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A typical tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is known as a strong carcinogen. We previously reported that metabolized NNK induced histone H2AX phosphorylation (γ-H2AX), a DNA damage-induced histone modification. In this study, we found that NNK globally acetylated histone H3, which affected γ-H2AX generation. Human lung adenocarcinoma A549 was treated with several doses of NNK. NNK induced dose-dependent global histone H3 acetylation (Ac-H3), at 2 to 12 h after the treatment, independent of the cell cycle. The Ac-H3 pattern was not affected by CYP2A13 overexpression unlike γ-H2AX, indicating no requirement of NNK metabolism to induce Ac-H3. Immunofluorescence staining of Ac-H3 was uniform throughout the nucleus, whereas γ-H2AX was formed as foci and did not coincide with Ac-H3. Nicotinic receptor antagonist methyllycaconitine inhibited Ac-H3 and also γ-H2AX. Phosphoinositide-3-kinase (PI3K)/Akt inhibitors, LY294002, wortmannin, and GSK690693, also suppressed both Ac-H3 and γ-H2AX, whereas KU-55933, an inhibitor of ataxia telangiectasia mutated (ATM) upstream of γ-H2AX, inhibited γ-H2AX but not Ac-H3. These results suggested that binding of NNK to the nicotinic acetylcholine receptor (α7nAChR) activated the PI3K/Akt pathway, resulting in Ac-H3. The activated pathway leading to Ac-H3 enhanced γ-H2AX, suggesting that NNK-induced DNA damage is impacted by the α7nAChR-mediated signal transduction pathway.

ADP-ribosylation of nuclear proteins is a critical feature of various DNA damage repair pathways. Histones, particularly H3 and H2B, are major targets of ADP-ribosylation and are primarily modified on serine with a single ADP-ribose unit following DNA damage. While the overall impact of PARP1-dependent poly-ADP-ribosylation is heavily investigated, very little is known about the specific roles of histone ADP-ribosylation. Here, we report the development of an efficient and modular semisynthetic route to full-length ADP-ribosylated histones H3 and H2B, chemically installed at specific serine residues. The modified histones were used to generate various chemically defined ADP-ribosylated chromatin substrates, which were employed in biophysical assays. These studies revealed that ADP-ribosylation of serine-6 of histone H2B (H2BS6ADPr) inhibits chromatin folding and higher-order organization; notably, this effect was enhanced by ADP-ribosylation of H3S10. In addition, ADP-ribosylated nucleosomes were utilized in biochemical experiments employing a panel of lysine methyltransferase enzymes, revealing a context-dependent inhibition of histone H3K9 methylation. The availability of designer ADP-ribosylated chromatin described here is expected to facilitate further biochemical and structural studies regarding the roles of histone ADP-ribosylation in the DNA damage response.

The nucleosome is an octamer containing DNA wrapped around one histone H3-H4 tetramer and two histone H2A-H2B dimers. Within the nucleosome, histones are decorated with post-translational modifications. Previous studies indicate that the H3-H4 tetramer is conserved during DNA replication, suggesting that old tetramers serve as a template for the modification of newly synthesized tetramers. Here, we present a method that merges bioorthogonal chemistry with mass spectrometry for the study of modifications on newly synthesized histones in mammalian cells. HeLa S3 cells are dually labeled with the methionine analog azidohomoalanine and heavy (13)C6,(15)N4 isotope labeled arginine. Heavy amino acid labeling marks newly synthesized histones while azidohomoalanine incorporation allows for their isolation using bioorthogonal ligation. Labeled mononucleosomes were covalently linked via a copper catalyzed reaction to a FLAG-GGR-alkyne peptide, immunoprecipitated, and subjected to mass spectrometry for quantitative modification analysis. Mononucleosomes containing new histones were successfully isolated using this approach. Additionally, the development of this method highlights the potential deleterious effects of azidohomoalanine labeling on protein PTMs and cell cycle progression, which should be considered for future studies utilizing bioorthogonal labeling strategies in mammalian cells.

