Assembly of newly replicated chromatin.

Mild staphylococcal nuclease digestions under isotonic conditions release fragments of a 200 Å diameter fiber from nuclei of Drosophila melanogaster tissue culture cells. These soluble fragments have high sedimentation coefficients (30–100S) and show tightly packed nucleosomes in the electron microscope. Under the same conditions, newly replicated chromatin is released as more slowly sedimenting fragments (14S). Within 20 min after DNA replication, the nascent chromatin gradually matures into compact supranucleosomal structures which are indistinguishable from bulk chromatin on the isokinetic sucrose gradients.We have used this fractionation technique to examine the question of the fate and assembly of the new histones. After short pulses with either ³⁵S-methionine or ³H-lysine, the radioactive histones do not co-sediment with the bulk chromatin but appear instead in the fractions where the newly replicated DNA is found. Furthermore, the various nascent histones appear in different fractions on the gradient: histones H3 and H4 in 10–15S structures, histones H2A and H2B in 15–50S structures and histone H1 in 30–100S structures. These results, together with the analysis of pulse and pulse-chase experiments of both nascent DNA and histones, strongly suggest that histones H3 and H4 are deposited first on the nascent DNA (during or slightly after the DNA is replicated), histones H2A and H2B are deposited next (2–10 min later) and histone H1 is deposited last (10–20 min after DNA replication). A high turnover 20,000 dalton protein is also associated with the newly replicated chromatin.     

The presence of F3-F2a1 dimers and F1 oligomers in chromatin.

The oligomeric structure of histones in nuclei and chromatin has been studied by crosslinking nuclei and chromatin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Crosslinked histones were detected as new high molecular weight components on SDS gels, and the protomers of the crosslinked histones were identified by their characteristic ¹²⁵I-fingerprints. The results show that a considerable portion of histones F3 and F2a1 exist in nuclei and chromatin as an F3-F2a1 dimer. Evidence is presented that histone F1 probably exists in chromatin as large oligomers.     

Nucleosomes and subnucleosomes: heterogeneity and composition

Previous studies (Varshavsky, Bakayev and Georgiev, 1976a) have shown that chromatin subunits (mononucleosomes) and their oligomers in a mild staphylococcal nuclease digest of chromatin display a heterogeneous content of histone H1. We now report that a mild staphylococcal nuclease digest of either chromatin or nuclei from mouse Ehrlich tumor cells contains mononucleosomes of three discrete kinds. The smallest mononucleosome (MN₁) contains all histones except H1 and a DNA fragment 140 base pairs (bp) long. The intermediate mononucleosome (MN₂) contains all five histones and a DNA fragment 170 bp long. The third mononucleosome (MN₃) also contains all five histones, but its DNA fragment is longer and more heterogeneous in size (180–200 bp). Most of the MN₃ particles are rapidly converted by nuclease into mononucleosomes MN₁ and MN₂ There exists, however, a relatively nuclease-resistant subpopulation of the MN₃ mononucleosomes. These 200 bp MN₁ particles contain not only histones but also nonhistone proteins, and are significantly more resistant to nuclease than the bulk of MN₃ particles and the smaller mononucleosomes MN₁ and MN₂.There are eight major kinds of staphylococcal nuclease-produced soluble subnucleosomes (SN). The SN₁ is a set of naked double-stranded DNA fragments ～20 bp long. The SN₂ is a complex of a specific basic nonhistone protein (molecular weight ～16,000 daltons) and a DNA fragment ～27 bp long. The SN₃ contains histone H4, the above-mentioned specific nonhistone protein and a DNA fragment ～27 bp long. The SN₄ contains histones H2a, H2b, H4 and a DNA fragment ～45 bp long. The SN₅ contains histones H2a, H2b, H3 and a DNA fragment ～55 bp long. The SN₆ is a complex of histone H1 and a DNA fragment ～35 bp long. Subnucleosomes SN₇ and SN₈ each contain all the histones except H1, and DNA fragments ～100 and ～120 bp long, respectively.Nuclease digestion of isolated mono- or dinucleosomes does not produce some of the subnucleosomes. These and related findings indicate that the cleavage required to generate these subnucleosomes result from some aspect of chromatin structure which is lost upon digestion to mono- and dinucleosomes.     

THE HISTONES ASSOCIATED WITH CONDENSED AND EXTENDED CHROMATIN OF MOUSE LIVER

Histones were extracted from isolated mouse liver nuclei, and from mouse liver condensed and extended chromatin. Mouse liver histones were found to be very similar to those of calf thymus in their solubility properties, relative electrophoretic mobilities, and molecular weights as determined on SDS-polyacrylamide gels. Quantitative analysis by high-resolution gel electrophoresis demonstrated a remarkable similarity between the histones of condensed chromatin and those of extended chromatin. However, minor differences were found. A unique subspecies was found only in condensed chromatin histone and the relative amounts of fractions F2A1 and F2A2 differed in the two types of chromatin. The ratio of the parental to the acetylated form of F2A1 was identical in the two chromatin samples. Since DNA extracted from the condensed chromatin fraction consisted of approximately 50% satellite DNA, the general similarities between the histones of condensed and extended chromatin make it likely that even this simple, highly repetitive DNA is complexed with a number of histone subfractions.     

