Histone Exchange Research Articles

Chromatin as a regulative architecture of the early developmental functions of mammalian embryos after fertilization or nuclear transfer.

Nuclear transfer of a somatic nucleus into an enucleated oocyte has demonstrated in several mammalian species that the chromatin of a differentiated nucleus can be reprogrammed so as to be able to direct the full development of the reconstructed embryo. This review focus on the timing of the early events that allow the return of somatic chromatin to a totipotent state. Our understanding of the modifications associated with chromatin remodeling is limited by the low amount of biological material available in mammals at early developmental stages and the fact that very few genetic studies have been conducted with nuclear transfer embryos. However, the importance of several factors such as the covalent modifications of DNA through the methylation of CpG dinucleotides, the exchange of histones through a reorganized nuclear membrane, and the interaction between cytoplasmic oocyte components and nuclear complexes in the context of nuclear transfer is becoming clear. A better characterization of the changes in somatic chromatin after nuclear transfer and the identification of oocyte factors or structures that govern the formation of a functional nucleus will help us to understand the relationship between chromatin structure and cellular totipotency.

Analysis of nucleosome assembly and histone exchange using antibodies specific for acetylated H4.

Using antibodies that specifically recognize the acetylated forms of histone H4, we show that it is possible to immunoprecipitate newly assembled (acetylated) nucleosomes. Newly replicated HeLa cell chromatin was labeled for 5-30 min with [3H]thymidine in the presence of sodium butyrate (thus inhibiting the deacetylation of newly deposited H4); bulk chromatin DNA was labeled for 24 h with [14C]thymidine. When soluble nucleosomes were incubated with immobilized antibodies, a comparison of the bound and unbound fractions showed up to a 65-fold enrichment for new chromatin DNA in the immunoprecipitate (bound), relative to the supernatant (unbound). No enrichment for new DNA was observed when preimmune control serum was used in a similar fashion. The enrichment for new DNA in the immunopellet was paralleled by a similar enrichment for all four newly synthesized histones. Acetylation was required for antibody recognition: When chromatin was replicated in the absence of butyrate (permitting histone deacetylation and chromatin maturation), equally low levels of new and old chromatin were immunoprecipitated, and no enrichment for new DNA was observed. Competition experiments confirmed these results. Analyses of histone deposition during the inhibition of DNA replication established that acetylated chromatin is the preferential target for H2A/H2B exchange. These experiments provide evidence for the highly selective assembly of newly synthesized H3, H2A, and H2B with acetylated H4, and for the involvement of histone acetylation in dynamic chromatin remodeling. In addition, immunoprecipitations of radiolabeled cytosolic extracts identified a possible somatic chromatin preassembly complex, containing newly synthesized H3 and new (acetylated) H4.

Poly[d(A · T)]and other synthetic polydeoxynucleotides containing oligoadenosine tracts form nucleosomes easily

Synthetic double-stranded polydeoxynucleotides of the general form poly[d(A n T) · d(AT n )], with n ranging from 3 to 11, have been synthesized. The conformation of the polymers was investigated by circular dichroism spectroscopy and the polymers were examined for their ability to form nucleosomes. Although spectra show that a circular dichroism band characteristic of poly[d(A · T)] appears in the polymer family for n ≥ 7, we demonstrate that even polynucleotides with the longest tracts of contiguous adenosine bases ( n = 11) are able to form nucleosomes when reconstituted using a histone exchange procedure. Thus resistance to nucleosome formation does not coincide with the appearance of features similar to that of poly[d(A · T)] over the bulk of the nucleosomal DNA. Furthermore, we show that an approximately 150 base-pair poly[d(A · T)] itself, long thought to be refractory to nucleosome formation, can assemble into such a protein-DNA complex when reconstituted by a low-salt exchange procedure. Competitive assays show that the homopolymer reconstitutes about as well as heterogeneous sequences DNA. Our work, therefore, suggests that highly adenosine-rich sequences in vivo apparently have a function that operates at a level other than that of nucleosome structure.

