Changes in chromatin and the phosphorylation of nuclear proteins during heat shock of Achlya ambisexualis.

Heat shock led to marked changes in the apparent levels of phosphorylation of nuclear proteins in the fungus Achlya ambisexualis. We characterized these heat shock-induced changes in nuclear proteins on two types of two-dimensional polyacrylamide gel systems. We report here that one of two Achlya H3 histones (H3.1) and also the oomycete histone alpha appear to be highly phosphorylated with heat shock. Additional changes observed in acid-soluble nuclear proteins included an apparent increase in the 32P labeling of a 43,000-molecular-weight protein and the dephosphorylation of a major group of Achlya phosphoproteins in the 30,000-to-32,000-molecular-weight range. The changes in protein phosphorylation were accompanied by striking changes in the morphology of Achlya nuclei. Nuclei in the heat-shocked cells, but not in control cells, exhibited marked chromatin condensation and contained bundles of filaments which were approximately 4 nm in diameter. Concomitantly, the bulk of chromatin from heat-shocked nuclei showed a decreased sensitivity to digestion with the enzyme DNase I relative to chromatin from control cells.     

The characterization of the monoclonal antibody Th-10a, specific for a nuclear protein appearing in the S phase of the cell cycle in normal thymocytes and its unregulated expression in lymphoma cell lines

A monoclonal antibody (Th-10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG_2a and had κ light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC-conjugated anti-BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G₁, G₂ and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th-10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characteize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analysed them by immunoblotting or***     

Isolation and biochemical characterization of nuclei from immature and mature guinea pig granulocytes

Intact nuclei were isolated in high yield from enriched fractions of immature and mature guinea pig granulocytic leukocytes. These nuclei were used to determine whether any changes in synthesis and content of nuclear proteins accompany the striking increase in chromatin condensation and the nuclear lobation which occur during granulocyte maturation. The results indicate that the synthesis of nuclear proteins and the nuclear RNA content decrease markedly during granulocyte maturation. The incorporation of l-[U-¹⁴C]leucine into the acid-soluble histone-rich fraction of chromatin from immature cells is about 25 times that of mature cells, and the incorporation into the acid-insoluble, nonhistone proteins of chromatin from immature cells is about 6 times that of mature cells. It appears that there is very little quantitative change with respect to the protein components of nuclei from immature and mature granulocytic leukocytes. No significant differences in the amounts of histone, nonhistone protein, or phosphoprotein between nuclei of immature and mature granulocytes could be detected. No major differences in gel electrophoretic patterns of histones or nonhistone proteins could be detected. The fact that the amount of the chromatin proteins remains relatively constant during cell maturation in spite of the pronounced decrease in the rate of synthesis suggests that the rate of turnover of these proteins decreases significantly as the maturation of granulocytic leukocytes proceeds.     

Characterization of apoptosis-like endonuclease activity in avian thymocytes.

In the current study the internucleosomal DNA cleavage activity associated with apoptosis was investigated in avian thymocytes. Thymocyte nuclear proteins from glucocorticoid-treated chickens were incubated with chicken red blood cell (cRBC) nuclei, and DNA degradation was analyzed by agarose gel electrophoresis and fluorescence-activated flow cytometry. The thymocyte nuclear extract contained an endonuclease activity that degraded cRBC chromatin at internucleosomal sites as detected by agarose gel electrophoresis. Flow cytometry analysis of cRBC nuclei that were treated with thymocyte nuclear proteins demonstrated a loss of cellular DNA as a function of the amount of added nuclease activity. Furthermore, it was demonstrated that the thymocyte nuclear extract contained a nuclease activity that was capable of degrading radiolabelled naked 32P-DNA into acid soluble DNA fragments. All three assay methods demonstrate that the thymocyte nuclease activity can be inhibited by EDTA, zinc ions and the nuclease inhibitor aurintricarboxylic acid. Based on the analysis of cofactor requirement of this nuclease activity and its susceptibility to inhibitors, the endonuclease activity present in avian apoptotic thymocytes appears to be identical to the mammalian counterpart.     

Non-histone chromatin proteins of B lymphocytes stimulated by lipopolysaccharide. II. Phosphorylation.

