Triiodothyronine nuclear receptor: Role of histones and DNA in hormone binding

The triiodothyronine (T₃) nuclear receptor was previously shown to lose rapidly its high affinity hormone-binding property after a partial purification from the nuclear extract. It was then found that histones + DNA added to the incubation medium with labeled T₃ could restore, at least in part, the high affinity T₃ binding. We now demonstrate that DNA alone increases the high affinity T₃ binding site concentration moderately, and only at low ionic strength where it can bind to the receptor. Total histones and all histone fractions studied (total core histones, F2a, H2B, H3, H4, H1) specifically increase, at low concentrations, the level of T₃ binding; but higher concentrations of some individualized histones, particularly arginine-rich histones, have an inhibitory effect. DNA, or several other polynucleotides, in the presence of histones increase the stimulating histone effect and reverse the inhibitory effect into a true activation. Histones increase the number of T₃ binding sites but decrease the affinity for T₃; addition of DNA restores the high affinity for T₃ and stabilizes the T₃-receptor complexes. Thus, some of the histone molecules could play a role in the maintenance of the T₃ binding site, but multiple interactions between histones or with DNA seem necessary to impair the negative effect exerted by other parts of the histone molecules. Whether these positive and negative effects of histones on the T₃ binding site are of biological relevance in the regulation of T₃ binding to its receptor remains to be determined.     

Histone acetyltransferase in chromatin. Extraction of transferase activity from chromatin

Previous work has attempted to localize nuclear histone acetyltransferase activity in the cromatin. Evidence was presented indicating that the transfer of ¹⁴C-acetate from ¹⁴C-acetyl CoA to histones in chromatin was an enzymatic process. We now report on the extraction of part of the histone acetyltransferase activity from rat liver chromatin, employing a procedure originally described for extraction of DNA-dependent RNA polymerase. The K_m of the extracted transferase activity for the substrate acetyl CoA was 5 × 10⁻⁷, the Q₁₀: 1.8 and the optimal pH: 7.1. Serum albumine, protamine and polylysine were poor substrates as compared to histones. Activity of extracted or heated chromatin was not restored upon incubation in the presence of extract. Also the selectivity exhibited by the transferase activity in unextracted chromatin towards arginine-rich histones, was much less pronounced in the extracts prepared from it. It is possible that the influence of steric factors contributing to this specificity in native chromatin is lost upon isolation of the enzyme from it. Alternatively, a less specific isoenzyme may have been extracted.     

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.     

A CYTOPHOTOMETRIC STUDY OF NUCLEIC ACIDS AND PROTEINS IN THE SHOOT APEX OF WHITE SPRUCE

During the annual three-phase growth cycle of white spruce [Picea glauca (Moench) Voss] the vegetative shoot apex changed in anatomical configuration. Relative amounts of DNA, histones, RNA, and proteins were measured in three cytohistological zones and were related to the anatomical changes during ontogeny. An extended period of DNA synthesis (S) and G₂ preceded an increase in the number of apical initial cells which were part of the mammillary apex. While DNA and histones were generally synchronous during ontogeny, the ratio of DNA to histone increased on June 20. This loss of histone and subsequent increases in RNA and cytoplasmic proteins preceded the appearance of needle primordia on next year's apex. We propose that induction of the apex to reorganize and form needle primordia occurred when the DNA was in a 2C condition, following the loss of histone on June 20.     

CYTOCHEMICAL AND BIOCHEMICAL DETERMINATIONS OF CHANGES IN NUCLEAR HISTONE CONTENT DURING DIFFERENTIATION OF BARLEY ROOT CELLS

Changes in nuclear histone content in barley root cells have been studied by cytochemical methods for identification of histone subtypes and by conjunction with standard biochemical extraction procedure for various histone fractions and alkaline fast green stainability. The results obtained by the cytochemical methods indicate that the nuclear histones in cell nuclei found in their terminal stages of cellular differentiation or elongation contain histones rich in arginine, whereas the nuclei in meristematic cells contain histones rich in lysine. Cytochemicaly intermediate or transitional types of nuclear histones have been observed in cell nuclei which are undergoing differentiation or elongation and in chromosomes of mitotic nuclei. Information obtained from the conjunction of methods of biochemical extraction procedures for various histone fractions and alkaline fast green stainability indicate that the nuclei in well-differentiated cells contain predominantly histones rich in arginine (f3), whereas the nuclei of meristematic cells contain both very lysine-rich histones (f1) and slightly lysine-rich histones (f2). These results suggest the replacement of lysine-rich histones in the nuclei of meristematic cells by arginine-rich histones during cellular differentiation.     

