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1.
The accessibility and role of histone regions in chromatin fibres were investigated using limited proteolysis with enzymes covalently bound to collagen membranes. The changes in chromatin conformation and condensation monitored by various biophysical methods, were correlated to the degradation of the histone proteins revealed by antibodies specific for histones and histone peptides. Upon digestion with trypsin and subtilisin, chromatin undergoes successive structural transitions. The cleavage of the C-terminal domains of H1, H2A and H2B, and of the N-terminal tail of H3 led to a decondensation of chromatin fibres, indicated by increases in electric birefringence and orientational relaxation times. It corresponds to a 15% increase in linear dimensions. The degradation of the other terminal regions of histones H3, H2A and H2B resulted in the appearance of hinge points between nucleosomes without alteration of the overall orientation of polynucleosome chains. Despite the loss of all the basic domains of H1, H3, H2A and H2B, no significant change in DNA-protein interactions occurred, suggesting that most of these protease-accessible regions interact weakly, if at all, with DNA in chromatin. Further proteolysis led to H4 degradation and other additional cleavages of H1, H2B and H3. This caused the relaxation of no more than 8% of the total DNA but resulted in changes in the ability of chromatin to condense at high ionic strength. More extensive digestion resulted in a total unravelling of nucleosomal chains which acquired properties similar to those of H1-depleted chromatin, although the globular part of H1 was still present. The data suggest that histone-histone interactions between H1 and core histone domains play a central role in stabilizing the chromatin fibres, and cuts in H3, H2A and H2B as well as H1, seem necessary for chromatin expansion. On the contrary, H4 might be involved in the stabilization of nucleosomes only.  相似文献   

2.
Abstract

The accessibility and role of histone regions in chromatin fibres were investigated using limited proteolysis with enzymes covalently bound to collagen membranes. The changes in chromatin conformation and condensation monitored by various biophysical methods, were correlated to the degradation of the histone proteins revealed by antibodies specific for histones and histone peptides.

Upon digestion with trypsin and subtilisin, chromatin undergoes successive structural transitions. The cleavage of the C-terminal domains of Hl, H2A and H2B, and of the N-terminal tail of H3 led to a decondensation of chromatin fibres, indicated by increases in electric birefringence and orientational relaxation times. It corresponds to a 15% increase in linear dimensions. The degradation of the other terminal regions of histones H3, H2A and H2B resulted in the appearance of hinge points between nucleosomes without alteration of the overall orientation of polynucleosome chains. Despite the loss of all the basic domains of HI, H3, H2A and H2B, no significant change in DNA-protein interactions occurred, suggesting that most of these protease-accessible regions interact weakly, if at all, with DNA in chromatin. Further proteolysis led to H4 degradation and other additional cleavages of Hl, H2B and H3. This caused the relaxation of no more than 8% of the total DNA but resulted in changes in the ability of chromatin to condense at high ionic strength. More extensive digestion resulted in a total unravelling of nucleosomal chains which acquired properties similar to those of Hl- depleted chromatin, although the globular part of HI was still present.

The data suggest that histone-histone interactions between HI and core histone domains play a central role in stabilizing the chromatin fibres, and cuts in H3, H2A and H2B as well as HI, seem necessary for chromatin expansion. On the contrary, H4 might be involved in the stabilization of nucleosomes only.  相似文献   

3.
Effects of ionic strength and proteolytic digestion on the conformation of chromatin fibers were studied by electric birefringence and relaxation measurements. The results confirm that at low ionic strength chromatin presents structural features reflecting those observed in the presence of cations. Soluble chromatin prepared from rat liver nuclei by brief nuclease digestion exhibits a positive birefringence. As the salt concentration is increased, the transition to a compact solenoidal structure is deduced from changes in electro-optical properties: the positive birefringence gradually decreases and the observed reduction in 40 mM NaCl is nearly 95%; the relaxation time decreases dramatically and the character of the kinetic changes since the decay of birefringence described initially by a spectrum of relaxation times becomes monoexponential. On digestion with proteases at low ionic strength we observe at first a rapid increase of the positive birefringence concomitant with an increase of the relaxation time. Then the birefringence decreases and becomes negative. Chromatin undergoes two successive transitions: the first transition is explained by a lengthening of nucleosomal chains without modification of the orientation of nucleosomes within the superstructure and the second one by the unwinding of the DNA tails and internucleosomal segments. When chromatin is digested at 30 mM NaCl we find a single unfolding transition characterized by the decrease of birefringence and a slight increase in the relaxation time. The results imply that the positive birefringence of chromatin does not depend on the presence of whole histone H1 and that a salt concentration of 30 mM NaCl is sufficient to modify the initial site or/and the effects of proteolytic attack.  相似文献   