Lysine methylation is an important post-translational modification of histone proteins that defines epigenetic status and controls heterochromatin formation, X-chromosome inactivation, genome imprinting, DNA repair, and transcriptional regulation. Despite considerable efforts by chemical biologists to synthesize modified histones for use in deciphering the molecular role of methylation in these phenomena, no general method exists to synthesize proteins bearing quantitative site-specific methylation. Here we demonstrate a general method for the quantitative installation of N(epsilon)-methyl-L-lysine at defined positions in recombinant histones and demonstrate the use of this method for investigating the methylation dependent binding of HP1 to full length histone H3 monomethylated on K9 (H3K9me1). This strategy will find wide application in defining the molecular mechanisms by which histone methylation orchestrates cellular phenomena.

The synthesis of a novel series of quinazolines substituted at C4 by five-membered ring aminoheterocycles is reported. Their in vitro structure-activity relationships versus Aurora A and B serine-threonine kinases is discussed. Our results demonstrate that quinazolines with a substituted aminothiazole at C4 possess potent Aurora A and B inhibitory activity and excellent selectivity against a panel of various serine-threonine and tyrosine kinases, as exemplified by compound 46. We found also that the position and nature of the substituent on the thiazole play key roles in cellular potency. Compounds with an acetanilide substituent at C5' have the greatest cellular activity. The importance of the C5' position for substitution has been rationalized by ab initio molecular orbital calculations. Results show that the planar conformation with the sulfur of the thiazole next to the quinazoline N-3 is strongly favored over the other possible planar conformation. Compound 46 is a potent suppressor of the expression of phospho-histone H3 in tumor cells in vitro as well as in vivo, where 46, administered as its phosphate prodrug 54, suppresses the expression of phospho-histone H3 in subcutaneously implanted tumors in nude mice.

Proteins of the ATF/CREB class of transcription factors stimulate gene expression of several cell growth-related genes through protein kinase A-related cAMP response elements. The promoter activity of cell cycle regulated histone H4 genes is regulated by at least four principal cis-acting elements which mediate G1/S phase control and/or enhancement of transcription during the cell cycle. Using protein-DNA interaction assays we show that the H4 promoter contains two ATF/CREB recognition motifs which interact with CREB, ATF1, and ATF2 but not with ATF4/CREB2. One ATF/CRE motif is located in the distal promoter at the nuclear matrix-associated Site IV, and the second motif is present in the proximal promoter at Site I. Both ATF/CRE motifs overlap binding sequences for the multifunctional YY1 transcription factor, which has previously been shown to be nuclear matrix associated. Subnuclear fractionation reveals that there are two ATF1 isoforms which appear to differ with respect to DNA binding activity and partition selectively between nuclear matrix and nonmatrix compartments, consistent with the role of the nuclear matrix in regulating gene expression. Site-directed mutational studies demonstrate that Site I and Site IV together support ATF1- and CREB-induced trans-activation of the H4 promoter. Thus, our data establish that ATF/CREB factors functionally modulate histone H4 gene transcription at distal and proximal promoter elements.

The histone H1t gene is expressed exclusively in testis primary spermatocytes. Previous studies indicate that accumulation of H1t mRNA occurs only in primary spermatocytes in normal rats and in transgenic mice bearing the rat H1t transgene. In this study, DNA sequences of human, monkey, mouse, and rat H1t genes were compared and found to be almost identical in the proximal promoter region extending from the H1/AC box through the TATAA box. In addition to conserved elements common to replication-dependent H1 promoters, the H1t promoter contains a unique TE element, and sequences within this element may contribute to enhanced expression of the gene in primary spermatocytes. Two imperfect inverted repeat sequences designated TE1 and TE2, that are located within the larger TE element, overlap a central GC-rich region and bind specifically to nuclear proteins derived from primary spermatocytes. Protein interactions characterized by methylation interference and UV cross-linking experiments indicate that a complex of proteins with a molecular mass of approximately 180 kDa binds TE1. The GC-rich region in H1t and in some replication dependent histone H1 promoters contains an Sp1 consensus sequence. Although the H1t/TE element that contains the GC-rich region binds nuclear proteins, it does not appear to bind Sp1 obtained from cell populations enriched in primary spermatocytes as determined by electrophoretic mobility supershift assays using polyclonal anti-Sp1 antibodies.

