Histone acetyltransferase activity during the cell cycle

Histone acetyltransferase activity was measured in isolated nuclei during the synchronous cell cycle of the myxomycete Physarum polycephalum. Nuclei were incubated with [14C]acetyl-coenzyme A and an excess of exogenous calf thymus histones. The activity is periodic during the cell cycle; it rises during the S-phase to reach a maximum in the early G2-period with a decline in mid and late G2. Comparison of the pattern of enzyme activity with the in vivo acetylation of histones during the cell cycle reveals that the enzyme activity does not wholly determine the acetylation state, indicating that other factors, including possibly the structural state of chromatin, are responsible for the observed cell cycle pattern of in vivo histone acetylation.     

Amphibian oocyte maturation induced by extracts of Physarum polycephalum in mitosis

The orderly progression of eukaryotic cells from interphase to mitosis requires the close coordination of various nuclear and cytoplasmic events. Studies from our laboratory and others on animal cells indicate that two activities, one present mainly in mitotic cells and the other exclusively in G1-phase cells, play a pivotal role in the regulation of initiation and completion of mitosis, respectively. The purpose of this study was to investigate whether these activities are expressed in the slime mold Physarum polycephalum in which all the nuclei traverse the cell cycle in natural synchrony. Extracts were prepared from plasmodia in various phases of the cell cycle and tested for their ability to induce germinal vesicle breakdown and chromosome condensation after microinjection into Xenopus laevis oocytes. We found that extract of cells at 10-20 min before metaphase consistently induced germinal vesicle breakdown in oocytes. Preliminary characterization, including purification on a DNA-cellulose affinity column, indicated that the mitotic factors from Physarum were functionally very similar to HeLa mitotic factors. We also identified a number of mitosis-specific antigens in extracts from Physarum plasmodia, similar to those of HeLa cells, using the mitosis-specific monoclonal antibodies MPM-2 and MPM-7. Interestingly, we also observed an activity in Physarum at 45 min after metaphase (i.e., in early S phase since it has no G1) that is usually present in HeLa cells only during the G1 phase of the cell cycle. These are the first studies to show that maturation-promoting factor activity is present in Physarum during mitosis and is replaced by the G1 factor (or anti-maturation-promoting factor) activity in a postmitotic stage. A comparative study of these factors in this slime mold and in mammalian cells would be extremely valuable in further understanding their function in the regulation of eukaryotic cell cycle and their evolutionary relationship to one another.     

Analysis of chromatin assembled in vivo using exogenous histones in Physarum polycephalum

Histones are involved in the regulation of almost all events within the eukaryotic cell nucleus that utilize DNA as a substrate. We have developed a novel approach for examining the function of histone proteins and specific domains of these proteins in these various nuclear processes, and in particular assembly of chromatin throughout the cell cycle. This approach exploits several unique characteristics of the slime mold Physarum polycephalum, including the natural synchrony of all (approximately 10(8)) nuclei throughout the cell cycle and the ability of this organism to take up exogenous proteins. Here, culture techniques and biochemical procedures for the incorporation of exogenous core histones into Physarum chromatin in vivo are described. The procedures for subsequent verification of the assembly of exogenous proteins into bona fide nucleosomes are also described.     

Cell-cycle dependence of two nuclear histone kinase enzyme activities.

Growth-associated histone kinases have been extracted from Physarum polycephalum nuclei and resolved into two components by ion-exchange chromatography. The two component activities have different substrate specificities and different times of appearance in the cell cycle. It is proposed that the enzyme(s) phosphorylate H1 histone in vivo in G2 phase, possibly sequentially in time at different sites in the H1 amino acid sequence.     

Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei

Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly casually involved in the formation of cell type specific heterochromatin compartments, composed of (peri)centromeric regions and chromosomal subregions from neighboring chromosomes territories, which contain silent genes.     

Histone H1 of Trypanosoma cruzi is concentrated in the nucleolus region and disperses upon phosphorylation during progression to mitosis

Phosphorylation of histone H1 is intimately related to the cell cycle progression in higher eukaryotes, reaching maximum levels during mitosis. We have previously shown that in the flagellated protozoan Trypanosoma cruzi, which does not condense chromatin during mitosis, histone H1 is phosphorylated at a single cyclin-dependent kinase site. By using an antibody that recognizes specifically the phosphorylated T. cruzi histone H1 site, we have now confirmed that T. cruzi histone H1 is also phosphorylated in a cell cycle-dependent manner. Differently from core histones, the bulk of nonphosphorylated histone H1 in G(1) and S phases of the cell cycle is concentrated in the central regions of the nucleus, which contains the nucleolus and less densely packed chromatin. When cells pass G(2), histone H1 becomes phosphorylated and starts to diffuse. At the onset of mitosis, histone H1 phosphorylation is maximal and found in the entire nuclear space. As permeabilized parasites preferentially lose phosphorylated histone H1, we conclude that this modification promotes its release from less condensed and nucleolar chromatin after G(2).     