4.
In order to understand how the phosphorylation of histones affects the chromatin structure, we used electron microscopy, sedimentation velocity, circular dichroism and electric birefringence to monitor the salt-induced filament reversible solenoid transition of phosphorylated and native chromatin. Phosphorylation in vitro of chicken erythrocyte chromatin by cyclic-AMP-dependent protein kinase from porcine heart led to the modification of the histones H3 and H5 only, which were modified at a level of one phosphate and about three phosphate groups per molecule, respectively. In contrast to circular dichroism and sedimentation studies, which tend to suggest that phosphorylation of H3 and H5 does not affect chromatin structure, electron microscopy reveals that phosphorylation causes a relaxation of structure at low ionic strength. Electric birefringence and relaxation time measurements clearly prove that local structural changes are induced in chromatin: we observe a decrease of the steady-state birefringence with the appearance of a negative contribution in the signal and a marked increase of the flexibility of fibres. The component with the negative birefringence presents very short relaxation times, like those exhibited by small DNA fragments or individual nucleosomes. Two possibilities are then suggested. First, the conformational change is consistent with what would be expected from the presence of DNA segments loosely associated with the core histone H3. That the length of such segments could correspond to about one to two base-pairs per nucleosome strongly suggests that phosphorylation induces changes affecting some specific H3-DNA interactions only. This result could corroborate previous observations indicating that the N-terminal region of H3, where the site of phosphorylation is located, plays a decisive role in maintaining the superstructure of chromatin. Second, phosphorylation could introduce hinge points between each nucleosome. In this case, the negative birefringence results from partial orientation of the swinging nucleosomes. A possible mode of action of phosphorylation might be to weaken structural restraints imposed by histone H3, thus facilitating further condensation of chromatin.  相似文献   

5.
Abstract

Effects of ionic strength and proteolytic digestion on the conformation of chromatin fibers were studied by electric birefringence and relaxation measurements. The results confirm that at low ionic strength chromatin presents structural features reflecting those observed in the presence of cations. Soluble chromatin prepared from rat liver nuclei by brief nuclease digestion exhibits a positive birefringence. As the salt concentration is increased, the transition to a compact solenoidal structure is deduced from changes in electro-optical properties: the positive birefringence gradually decreases and the observed reduction in 40 mM NaCl is nearly 95%; the relaxation time decreases dramatically and the character of the kinetic changes since the decay of birefringence described initially by a spectrum of relaxation times becomes monoexponential.

On digestion with proteases at low ionic strength we observe at first a rapid increase of the positive birefringence concomitant with an increase of the relaxation time. Then the birefringence decreases and becomes negative. Chromatin undergoes two successive transitions: the first transition is explained by a lengthening of nucleosomal chains without modification of the orientation of nucleosomes within the superstructure and the second one by the unwinding of the DNA tails and internucleosomal segments.

When chromatin is digested at 30 mM NaCl we find a single unfolding transition characterized by the decrease of birefringence and a slight increase in the relaxation time. The results imply that the positive birefringence of chromatin does not depend on the presence of whole histone HI and that a salt concentration of 30 mM NaCl is sufficient to modify the initial site or/and the effects of proteolytic attack.  相似文献   