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase in reconstituted Simian virus 40 chromatin

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.

Conservative Segregation of Tetrameric Units of H3 and H4 Histones during Nuclesome Replication

We have specifically investigated the behavior of H3 and H4 histones during the replication cycle of MH-134SC cells. Mononucleosomes obtained from cells density-labeled with IdU or dense amino acids in the presence of appropriate radiolabeled precursors were applied to sucrose gradients containing 0.3 M NaCl and 4 M urea for rate zonal centrifugation. This allowed the resolution of dense and normal subnucleosome particles composed of DNA and two molecules each of H3 and H4 without any measurable interparticle histone exchange. On labeling with dense amino acids and radiolabeled lysine, a distinct peak of radiolabeled dense particles was obtained. In contrast, pre-radiolabeled H3 and H4 remained in the normal subnucleosome peak region even after one generation time of culturing with dense amino acids. These data indicate the formation of (H3-H4)2 tetramers composed entirely of new H3 and H4 molecules as well as the conservation of pre-existing tetramers. Density labeling for 1 h with IdU in the presence of radiolabeled lysine yielded a distinct peak of radiolabeled dense particles, indicating the deposition of new tetramers on newly replicated DNA. Similar rate zonal analysis of subnucleosome particles obtained from cells prelabeled for 1 h with radiolabeled lysine followed by various IdU-labeling schedules in nonisotopic media yielded data suggesting that tetramers once deposited do not move about randomly during the replication cycle. A possible mode of nucleosome replication is discussed in the light of the present data.

Deposition of newly synthesized histones: hybrid nucleosomes are not tandemly arranged on daughter DNA strands.

Density labeling procedures have been utilized to study the dynamics of histone-histone interactions in vivo. Cells were labeled for 60 min with dense amino acids, and the label was chased for up to 22 h (two replication events for these cells). Nuclei were isolated and treated with formaldehyde to stabilize the histone-histone interactions with a covalent cross-link that produces an octameric complex of two each of H3, H2B, H2A, and H4. This complex was then extracted from the DNA and analyzed on density gradients. The results indicate that new H3,H4 deposits as a tetramer and does not dissociate in the subsequent chases. New H2A,H2B deposited as a dimer and also does not dissociate in subsequent chases. These new histones form hybrid octamers with old histones. On the basis of the new:old ratio in the hybrid octamers, we propose that additional old H2A,H2B from elsewhere in the genome interacts with tetramers of new H3,H4 to form the newly synthesized nucleosomes. It is also observed that 5% of the cross-linked complexes produced by formaldehyde are octamer-octamer (dioctamer). Upon analysis of the density of the dioctamer, the hybrid octamers were found adjacent to octamers that were homogeneous with respect to containing normal density histones. Control experiments are presented to demonstrate that the octamer-octamer cross-links are a product of intrastrand and not interstrand interactions between nucleosomes. These same control experiments also indicate that these procedures do not induce histone exchange during the preparative procedure prior to density gradient analysis. The significance of these results with regard to the dynamics of histone-histone interactions at the replication fork and the potential role in the maintenance of differentiation is discussed.

Exchange of H1 histone depends on aggregation of chromatin, not simply on ionic strength.

Chromatin solubility was observed at several concentrations of various cations. Spermine and spermidine precipitated (50%) chromatin at about 0.2 mM, Ca2+ and Mg2+ at about 1-2 mM, and Na+ at about 100 mM. Further increases in cation concentration induced more aggregation, but eventually excess cation increased chromatin solubility so that 50% solubility was observed again at 60 mM Mg2+ and 180 mM Na+. H1 histone was 50% released by 80 mM MgCl2 or 425 mM NaCl. Combinations of MgCl2 and NaCl showed that Mg2+ and Na+ are synergistic in the induction of aggregation in lower concentrations (less than 2 mM) of Mg2+ but antagonistic at higher concentrations, and a similar effect of NaCl on spermidine-induced precipitation was shown below and above about 0.2 mM spermidine. At 5 mM, MgCl2 proved capable of precipitating chromatin depleted of H1 histone, but no concentration of NaCl was capable of doing so. These phenomena can be rationalized by supposing that neutralization of chromatin by any cation (including H1 histone) favors aggregation and also that cross-linking of chromatin fibers by multivalent cations (including H1 histone) is also critically important. The exchange of H1 histone between chromatin fragments was tested in various concentrations of different salts. H1 exchange was correlated with chromatin aggregation rather than with ionic strength and thus appears to depend on fiber to fiber contact. Under conditions where H1 exchanges between chromatin fibers that are permitted to make contact with each other, no H1 exchange occurred between chromatin inside the nucleus and chromatin outside, even though H1 histone is capable of passage through the nuclear membrane.