Acceptor proteins for (ADP-ribose)n in the HeLa S3 cell cycle

The acceptor proteins for (ADP-ribose)n were investigated by using nuclei or chromosomes isolated from specific phases of the cell cycle of HeLa S3 cells. Analysis of HMG proteins and histone H1 by acetic acid/urea polyacrylamide gel electrophoresis demonstrated that the (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 increased by 12- and 5-fold, respectively, in the metaphase chromosomes as compared with that in the G1 phase cell nuclei. The degree of (ADP-ribosyl)n-ation of these proteins in the S phase cell nuclei was as low as that in G1 phase cell nuclei. In the G2 phase cell nuclei, the degrees of (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 were about 5- and 2-fold greater, respectively, as compared with that in the G1 phase cell nuclei. The (ADP-ribosyl)n-ation of HMG 1 and 2 was constant through the cell cycle except for a slight decrease in the S phase. The data may imply that the (ADP-ribosyl)n-ation of HMG 14 and 17 and histone H1 is linked to chromatin structural changes in mitosis.     

Cycle specific association of nascent chromatin with nuclear envelope components in Physarum polycephalum.

The action of heparin on isolated nuclei derived from different phases of the mitotic cycle in plasmodia of Physarum polycephalum was studied. Heparin addition at two-fold excess over DNA concentration to nuclei in Mg-free low ionic strength buffer (10 mM TRIS-HC1, 10 mM Na2 HPO4, pH = 8) releases 60-80% of chromatin from S, G2, and mitotic phase nuclei. The RNA/protein ratio of herparin-solubilized cromatin is constant through S and G2 phases, but rises about two-fold at early prophase coincident with nucleolar breakdown. Purified nuclear envelopes were obtained from heparin-treated nuclei by sedimentation according to Bornens procedures (Nature 244, 28, 1973), and examined by transmission electron microscopy. Residual chromatin is seen at all stages with fine network of DNA fibrils in contact with the envelop. Regardless of time in S, 80% of 3H-labeled DNA was released into soluble chromatin with identical 3H/14C ratios. The residual chromatin in nuclear envelopes exhibited a preferential association of early S-DNA in nuclei engaged in early S replication, and late S preferential association in nuclei engaged in late S replication.     

Cell cycle regulation of chromatin at an origin of DNA replication

Selection and licensing of mammalian DNA replication origins may be regulated by epigenetic changes in chromatin structure. The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) uses the cellular licensing machinery to regulate replication during latent infection of human cells. We found that the minimal replicator sequence of OriP, referred to as the dyad symmetry (DS), is flanked by nucleosomes. These nucleosomes were subject to cell cycle-dependent chromatin remodeling and histone modifications. Restriction enzyme accessibility assay indicated that the DS-bounded nucleosomes were remodeled in late G1. Remarkably, histone H3 acetylation of DS-bounded nucleosomes decreased during late G1, coinciding with nucleosome remodeling and MCM3 loading, and preceding the onset of DNA replication. The ATP-dependent chromatin-remodeling factor SNF2h was also recruited to DS in late G1, and formed a stable complex with HDAC2 at DS. siRNA depletion of SNF2h reduced G1-specific nucleosome remodeling, histone deacetylation, and MCM3 loading at DS. We conclude that an SNF2h-HDAC1/2 complex coordinates G1-specific chromatin remodeling and histone deacetylation with the DNA replication initiation process at OriP.     

Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.     

Reactivation of DNA replication in erythrocyte nuclei by Xenopus egg extract involves energy-dependent chromatin decondensation and changes in histone phosphorylation.

Reactivation of chicken erythrocyte nuclei for DNA replication in Xenopus egg extracts involves two phases of chromatin remodelling: a fast decondensation leading to a small volume increase and chromatin dispersion occurring within a few minutes (termed stage I decondensation), followed by a slower membrane-dependent decondensation and enlargement of up to 40-fold from the initial volume (stage II decondensation). Chromatin decondensation as measured by nuclear swelling and micrococcal nuclease digestion required ATP. We observed a characteristic change in the phosphorylation pattern of erythrocyte proteins upon incubation in egg extract. While histones H5, H2A, and H4 became selectively phosphorylated during decondensation, the phosphorylation of histone H3 and of several nonhistone proteins was prevented. Furthermore, histone H5 was selectively released from erythrocyte nuclei in an energy-dependent reaction. These molecular changes already occurred during stage I decondensation and they persisted during stage II decondensation. DNA replication was confined to nuclei of stage II decondensation which incorporated lamin LIII from the egg extract. These results show that initiation of DNA replication in chicken erythrocytes requires in addition to ATP-dependent chromatin remodelling (stage I), further changes in chromatin structure that correlates with lamin LIII incorporation, and stage II decondensation.     

多头绒泡菌类cyclin B1蛋白在细胞周期中的变化

以自然同步化的多头绒泡菌(Physarum polycephalum L.)为材料,经抗cyclin B1抗体的免疫印迹和免疫电镜实验观察结果表明,多头绒泡菌中含有类cyclin B1蛋白,该蛋白的含量和细胞内位置在细胞周期进程中存在着动态变化:类cyclin B1蛋白在S期开始合成并在细胞质中积累,G2晚期开始进入细胞核,该蛋白在细胞质和细胞核中含量逐渐增加,有丝分裂中期时达最大值,后末期时骤然消失.在G2晚期到有丝分裂中期期间,类cyclin B1蛋白既是细胞核蛋白又是细胞质蛋白,细胞质是类cyclin B1蛋白的主要存在区域,细胞核中的类cyclin B1蛋白主要结合于染色体和核仁区域.