UV-induced pyrimidine dimers and trimethylpsoralen cross-links do not alter chromatin folding in vitro

We have examined the ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro following DNA damage by two different agents: UV irradiation at 254 nm and trimethylpsoralen plus near-UV light. Both agents damage DNA specifically, yet cause different degrees of unwinding (and possibly bending) of the DNA helix. In addition, trimethylpsoralen forms interstrand DNA cross-links. The structural transitions of intact and histone H1 depleted chromatin fibers, induced by NaCl, were monitored by analytical ultracentrifugation, light scattering, and circular dichroism. Our results indicate that when chromatin fibers contain even large, nonphysiological amounts of DNA photodamage by either agent, the salt-induced folding of these fibers into higher ordered structures is unaffected. The compact 30-nm fiber must therefore be able to accommodate a large amount of DNA photodamage (greater than one UV-induced photoproduct or trimethylpsoralen interstrand cross-link per nucleosome) with little or no change in the overall size or compaction of this structure.     

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.     

Atomic force microscopy of mammalian sperm chromatin

We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected bull sperm nucleus have revealed that the DNA is organized into large nodular subunits, which vary in diameter between 50 and 100 nm. Other images of partially decondensed mouse sperm chromatin show that the nodules are arranged along thick fibers that loop out away from the nucleus upon decondensation. These fibers appear to stretch or unravel, generating narrow smooth fibers with thicknesses equivalent to a single DNA-protamine complex. High resolution AFM images of the nodular subunits suggest that they are discrete, clipsoid-shaped DNA packaging units possibly only one level of packaging above the protamine-DNA complex.     

Structural organization of rat hepatocyte chromatin as visualized in thin frozen sections selectively stained for DNA

We studied the structure of rat hepatocyte chromatin in situ using thin frozen sections selectively stained for DNA after aldehyde fixation. Our results indicate that intranucleolar chromatin is arranged into three different organization levels, confirming the observations on Epon-embedded chromatin. These are: completely extended DNA filaments, with a thickness of approximately 3 nm, clustered in loose, roundish agglomerates, very long fibers with a thickness ranging from 15 to 35 nm and compact chromatin clumps. Both the fibers and the chromatin clumps frequently appeared to be composed of nucleosome-like particles. In the extranucleolar chromatin, agglomerates of extended DNA filaments and long fibers were never visualized. In contrast to data from Epon-embedded chromatin, we noticed that in frozen sections neither the nucleolar nor the extranucleolar compact chromatin appear to be organized into discrete, 20 to 30 nm superordered fibers.     

Psoralen-crosslinking of soluble and of H1-depleted soluble rat liver chromatin

We purified soluble rat liver chromatin and H1-depleted chromatin and photocrosslinked its DNA with psoralen at pH 7. Digestion of this chromatin with micrococcal nuclease produced a normal nucleosomal repeat. Chromatin was photoreacted in the presence of 0 to 700 mM-NaCl and was fractionated in sucrose gradients containing the same NaCl concentrations. The dissociation of H1 occurred as in the non-crosslinked controls and no preferential dissociation of core histones was observed. The samples between 100 and 500 mM-NaCl showed precipitation. In the electron microscope, the fibers appeared indistinguishable from the controls at low ionic strength. In the presence of 40 mM-NaCl, the fibers of the photoreacted chromatin were slightly more compact than the controls, and at 500 mM-NaCl, despite the complete dissociation of H1, there were still apparently intact fibers at this ionic strength. The disruption of the psoralen-treated chromatin fibers occurred only in 600 mM-NaCl, as opposed to 500 mM-NaCl in controls. The DNA of all the photoreacted samples was spread for electron microscopy under denaturing conditions. They revealed, for all the samples, single-stranded bubbles corresponding to 200 to 400 base-pairs in size. H1-depleted chromatin containing stoichiometric amounts of core histones was photoreacted at pH 10 and very low ionic strength. Under these conditions many of the nucleosomes appeared to be unraveled, although to a variable extent. In the electron microscope, the purified DNA from these samples showed extensive crosslinking when spread under denaturing conditions. These observations show that histone-DNA interactions different from those in intact nucleosomes may be created, which allow extensive access of psoralen to the DNA.     

The use of ultraviolet light in the fractionation of chromatin containing unsubstituted and bromodeoxyuridine-substituted DNA.

