Chromatin Condensation State Research Articles

Chromosomal organization of viral integration sites in human papillomavirus-immortalized human keratinocyte cell lines

The target specificity of viral integration is essential to determining the biologic significance of this integration to various pathologic conditions, including cancer. In this study the chromosomal features of several human papillomavirus (HPV)-16 integration sites mapped by in situ hybridization in human keratinocyte lines were visualized directly by G-banding and differential labeling with bromodeoxyuridine of later replicating domains. G-negative chromosomal bands exhibiting late replication were selectively targeted by HPV-16, suggesting that the structural and functional relationship of the state of chromatin condensation and replication is critical in accessibility to virus integration.

Flow cytometric probing of chromatin condensation in maize diploid nuclei

SUMMARYNuclei were isolated from stems, mesocotyls, nodal roots and root tips of two maize hybrids. The fluorochrome, propidium iodide (PI), was used to stain the nuclei that were passed through a flow cytometer‐cell sorter. Significant differences in fluorescence intensities were observed among the diploid nuclei from the different plant organs. These observations, in conjunction with previously reported DAPI fluorescence data, reflect the state of chromatin condensation in diploid nuclei isolated from meristematic and differentiating tissues. Nuclei from root meristematic tissues showed no or very little chromatin condensation. Mesocotyls and nodal roots, 1‐wk‐old, appeared to contain chromatin in a more diffuse state owing to completed differentiation which allows more DNA to be accessible to PI. Mesocotyls, 2‐wk‐old owing to senescence, contained degraded chromatin in which DNA was more accessible to PI because it lacked associated proteins. The diploid nuclei from stems had the highest amount of chromatin condensation. The results show the usefulness of flow cytometry for analyzing chromatin condensation in plant nuclei. The study also shows the importance of selecting a particular type of fluorochrome in the analysis of nuclear chromatin.

Protein phosphorylation is involved in the regulation of chromatin condensation during interphase

Loci affecting the condensation state of interphase chromatin have been previously identified from analysis of suppression and enhancement of position effect variegation (PEV) in Drosophila. Here we show that Su-var(3)6 and an allelic mutant, e078, which both show suppression of PEV in the heterozygous state, have point mutations (Gly 220→Ser and Gly 220→Asp, respectively) in a protein phosphatase 1 catalytic subunit located at 87B (PP1 87B). The mutated glycine is conserved in all known protein serine/threonine phosphatases in the same gene family, and its substitution decreases PP1 activity. We conclude that protein dephosphorylation by PP1 87B regulates the condensation state of chromatin during interphase.

Effects of 6-dimethylaminopurine on microtubules and putative intermediate filaments in sea urchin embryos

The effects of 6-dimethylaminopurine (6-DMAP) (a putative phosphorylation inhibitor) on the state of assembly of microtubules and intermediate filaments have been studied during the first cell cycle of the sea urchin Strongylocentrotus droebachiensis. Changes in the spatial organization of cytoskeletal structures were studied by indirect immunofluorescence with anti-tubulin and anti-IFa antibodies. The rates and patterns of protein phosphorylation in control and treated eggs were also investigated. The transfer of fertilized eggs to 600 microM 6-DMAP within 4 min following insemination inhibits pronuclear migration and syngamy. This also prevents male pronuclear decondensation, while chromatin condensation and nuclear envelope breakdown do not occur in the female pronucleus. Immunolabeling with anti-tubulin antibodies reveals the presence of cortical microtubules as early as 15 min after fertilization in both control and treated eggs. However, no sperm astral microtubules could be detected in the treated eggs. At later stages, from syngamy (40 min) up to nuclear envelope breakdown (90 min), 6-DMAP affects neither cortical microtubule organization nor the state of chromatin condensation but it precludes nuclear envelope breakdown and entry into mitosis. Treatment of the fertilized eggs after nuclear envelope breakdown induces permanent chromosome decondensation and premature disappearance of the mitotic apparatus. This last event involves disruption of the spatial organization of both microtubules and putative intermediate filaments. Quantitative measurements of protein phosphorylation show that 6-DMAP efficiently and reversibly inhibits 32P incorporation into proteins. Qualitative analysis of the autoradiograms of 32P-labeled proteins separated by SDS-PAGE reveals that a major protein band, migrating with an apparent molecular weight of 31 x 10(3)Mr, is specifically dephosphorylated in eggs treated with 6-DMAP. This study suggests that protein phosphorylation is required for sperm aster microtubule growth and migration, but not for cortical microtubule polymerization. It also strengthens the hypothesis that, in sea urchin eggs, putative intermediate filaments are tightly associated with spindle microtubules. Finally, it confirms that inhibiting protein phosphorylation before nuclear envelope breakdown reversibly prevents the entry into mitosis.