In the following report the relationship between histone methylation and histone acetylation has been examined in HeLa cells to better define the distribution of these two modifications. By labeling methylated histones in the presence or absence of sodium butyrate, we have found that the methylation of H3 is much more targeted to rapidly acetylated chromatin than is the methylation of H4, which largely involves the unacetylated subtype even in the presence of butyrate. Newly methylated H3 is highly likely to be complexed in nucleosomes that contain acetylated H4, as determined by immunoprecipitating radiolabeled chromatin with antibodies specific for acetylated H4 isoforms. In contrast, dynamically methylated H4 is underrepresented in acetylated chromatin, relative to newly methylated H3. The preferential methylation of acetylated H3 continues after pretreatment of cells with cycloheximide, indicating that not all acetylation-related methylation is associated with histone synthesis. This was confirmed by analyzing histone methylation in cells arrested at the G1/S boundary, in which histone synthesis was sharply lowered (relative to randomly cycling cells): under these conditions H3 methylation declined only approximately 4-fold, although ongoing methylation of H4 decreased approximately 20-fold. The continuing methylation of H3 in arrested cells included all H3 sequence variants, was selective for acetylated H3, and coincided with methyl group turnover that could not be ascribed to histone replacement synthesis. Most newly methylated H3 in arrested cells was complexed with acetylated H4 in chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)

The rates of synthesis of histone H1 subtypes in synchronized mouse NIH 3T3 fibroblasts were compared with those of rat C6 glioma cells during the G0, G1, and S phases by using a combination of HPLC techniques and conventional gel electrophoresis. In the mouse cell line, all H1 subtypes, H1a-H1e including histone H1(0), were detectable. In the rat cell line, however, no histone H1a was found. H1c and H1e from both cell lines show in the quiescent state a relatively high specific activity comparable with that of H1(0). After release from the G0/G1 block, the synthesis of H1(0) and likewise that of H1c and H1e increase for a short period. All H1 subtypes have their maximum specific activity at the same time after stimulation. The percentage of total H1 specific activity of H1a, H1b, and H1d increases, those of H1c and H1e remain relatively constant, and that of H1(0) decreases while cells cycle from the G0/G1 to the S phase. These findings support our assumption that H1 subtypes could be classified into three groups with common metabolic characteristics: one consists of H1a, H1b, and H1d; another of H1c and H1e; and a third of H1(0) histone. Moreover, the corresponding H1 subtypes from two different species seem to have similar specific activities during the G1 and S phases.

The studies presented were undertaken to clarify the unsettled question whether the synthesis of histones with a N-acetylserine residue at the amino terminal end is initiated with methionine. Histones were synthesized in vitro in a rabbit reticulocyte lysate, primed with a mRNA preparation from ascites cells. Initiation of polypeptide synthesis was investigated by using N-formyl[35S]met-tRNAfMet from yeast to label the N-termini. N-Formylmethionine was incorporated into histones H1 and H4 whose N-terminal amino acid is alpha-N-acetylserine. By comparison of tryptic peptides derived from these two histones labeled either with methionine or formylmethionine and from Edman degradation it is shown that N-terminal acetylated histones are initiated with methionine, as is the case for other eukaryotic and bacterial proteins.

We have reinvestigated studies using Lomant's reagent to cross-link newly synthesized density-labeled histones into octameric complexes to determine the nature of histone deposition. The analysis has additionally included procedures for reversal of the cross-link in order to analyze the individual histones in these complexes. These studies indicate that density-labeled, newly synthesized histones form hybrid octameric structures composed of both new and old histones. These studies also suggest that previous interpretations by other investigators for the production of homogeneous complexes (100% dense octamer containing 100% new histones) are misinterpretations due to the presence of non-histone protein that contaminates the preparation, even under conditions where much of this non-histone protein is removed by use of ion-exchange resins. These non-histone proteins can be fractionated on density gradients as un-cross-linked proteins with molecular weights that mimic those of cross-linked histone complexes.

