In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.     

Visualization of chromatin distribution in living PTO cells by Hoechst 33342 fluorescent staining

Chromatin distribution was visualized in living cells with the selective DNA fluorochrome Hoechst 33342. This dye was shown to be non-toxic on the rat kangaroo PTO cell line by measuring the labelled cell growth rate. The aim of this work was firstly to visualize chromatin distribution without fixation or dehydration and secondly to demonstrate that quantitative determination of DNA content was possible under these non-toxic labelling conditions. During interphase, condensed, decondensed and thin network chromatin configurations were visualized. In nucleolar regions the fluorochrome revealed well-defined chromocentres. During mitosis, fluorescent chromosome banding was observed in vital conditions and chromocentres on fixed chromosomes. Chromatin segregation was visualized after micronucleation, which induced chromosomal set distribution in individual micronuclei. By this means, we demonstrated that the chromocentres observed in interphase nuclei were part of nuclear organizer region (NOR)-bearing chromosomes. This vital staining of chromatin was shown to be compatible with the quantitative determination of DNA content, both in living PTO cells and in isolated nuclei.     

Visualization by atomic force microscopy and FISH of the 45S rDNA gaps in mitotic chromosomes of Lolium perenne

The mitotic chromosome structure of 45S rDNA site gaps in Lolium perenne was studied by atomic force microscope (AFM) combining with fluorescence in situ hybridization (FISH) analysis in the present study. FISH on the mitotic chromosomes showed that 45S rDNA gaps were completely broken or local despiralizations of the chromatid which had the appearance of one or a few thin DNA fiber threads. Topography imaging using AFM confirmed these observations. In addition, AFM imaging showed that the broken end of the chromosome fragment lacking the 45S rDNA was sharper, suggesting high condensation. In contrast, the broken ends containing the 45S rDNA or thin 45S rDNA fibers exhibited lower density and were uncompacted. Higher magnification visualization by AFM of the terminals of decondensed 45S rDNA chromatin indicated that both ends containing the 45S rDNA also exhibited lower density zones. The measured height of a decondensed 45S rDNA chromatin as obtained from the AFM image was about 55–65 nm, composed of just two 30-nm single fibers of chromatin. FISH in flow-sorted G2 interphase nuclei showed that 45S rDNA was highly decondensed in more than 90% of the G2/M nuclei. Our results suggested that a failure of the complex folding of the chromatin fibers occurred at 45S rDNA sites, resulting in gap formation or break.     

Characterizing locus specific chromatin structure and dynamics with correlative conventional and super-resolution imaging in living cells

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution photoactivated localization microscopy (PALM) precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres move faster and have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule sensitivity and yields unprecedented insights across length and time scales.     

Chromatin Compaction Protects Genomic DNA from Radiation Damage

Genomic DNA is organized three-dimensionally in the nucleus, and is thought to form compact chromatin domains. Although chromatin compaction is known to be essential for mitosis, whether it confers other advantages, particularly in interphase cells, remains unknown. Here, we report that chromatin compaction protects genomic DNA from radiation damage. Using a newly developed solid-phase system, we found that the frequency of double-strand breaks (DSBs) in compact chromatin after ionizing irradiation was 5–50-fold lower than in decondensed chromatin. Since radical scavengers inhibited DSB induction in decondensed chromatin, condensed chromatin had a lower level of reactive radical generation after ionizing irradiation. We also found that chromatin compaction protects DNA from attack by chemical agents. Our findings suggest that genomic DNA compaction plays an important role in maintaining genomic integrity.     

Random Motion of Chromatin Is Influenced by Lamin A Interconnections

Using fluorescence correlation spectroscopy in single-plane illumination microscopy, we investigated the dynamics of chromatin in interphase mouse adult fibroblast cell nuclei under the influence of the intermediate filament protein lamin A. We find that 1) lamin A-eGFP and histone H2A-mRFP show significant comobility, indicating that their motions are clearly interconnected in the nucleus, and 2) that the random motion of histones H2A within the chromatin network is subdiffusive, i.e., the effective diffusion coefficient decreases for slow timescales. Knocking out lamin A changes the diffusion back to normal. Thus, lamin A influences the dynamics of the entire chromatin network. Our conclusion is that lamin A plays a central role in determining the viscoelasticity of the chromatin network and helping to maintain local ordering of interphase chromosomes.     