Kinetics of DNA Double-strand Break Repair Throughout the Cell Cycle as Assayed by Pulsed Field Gel Electrophoresis in CHO Cells

Repair of DNA double-strand breaks (dsb) was measured in exponentially growing, plateau-phase and synchronized G1, G1/S, early S, mid-S, late S, G2 + M and mitotic CHO cells. Cells were exposed to 50 Gy X-rays and allowed to repair (up to 4 h) in suspension at 37 degrees C. The dsb rejoining was measured by means of asymmetric field inversion gel electrophoresis (AFIGE), a pulsed-field gel electrophoresis technique. The fraction of DNA-associated [14C]thymidine activity released from the plug (FAR) during electrophoresis was used as a parameter to determine the number of dsb present in the DNA. The assay had been previously calibrated using 125I-decay and its sensitivity in detecting dsb throughout the cell cycle established. Biphasic kinetics of dsb rejoining with a fast and a slow component were obtained throughout the cell cycle, including mitosis, as well as in the various stages of growth. Repair kinetics were described mathematically as the sum of two exponential functions. The repair half-times calculated for the fast component by least-square fitting to the data were in the range of 7-14 min, with the shorter values reached in mid-S and late S and the longer ones during G1 and mitosis. The repair half-times of the slow component were in the range between 60 and 90 min and did not show consistent fluctuations throughout the cell cycle. The fraction of dsb repaired by the slow component ranged from 18% to 37% and did not show consistent variation throughout the cell cycle either. These results suggest that the state of chromatin condensation has only a limited impact on the ability of the cells to rejoin dsb, and indicate that the cell cycle-dependent fluctuations in radiosensitivity cannot be explained by alterations in the rate of rejoining of dsb. The repair half-times of the slow component of dsb rejoining were similar to the half-times of rejoining of chromosome breaks as visualized by the technique of premature chromosome condensation, suggesting a cause-effect relationship between rejoining of this subject of dsb and rejoining of chromosome breaks.

Chromatin superstructure-dependent crosslinking with DNA of the histone H5 residues Thr1, His25 and His62

The points of histone H5 interactions with DNA within nucleosomes and chromatin at different levels of compaction are delineated by identification of H5 amino acid residues that can be covalently bound to DNA. Three major crosslinkable points of H5 are His25, His62 (both within the globular part of the molecule), and N-terminal Thr1. His25 interacts with the terminal regions of nucleosomal DNA; His62 appears to bind more distal segments of the linker DNA. The His25-DNA crosslink predominates in the isolated mononucleosomes and persists throughout the chromatin condensation states studied, from extended oligonucleosomal chains to nuclei. His62 is the strongest crosslinking site in nuclei; in oligonucleosomes, the predominance of the His62-DNA crosslink requires the number of nucleosomes in the chain to be above some critical value. The Thr1-DNA crosslink is generated only in decondensed poly- or oligonucleosomes, but not in mononucleosomes. Thus, underlying the higher-order folding transitions of the nucleosomal chain is the restructuring of H5-DNA interactions.