6.
7.
We have observed that three proteolytic enzymes with widely different specificities produce a very similar pattern in terms of the order of digestion of the various histone fractions in chromatin. Histone H2A is most resistant to proteolytic attack by trypsin, chymotrypsin, or Pronase. H2B is next most resistant, followed by H3. Histone H1 is least resistant and is rapidly hydrolyzed by each of these enzymes. The behavior of histone H4 differs for the various enzymes. It is as resistant as H2A to digestion by trypsin and chymotrypsin but is readily hydrolyzed by Pronase. A comparison of the rates of digestion of the various histone fractions in chromatin with the rates in a DNA-free histone mixture and a study of the degradation products which result from digestion indicate that histone conformation and histone-histone and DNA-histone interactions are all involved in protecting histones from attack by proteolytic enzymes. From the results of our studies we have concluded that histones H1 and H3 are located in superficial positions of the chromosomal substructures (or nu bodies) while H2A is buried inside. Since histone H2B is relatively resistant to digestion but more readily degraded in chromatin than in a DNA-free histone mixture, it is difficult to determine its chromosomal location. Histone H4 behaves as if a large portion of the molecule is located in the major groove of the DNA helix.  相似文献   

8.
A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein.  相似文献   

9.
The role of histone H1 in the actual interactions bringing about chromatin folding is investigated by studying the reversibility of its dissociation. H1 was dissociated by increase of the NaCl concentration and reassociated by dialysis, without removal from the dialysis bag. To scrutinize the fidelity of this stoichiometric form of chromatin reconstitution, we use circular dichroism, nuclease digestion, thermal denaturation and the sensitive electric birefringence method. No alteration of the repeat length and no nucleosomal sliding are observed upon the reassociation procedure. However, under all the different conditions investigated, the original value of the positive electric birefringence is never recovered, indicating an irreversible change of structure. CD and melting profiles confirm that DNA-protein interactions are modified, and orientational relaxation time measurements indicate that these structural perturbations affect the salt-induced transition of polynucleosomal fibers. The striking conclusion of these studies is that variations of ionic concentration are sufficient to induce irreversible structural alterations affecting the higher-order folding of chromatin. It is of interest that the only sample which exhibits behavior upon reassociation comparable to that of native chromatin is the one which experienced the fastest salt transitions. We suggest that these conformational changes arise from the unbinding to DNA of certain basic tails of histone(s), and that a competition for DNA binding locations exists upon the reassociation. These results are then additional arguments (Mazen, A., Hacques, M.F. and Marion, C.,J. Mol. Biol. 194, 741-745 (1987)), to suggest that dissociation of H1 might modify a direct interaction between basic tails of core histones and H1.  相似文献   

10.
Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.  相似文献   

11.
BACKGROUND: The discovery of histone-like proteins in Archaea urged studies into the possible organization of archaeal genomes in chromatin. Despite recent advances, a variety of structural questions remain unanswered. RESULTS: We have used the atomic force microscope (AFM) with traditional nuclease digestion assays to compare the structure of nucleoprotein complexes reconstituted from tandemly repeated eukaryal nucleosome-positioning sequences and histone octamers, H3/H4 tetramers, and the histone-fold archaeal protein HMf. The data unequivocally show that HMf reconstitutes are indeed organized as chromatin fibers, morphologically indistinguishable from their eukaryal counterparts. The nuclease digestion patterns revealed a clear pattern of protection at regular intervals, again similar to the patterns observed with eukaryal chromatin fibers. In addition, we studied HMf reconstitutes on mononucleosome-sized DNA fragments and observed a great degree of similarity in the internal organization of these particles and those organized by H3/H4 tetramers. A difference in stability was observed at the level of mono-, di-, and triparticles between the HMf particles and canonical octamer-containing nucleosomes. CONCLUSIONS: The in vitro reconstituted HMf-nucleoprotein complexes can be considered as bona fide chromatin structures. The differences in stability at the monoparticle level should be due to structural differences between HMf and core histone H3/H4 tetramers, i.e., to the complete absence in HMf of histone tails beyond the histone fold. We speculate that the existence of core histone tails in eukaryotes may provide a greater stability to nucleosomal particles and also provide the additional ability of chromatin structure to regulate DNA function in eukaryotic cells by posttranslational histone tail modifications.  相似文献   