Biochemical and immunological characterization of a histone variant associated with spermatogenesis in the mouse

A chromosomal histone, H2S, specific to the mouse testis has been purified. Amino acid analysis indicated lack of cysteine and a high basic amino acid content typical of histones. Specific antibodies against histones H2S have been generated in rabbits and partially purified using (NH₄)₂SO₄ precipitation and ion-exchange chromatography. Protein transfer experiments indicate presence of antigenically similar histones in the rat and rabbit testes but not in the guinea pig and dog testes. In addition, histone complement of somatic tissues such as lung, kidney, liver and spleen lacked antigenically similar proteins. Immunocytochemical studies using peroxidase-antiperoxidase complex indicated presence of immunoreactive cells in the seminiferous epithelium which were lacking in the interstitium. These data demonstrate histone H2S to be a unique histone associated with spermatogenesis in the mouse.     

Cell cycle regulation during growth-dormancy cycles in pea axillary buds

Accumulation patterns of mRNAs corresponding to histones H2A and H4, ribosomal protein genes rpL27 and rpL34, MAP kinase, cdc2 kinase and cyclin B were analyzed during growth-dormancy cycles in pea (Pisum sativum cv. Alaska) axillary buds. The level of each of these mRNAs was low in dormant buds on intact plants, increased when buds were stimulated to grow by decapitating the terminal bud, decreased when buds ceased growing and became dormant, and then increased when buds began to grow again. Flow cytometry was used to determine nuclear DNA content during these developmental transitions. Dormant buds contain G₁ and G₂ nuclei (about 3:1 ratio), but only low levels of S phase nuclei. It is hypothesized that cells in dormant buds are arrested at three points in the cell cycle, in mid-G₁, at the G₁/S boundary and near the S/G₂ boundary. Based on the accumulation of histone H2A and H4 mRNAs, which are markers for S phase, cells arrested at the G₁/S boundary enter S within one hour of decaptitation. The presence of a cell population arrested in mid-G₁ is indicated by a second peak of histone mRNA accumulation 6 h after the first peak. Based on the accumulation of cyclin B mRNA, a marker for late G₂ and mitosis, cells arrested at G₁/S begin to divide between 12 and 18 h after decapitation. A small increase in the level of cyclin B mRNA at 6 h after decapitation may represent mitosis of the cells that had been arrested near the S/G₂ boundary. Accumulation of MAP kinase, cdc2 kinase, rpL27 and rpL34 mRNAs are correlated with cell proliferation but not with a particular phase of the cell cycle.     

Triiodothyronine nuclear receptor. Role of histones and DNA in hormone binding

The triiodothyronine (T3) nuclear receptor was previously shown to lose rapidly its high affinity hormone-binding property after a partial purification from the nuclear extract. It was then found that histones + DNA added to the incubation medium with labeled T3 could restore, at least in part, the high affinity T3 binding. We now demonstrate that DNA alone increases the high affinity T3 binding site concentration moderately, and only at low ionic strength where it can bind to the receptor. Total histones and all histone fractions studied (total core histones, F2a, H2B, H3, H4, H1) specifically increase, at low concentrations, the level of T3 binding; but higher concentrations of some individualized histones, particularly arginine-rich histones, have an inhibitory effect. DNA, or several other polynucleotides, in the presence of histones increase the stimulating histone effect and reverse the inhibitory effect into a true activation. Histones increase the number of T3 binding sites but decrease the affinity for T3; addition of DNA restores the high affinity for T3 and stabilizes the T3-receptor complexes. Thus, some of the histone molecules could play a role in the maintenance of the T3 binding site, but multiple interactions between histones or with DNA seem necessary to impair the negative effect exerted by other parts of the histone molecules. Whether these positive and negative effects of histones on the T3 binding site are of biological relevance in the regulation of T3 binding to its receptor remains to be determined.     