In vivo influence of androgens on the cell kinetics and chromatin pattern of the MXT mouse mammary tumor treated or not by aminogluthetimide

We characterized the influence of androstenedione, 5-alpha-dihydrotestosterone and 17-beta-estradiol on the chromatin organization and the cell kinetics (distribution of the cells into the G0-G1, S and G2+ M fractions) of MXT mouse mammary tumors grafted onto animals that were left intact or that were oophorectomized and/or treated with aminoglutethimide. The cell kinetics and chromatin pattern were monitored by analyzing Feulgen-stained MXT imprint smears through a cell image processor, the SAMBA 200 system. We have also assayed the estrogen and progesterone receptors of these MXT tumors, which appeared to possess both of these biological markers. Our results show that ovariectomy or aminoglutethimide treatment slowed down the MXT tumor growth without any additive effect between them. Androstenedione exerted a stimulating influence on the cell kinetics, which were very similar to those observed after estradiol administration; the treatment of castrated animals with aminoglutethimide completely abolished this androstenedione-induced stimulating influence, however. This androgen lacked any apparent efficiency to transform cell nuclei from a state of castration-induced chromatin condensation into a thinly textured and decondensed state, as did estradiol. Dihydrotestosterone was able to activate the cell kinetics of MXT tumor grafted onto castrated animals as well as those of MXT neoplasms grafted on oophorectomized mice treated with aminoglutethimide. Dihydrotestosterone also possesses the property to transform condensed chromatin into decondensed chromatin. It thus appeared that androstenedione and dihydrotestosterone could activate MXT cell proliferation, as did estradiol, although it appeared that their mechanisms of action were quite distinct from each other.

Sister chromatid exchanges in the human active and inactive X chromosomes.

Four human female fibroblast strains with an i(Xq) or derivative X chromosome as a cytological marker for the inactive X chromosome were used to determine the frequency of sister chromatid exchanges (SCEs) in the active and inactive X chromosomes. No significant difference in SCE frequency between the active and inactive X chromosomes was observed. Therefore, the state of chromatin condensation and the late DNA replication in the facultative heterochromatin of the inactive X chromosome do not appear to influence the SCE frequency.

Chromosome Structure: Euchromatin and Heterochromatin

Publisher Summary This chapter discusses the terms “euchromatin” and “heterochromatin” that have long been familiar for expressing primarily the state of chromatin condensation during the cell cycle. The term “heterochromatin,” in particular, has been used to describe such chromatin or chromosome segments that did not go through the uncoiling process toward the end of mitosis and so remained condensed throughout interphase. Usually this condensed state of chromatin results in a heteropyknotic property. On the other hand, the chromatin that uncoils in the subsequent interphase is considered euchromatin. The earlier studies on different chromatintypes were based on the staining variation in interphase nuclei. Similar studies using metaphase chromosomes were limited to those species where the heterochromatic segments were recognizable from the staining differences by conventional methods. However, recent advances in banding technology and in sequencing DNA replication using isotype-labeled nucleotides and BrdU have rendered the metaphase chromosomes amenable for such investigations. In metaphase chromosomes, the regions stained by the C-banding method and the latereplicating X chromosome (in females) are regarded as heterochromatic. The remaining regions are referred to as “euchromatic.”

5-azacytidine-induced decrease in the frequency of Barr body in human fibroblasts

Summary5-azacytidine-treated human fibroblasts exhibit a significant decrease in the frequencies of Barr body+ cells as compared to nontreated cultures. This presumably indicates that 5-azacytidine can induce a change in the degree of condensation of the Barr body. It is suggested that the state of chromatin condensation of the Barr body may be related to the reactivation process by 5-azacytidine of gene loci in the inactive X.

