structure is important to the preferential DNase I sensitivity of active chromatin, this has so far not been tested. Here we show that the structural distinction between DNase I sensitive and resistant chromatin is remarkably stable to digestion by trypsin. Chick embryonic red blood cell nuclei were subjected to increasing levels of trypsin digestion and then assayed in the following three ways: (1) by gel electrophoresis for histone cleavage, (2) by sedimentation and nuclease digestion for loss of higher order structure, and (3) by dot-blot hybridization to globin and ovalbumin probes for disappearance of preferential DNase I sensitivity. We have found that chromatin higher order structure is lost concomitantly with the cleavage of histones H1, H5, and H3. In contrast, the preferential sensitivity of the globin domain to DNase I persists until much higher concentrations of trypsin, and indeed is not completely abolished even by the highest levels of trypsin we have used. We therefore conclude that the structural distinction of active chromatin, recognized by DNase I, does not reside at the level of higher order structure.     

Perturbation of chromatin structure in the region of the adult beta-globin gene in chicken erythrocyte chromatin

An EcoRI chromatin fragment containing the adult beta-globin gene and flanking sequences, isolated from chicken erythrocyte nuclei, sediments at a reduced rate relative to bulk chromatin fragments of the same size. We show that the specific retardation cannot be reversed by adding extra linker histones to native chromatin. When the chromatin fragments are unfolded either by removing linker histones or lowering the ionic strength, the difference between globin and bulk chromatin fragments is no longer seen. The refolded chromatin obtained by restoring the linker histones to the depleted chromatin, however, exhibits the original sedimentation difference. This difference is therefore due to a special property of the histone octamers on the active gene that determines the extent of its folding into higher-order structure. That it is not due to the differential binding of linker histones in vitro is shown by measurements of the protein to DNA ratios using CsCl density-gradients. Both before and after selective removal of the linker histones, the globin gene fragment and bulk chromatin fragments exhibit only a marginal difference in buoyant density. In addition, we show that cleavage of the EcoRI fragment by digestion at the 5' and 3' nuclease hypersensitive sites flanking the globin gene liberates a fragment from between these sites that sediments normally. We conclude that the hypersensitive sites per se are responsible for the reduction in sedimentation rate. The non-nucleosomal DNA segments appear to be too long to be incorporated into the chromatin solenoid and thus create spacers between separate solenoidal elements in the chromatin, which can account for its hydrodynamic behaviour.     

The binding of histones H1 and H5 to chromatin in chicken erythrocyte nuclei.

The binding curves of histones H1 and H5 to chromatin in nuclei have been determined by a novel method which utilises the differential properties of free and bound histones on cross-linking with formaldehyde. The dissociation is thermodynamically reversible as a function of [NaCl]. The binding curves are independent of temperature over the range 4 degrees - 37 degrees C and independent of pH over the range 5.0 to 9.0. The curves are sigmoid, indicating co-operative dissociation with NaCl. The standard free energy of dissociation in 1 M NaCl for H1 is 0.5 Kcals/mole and for H5 is 3.5 Kcals/mole.     

Alteration of domain architecture of chicken erythrocyte chromatin

The higher-order organisation of chromatin in chicken erythrocyte nuclei as a function of the ionic strength of the nuclear suspension buffer and also of the time of incubation in this buffer prior to nuclease digestion has been investigated. This organisation is described in terms of a physical parameter called the domain length. The 45-kbp-long domains of control nuclei were unravelled to give rise to domains of length 150 kbp on overnight equilibration at 0 degree C of the nuclei in standard isolation buffer containing 0.135 M NaCl prior to nuclease digestion. However, transition to the equilibrium state was preceded by a metastable and irregular domain architecture when the nuclei were incubated for only 1 h. In contrast, the domain length remained unchanged when nuclei were incubated in the isolation buffer alone for identical periods of time. The proteins dissociated at the higher ionic strength were characterised and their role in stabilising the domain structure is discussed.     

Estimation of domain length of chicken erythrocyte chromatin

Chromatin of chicken erythrocyte nuclei was extracted by digestion with micrococcal nuclease. The length distribution of the soluble chromatin was determined by gel electrophoresis and electron microscopy. These results were fitted with a theoretical distribution which was an outcome of the domain model proposed by Igo-Kemenes and Zachau (Igo-Kemenes, T. and H.G. Zachau (1977) Cold Spring Harbour Symp. Quant. Biol. 42, 109–118). A domain length of 45 kbp was obtained.     

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.