12.
Abstract

The role of histone H1 in the actual interactions bringing about chromatin folding is investigated by studying the reversibility of its dissociation. HI was dissociated by increase of the NaCl concentration and reassociated by dialysis, without removal from the dialysis bag. To scrutinize the fidelity of this stoichiometric form of chromatin reconstitution, we use circular dichroism, nuclease digestion, thermal denaturation and the sensitive electric birefringence method. No alteration of the repeat length and no nucleosomal sliding are observed upon the reassociation procedure. However, under all the different conditions investigated, the original value of the positive electric birefringence is never recovered, indicating an irreversible change of structure. CD and melting profiles confirm that DNA-protein interactions are modified, and orientational relaxation time measurements indicate that these structural perturbations affect the salt- induced transition of poly nucleosomal fibers. The striking conclusion of these studies is that variations of ionic concentration are sufficient to induce irreversible structural alterations affecting the higher-order folding of chromatin. It is of interest that the only sample which exhibits behavior upon reassociation comparable to that of native chromatin is the one which experienced the fastest salt transitions. We suggest that these conformational changes arise from the unbinding to DNA of certain basic tails of histone(s), and that a competition for DNA binding locations exists upon the reassociation. These results are then additional arguments (Mazen, A., Hacques, M.F. and Marion, C., J. Mol. Biol. 194, 741–745 (1987)), to suggest that dissociation of H1 might modify a direct interaction between basic tails of core histones and H1.  相似文献   

13.
14.
The complete amino acid sequence (123 residues) of histone H2A from erythrocytes of the marine worm Sipunculus nudus, has been established from data provided by automated sequence analysis of large fragments generated by V8 staphylococcal protease digestion of histone H2A and by limited hydrolysis of the protein with alpha-chymotrypsin and from structural studies of tryptic peptides of the protein. By comparison with calf homologous histone, the sipunculid histone H2A shows 6 deletions and 13 substitutions. Six of the substitutions are non-conservative. Most of the evolutionary changes are mainly observed in the basic amino-terminal and carboxy-terminal regions of the molecule, which are the primary DNA-binding sites. Few conservative point changes are observed in the central region (residues 18-118) which interacts strongly with histone H2B to form the dimer H2A-H2B. 60% of the H2A molecules were found phosphorylated on the amino-terminal residue, N-acetyl-serine. The high content of phosphorylated histone H2A in the sipunculid erythrocyte chromatin could probably be related to smaller repeat length (177 +/- 5 base pairs) of nucleosomal DNA and to nuclear inactivation and chromatin condensation.  相似文献   

15.
After removal of histone H1 about 40% of DNA in chromatin acquires the sensitivity of naked DNA to DNAse I. Digestion of H1-depleted chromatin with DNAse I leads to a qualitative change in the digestion pattern, generating DNA fragments of approx. 200 b.p. and multiples, similar to those obtained with micrococcal nuclease. Both effects are reversed upon reconstitution of purified H1 to H1-depleted chromatin.  相似文献   

16.
Submaxillaris protease, reportedly specific for arginine residues, was used to probe accessible arginines of chicken erythrocyte nucleosome cores. The relative rates of digestion of histones in nucleosome cores by this protease were H3 greater than H2b greater than H4 much greater than H2a. At most, 8 of 52 total arginines among the four core histones were reasonably accessible to attack. Sites most rapidly cleaved were Arg-26 in H3, a site approximately 13 residues from the NH2 terminus of H2b, and Lys-12 or Arg-17 in H4. Five sites attacked more slowly were Arg-8, -128, or -129 and Arg-49, -52, or -53 in H3; Arg-3 and Arg-17 or -19 in H4; and a site near one terminus of H2b or H3. H2a and fragments resulting from the above cleavages were highly resistant to attack, even after prolonged incubation. Similar results were obtained upon digestion of histones in intact chromatin. H1 and H5 in whole chromatin were attacked at rates comparable to H3. Seven of the eight accessible sites lie outside of structure-forming histone sequences, i.e. sequences that are immobilized in histone complexes, indicating that these sequences are inaccessible in nucleosome cores. The single exceptional site noted, approximately 50 residues from the NH2 terminus of H3, is consistent with previous observations that Glu-51 and Glu-60 of H3 in nucleosomes are accessible to attack by S. aureus protease (Rill, R. L., and Oosterhof, D. K. (1981). J. Biol. Chem. 256, 12687-12691). The relationships of these protease accessibilities to NMR assignments of mobile histone tails in nucleosome cores are discussed.  相似文献   

