Tightly-bound form of poly(ADP-ribose)polymerase in the higher order of chromatin organization

A tightly-bound form of poly(ADP-ribose)polymerase is present, within the third level of rat testis chromatin structure, both in the loops and in chromatin matrix. When chromatin matrix was extensively digested with DNAaseI, only little residual enzymatic activity remained in the insoluble fraction, the extent of DNA hydrolysis being well correlated to the progressive loss of the poly(ADP-ribose)polymerase activity. These findings suggest that the tightly-bound form of the enzyme is not an intrinsic protein component of chromatin matrix but is only indirectly located in this structure, being rather associated to the attachment points of loop DNA on the matrix.     

Methylation of chromatin DNA.

E. coli DNA methylase has been used to methylate chromatin DNA in vitro. At saturation only 50% of the chromatin DNA becomes methylated. The methylated regions of chromatin correspond to that fraction of the chromatin which is sensitive to staphylococcal nuclease. Using in vitro methylated chromatin followed by nuclease digestion movement of chromatin proteins along the DNA can be detected. By this criterion, sonication of chromatin or precipitation with MnCl2 causes 10% of the previously uncovered methylated regions to become covered by protein. Reconstitution of methylated chromatin results in the randomization of the chromatin proteins. Using nuclei which were methylated in vitro we have demonstrated that a small degree of protein sliding does occur during the preparation of chromatin from nuclei. Finally, we have prepared open region DNA by polylysine titration. This procedure does not cause displacement of chromatin proteins.     

Postirradiation alterations of neuronal chromatin structure

Previous work from our laboratory suggested that neuronal chromatin structure may be altered immediately after exposure to ionizing radiation. In the present study, whole brains of 4-month-old male Fisher 344 rats were irradiated with a dose of 25 Gy. The kinetics of restoring the chromatin structure to its unirradiated state was investigated in rat cerebellar neurons using three different approaches: (1) measurement of changes in the DNA superhelical structure by the fluorescent halo assay, (2) measurement of changes in chromatin accessibility to digestion by micrococcal nuclease, and (3) measurement of changes in the accessibility of the nuclear-matrix-associated DNA to digestion by DNase I. Immediately after irradiation, the topological constraints on the DNA loops were altered, the chromatin was more accessible to m. nuclease digestion, and the DNA associated with the nuclear matrix was more resistant to digestion by DNase I. Return of the chromatin structure to its unirradiated state as measured by each of the three methods followed biphasic kinetics with the fast phase having a half-time of several minutes and the slow phase having a half-time of several hours. The kinetics are similar to that previously reported for repair of radiation-induced DNA damage in mammalian cells. Although the independent assays used in this study seemed to follow the same kinetics, their relationship at the molecular level remains to be determined.     

Isolation and characterisation of a 167 bp core particle isolated from stripped chicken erythrocyte chromatin

We have digested chicken erythrocyte soluble chromatin, both unstripped and stripped of histones H1 and H5 with either 0.6 M NaCl or DNA-cellulose, with micrococcal nuclease (MNase). Digestion of unstripped chromatin to monomeric particles initially paused at 188 bp DNA; continued digestion resulted in another pause at 177 before the 167 bp chromatosome and 146 bp core particle were obtained. Digestion of stripped chromatin to monomeric particles paused transiently at 177 bp; continued digestion resulted in marked pauses at 167 and 156 before the 146 bp core particle was obtained. These results suggested that 167 bp DNA representing two complete turns are bound to the histone octamer. Histone H1/H5 binds an additional two helical turns of DNA, thereby protecting up to 188 bp DNA against nuclease digestion. Monomeric particles containing 167 bp DNA were isolated from stripped chromatin and found by DNase I digestion to be a homogeneous population with a 10 bp DNA extension to either end relative to the 146 bp core particle. Thermal denaturation and circular dichroism spectroscopy showed stronger histone-DNA interactions and increased DNA winding as the length of DNA attached to the core histone octamer was decreased. Thermal denaturation also showed three classes of histone-DNA interaction: the core particle containing 167 bp DNA had tight binding of ten helical turns of DNA, intermediate binding of two helical turns and looser binding of four helical turns.     