Two procedures are described for the fractionation of chromatin containing unsubstituted (LL) DNA and DNA unifilarly substituted with bromodeoxyuridine (HL). The two procedures rely upon the sensitivity of bromodeoxyuridine-containing DNA to UV light to induce either strand breakage or protein crosslinking. When a mixture of LL and HL chromatin is irradiated with UV light, the HL DNA fragments into molecules of smaller molecular weight than the LL DNA and crosslinks more chromosomal protein than the LL DNA. LL and HL chromatin can be fractionated on the basis of size by centrifuging through a neutral sucrose gradient. The HL DNA-protein adducts that are generated by the UV light have a unique buoyant density and may be isolated by isopycnic centrifugation in CS2SO4. The ability to fractionate LL and HL chromatin permits certain studies on the structure of replicating chromatin.     

Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA

Eukaryotic topoisomerases I and II efficiently remove helical tension in naked DNA molecules. However, this activity has not been examined in nucleosomal DNA, their natural substrate. Here, we obtained yeast minichromosomes holding DNA under (+) helical tension, and incubated them with topoisomerases. We show that DNA supercoiling density can rise above +0.04 without displacement of the histones and that the typical nucleosome topology is restored upon DNA relaxation. However, in contrast to what is observed in naked DNA, topoisomerase II relaxes nucleosomal DNA much faster than topoisomerase I. The same effect occurs in cell extracts containing physiological dosages of topoisomeraseI and II. Apparently, the DNA strand-rotation mechanism of topoisomerase I does not efficiently relax chromatin, which imposes barriers for DNA twist diffusion. Conversely, the DNA cross-inversion mechanism of topoisomerase II is facilitated in chromatin, which favor the juxtaposition of DNA segments. We conclude that topoisomerase II is the main modulator of DNA topology in chromatin fibers. The nonessential topoisomerase I then assists DNA relaxation where chromatin structure impairs DNA juxtaposition but allows twist diffusion.     

The morphological relationship in electron microscopy between NOR-silver proteins and intranucleolar chromatin

The relative distribution of NOR proteins and chromatin fibers in the nucleoli was visualized in human cell line. The chromatin was revealed by a Feulgen-like procedure using osmium-ammine as DNA tracer. This selective staining was combined with NOR-silver staining. We provide morphological evidence for constant overlapping of the silver deposit sites with dispersed intranucleolar chromatin fibers. Silver stained proteins were sometimes observed in contact with the chromatin fibers, suggesting that at least some of the Ag-NOR proteins might be closely connected with the dispersed nucleolar DNA.     

Radial density distribution of chromatin: evidence that chromatin fibers have solid centers

Fiber diameter, radial distribution of density, and radius of gyration were determined from scanning transmission electron microscopy (STEM) of unstained, frozen-dried chromatin fibers. Chromatin fibers isolated under physiological conditions (ionic strength, 124 mM) from Thyone briareus sperm (DNA linker length, n = 87 bp) and Necturus maculosus erythrocytes (n = 48 bp) were analyzed by objective image-processing techniques. The mean outer diameters were determined to be 38.0 nm (SD = 3.7 nm; SEM = 0.36 nm) and 31.2 nm (SD = 3.6 nm; SEM = 0.32 nm) for Thyone and Necturus, respectively. These data are inconsistent with the twisted-ribbon and solenoid models, which predict constant diameters of approximately 30 nm, independent of DNA linker length. Calculated radial density distributions of chromatin exhibited relatively uniform density with no central hole, although the 4-nm hole in tobacco mosaic virus (TMV) from the same micrographs was visualized clearly. The existence of density at the center of chromatin fibers is in strong disagreement with the hollow-solenoid and hollow-twisted-ribbon models, which predict central holes of 16 and 9 nm for chromatin of 38 and 31 nm diameter, respectively. The cross-sectional radii of gyration were calculated from the radial density distributions and found to be 13.6 nm for Thyone and 11.1 nm for Necturus, in good agreement with x-ray and neutron scattering. The STEM data do not support the solenoid or twisted-ribbon models for chromatin fiber structure. They do, however, support the double-helical crossed-linker models, which exhibit a strong dependence of fiber diameter upon DNA linker length and have linker DNA at the center.     