H1 histone exchange is limited to particular regions of chromatin that differ in aggregation properties.

Chromatin fragments produced by mild nuclease digestion were chromatographed on Bio-Gel A-50m to give fractions ranging in size from 0.4 to 30 kilobase pair-DNA. The fragments that were larger than about 8-10 nucleosomes accounted for 80% of the chromatin, and the H1/core histone ratio was constant throughout these fractions. When adjusted to 150 mM NaCl, aggregates precipitated in each fraction, the largest fragments yielding 60% and the smallest 25%. In all of these fractions, after aggregation was induced by NaCl, the H1/core histone ratio in the aggregation-resistant chromatin (S) was 0.7 that in the aggregated chromatin (P). To show that the H1 deficiency and aggregation resistance were not produced by transfer of H1 from little fragments to bigger one, big aggregation-resistant fragments were incubated with little aggregation-prone fragments in 75 mM NaCl for 2 h, and readjusted to 150 mM. The little aggregation-prone fragments retained their aggregatibility after exposure to big aggregation-resistant fragments. By mixing [3H]P with [14C]S and vice versa, incubating at 75 mM NaCl for 2 h, and separating P from S with 150 mM NaCl, it was demonstrated that H1 histone did not equilibrate between S and P. Similarly, mixing combinations of radioactive and unlabeled, big and little, S and P fractions, and fractionating by size after 2 h or more incubation at 75 mM NaCl, it was shown that H1 equilibrates between different S fragments, and between different P fragments, but not between S and P. The distribution of H1 variants between S and P fractions was not correlated with the affinity of the variants for DNA. The order of binding affinities was H10 greater than H1ab = H1c, but the deficits of H1's in the aggregation-resistant S fractions were ranked H1ab greater than H1c greater than H10. It is suggested that chromatin is a mosaic of aggregation-resistant and aggregation-prone regions which differ in H1 content quantitatively and qualitatively.

Histone synthesis and deposition in the G1 and S phases of hepatoma tissue culture cells.

Hepatoma tissue culture cells were synchronized in G1 and in S phase in order to examine the level of synthesis of different histone types and to determine the rate, timing, and location of their deposition onto DNA. We observe a basal level of synthesis in G1 (5% of that seen in S phase) for H2A.1, H2A.2, H3.2, H2B, and H4. The minor histone variants X and Z are synthesized at 30% of the rate observed in S cells. The rate of synthesis of the ubiquinated histones uH2A.1,2 is not as depressed in G1 cells as seen for H2A.1 and H2A.2. Histones synthesized in G1 are not deposited on the DNA of these cells at equivalent rates. Thus, histones H3.2 and H4 are not deposited significantly until S phase begins, at which time deposition occurs selectively on newly synthesized DNA. The deposition of H2A.1, H2A.2, H2B, X, and Z proceeds in G1; however, it occurs to a 2-4-fold lower extent than seen for the deposition of H1, HMG 14, and HMG 17. The deposition of all histones synthesized in S phase occurs rapidly, but there are variations in the sites of deposition. Thus, newly synthesized H3.1, H3.2, and H4 deposit primarily on newly replicated DNA whereas H2A.1, H2A.2, uH2A.1, 2, and H2B deposit only partially on new DNA (30%) and mostly on old. H1, HMG 14, and HMG 17 are deposited in an apparently fully random manner over the chromatin. To interpret these observations, we propose a model which includes a measure of histone exchange on the chromatin fiber. The model emphasizes the dynamics of histone-histone and histone-DNA interactions in regions of active genes and at replication forks.