Pulsed-field gel electrophoresis analysis of higher-order chromatin structures of Zea mays. Highly methylated DNA in the 50 kb chromatin structure

We have investigated the presence of higher-order chromatin structures in different maize tissues. Taking advantage of the pulsed-field gel electrophoresis technique to analyse large DNA fragments from intact nuclei and cells, we have determined the size distribution of the high-molecular-weight DNA fragments obtained from chromatin degradation by endogenous nucleases in isolated nuclei. Chromatin digestion leads to the appearance of stable DNA fragments of about 50 kb in all the tissues examined, suggesting the folding of DNA in higher-order chromatin domain structures. It has been reported that such chromatin domains are formed by loops of the 30 nm fibres anchored to the nuclear matrix by a complex set of proteins, including DNA topoisomerase II. Treatment of maize protoplasts with the calcium ionophore A23187 and the antitumour drug VM-26, which specifically inhibit the religation of the cleaved DNA in the topoisomerase II reaction, also produces the 50 kb structure. Analysis of the DNA contained in the 50 kb chromatin structure shows a higher degree of methylation than in bulk maize chromosomal DNA. The role of methylated DNA in the chromatin folding is discussed.     

Cytochemical localization of DNA loop attachment sites to the nuclear lamina and to the inner nuclear matrix

Summary The rat liver nuclear matrix, obtained by endogenous nuclease digestion and extraction with low and high lonic strength media, contains residual DNA fragments that are considered to represent the attachment sites of the chromatin domains to the nucleoskeleton. These sites, protected against nuclease digestion by their binding with the nucleoskeleton proteins, should be either mainly linked to the peripheral lamina or to the inner nuclear matrix. The DNA fragment distribution at the level of the different components of the nuclear matrix has been evaluated in samples embedded in Epon and in hydrophilic resins by means of the DNase-gold technique. The labeling obtained suggests that the chromatin loops are prevailingly associated with the interior of the matrix; in fact about twice of the label is present in the inner matrix with respect to the peripheral lamina area.These results confirm the hypothesis that in interphase the chromatin maintains an organization similar to that of chromosomes, with loops radiating from a central scaffold, instead of being mainly attached to the lamina as otherwise suggested.     

Periodicity of DNA folding in higher order chromatin structures.

Each level of DNA folding in cells corresponds to a distinct chromatin structure. The basic chromatin units, nucleosomes, are arranged into solenoids which form chromatin loops. To characterize better the loop organization of chromatin we have assumed that the accessibility of DNA inside these structures is lower than on the outside and examined the size distribution of high mol. wt DNA fragments obtained from cells and isolated nuclei after digestion with endogenous nuclease or topoisomerase II. The largest discrete fragments obtained contain 300 kbp of DNA. Their further degradation proceeds through another discrete size step of 50 kbp. This suggests that chromatin loops contain approximately 50 kbp of DNA and that they are grouped into hexameric rosettes at the next higher level of chromatin structure. Based upon these observations a model by which the 30 nm chromatin fibre can be folded up into compact metaphase chromosomes is also described.     

Ribosomal DNA sequences attached to the nuclear matrix

The organization of rat liver ribosomal DNA (rDNA) as matrix-attached DNA loops was examined using a protocol which fractionates chromatin from discrete regions of DNA loops. Southern blot analysis of matrix-attached and solubilized chromatin DNA fragments demonstrated that rDNA is associated with the matrix via its 5' and 3' nontranscribed spacer sequences (NTS). Although the 45 S rRNA coding sequences were approximately threefold enriched in matrix preparations, the recovery of this DNA (unlike the NTS) was dependent on the extent of nuclease digest and proportional to the length of the matrix-attached DNA fragments. The data suggest that rDNA is organized as matrix-attached DNA loops and only the NTS are directly involved in matrix binding. Further, we demonstrated that while the kinetics and extent of nuclease digestion were similar in all regions of the DNA loops, the nuclease digestion pattern of bulk nuclear and matrix DNA showed a typical nucleosome organization, but the rDNA fragments retained with the nuclear matrix did not.     