Separation of nucleosomes containing histones H1 and H5

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165–180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170–190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.     

NUCLEAR HISTONE PROTEINS OF GAMETES IN AN OOGAMOUS AND TWO ISOGAMOUS BROWN ALGAE1

Nuclear basic proteins (histones) were studied in male and female gametes of the isogamous brown algae, Colpomenia bullosa (Saunders) Yamada and Analipus japonicus (Harvey) Wynne and sperm of the oogamous Cystoseira hakodatensis (Yendo) Fensholt by using SDS‐ and AUT‐PAGE. Four major core histones and several linker histone H1s were detected by electrophoresis. Each of the core histones was identified by amino acid sequence analysis and peptide mapping. Electrophoresis patterns of histones were the same in male and female gametes and quite similar between the two species. The composition of histone H1s in conspicuously condensed sperm nuclei of C. hakodatensis was different from that in isogamous gametes. Electrophoresis after micrococcal nuclease digestion of chromatin in male and female gamete nuclei of C. bullosa and A. japonicus and sperm of C. hakodatensis resulted in regular ladder patterns of DNA fragments (ca. 200 base pair). The chromatin of the brown algal gametes thus has the typical nucleosome structure. These results showed that chromatin condensation in sperm nuclei of C. hakodatensis was associated with a modification of linker histone H1 but not by change of core histones, replacement by other basic proteins, changes of repeating patterns, or disappearance of nucleosomes.     

Comparison of the primary structure of reconstructed minimal nucleosomes and nucleosomes isolated from the chromatin

We have reconstructed nucleosomes from a histone octamer (H2A, H2B, H3, H4)2 and DNA 146 b.p. or 2-3 thousands b.p. in length. Comparison by means of DNA-histone cross-links of the primary organization of minimal nucleosomes obtained by reconstruction or isolated from chromatin of chicken erythrocyte nuclei has demonstrated a high similarity in histone location on their DNAs. Simultaneously, there have been observed some variations in the character of interaction for all core histones with DNA on nucleosomes. Thus, the cross-link of histone H4 with DNA of a core particle at H4 sites (65), unlike H4(55) and H4(88) sites, significantly depends on the superstructure of chromatin, ionic strength of solution and the presence of denaturating agents. All these differences are expected to probe the existence of conformational isomers for core particles. (Bracketed is the distance from the histone interaction site with the DNA of the core particle to the DNA 5'-terminus.)     

Replication-independent nucleosome exchange is enhanced by local and specific acetylation of histone H4

We used a novel single-cell strategy to examine the fate of histones during G₂-phase. Consistent with previous results, we find that in G₂-phase, the majority of nuclear histones are assembled into chromatin, whereas a small fraction comprises an unassembled pool. Small increases in the amount of histones within the free pool affect the extent of exchange, suggesting that the free pool is in dynamic equilibrium with chromatin proteins. Unexpectedly, acetylated H4 is preferentially partitioned to the unassembled pool. Although an increase in global histone acetylation did not affect overall nucleosome dynamics, an H4 containing lysine to glutamine substitutions as mimics of acetylation significantly increased the rate of exchange, but did not affect the acetylation state of neighbouring nucleosomes. Interestingly, transcribed regions are particularly predisposed to exchange on incorporation of H4 acetylation mimics compared with surrounding regions. Our results support a model whereby histone acetylation on K8 and K16 specifically marks nucleosomes for eviction, with histones being rapidly deacetylated on reassembly.     

The histones of rat testis

Polyacrylamide gel electrophoresis of the histones of the nuclei of seminiferous epithelial cells of rat testis revealed the five principal histone fractions which are found in liver and other somatic tissues, but, in addition, three unusual bands (desginated X₁, X₂, and X₃) were observed. Fraction X₁ had a mobility slightly less than that of F1 and was isolated with F1 in the fractionation procedure of Johns. F1 and X₁ were separated by chromatography on carboxymethylcellulose, and they were shown by amino acid analyses to be closely related lysine-rich histones. However, X₁ had lower content of lysine and alanine and higher content of arginine, aspartic acid, serine, proline, valine and leucine than F1. Both of these fractions had blocked amino-terminal residues, and both had a lysine residue at the carboxyl terminus. These fractions had similar molecular weights by electrophoresis on sodium dodecyl sulfate gels.Fraction X₂ migrated between histone fractions F1 and F3 on electrophoresis while X₃ migrated between fractions F2b and F3. Fraction X₃ was isolated with F2b during fractionation by the Johns procedure. Fraction X₂ has received minimal study, and this fraction may not be unique to the testis inasmuch as a faint band in approximately the position of X₂ can be seen in electrophoretic patterns of rat liver histones.The results of the treatment of the histone fractions with alkaline phosphatase indicated that the electrophoretic differences between X₁ and F1, or X₃ and F2b are not attributable to phosphorylation.     