We have asked whether histones synthesized in the absence of DNA synthesis can exchange into nucleosomal structures. DNA synthesis was inhibited by incubating hepatoma tissue culture cells in medium containing 5.0 mM hydroxyurea for 40 min. During the final 20 min, the cells were pulsed with [3H]lysine to radiolabel the histones (all five histones are substantially labeled under these conditions). By two electrophoretic techniques, we demonstrate that histones H1, H2A, and H2B synthesized in the presence of hydroxyurea do not merely associate with the surface of the chromatin but instead exchange with preexisting histones so that for the latter two histones there is incorporation into nucleosome structures. On the other hand, H3 and H4 synthesized during this same time period appear to be only weakly bound, if at all, to chromatin. These two histones have been isolated from postnuclear washes and purified. Some possible implications of in vivo exchange are discussed.

The phosphorylation of H1 histone subfractions was measured in mouse neuroblastoma cells stopped from dividing by three treatments that block cell division: 5 mM butyrate, 2% dimethyl sulfoxide, and serum withdrawal. H1 histone phosphorylation decreased in response to all three treatments, but the response differed in its timing and its extent for the different H 1 subfractions. The different decreases in phosphorylation correlated well with the differential decreases in biosynthesis of the individual H1 subfractions; however, an exception to this parallel decrease in synthesis and phosphorylation was observed in the case of histone H1(0). Phosphorylation of H1(0) was absent in each of the three treatments after 2 days, despite the continued synthesis and deposit of H1(0) on the chromatin. Thus, despite the fact that H1(0) was being synthesized and that the other newly synthesized H1 subfractions were phosphorylated at this time, the phosphorylation of H1(0) became uncoupled from its synthesis after prolonged treatments blocking cell division.

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.

The phosphorylating of the lysine-rich histone at various stages in the cell cycle has been studied. In rapidly dividing cell populations the lysine-rich histone is phosphorylated rapidly after synthesis and more slowly once bound to the chromosome. The half-life of hydrolysis of such interphase phosphorylation in 5 hr except during mitosis when the phosphata hydrolysis increases almost three-fold. During mitosis there is extensive phosphorylation at sites different from those phosphorylated during interphase and a smaller measure of sites common to both mitotic and interphase cells. The sites of mitotic phosphorylation are most critically distinguished from those phosphorylated in interphase by the rapidly hydrolysis of M-phase phosphohistone when the cells divide and enter the G1 phase of the cell cycle.

We have reinvestigated the degree of coupling between DNA and histone synthesis in mammalian cells. In at least one cell line (HTC cells), the coupling is not nearly as tight as had previously been inferred from experiments with HeLa cells. The site of deposition of such histones which continue to be made in the presence of sufficient hydroxyurea to depress DNA synthesis almost totally has been studied. Deposition seems to be on material which absorbs at 260 nm. This material is not a part of the bulk chromatin and binds histone in a relatively tight manner. The possible role of such a material in histone synthesis and deposition is discussed.

A cell free system programmed by endogenous mRNA and active in initiation of protein synthesis has been obtained from HeLa cells by adding 25-100 muM hemin to the medium used to homogenize the cells. Hemin stabilizes the initiation activity of the extract, which otherwise decays rapidly even at 0 degrees C. The role of hemin in promoting initiation has been examined by fractionating the extracts into ribosomes and postribosomal supernatant (S150). An extract prepared without hemin or the S150 obtained from this extract prepared without hemin or the S150 obtained from this extract inhibits protein synthesis of the extract containing hemin by about 30%. The ribosomes prepared from extracts containing hemin are active in initiation of protein synthesis, whereas the ribosomes obtained from the extracts prepared without hemin show little or no initiation. These results have suggested that addition of hemin prevents the formation of an inhibitor of initiation in the S150 and at the same time protects from inactivation an initiation factor associated with ribosomes or ribosomal subunits. Addition of 2 mM GTP to HeLa extracts stabilizes the initiation activity, though to a smaller degree than hemin. The effects of hemin and GTP are not additive, suggesting that they may act on the same target molecule, though possibly by different mechanisms. The mechanism of action of GTP is discussed in view of similar observations made in the rabbit reticulocyte cell free system.