Stimulation of mouse lymphocytes with the B lymphocyte specific mitogen lipopolysaccharide results in an increased rate of phosphorylation of non-histone chromatin proteins. An initial small increase in phosphorylation occurs during the first 2 h and a much larger increase after 24 h of culture with mitogen. The phosphorylated nuclear and cytoplasmic proteins were analysed by polyacrylamide gel electrophoresis and the stimulation index of each prominent peak measured. It was inferred that selective stimulation of the phosphorylation of individual proteins had occurred from: (1) the range of stimulation indices for different proteins, and (2) the appearance, after 8 h stimulation of an apparently newly phosphorylated non-histone chromatin protein of molecular weight 115 000. The pool size of ATP was monitored and showed only small changes during the first 24 h of exposure to lipopolysaccharide. Phosphatase activity was found to be associated with lymphocyte chromatin and nucleoplasm and may help to regulate the level of phosphorylation of non-histone chromatin proteins in vivo. To preserve phosphorylated proteins during their isolation phosphatase activity was inhibited by Na2MoO4. The selective changes in phosphorylation of nuclear proteins precede, and continue during, the stimulation of immunoglobulin and DNA synthesis. Our results are thus consistent with the hypothesis that phosphorylation of non-histone chromatin proteins plays a role in the regulation of gene expression in B lymphocytes.     

Protein-linked isoprenoid lipids in dexamethasone-treated human lymphoid lines in culture.

Accumulation of isoprenoids was studied in two cell lines derived from acute T-cell leukemia: CEM-C7 cells, whose growth is inhibited by the glucocorticoid dexamethasone, and CEM-C1 cells, which are resistant to this steroid. Isoprenoids were measured by growing the cells in serum-free medium in the presence of lovastatin, which blocks synthesis of mevalonate, and then labeling with exogenous [3H]mevalonolactone. In both cell lines, isoprenoids associated with proteins were detected in cytoplasm, nucleus, and chromatin, and in the chromatin residue that remains after extraction of histone and nonhistone proteins. Differences in labeling were detected after treatment with dexamethasone in the CEM-C7 line, showing a decrease in the cytoplasmic fraction with a corresponding increase in both the nuclear and chromatin fractions as compared with untreated cells. No change was seen in the CEM-C1 line. In both cell lines, 25-30% of the incorporated label was released by treatment with acid or alkali. However, the majority of the label required treatment with methyl iodide for the release of organic-soluble tritiated products. After extraction with chloroform, the lipid fractions contained farnesol, geraniol, dolichols, and possibly nerolidol.     

Early events in thymocyte activation. I. Stimulation of protein synthesis by a thymus-dependent human serum factor.

Human serum contains a thymus-dependent factor that raises cyclic AMP levels in thymocytes. We found that this factor stimulates protein synthesis in thymocytes cultured in vitro. This activity of serum factor is thymus-dependent, because it is absent in sera from thymectomized donors; furthermore, this effect is predominantly found on precursors of mature T cells. Incubation of thymocytes with other agents that increase cyclic AMP, induces an increase in protein synthesis similar to that observed with serum factor. Most likely, the increase in protein synthesis is one of the events following stimulation of adenylate cyclase in thymocytes that leads to cell differentiation.     

Effect of cell trypsinization on nuclear proteins of WI-38 fibroblasts in culture.

When resting confluent monolayers of WI-38 fibrolasts are trypsinized and replated at a lower density they are stimulated to proliferate again with an interval of 18 hours between replating and the onset of DNA synthesis. Trypsinization of resting cells causes a 40% loss of nuclear proteins as well as of cytoplasmic proteins. The amount of nuclear proteins remains low for the first six hours after the cells have been replated and then it increases rapidly, reaching the same level of non-trypsinized resting cells by ten hours after plating. The proteins that are lost from the nucleus immediately after trypsinization are chromatin-associated proteins and most of them are non-histone chromosomal proteins, although a modest loss of histones cannot be ruled out. The loss of non-histone chromosomal proteins from cells that have been trypsinized causes changes in the structure of chromatin that can be detected by circular dichroism and by viscosity measurements. These results show that cell trypsinization causes an extensive loss of proteins from chromatin and that the loss is restored only several hours after the cells have been replated at a lower density.     

Relationship between chromatin structure and replication in mouse L-cells

Mouse L-cells treated with cytosine arabinoside, hydroxyurea, fluorodeoxyuridine, methotrexate, or mitomycin C rapidly cease DNA synthesis and stop dividing. Such inhibition of DNA replication is followed by interruption of formation of lysine- and arginine-containing proteins, including chromatin-bound histones, and by a major reorganization of the heterochromatin of the central nucleoplasm, manifest as disaggregation of large clumps of this condensed chromatin. Morphometric analysis revealed both cell and nuclear enlargement in cells treated with such antimetabolites of DNA replication. These observations are in contrast to those made with WT-4 cells starved of isoleucine or treated with cycloheximide. Isoleucine depletion was associated with inhibition of DNA synthesis and continued increase of cell and nuclear volume, but not with massive disaggregation of heterochromatin. Cycloheximide produced inhibition of DNA synthesis and protoplasmic growth, and also prevented structural reorganization of chromatin. A model is presented which suggests that initiation of chromatin replication is associated with a process, dependent upon de novo protein synthesis, which results in chromatin disaggregation. This can be revealed by inhibition of the correct replication of chromatin DNA and chromatin protein.     