DNA replication and H5 histone exchange during reactivation of chick erythrocyte nuclei in heterokaryons.

Fusion of terminally differentiated chick erythrocytes (CE) with replicating quail myoblasts or established L6J1 rat myoblasts results in reactivation of DNA synthesis in the dormant CE nuclei and in suppression of DNA synthesis in the myoblast nuclei. The nuclei of primary quail myoblasts are more effectively inhibited than the nuclei of established rat myoblasts. Inhibition of DNA replication occurs not only by preventing G1 nuclei from entering S-phase but also by blocking nuclei in S-phase and by delaying nuclei in G2 from undergoing mitosis and starting a new DNA replication cycle. No inhibition of DNA synthesis could be observed when mouse erythrocytes, i.e., erythrocytes lacking nuclei, were fused with rat myoblasts to generate mouse-globin-containing L6J1 cybrids.--Reactivation of CE nuclei is associated with a loss of the tissue-specific H5 histone variant. Complete elimination of H5 histone, however, does not seem to be a necessary prerequisite for the initiation or completion of DNA replication in CE nuclei since H5 antigens are found on reactivated G1, S, and G2 nuclei.

Control of RNA polymerase binding to chromatin by variations in linker histone composition

We have measured the frequency of initiation sites in chromatin for RNA polymerase in vitro as a function of the composition of linker histones (H1 and its analogues). In linker histone-depleted chromatin, RNA chain initiation appears to be restricted to the exposed linker DNA. On titration with purified linker histones, initiation is further restricted to an extent determined by the amount and type of linker histone, and the source of depleted chromatin. The extent of repression is correlated with the capacity of linker histones to induce the formation of higher-order structure in the complex. The results suggest that the effects of linker histones are mediated through the higher-order structure of chromatin, which prevents access of polymerase to the linker DNA. Accordingly, we find that structures imposed by the linker histones after polymerase binding are not inhibitory. Microscopy reveals that the higher-order structure in partially condensed chromatin is discontinuous, with solenoidal units spaced by sections of unravelled nucleosomes. Since salt stimulation of linker histone exchange does not result in derepression of linkers in our assay, we conclude that the distribution of higher-order units in chromatin is static and that the linker histones exchange between high-affinity sites in established units. We have previously shown that the globin gene is selectively unfolded in tissues that express the gene. The present results suggest that the transcriptional activity of specific genes is maintained by differential linker histone binding within chromatin.

Effects of ionic strength and state of assembly on kinetics of hydrogen exchange of calf thymus histones.

The kinetics of hydrogen exchange of calf thymus histone H2A-H2B dimers and (H3-H4)2 tetramers at pH 7 have been examined at low (0.16 M NaCl) and high (2 M NaCl) ionic strengths and after incorporation into (H2A-H2B-H3-H4)2 octamers. The similarity of the results for both species is noteworthy. Approximately 60% of the backbone amide protons are detectable in both low and high salt, and at least three kinetic phases can be distinguished. Increasing the ionic strength from 0.16 to 2 M accelerates exchange of some of the rapidly exchanging protons in both dimers and tetramers, while slowing exchange of others. Exchange of the more slowly exchanging protons is virtually unaffected. Incorporation of dimers into octamers accelerates exchange of approximately 40 protons to such an extent that they can no longer be detected. The effects of assembly upon the tetramer are qualitatively similar. These results indicate that both high ionic strengths and assembly destabilize some regions of the structure while stabilizing others. For both dimers and tetramers, the effects of ionic strength are dramatic, while those of assembly are more subtle. Higher resolution studies aimed at identifying the responsive protons would be of interest.

Histone exchange in chromatin of hydroxyurea-blocked Ehrlich ascites tumour cells.