The role of histone H1 and non-structured domains of core histones in maintaining the orientation of nucleosomes within the chromatin fiber

Calf thymus chromatin was digested with trypsin and the structural alterations which occured were followed by flow linear dichroism. After a sharp initial increase, the amplitude of the positive signal gradually decreased followed by a change of the sign of the dichroism and further increase of the negative signal up to a plateau. These changes of the dichroism were compared to the respective changes in the histone pattern. It was shown that the positive dichroism of chromatin did not depend on the condensation state of chromatin, and that the orientation of the nucleosomes along the chromatin fiber was maintained by the globular domain of H1 and the non-structured parts of core histones.

Micrococcal nuclease cleavage of chromatin displays nonrandom properties.

A statistical analysis of the products of digestion of chicken erythrocyte chromatin by micrococcal nuclease was used to test for randomness of the cutting process. DNA fragment size classes corresponding to mononucleosome, dinucleosome, trinucleosome, tetranucleosome, and all fragments larger than tetranucleosome were evaluated. In every case, fragments in the mononucleosome and greater-than-tetranucleosome classes were produced in excess of the level expected on the basis of random cleavage while those in the dinucleosome-tetranucleosome classes exhibited a shortage. The pattern of nonrandomness appears to depend on substrate size: the magnitude of deviations from randomness was large when substrates of genomic size are compared with polynucleosomal segments whereas the direction of deviation is identical. Nonrandomness was independent of ionic conditions known to affect the state of chromatin condensation and also appeared to be unaffected by depletion of histones H1 and H5. The possible universality of nonrandom cleavage was suggested when other data from the literature was analyzed. Some possible mechanisms to account for this property are discussed.

Fluorimetric measurements and chromatin condensation patterns of nuclei from 3T3 cells throughout G1.

Using two cytological methods based on nuclear morphology, quinacrine dihydrochloride (QDH) staining and premature chromosome condensation (PCC), it has been possible to identify cell cycle positions within G1 of growing and arrested 3T3 cells. The fluorescent intensity of QDH-stained interphase cells appears to decrease as the cells pass from mitosis to S phase. Likewise, the length and thickness of prematurely condensed chromatids can be related to the cells; position within the G1 period. Data are presented that deal with three interrelated topics: 1) We determined by fluorometric measurements of nuclei from 3T3 cells that the visual observation of the decrease in QDH fluorescence during G1 reflects an actual decrease in total fluorescence and not a dispersion of the fluorescent chromatin in a larger nuclear area. 2) We correlated the results obtained by QDH staining with those of PCC on the same cell samples blocked in G1 by different conditions. Serum-starved and contact-inhibited cell nuclei had the highest intensity, hydroxyurea-treated ones had the lowest intensity, while that of isoleucine-deprived cells was in between. The same relative order of G1 positions was obtained based on PCC morphology. Thus, both methods monitor the state of chromatin condensation and can be used to identify cell cycle position within G1. 3) We showed with both methods that the states of chromatin resulting from the various G1 blocking conditions differ from each other.

Toluidine blue binding capacity of heterochromatin and euchromatin of Triatoma infestans Klug.

The localization of the absorption peaks of toluidine blue-stained heterochromatin (Ho and Hs) and euchromatin (Eo and Es) of the Malpighian tubules of the blood-sucking hemipteran, Triatoma infestans Klug was studied cytophotometrically (Ho and Eo: hetero- and euchromatin of nuclei with one single chromocenter; Hs and Es: hetero- and euchromatin of nuclei with several chromocenters). shifts of the absorption peaks to longer wave-lengths were found for the euchromatin fractions and Hs after RNAse treatment. The differences in the localization of absorption peaks after RNAse digestion when comparing the various chromatin types with one another are assumed to be due to differences in the number and proximity of DNA phosphates unattached to proteins and hence available to dye binding. The localization of the absorption peak of Ho at a relatively short wavelength (lambda = 570 nm) as compared with those for Hs, Eo, and Es, is attributed to a special state of chromatin condensation and practically absence of RNA in Ho. Also a certain removal of histones (probably, Hl) by the acetic ethanol "fixation" is speculated to play a role in the absorption curve properties of Ho subjected to TB staining.