Nuclear organization of DNA replication initiation proteins in mammalian cells.

Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Here we show that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells using an in vivo chemical cross-linking method. CDC6 proteins were also found to assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder binds to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. On the other hand, our results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed.     

Synthesis and phosphorylation of chromatin-associated proteins in cAMP-induced "differentiated" neuroblastoma cells in culture.

Prostaglandin E₁ and a cAMP phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, RO20-1724, were used to induce differentiation in mouse neuroblastoma cells in culture. The incorporation of amino acids and phosphate into nuclear proteins of control and drug-treated cells (1 h and 3 days after treatment) was examined using double radioisotopic techniques. A marked decrease in histone synthesis and H1-histone phosphorylation were observed in ‘differentiated’ neuroblastoma cells after 3 days of prostaglandin E₁ and RO20-1724 treatment, but only small differences were noted in the synthesis and phosphorylation of non-histone chromatin associated proteins after 3 days of drug treatment. Minimal changes were observed in the labeling of histone and non-histone nuclear proteins if the cells were treated for 1 h with prostaglandin E₁ and RO20-1724.     

The effect of gamma-irradiation on the poly(ADP-ribosylation) of nuclear proteins in rat thymocytes

Differences in the spectra of modified nuclear proteins of thymocytes of control and irradiated rats were investigated using antibodies specific for poly(ADP-ribose) and incorporation of a label from 14C-NAD in vitro. Two classes of modified proteins were identified differing in the rate of the polymer metabolism and the degree of poly(ADP-ribosylation). No postirradiation changes were detected in poly(ADP-ribosylation) of the nuclear sap proteins and chromatin. A pronounced increase in modification of proteins with the molecular mass of 72 and 83 kD and a sharp decrease in poly(ADP-ribosylation) of a protein group with the molecular mass of 47 to 65 kD were detected within the nuclear matrix by the second hour following irradiation. A study was made of the localization of modified proteins in polydeoxynucleotide fractions of different sizes (mononucleosomes and their oligomers).     

Synthesis of nuclear acidic proteins in density-inhibited fibroblasts stimulated to proliferate

Previous work from this laboratory (Rovera and Baserga, 1971) has shown that, when density-inhibited WI-38 human diploid fibroblasts are stimulated to proliferate by a change of medium, the synthesis of nuclear acidic proteins increases within 30 minutes after stimulation; several hours before DNA synthesis begins to increase. Similar results have now been obtained with density-inhibited 3T6 mouse fibroblasts, also stimulated by a change of medium. Gel electrophoretic analysis of nuclear acidic proteins in both WI-38 human diploid fibroblasts and 3T6 mouse fibroblasts stimulated to proliferate indicates that the increased synthesis of nuclear acidic proteins is limited to certain classes of proteins while other classes are totally unaffected. The increase in nuclear acidic proteins synthesis is inhibited when WI-38 cells or 3T6 cells are stimulated in the presence of 5-azacytidine (10 μg/ml), a treatment which also inhibits the subsequent stimulation of DNA synthesis. These results, confirming and extending similar findings previously reported in other models of stimulated DNA synthesis, lend further support to the hypothesis that nuclear acidic proteins may play a critical role in the control of DNA synthesis and cell division in mammalian cells.     

Degree of chromatin fragmentation and frequency of nuclear pyknosis in Percoll-fractionated thymocytes of irradiated rats

The relationship between nuclear chromatin degradation to polydeoxyribonucleotides (PDN) and other features of interphase death were studied using thymocytes of normal and X-irradiated rats. Fractionation of the thymic cells in Percoll gradients was performed in order to separate dead from intact cells. The degree of radiation-induced chromatin fragmentation, as assessed by electrophoresis, was similar for PDN from all Percoll bands. Following irradiation 87-98 per cent of 'heavy' thymocytes were pyknotic and almost devoid of receptors to autologous erythrocytes thus comprising a dead cell population. A direct relationship between PDN content and nuclear pyknosis was noted throughout the gradient. The loss of autologous rosette-forming ability was directly related to other indices of interphase death. The possibility of PDN originating from pyknosis-prone cells and the capacity of radiosensitive thymocytes to form autologous rosettes are discussed.     

Effect of cyclic AMP on chromatin-bound protein kinases in WI-38 fibroblasts stimulated to proliferate.