It is well established that DNA and histone synthesis are tightly coupled. Nevertheless, these two processes can be partially uncoupled by drugs specifically inhibiting protein or DNA synthesis, and also during n-butyrate-induced differentiation of Friend cells. The fate of the histones synthesized in the absence of DNA synthesis is unknown; they could be: (1) degraded without joining chromatin; (2) deposited on chromatin as extra histones; or (3) replace original chromatin histones. The only data concerning this problem are a recent report supporting the second possibility. We present evidence here in favour of the third possibility by showing that the histones synthesized in the absence of DNA synthesis enter chromatin and become organized in nucleosomes.

Structural changes in simian virus 40 chromatin as probed by restriction endonucleases

The structure of simian virus 40 (SV40) chromatin was probed by treatment with single- and multiple-site bacterial restriction endonucleases. Approximately the same fraction of the chromatin DNA was cleaved by each of three different single-site endonucleases, indicating that the nucleosomes do not have unique positions with regard to specific nucleotide sequences within the population of chromatin molecules. However, the extent of digestion was found to be strongly influenced by salt concentration. At 100 mM NaCl-5 mM MgCl2, only about 20% of the simian virus 40 (SV40) DNA I in chromatin was converted to linear SV40 DNA III. In contrast, at lower concentrations of NaCl (0.05 or 0.01 M), an additional 20 to 30% of the DNA was cleaved. These results suggest that at 100 mM NaCl only the DNA between nucleosomes was accessible to the restriction enzymes, whereas at the lower salt concentrations, DNA within the nucleosome regions became available for cleavage. Surprisingly, when SV40 chromatin was digested with multiple-site restriction enzymes, less than 2% of the DNA was digested to limit digest fragment, whereas only a small fraction (9 to 15%) received two or more cuts. Instead, the principal digest fragment was full-length linear SV40 DNA III. The failure to generate limit digest fragments was not a consequence of reduced enzyme activity in the reaction mixtures or of histone exchange. When the position of the principal cleavage site was mapped after HpaI digestion, it was found that this site was not unique. Nevertheless, all sites wree not cleaved with equal probability. An additional finding was that SV40 chromatin containing nicked-circular DNA II produced by random nicking of DNA I was also resistant to digestion by restriction enzymes. These results suggest that the initial cut which causes relaxation of topological constraint in SV40 chromatin DNA imparts resistance to further digestion by restriction enzymes. We propose that this may be accomplished by either "winding" of the internucleosomal DNA into the body of the nucleosome, or as suggested by others, by successive right-hand rotation of nucleosomes.

Histone dimers: a fundamental unit in histone assembly.

Histone interactions which occur, at moderate ionic strengths, when several types of purified, renatured histones are mixed at equimolar ratios have been studied. The four histones H2A,H2B,H3 and H4 complex and form dimers. Histone H1 does not interact with the other four histone types and does not form dimers. Mixing of single histone species with preformed histone pairs as well as mixing of two different types of histone pairs, leads to exchange of histones among the pairs and formation of dimers. No trimers are formed. The dimers are in equilibrium with high-molecular weight histone structures. The results indicate that histone dimers may serve as a stable intermediate in histone assembly. Because each histone type (except H1) can interact with itself as well as with each of the other three histone types we suggest that each histone type should be considered as an interchangeable subunit of a multichain protein in which the dimer species is the most stable structure.

Nature of protein association with chromatin

The reality of the nonrandom distribution of histones along the chromatin strand was investigated in several ways. It does not appear to derive from histone exchange during shearing and is evident in chromatin fixed with formaldehyde prior to shearing. Endogenous or Escherichia coli polymerase are preferentially associated with regions of chromatin with a low protein/DNAratio. Although RNA polymerase and histones are fixed to chromatin after formaldehyde treatment with high efficiency, only a minor fraction of non-histone protein is fixed under similar conditions. Even after washing in high salt to minimize adventitious association, most remaining non-histone protein fails to be fixed. The utility of this approach for defining chromosomal proteins is discussed.