Visualization of chromatin domains created by the gypsy insulator of Drosophila

Insulators might regulate gene expression by establishing and maintaining the organization of the chromatin fiber within the nucleus. Biochemical fractionation and in situ high salt extraction of lysed cells show that two known protein components of the gypsy insulator are present in the nuclear matrix. Using FISH with DNA probes located between two endogenous Su(Hw) binding sites, we show that the intervening DNA is arranged in a loop, with the two insulators located at the base. Mutations in insulator proteins, subjecting the cells to a brief heat shock, or destruction of the nuclear matrix lead to disruption of the loop. Insertion of an additional gypsy insulator in the center of the loop results in the formation of paired loops through the attachment of the inserted sequences to the nuclear matrix. These results suggest that the gypsy insulator might establish higher-order domains of chromatin structure and regulate nuclear organization by tethering the DNA to the nuclear matrix and creating chromatin loops.     

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.     

A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease.

We have followed the kinetics of staphylococcal nuclease digestion of duck reticulocyte nuclei and chromatin from early stages to the digestion limit. We confirm that partial digestion of nuclei produces discrete DNA bands which are multiples of a monomer, 185 base pairs in length. The multimers are shown to be precursors of the monomer, which is next digested to a homogeneous, 140 base pair fragment. This fragment in turn gives rise to an array of nuclear limit digest DNA bands, which is almost identical with the limit digest pattern of isolated chromatin. As in the case of chromatin, half the DNA of nuclei is acid soluble at this limit. While the DNA limit digest patterns of nuclei and chromatin are similar, the large multimeric structures present as intermediates in nuclear digestion are absent in chromatin digestion. Alternate methods of chromatin gel preparation appear to leave more of the higher order structure intact, as measured by the production of these multimeric bands. Our results are consistent with the "beads on a string" model of chromatin proposed by others.     

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.     

S(T)PXX motifs promote the interaction between the extended N-terminal tails of histone H2B with "linker" DNA.

The assembly of hybrid core particles onto long chicken DNA with histone H2B in the chicken histone octamer replaced with either wheat histone H2B(2) or sea urchin sperm histone H2B(1) or H2B(2) is described. All these histone H2B variants have N-terminal extensions of between 18 and 20 amino acids, although only those from sea urchin sperm have S(T)PXX motifs present. Whereas chicken histone octamers protected 167 base pairs (bp) (representing two full turns) of DNA against micrococcal nuclease digestion (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813), all the hybrid histone octamers protected an additional 17-bp DNA against nuclease digestion. This protection was more marked in the case of hybrid octamers containing sea urchin sperm histone H2B variants and similar to that described previously (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813) for hybrid histone octamers containing wheat histone H2A variants all of which also have S(T)PXX motifs present. Continued micrococcal nuclease digestion reduced the length of DNA associated with the core particle via 172-, 162-, and 152-bp intermediates until the 146-bp core particle was obtained. These DNA lengths were approximately 5 bp or half a helical turn longer than those reported previously for stripped chicken chromatin and for core particles containing histone octamers reconstituted using "normal" length histone H2B variants. This protection pattern was also found in stripped sea urchin sperm chromatin, demonstrating that the assembly/digestion methodology reflects the in vivo situation. The interaction between the N-terminal histone H2B extension and DNA of the "linker" region was confirmed by demonstrating that stripped sea urchin sperm chromatin precipitated between 120 and 500 mM NaCl in a manner analogous to unstripped chromatin whereas stripped chicken chromatin did not. Tryptic digestion to remove all the histone tails abolished this precipitation as well as the protection of DNA outside of the 167-bp core particle against nuclease digestion.     

Isolation and characterization of the DNA fraction of rat liver chromatin which binds polylysine.