When resting confluent monolayers of WI-38 fibroblasts are stimulated to proliferate by serum, DNA synthesis begins to increase between 15-18 h after stimulation. Chromatin-bound protein kinase activity increases in stimulated cells within 1 h after the nutritional change, concomitant with an increase in the template activity of nuclear chromatin. Addition of dibutyryl 3' : 5'-cyclic adenosine monophosphate (dibutyryl cyclic) AMP to the stimulating medium inhibits the entrance of cells into S phase, but only if dibutyryl cyclic AMP (5-10(-4) M) is added before the onset of DNA synthesis. The increases in chromatin template activity and in the chromatin-bound kinase activity are not inhibited by dibutyryl cyclic AMP in the early hours after stimulation, but are completely inhibited after the 5th hour from the nutritional change. This seems to indicate that in stimulated WI-38 cells, dibutyryl cyclic AMP exerts its inhibitory action somewhere between 5 and 12 h after stimulation. A number of protein kinase activities were extracted from chromatin with 0.3 M NaCl and partially resolved on a phosphocellulose column. Two distinct peaks of protein kinase activity appeared to be markedly increased in WI-38 cells 6 h after serum stimulation. Both peaks of increased activity were inhibited by dibutyryl cyclic AMP in vivo. Adenosine, sodium butyrate and adenosine 5'-monophosphate (AMP) do not inhibit the increase in DNA synthesis nor the increase in protein kinase activity. The results suggest that stimulation of cell proliferation in confluent monolayers of WI-38 cells causes an increase (or the new appearance) of certain chromatin-bound protein kinases, and that this increase is inhibited by cyclic AMP in vivo.     

Immunohistochemical demonstration of nuclear S1 proteins in various cells

Summary S1 proteins are present in the nuclear structures sensitive to DNases and RNase. To examine localization of these proteins, an antibody was raised in a rabbit. Indirect immunofluorescence staining revealed that S1 proteins located in the extranucleolar nuclear regions of quiescent myocardial and cerebellar cells as well as actively duplicating mouse 3T3 fibroblasts. They located in euchromatin regions of thymus lymphocytes, with a characteristic aster-like immunofluorescence pattern, and on the border of condensed chromatin areas by deposition of immunogold particles in ultrathin sections of thymus. Thus, S1 proteins may be in a nuclear function assigned to the border of heterochromatin areas, and other than synthesis of DNA or of ribosomal RNA. Possible involvement of S1 proteins in the extranucleolar RNA synthesis is discussed.     

Fanconi anemia proteins localize to chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner

Fanconi anemia (FA) is a genetic disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. Cells from patients with FA exhibit genomic instability and hypersensitivity to DNA cross linking agents such as mitomycin C. Despite the identification of seven complementation groups and the cloning of six genes, the function of the encoded gene products remains elusive. The FancA (Fanconi anemia complementation group A), FancC, and FancG proteins have been detected within a nuclear complex, but no change in level, binding, or localization has been reported as a result of drug treatment or cell cycle. We show that in immunofluorescence studies, FancA appears as a non-nucleolar nuclear protein that is excluded from condensed, mitotic chromosomes. Biochemical fractionation reveals that the FA proteins are found in nuclear matrix and chromatin and that treatment with mitomycin C results in increase of the FA proteins in nuclear matrix and chromatin fractions. This induction occurs in wild-type cells and mutant FA-D (Fanconi complementation group D) cells but not in mutant FA-A cells. Immunoprecipitation of FancA protein in chromatin demonstrates the coprecipitation of FancA, FancC, and FancG, showing that the FA proteins move together as a complex. Also, fractionation of mitotic cells confirms the lack of FA proteins in chromatin or the nuclear matrix. Furthermore, phosphorylation of FancG was found to be temporally correlated with exit of the FA complex from chromosomes at mitosis. Taken together, these findings suggest a role for FA proteins in chromatin and nuclear matrix.     

Immunohistochemical demonstration of nuclear S1 proteins in various cells

S1 proteins are present in the nuclear structures sensitive to DNases and RNase. To examine localization of these proteins, an antibody was raised in a rabbit. Indirect immunofluorescence staining revealed that S1 proteins located in the extranucleolar nuclear regions of quiescent myocardial and cerebellar cells as well as actively duplicating mouse 3T3 fibroblasts. They located in euchromatin regions of thymus lymphocytes, with a characteristic aster-like immunofluorescence pattern, and on the border of condensed chromatin areas by deposition of immunogold particles in ultrathin sections of thymus. Thus, S1 proteins may be in a nuclear function assigned to the border of heterochromatin areas, and other than synthesis of DNA or of ribosomal RNA. Possible involvement of S1 proteins in the extranucleolar RNA synthesis is discussed.