The distribution of [3H]methyl radioactivity in cytoplasmic histone mRNA, isolated during the DNA synthetic (S) phase of the HeLa S3 cell cycle, has been investigated. Evidence is presented that approximately 30% of the radioactivity is in m7GpppXmpYp oligonucleotides, where Xm represents 2'-O-methylated adenosine and guanosine with a molar ratio of 4:1, respectively. The remainder of the radioactivity is present as m7GpppCmpYmpZp oligonucleotides, where Xm is again 2'-O-methylated adenosine and guanosine (4:1) and where ym represents 2'-O-methylated adenosine, guanosine, cytidine, and uridine with ratios of 2:1:1:1, respectively. While 48.6% of the [3H]methyl radioactivity was present as N6-methyladenosine in poly(adenylic acid)-terminated mRNA from S-phase cells, no evidence for N6-methyladenosine was found in histone mRNA. It thus appears that histone mRNA which lacks 3'-terminal poly(adenylic acid) sequences and functions on cytoplasmic polyribosomes during a limited protion of the cell cycle is capped but lacks internal-modified nucleosides.

During avian erythropoiesis, the blast cells of the bone marrow mature into polychromatic erythrocytes (late stages knwon as reticulocytes) and then into mature red blood cells. When chickens are made anemic, the proportion of immature cells in the anemic bone marrow increases dramatically. The level of the lysine-rich histones. H1 and H5, has been found to be constant in the blood and bone marrow of normal and anemic chickens. This implies that H5 replaces H1 quantitatively. Urea-aluminum-lactate starch gel electrophoresis of H5 from these sources show that the degree of phosphorylation of H5 is proportional to the number of immature cells. About 70% of the H5 from the most immature bone marrow is phosphorylated, while 50% of the H5 from anemic blood is phosphorylated and H5 in normal blood is almost completely devoid of phosphate. When immature cells of the anemia bone marrow are incubated in the presence of inorganic 32P and [3H]lysine and [3H]arginine, extensive 32P incorporation is found in the phospho species. A minimum of nine phosphorylated components have been demonstrated by starch gel electrophoresis. The incorporation of 3H is time dependent. After 1.5 h of labeling, 3H is found in H5 containing 0, 1, 2, and 3 phosphates. tthe combined data suggest that newly synthesized H5 becomes progressively phosphorylated and that at the terminal stage of development, the phosphorylated H5 is completely dephosphorylated. These events may be important in controlling the timing of chromatin condensation.

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.

We describe a rapid and simple method for the purification of biologically active messenger RNAs. The method allows the isolation in a few hours of specific mRNAs from either whole cell or polysomal RNA even if the RNA represents less than 1% of the starting molecules. We used, as a model, cloned sea urchin (Strongylocentrotus purpuratus) histone gene fragments linked to cellulose by the method of B. E. Noyes & G. R. Stark (1975) Cell 5, 301--310) as hybridization probes to isolate specific histone mRNAs from whole cell and polysomal RNA extracts. RNAs isolated in this manner maintain their biological activity, serving as templates for histone proteins in a wheat-germ, cell-free protein translation system. In addition, radiolabeled histone-specific RNA purified from cleavage stage sea urchin embryos, pulse labeled for short periods of time and analyzed on denaturing polyacrylamide gels, was the same size as mature histone mRNA's.

During embryogenesis in the sea urchin Strongylocentrotus purpuratus, there is a shift from one histone mRNA population to another. The early and late embryonic histone mRNAs, previously shown to differ considerably in sequence from each other by hybrid melting studies, are shown here to differ also in electrophoretic mobility on polyacrylamide gels as the positions of the early and late mRNAs are completely noncoincident. The various species of both early and late samples are identified as particular histone mRNAs by hybridization to cloned histone DNAs containing part of the early-type repeat unit or to restriction enzyme fragments derived from these unit. Four bands in the early mRNA sample are identified as H1, H3, H2A " H2B, and H4 mRNA while at least 10 bands can be seen in the late mRNA preparation with unambiguous identification of H1, H2B, and H4 mRNAs. A cluster of late species is shown to contain both H3 and H2A mRNA. When a polysomal RNA preparation from the 26-h embryo is hybridized to the histone DNA, eluted, and then translated in vitro in a wheat germ system, the histone products migrate in the position of late histones when subjected to electrophoresis on Triton X-urea gels. Using DNA which contains genes for H2A + H3 or H2A alone, we demonstrate the specificity of the early-type DNA probes for these two late histones. Therefore, by hybridization of newly synthesized RNAs and translation of the total polysomal RNA present in the late embryo, it is shown that mRNAs for all five histone classes may cross-react with the cloned early-type DNA. The hybrids formed, however, are much less stable than those formed with the early histone mRNA. In vitro translation of total cytoplasmic RNA from various embryonic stages indicates that transition between the two classes occurs during most of the blastula period.