Interactions of mercury and copper with constitutive heterochromatin and euchromatin in vivo and in vitro.

Mouse liver nuclei were fractionated into (condensed) heterochromatin and (noncondensed) euchromatin by differential centrifugation of sonicated nuclei. The fractions were subsequently characterized as unique nuclear species by thermal denaturation derivative profile analysis, which revealed the heterochromatin fraction enriched in satellite DNA and by endogenous metal content, which displayed partitioning of mercury in euchromatin over heterochromatin by a 10:1 ratio, with a comparatively uniform distribution of copper in both fractions. Fractionation of nuclei following in vivo challenge with copper showed enrichment of copper in heterochromatin, relative to euchromatin, while in vivo exposure to mercury resulted in a 20-fold accumulation of mercury in euchromatin, relative to heterochromatin. Using gel filtration and equilibrium dialysis to measure in vitro binding under relatively physiologic conditions of pH (6.0-7.0) and ionic strength (standard saline citrate or saline), the condensed and noncondensed chromatin fractions exhibited binding specificities toward mercury and copper similar to that observed in the in vivo metal challenge experiments. The level of mercury which binds to euchromatin in vitro, when measured either in physiologic [standard saline citrate (SSC)] or in dilute (1:100 SSC) salt solutions, was comparable (approximately 3 mug of Hg/mg of DNA) to that of in vivo euchromatin-bound mercury after 1 month of challenge with dietary metal. In contrast, copper showed little or no preference for the nuclear fractions in dilute salt solutions and displayed patterns which mimic in vivo binding only at higher ionic strengths (saline). Removal of proteins from the chromatin fractions resulted in a loss of binding specificity toward both metals. Therefore, the binding selectivity of condensed and noncondensed chromatin toward both mercury and copper appears to arise from protein or from protein-DNA associations. The state of chromatin condensation is especially critical in the case of copper.

Influence of infra-optimal breeding temperature on spermiogenesis of the locust Locusta migratoria: II. Abnormalities in differentiation of the nucleus

The sterile males of the Migratory locust that have been raised under infra-optimal temperature conditions produce spermatids with highly abnormal nuclei. These are spherical in young spermatids but later they do not elongate uniformly and they show deep invaginations into which cytoplasmic organelles penetrate. Condensation of the chromatin also is disturbed and the typical honeycomb pattern of the locust spermatid is never achieved. The most advanced stage of condensation that can be reached corresponds to stage 6 of the normal process. Nuclei demonstrating a “stage 6” pattern of chromatin condensation are never thin and slender, as they normally should be at this stage. The shape of the nucleus in these experimental animals seems to be correlated with the pattern of perinuclear microtubules rather than with the state of chromatin condensation. Nuclear abnormalities as well as those regarding the cytoplasmic organelles are compared to several spermiogenetic defects reported in various sterile mutants of Drosophila or in insects submitted to heat-shocks. A total sterility at temperatures only slightly inferior to the usual breeding ones has never yet been reported in insects other than locusts; temperature is thus demonstrated to be a very critical factor for fertility not only when it is too high but also when it does not reach an optimal level.

DNA Synthesis and Cell Division in Germinating Onion: III. Variation in Apparent Feulgen-DNA Content with Nuclear area Change

SUMMARYCareful microspectrophotometric analysis of the 2C (Gl) nuclear population of the onion during the critical period of decompaction from the quiescent to the functional state reveals an increase in apparent DNA content correlated with increased nuclear area. This finding is demonstrated not to depend on technical flattening of nuclei, but on the state of chromatin condensation. The probable identity of this phenomenon with that recently reported from mammalian systems is discussed, as are the possible interpretations of cause. It is provisionally concluded that current evidence favors the view that the relationship reflects non-stoichiometric stain-substrate interaction. The relative advantages of the onion system for future work in this area are discussed.