The structure of eukaryotic chromatin has been investigated by isolating and analyzing the "accessible" DNA fraction of rat liver chromatin. This DNA fraction has been isolated by titrating the chromatin with the protese-resistant D isomer of polylysine to bind the "accessible" DNA sites. After removal of chromosomal proteins by digestion with pronase, all DNA not protected from attack by bound polylysine was removed by digestion with DNase. Even after exhaustive treatment with pronase and DNase approximately 30% of the chromatin DNA remains resistant to nuclease attack. Analysis of the isolated DNA shows it to be mainly double-stranded with an average size of 200-250 base pairs. The DNA is slightly A-T rich and contains both repetitive and "single-copy" nuleotide sequences. The results suggest that there are extensive regions in chromatin where the DNA is not tightly complexed with protein. Furthermore, the DNA of these regions is similar in gross properties to the DNA of the total genome.     

Evidence for a subunit structure of chromatin in mouse myeloma cells

If micrococcal nuclease is allowed to digest chromatin as it exists inside intact nuclei isolated from mouse myeloma tissue culture cells, more than 60% of the DNA can be isolated as a homogeneous fragment on a sucrose gradient. Analytical ultracentrifugation indicates that the protected DNA is native, unnicked, and about 140 +/- 10 base pairs long. After less extensive nuclease digestion, the protected DNA migrates in gels in lengths which are integral multiples of this 140 base pair "monomer" band. A submonomer band, 105 "/- 10 base pairs long, can also be detected. Similar digestion patterns were obtained by two different nuclear isolation procedures and even when intact cells were gently lysed directly in the digestion medium. These results confirm and extend the chromatin digestion studies of previous investigators and provide support for a subunit model for eukaryotic chromatin. The single strand specific S1 nuclease did not digest intranuclear chromatin under the conditions used.     

Isolation and characterization of the nuclear matrix in Friend erythroleukemia cells: chromatin and hnRNA interactions with the nuclear matrix.

Nuclear matrices from undifferentiated and differentiated Friend erythroleukemia cells have been obtained by a method which removes DNA in a physiological buffer. These matrices preserved the characteristic topographical distribution of condensed and diffuse "chromatin" regions, as do nuclei in situ or isolated nuclei. Histone H1 was released from the nuclear matrix of undifferentiated cells by 0.3 M KCl; inner core histones were released by 1 M KCl. Nuclear matrix from differentiated cells did not maintain H1, and histone cores were fully released in 0.7 M KCl. KCl removed the core histones as an octameric structure with no evidence of preferential release of any single histone. Electron microscopy of KCl-treated matrix revealed no condensed regions but rather a network of fibrils in the whole DNA-depleted nuclei. When nuclear matrices from both types of cell were exposed to conditions of very low ionic strength, inner core histones and condensed regions remained. These observations support the contention that inner core histones are bound to matrix through natural ionic bonds or saline-labile elements, and that these interactions are implicated in chromatin condensation. hnRNA remained undegraded and tenaciously associated to the matrix fibrils, and was released only by chemical means which, by breaking hydrophobic and hydrogen bonds, produced matrix lysis. Very few nonhistone proteins were released upon complete digestion of DNA from either type of nuclei. The remaining nonhistone proteins represent a large number of species of which the majority may be matrix components. The molecular architecture in both condensed and diffuse regions of interphase nuclei appears to be constructed of two distinct kinds of fibers; the thicker chromatin fibers are interwoven with the thinner matrix fibers. The latter are formed by a heteropolymer of many different proteins.     

Structural heterogeneity of chromatin preparations at the level of DNA topology

The structural heterogeneity of calf thymus chromatin preparations was studied at the level of DNA topology by analysing the influence of ethidium bromide on the chromatin viscosity in deproteinizing medium. In 0.7 M NaCl the chromatin was separated into the fractions with linear DNA (3--36% in various preparations) and with supercoiled circular DNA (scc DNA), which differ from each other in their adhesive properties. Reduction of disulfide bonds in residual chromatin protein with 5% mercaptoethanol linearized scc DNA, present in chromatin preparations as nuclear matrix subunits containing some loops of scc DNA on the protein globule.