17.
High molecular weight (HMW) fragmentation of nuclear chromatin was studied in cultured rat oligodendrocytes (OL) exposed to hydrogen peroxide (H2O2). Intact genomic DNA was isolated by agarose embedding, and analyzed by field inversion gel electrophoresis, with and without S1 endonuclease digestion to detect and discriminate between single and double stranded fragmentations, respectively. The exposure of OL to H2O2 resulted in a very rapid degradation of chromosomal DNA into HMW fragments that reflect native chromatin structure. Hence, within 10 min after the addition of 1 mM H2O2, a discrete pool representing approximately 45% of the nuclear chromatin underwent single strand digestion into >400 kb fragments likely at AT-rich matrix attachment regions. Subsequent accumulation of single stand breaks at these regions led to bifilar scission. Ultimately, chromatin within this susceptible pool was cleaved at remaining matrix attachment regions into 50-200 kb fragments. Chromatin digestion could be elicited with H2O2 concentrations as low as 50 microM. After the removal of H2O2, most >400 kb fragments were religated within 2 h; however, digestion into 50-200 kb fragments was irreversible. The DNA digestion was not accompanied by the degradation of nuclear proteins, i.e., lamins A/C and poly (ADP-ribose) polymerase indicating that chromatin fragmentation is unlikely to be mediated by proteolysis. In conclusion, H2O2 at pathologically relevant concentrations induces a very rapid and extensive digestion of OL chromatin into HMW fragments. Because the chromatin fragmentation is only partly reversible, it may be a decisive factor in committing oxidatively stressed OL to degeneration and/or death.  相似文献   

18.
The sites of deposition of newly synthesized histone.   总被引:4,自引:2,他引:2       下载免费PDF全文
The chromosomal fragments produced by nuclease digestion of freshly replicated chromatin migrate more rapidly relative to bulk chromatin when analyzed in nucleoprotein gels. The cause of the anomalous migration has been studied and the evidence indicates that rather than reflecting a shorter nucleosomal repeat in vivo that it may be a consequence of nucleosome sliding during the digestion itself. The distinct electrophoretic characteristics of nucleosomal material containing newly replicated DNA have enabled us to examine their histone composition by two dimensional electrophoresis. We find that nucleosomes containing new DNA also contain newly synthesized histones H3 and H4. In contrast more than 50% of newly synthesized H2A and H2B, and essentially all of new H1, are deposited at sites on the bulk chromatin distinct from that material containing newly replicated DNA. In addition we show that newly synthesized histones H3 and H4 are bound unusually weakly when they first become associated with the chromatin.  相似文献   

19.
We describe several morphological and functional modifications in isolated rat liver nuclei incubated in the presence of phosphatidylserine (PS) multilamellar vesicles (MLV). These effects, which occur through the release of histone H1, induce chromatin decondensation, as shown by electron microscopy and nuclease digestion. Flow cytometry was employed to monitor these changes in chromatin structure in isolated nuclei by means of perpendicular light scatter (PLS) and fluorescence signals. Chromatin decondensation induced by PS or by low pH treatment was accompanied by an increase in perpendicular light scatter and by less efficient binding of ethidium bromide. These flow cytometric findings are peculiar to chromatin decondensation induced by displacement of histone H1. Conversely, chromatin decondensation caused by lowering of the divalent ion concentration, without displacement of histone H1, is characterized only by an increase in perpendicular light scatter.  相似文献   

20.
Trypsinolysis of histone H5 in solution and as a component of chromatin with different level of compactization was studied. It was demonstrated that the existence of supernucleosomal organization leads to a significant decrease of the degradation rate of histones H1 and H5 in comparison with histones H2A, H2B, H3 and H4. Analysis of trypsinolysis electrophoretic spectra of histone H5 revealed the existence of protease-resistant fragments in chromatin, but not in solution. These fragments contain not only the globular domain of histone H5 but also small-sized unstructured N- and/or C-terminal regions. The peptides were identified with the help of an immune serum specific for the globular region of histone H5. The possible role of resistant fragments in the nucleosomal organization of chromatin is discussed.  相似文献   

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