Following a labeling period of 2 min, HeLa histones continue to accumulate in chromatin for 10 min, indicating the presence of a histone pool. During the accumulation period, H2A and H2B enter chromatin immediately, while entry of H3 and H4 is more prolonged. Association of newly synthesized core histones with chromatin does not necessarily indicate assembly. When 2-min [3H]lysine-labeled chromatin is exposed to 0.45 M NaCl, nearly half of the newly synthesized histones are dissociated, while mature core histones are stable. H2A and 70% of H2B are salt stable and remain with newly synthesized polynucleosomes. About 30% of H2B, 50% of H4, and all of H3 are salt labile; thus, both the new nucleosomal core histones and salt-labile new core histones are nonstoichiometric. Pulse-labeled core histones are more trypsin-sensitive than mature histones. When the salt-labile, newly synthesized histones are removed, the remaining proteins have the same trypsin sensitivity as bulk core protein. Examination of the tryptic peptides indicated that the increased trypsin sensitivity was due to complete destruction of the loosely associated core histones which undergo a lag prior to assembly. The altered order of appearance of two peptides in stripped, newly assembled nucleosomes indicates that the conformation in these particles is different from that in mature chromatin.

We have employed high-performance liquid chromatography (HPLC) to investigate the syntheses of histones H1 and H1o as synchronized cells traverse from mitosis to S phase. Chinese hamster (line CHO) cells were synchronized by mitotic selection, and, at appropriate times, they were pulse labeled for 1 h with [3H]lysine. Histones H1 and H1o were extracted by blending radiolabeled and carrier cells directly in 0.83 M HC1O4; the total HC1O4-soluble, Cl3CCO2H-precipitable proteins were then separated by a modification of an HPLC system employing three mu Bondapak reversed-phase columns [Gurley, L. R., D'Anna, J. A., Blumenfeld, M., Valdez, J. G., Sebring, R. J., Donahue, D. K., Prentice, D. A., & Spall, W. D. (1984) J. Chromatogr. 297, 147-165]. These procedures (1) produce minimally perturbed populations of synchronized proliferating cells and (2) maximize the recovery of radiolabeled histones during isolation and analysis. Measurements of rates of synthesis indicate that the rate of H1 synthesis increases (3.6 +/- 0.5)-fold as cells traverse from early to mid G1; as cells enter S phase, the rate of H1 synthesis increases an additional congruent to 22-fold and is proportional to the number of S-phase cells. In contrast to H1, the rate of H1o synthesis is nearly constant throughout G1. As cells progress into S phase, the rate of H1o synthesis increases (3.1 +/- 0.2)-fold so that it also appears to be proportional to the number of S-phase cells. Except for the first 1-2 h after mitotic selection, these results are similar to those obtained when cells are synchronized in G1 with the isoleucine deprivation procedure.

Incubation of MSB-1 chicken lymphoblastoid cells with hydroxyurea leads to a rapid 25-fold decrease in the incorporation of [3H]thymidine into DNA and a 5-fold decrease [3H]lysine into the nucleosome core histones. I have investigated whether the distortion in the normal proportion of histone-DNA synthesis results in alterations in the nucleosome assembly process and find that neither the stoichiometry of new histone synthesis nor the deposition is appreciably changed during hydroxyurea incubation. Protein cross-linking and micrococcal nuclease digestion show that the histones synthesized during hydroxyurea treatment form octamer structures and are assembled into typical nucleosome particles. Minor nucleosome subpopulations are found which exhibit altered sensitivity to nuclease digestion and which are depleted in new histones H3 and H4. When MSB-1 cells incubated in hydroxyurea are pulsed briefly with density-labeled amino acids and [3H]lysine, the radiolabeled core histone octamers formed are as dense as individual monomer histones. These results suggest that the newly synthesized histone octamers are uniformly dense and do not contain mixtures of new and old histones. Thus, histones synthesized during hydroxyurea incubation are deposited nonrandomly and do not exchange with preexisting histones.