Histone synthesis in early amphibian development: histone and DNA syntheses are not co-ordinated

The synthesis of basic proteins has been studied in the oocytes, eggs and embryos of the South African clawed frog, Xenopus laevis. A group of newly synthesized proteins has been identified as histones by the following criteria: solubility properties; incorporation of [³H]lysine and [³H]arginine in the correct proportions, but lack of incorporation of [³H]tryptophan; co-cleotrophoresis with marker histones in various types of polyacrylamide gels, including a type run in two dimensions; peptide analysis of the arginine-rich fraction, F2A1. The four main histone fractions other than F1 were found to be synthesized at all stages of development. F1 histone synthesis was first detected at the late blastula stage.Rates of histone synthesis were estimated for the different stages of development and it was concluded that histone synthesis was not co-ordinated with DNA synthesis either temporally or quantitatively. Histone synthesis was unusual in the following major respects: histones were synthesized in oocytes, and yet in these cells DNA replication had not occurred for several months; histones were synthesized in activated or fertilized eggs at a rate far in excess (about 500 times) of the immediate requirements. We suggest that in order to provide enough histones for the late blastula embryo a store of histone is accumulated during the early cleavage stages and possibly during oogenesis.     

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1⁰ protein was predominant in G₀ cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.     

Initial steps in the large-scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase

Histones from exponential and stationary-phase mouse L-cells were quantitated after acrylamide gel electrophoresis in order to investigate cell cycle-dependent changes in the mode of binding of the various fractions in chromatin. By introducing various concentrations of citrate and divalent cations in the medium used for cell lysis and isolation of nuclei prior to histone extraction it was possible to demonstrate that certain histone fractions are preferentially retained in either exponential or stationary-phase nuclei. Differential retention of lysine-rich F₁ was most evident when the lysing medium contained 1 mm Mg²⁺ and Ca²⁺ and 5 mm citrate (pH 2.75). In these conditions twice as much F₁ is retained in stationary as in exponential nuclei. Differential retention of arginine-rich histones was most evident when the lysing medium contained 10 mm Mg²⁺ and Ca²⁺ and no citrate. In these conditions more F_{2a 1} is retained in exponential than in stationary nuclei while the opposite is true for F₃. However, the total amount of arginine-rich fractions (F_{2a 1} + F₃) retained was found to be the same in both cell phases. The results are discussed in relation to known structural features of the histones.     

Characterization of pea histone deacetylases

The present paper is the first report on histone deacetylases from plants. Three enzyme fractions with histone deacetylase activity (HD0, HD1 and HD2) have been partially purified from pea (Pisum sativum) embryonic axes. They deacetylate biologically acetylated chicken histones and, to a lesser extent, chemically acetylated histones, this being a criterion of their true histone deacetylase nature. The three enzymes are able to accept nucleosomes as substrates. HD1 is not inhibited by n-butyrate up to 50 mM, whereas HD0 and HD2 are only slightly inhibited, thereby establishing a clear difference to animal histone deacetylases. The three activities are inhibited by acetate, Cu²⁺ and Zn²⁺ ions and mercurials, but are only scarcely affected by polyamines, in strong contrast with yeast histone deacetylase. Several criteria have been used to obtain cumulative evidence that HD0, HD1 and HD2 actually are three distinct enzymes. In vitro experiments with free histones show that HD0 deacetylates all four core histones, whereas HD1 and HD2 show a clear preference for H2A and H2B, the arginine-rich histones being deacetylated more slowly.     

THE SYNTHESIS OF ACIDIC CHROMOSOMAL PROTEINS DURING THE CELL CYCLE OF HELA S-3 CELLS : I. The Accelerated Accumulation of Acidic Residual Nuclear Protein before the Initiation of DNA Replication

The synthesis and accumulation of acidic proteins in the tightly bound residual nuclear fraction goes on throughout the cell cycle of continuously dividing populations of HeLa S-3 cells; however, during late G₁ there is an increased rate of synthesis and accumulation of these proteins which precedes the onset of DNA synthesis. Unlike that of the histones, whose synthesis is tightly coupled to DNA replication, the synthesis of acidic residual nuclear proteins is insensitive to inhibitors of DNA synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of acidic residual nuclear proteins shows different profiles during the G₁, S, and G₂ phases of the cell cycle. These results suggest that, in contrast to histones whose synthesis appears to be highly regulated, the acidic residual proteins may have a regulatory function in the control of cell proliferation in continuously dividing mammalian cells.     