The effect of histone hyperacetylation on the nuclease sensitivity and the solubility of chromatin

We have examined the effects of histone hyperacetylation upon nuclease digestion of nuclei and subsequent fractionation of chromosomal material in the presence of MgCl2. DNase I shows a maximum sensitivity towards hyperacetylated nuclei at somewhat elevated ionic strengths (150-200 mM NaCl), whereas micrococcal nuclease exhibits no specificity for acetylated nuclei over a broad range of ionic strengths. Fractionation in the presence of MgCl2 of hyperacetylated nuclei digested with micrococcal nuclease results in a substantial increase in the amount of soluble chromatin relative to that obtained with control nuclei. This increased yield of Mg2+-soluble chromatin results from the recruitment into this fraction of oligonucleosomes containing extremely hyperacetylated histones. These results suggest that contiguous nucleosomes containing highly acetylated histones may be altered in their ability to interact with themselves and with other nucleosomes.     

The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.     

Multistep chromatin assembly on supercoiled plasmid DNA by nucleosome assembly protein-1 and ATP-utilizing chromatin assembly and remodeling factor

We examine in vitro nucleosome assembly by nucleosome assembly protein-1 (NAP-1) and ATP-utilizing chromatin assembly and remodeling factor (ACF). In contrast to previous studies that used relaxed, circular plasmids as templates, we have found that negatively supercoiled templates reveal the distinct roles of NAP-1 and ACF in histone deposition and the formation of an ordered nucleosomal array. NAP-1 can efficiently deposit histones onto supercoiled plasmids. Furthermore, NAP-1 exhibits a greater affinity for histones H2A-H2B than does naked DNA, but in the presence of H3-H4, H2A-H2B are transferred from NAP-1 to the plasmid templates. These observations underscore the importance of a high affinity between H2A-H2B and NAP-1 for ordered transfer of core histones onto DNA. In addition, recombinant ACF composed of imitation switch and Acf1 can extend closely packed nucleosomes, which suggests that recombinant ACF can mobilize nucleosomes. In the assembly reaction with a supercoiled template, ACF need not be added simultaneously with NAP-1. Regularly spaced nucleosomes are generated even when recombinant ACF is added after core histones are transferred completely onto the DNA. Atomic force microscopy, however, suggests that NAP-1 alone fails to accomplish the formation of fine nucleosomal core particles, which are only formed in the presence of ACF. These results suggest a model for the ordered deposition of histones and the arrangement of nucleosomes during chromatin assembly in vivo.     

Proteolytic digestion of histones indicates that the nucleosomes of nuclei and of sheared chromatin are similar

Calf thymus nuclei were treated with trypsin, chymotrypsin or Pronase, and the rate of digestion of the various histone fractions was determined. The results differed from those obtained by digestion of DNA-free histones with the same set of enzymes but were identical to those obtained by digestion of calf thymus chromatin. Because these enzymes have such different specificities, the results of these digestions indicate that the histone fractions have similar locations in the chromosomal substructures of nuclei and chromatin, $\text{i.e.}$ that the structure of the nucleosomes which exist within nuclei is not changed markedly when chromatin is isolated from nuclei by a method which involves shearing.     

Enzymic acetylation of nucleosome histone.

Rat liver chromatin prepared from purified nuclei catalyzed the acetylation of histones in nucleosomes at the same level as that of nuclei. The activity of histone acetyltransferase in chromatin was destroyed by heat treatment at 65 degrees C for 5 min. Histones in exogenously added nucleosomes also served as substrate for the enzyme. The sites of acetylation in the nucleosomes appeared to be in the trypsin-digestable N-terminal regions of histones H4, H3, and H2A, as has been reported in an in vivo system.     

Nucleosomal organization of a part of chromatin in mollusc sperm nuclei with a mixed basic protein composition

The structural organization of mature sperm chromatin from three representatives of theMytilidae family has been studied. The acid-soluble proteins in these species nuclei are primarily sperm-specific (approximately 80%) with the remainder being core histones. Previously, we have shown that the mature sperm nuclei of these molluscs are compact, dense structures formed by interaction of the spermspecific proteins with DNA (1). Here we show that: a) although the histones are minor chromatin protein fraction, they still organize a part (20–25%) of the total DNA into nucleosomes; b) one of the sperm-specific proteins, different from somatic H1 or H5 histones participates in the formation of the beaded structures.     

The structure of Sipunculus nudus erythrocyte chromatin

The structure of the Sipunculus erythrocyte chromatin has been characterized by electron microscopy and nuclease digestion (staphylococcal nuclease and pancreatic nuclease). Contrary to previous results [1], we were able to isolate and characterize a histone H_2B in sipunculid nuclei. Though the histones H_2A and H_2B were markedly different from their vertebrate homologues, the subunit structure of the chromatin is the same. But the length of the repeat unit of DNA in the chromatin, is 177 ± 5 bp for the sipunculid erythrocyte nuclei, close to that reported for the chromatin of some lower eukaryotes.