We have developed procedures to study histone-histone interactions during the deposition of histones in replicating cells. Cells are labeled for 60 min with dense amino acids, and subsequently, the histones within the nucleosomes are cross-linked into an octameric complex with formaldehyde. These complexes are sedimented to equilibrium in density gradients and octamer and dioctamer complexes separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With reversal of the cross-link, the distribution of the individual density-labeled histones in the octamer is determined. Newly synthesized H3 and H4 deposit as a tetramer and are associated with old H2A and H2B. Newly synthesized H2A and H2B deposit as a dimer associated with old H2A, H2B, H3, and H4. The significance of these results with respect to the dynamics of histone interactions in the nucleus is discussed. Control experiments are presented to test for artifactual formation of these complexes during preparative procedures. In addition, reconstitution experiments were performed to demonstrate that the composition of these octameric complexes can be determined from their distribution on density gradients.

The positions of the several sea urchin histone genes on the eukaryotic fragments of the chimeric plasmids pSp2 and pSp17 have been mapped relative to the Eco RI and Hind III restriction endonuclease sites on the plasmids. Two principal mapping methods using the electron microscope have been used: (a) the R-loop procedure and a new modification thereof to map the genes on duplex DNA; (b) the gene 32-ethidium bromide technique to visualize RNA-DNA hybrids on single strands of DNA. It is known that there are two histone genes, H3 and H2A, on pSp17. There are two Eco RI sites at the two junctions of the procaryotic segment with the eucaryotic segment on the plasmid. We show, by an electron microscope method, that for H2A, with a length of 0.52 kilobases (kb), one end of the gene is situated 0.02 to 0.03 kb from one RI site, and that there is a Hind III site within this gene at about 0.13 kb from the end phe other RI site of this plasmid. The H4 gene lies between H2B and H1. The ms the incubation temperature is raised up to a temperature just below that at which strand dissociation of the duplex DNA occurs.

The synthesis and enzymatic modifications of histones by phosphorylation, acetylation, and methylation during erythroid cell maturation have been studied. All newly synthesized histones, H1, H5, H2a, h2b, h3, and H4 undergo phosphorylation; histones H2a, H2b, H3, and H4, are acetylated and histones H3 and H4 are methylated. This type of histone metabolism is common to all dividing cells and therefore may be related to the assembly of histones into chromatin subunits. In the nondividing reticulocytes, the synthesis of histone H5 continues, while all the other histones show negligible incorporation of [3H]amino acids. Furthermore, the reticulocytes show a unique pattern of enzymatic modification: phosphorylation of histone H2b, acetylation of histones H2a, H2b, H3, and H4, and methylation of histones H3 and H4. These "differentiation-linked" modifications are not dependent on histone synthesis, nor related to RNA synthesis, but may be related to the reorganization of chromatin in preparation for genomic inactivation.

Procedures are presented which permit the identification and analysis of cellular histone that is not bound to chromatin. This histone, called soluble histone, could be distinguished from that bound to chromatin by the state of H4 modification and the lack of H2A ubiquitination. Changes in the levels of newly synthesized soluble histone were analyzed with respect to the balance between histone and DNA synthesis in hamster ovary cells. Pulse-chase protocols suggested that the chase of newly synthesized histone from the soluble fraction into chromatin may have two kinetic components with half-depletion times of about 1 and 40 min. When protein synthesis was inhibited, the pulse-chase kinetics of newly synthesized histone from the solubl fraction into chromatin were not significantly altered from those of the control. However, in contrast to the control, when protein synthesis was inhibited, DNA synthesis was also inhibited with kinetics similar to those of the chase of newly synthesized histone from the soluble fraction. There was a rapid decrease in the rate of DNA synthesis with a half-deceleration time of 1 min down to about 30% of the control rate, followed by a slower decrease with an approximate half-deceleration time of 40 min. When DNA synthesis was inhibited, newly synthesized histone accumulated in the soluble fraction, but H2A and H2B continued to complex with chromatin at a significant rate. Soluble histone in G1 cells showed the same differential partitioning of H4/H3 and H2A/H2B between the soluble and chromatin-bound fractions as was found in cycling cells with inhibited DNA synthesis. These results support a unified model of reciprocal regulatory mechanisms between histone and DNA synthesis in the assembly of chromatin.