Acridine orange-induced precipitation of mouse testicular sperm cell DNA reveals new patterns of chromatin structure

Precipitate resulting from interaction between certain intercalators, such as acridine orange (AO), and nucleic acids can be detected by electron microscopy. Formation of precipitate in nuclei of live cells is modulated by chromatin structure. Susceptibility of in situ DNA to precipitation was studied in mouse testicular germ cells during various stages of sperm maturation. DNA in round spermatid chromatin, similar to somatic cell euchromatin, was rather resistant to precipitation; the electron-dense precipitate was granular and randomly distributed. DNA in elongated spermatids was more susceptible to precipitation; the products were in the form of fibers. At early stages of spermatid maturation these fibers were distributed uniformly throughout the entire nucleus. At later stages, the products appeared as approximately 25-nm-thick fibers arranged longitudinally in arrays within the nucleus. With further cell maturation, fibers in the anterior portion of the nucleus appeared to fuse, forming homogeneously dense product. These fibrous products likely represent AO interactions with DNA in chromatin in which transition proteins had replaced histones. Changing patterns of these precipitated fibers likely reflect progressive stages of chromatin condensation, which starts at the center and anterior portion of the nucleus where the fibers coalesce. Mature sperm cell DNA, known to be complexed with protamines, was more resistant to AO-induced precipitation. The data suggest that precipitation induced by AO and monitored by electron microscopy may be a useful probe of nuclear chromatin structure.     

Polyamine-DNA interactions. Condensation of chromatin and naked DNA

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin by polyamines, such as spermine, spermidine and its analogs designated by formula NH3+(CH2)iNH2+(CH2)jNH3+, where i = 2,3 and j = 2,3, putrescine, cadaverine and MgCl2. It has been found that the different polyamines affect DNA and chromatin in a similar way. The level of compaction of the chromatin fibers induced by spermine, spermidine and the triamines NH3+(CH2)3NH2+(CH2)3NH3+ and NH3+(CH2)3NH2+(CH2)2NH3+ and MgCl2 is found to be identical. The triamine NH3+(CH2)3NH2+(CH2)2NH3+ and the diamines studied condense neither chromatin nor DNA. This drastic difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations might play essential roles in their interactions with DNA and chromatin. It is shown that a mixture of mono- and multivalent cations affect DNA and chromatin condensation competitively, but not synergistically, as claimed in a recent report by Sen and Crothers (Biochemistry 25, 1495-1503, 1986). We have also estimated the extent of negative charge neutralization produced by some of the polyamines on their binding to chromatin fibers. The stoichiometry of polyamine binding at which condensation of chromatin is completed is found to be two polyamine molecules per DNA turn. The extent of neutralization of the DNA phosphates by the histones in these compact fibers is estimated to be about 55%. The model of polyamine interaction with chromatin is discussed.     

Sperm-chromatin maturation in the mouse. A cytochemical approach

Cytochemical techniques were used to study chromatin during spermiogenesis and sperm maturation in the mouse, starting from the stages at which the substitution of somatic histones by testis-specific proteins occurs. It was possible to distinguish and analyze the different temporal incidence of two processes involved in sperm maturation, i.e. chromatin condensation (a tridimensional highly compacted arrangement) and chromatin stabilization (a tough structure, which protects the genome DNA). The first process, involving a reduction in the nuclear size and a decrease in the amount of sperm DNA accessible to specific cytochemical reactions and stainings, was found to reach its maximum in caput-epididymidis spermatozoa, in which electron microscopy revealed that the sheared chromatin was mainly organized into 120-A-thick knobby fibers. No further changes were found in sperm up to their appearance in the fallopian tubes. On the contrary, chromatin stabilization, the onset of which occurs in the testis (at the late spermatid stage) via the formation of -S-S- cross-links, is completed in the vas deferens, where chromatin has a superstructure consisting of thicker fibers, with diameters of 210 and 350 A. The reductive cleavage of disulfides in vas-deferens spermatozoa does not completely destroy the superstructure of sperm chromatin, which could indicate 'coiling' of the basic knobby fiber. In fact, when the ion concentration was increased, the chromatin of vas-deferens spermatozoa appeared to be organized into fibers with diameters similar to those of the caput epididymidis. This unique organization of mature sperm chromatin should have an essential role in the fast swelling of spermatozoa during fertilization.     

CpG methylated minichromosomes become inaccessible for V(D)J recombination after undergoing replication.

The physical parameters controlling the accessibility of antigen receptor loci to the V(D)J recombination activity are unknown. We have used minichromosome substrates to study the role that CpG methylation might play in controlling V(D)J recombination site accessibility. We find that CpG methylation decreases the V(D)J recombination of these substrates more than 100-fold. The decrease correlates with a considerable increase in resistance to endonuclease digestion of the methylated minichromosome DNA. The minichromosomes acquire resistance to both the intracellular V(D)J recombinase and exogenous endonuclease only after DNA replication. Therefore, CpG methylation specifies a chromatin structure that, upon DNA replication, is resistant to eukaryotic site-specific recombination. These findings are important to V(D)J recombination as well as to the chromatin assembly of methylated DNA during replication.