Histones,Chromatin Structure and RNA Synthesis

THE primary structure of the histone molecules is very irregular in the spacing of the basic amino-acids¹. In various histone fractions, there are some parts of the polypeptide chains with five basic amino-acids together and parts with up to fourteen non-basic amino-acids together^2,3. Nevertheless, this irregularity is extremely specific, for it has been shown for one of the arginine-rich histones that the amino-acid sequence has remained virtually unchanged since the divergence of plants and animals⁴. This indicates that this histone has an extremely important structural function in which the whole molecule is important. Although histones are known to be gene repressors, it is important to consider this role in relation to the other possibility that they are structural proteins.     

METHODS FOR THE PURIFICATION OF THYMUS NUCLEI AND THEIR APPLICATION TO STUDIES OF NUCLEAR PROTEIN SYNTHESIS

Procedures are described for the purification of calf thymus nuclei using mild hypotonit shock to break intact cells, and layering techniques to remove cytoplasmic debris. Ficolc (a high polymer of sucrose) was dissolved in isotonic sucrose to give dense solutions suitable for gradient centrifugation. The method yields nuclei which can incorporate amino acids in vitro. Thymus nuclei isolated under isotonic conditions were incubated with C¹⁴-amino acids and later purified by centrifugation through dense sucrose solutions. The distribution of radioactivity in different nuclear proteins was measured and it was found that isotopic amino acids are actively incorporated into characteristically chromosomal proteins, such as the arginine-rich and lysine-rich histones. Protein synthesis in the nucleus is markedly inhibited by puromycin and by agents, such as 2,4-dinitrophenol, which inhibit ATP synthesis. The synthesis of histones is also inhibited by puromycin, but the uptake of several amino acids into the lysine-rich histone fraction seems less sensitive to puromycin inhibition than is uptake into the arginine-rich histones or other proteins of the nucleus. High resolution autoradiography using tritiated leucine and observing grain distribution over thin sections of isolated nuclei and whole cells shows that amino acid incorporation occurs within the nucleus and is not due to cytoplasmic contamination.     

On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes

In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972;Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly(A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70°C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly(A)^? RNA (7S–14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.     

On the infrastructural localization of basic proteins in the nucleus and nucleolus of cells in pea axillary meristems submitted to or released from apical dominance

Summary In pea axillary meristems submitted to or released from apical dominance, basic nuclear proteins and their fractions (lysine or arginine-rich) were localized at the infrastructural level using convergent methods. In the inhibited nuclei, the condensed chromatin and the nucleoli are the most reactive regions to alcoholic solution of phosphotungstic acid and to ammoniacal silver nitrate. It is the same in the reactivated nuclei after the release from dominance, but the increase in diameter of the nucleoli is accompanied by the creation of a granular component which is observed around the nucleoli during the G₁ S or G₂ phases. This structure is built up essentially by a lysin-rich ribonucleoprotein complex characteristic of active nuclei.     

Histone acetylation and heterochromatin content of cultured Peromyscus cells

In order to explore the relationship between unacetylated arginine-rich histones and condensed chromatin structure, the extent of histone acetylation was examined in cultured cell lines derived from three species of deer mice. These species differ considerably in their genomic content of heterochromatin but contain essentially the same euchromatin content. Cells of Peromyscus eremicus, containing 34–36% more constitutive heterochromatin than Peromyscus boylii or Peromyscus crinitus cells were found to contain 28–35% more unacetylated histone H4, 22–29% more unacetylated histone H3, and 18–22% more unacetylated histone H2B. This relationship between unacetylated histones and heterochromatin content was further explored by inducing hyperacetylation of P. eremicus and P. boylii histones through treatment of cells with 15 mM sodium butyrate for 24 h. It was found that the percentages of unacetylated histones H3 and H4 remaining after butyrate treatment were proportional to the amount of constitutive heterochromatin in the genome. These data support the concept that a small core of histones in constitutive heterochromatin is inaccessible to acetylation. It was also found that the acetylated state of isolated histones was sensitive to the method of histone extraction. Thus concern must be given to preparative procedures when studying histone acetylation in order to minimize these acetate losses.