Chromocenters of interphase nuclei in the mouse liver contain satellite DNA]

In isolated interphase mouse liver nuclei after hypotonic treatment only the chromocenters belonging to the pericentromeric heterochromatin remain in dense form while the main mass of a chromatin is completely decondensed. The centromeric nature of these chromocenters is demonstrated by their capability for C-banding and for hybridization with a satellite mouse DNA.     

Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation

The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.     

Chromosome condensation and sister chromatid pairing in budding yeast

We have developed a fluorescent in situ hybridization (FISH) method to examine the structure of both natural chromosomes and small artificial chromosomes during the mitotic cycle of budding yeast. Our results suggest that the pairing of sister chromatids: (a) occurs near the centromere and at multiple places along the chromosome arm as has been observed in other eukaryotic cells; (b) is maintained in the absence of catenation between sister DNA molecules; and (c) is independent of large blocks of repetitive DNA commonly associated with heterochromatin. Condensation of a unique region of chromosome XVI and the highly repetitive ribosomal DNA (rDNA) cluster from chromosome XII were also examined in budding yeast. Interphase chromosomes were condensed 80-fold relative to B form DNA, similar to what has been observed in other eukaryotes, suggesting that the structure of interphase chromosomes may be conserved among eukaryotes. While additional condensation of budding yeast chromosomes were observed during mitosis, the level of condensation was less than that observed for human mitotic chromosomes. At most stages of the cell cycle, both unique and repetitive sequences were either condensed or decondensed. However, in cells arrested in late mitosis (M) by a cdc15 mutation, the unique DNA appeared decondensed while the repetitive rDNA region appeared condensed, suggesting that the condensation state of separate regions of the genome may be regulated differently. The ability to monitor the pairing and condensation of sister chromatids in budding yeast should facilitate the molecular analysis of these processes as well as provide two new landmarks for evaluating the function of important cell cycle regulators like p34 kinases and cyclins. Finally our FISH method provides a new tool to analyze centromeres, telomeres, and gene expression in budding yeast.     

Interactions between metaphase and interphase factors in heterokaryons produced by fusion of mouse oocytes and zygotes

The cytoplasmic factor responsible for chromosome condensation was introduced into mouse zygotes at different times after fertilization by fusion of the zygotes with metaphase I oocytes. In 72% of heterokaryons obtained after fusion of early zygotes (14-18 hr post-human chorionic gonadotrophin (HCG) with oocytes, the male and female pronuclei of the zygote decondensed. At the same time, the oocyte chromosomes became enclosed in a nuclear envelope and decondensed to an interphase state. However, in the rest of the heterokaryons, the chromatin of the pronuclei condensed to metaphase chromosomes, thus resulting in three sets of chromosomes. Fusion of zygotes that had begun DNA synthesis (20-22 hr post-HCG) with oocytes induced chromosome condensation of the pronuclei in 76% of the cases. In some heterokaryons, however, the oocyte chromosome decondensed to an interphase state similar to the zygote pronuclei. Fusion between late zygotes (27-29 hr post-HCG) with oocytes resulted in chromosome condensation of the pronuclei in all heterokaryons. On the basis of these results, the formation of the pronuclei and their progression toward mitosis in the zygote may be explained by changing levels of a metaphase factor in the cell, or by a balance between interphase and metaphase factors.     

Parathymosin affects the binding of linker histone H1 to nucleosomes and remodels chromatin structure

Linker histone H1 is the major factor that stabilizes higher order chromatin structure and modulates the action of chromatin-remodeling enzymes. We have previously shown that parathymosin, an acidic, nuclear protein binds to histone H1 in vitro and in vivo. Confocal laser scanning microscopy reveals a nuclear punctuate staining of the endogenous protein in interphase cells, which is excluded from dense heterochromatic regions. Using an in vitro chromatin reconstitution system under physiological conditions, we show here that parathymosin (ParaT) inhibits the binding of H1 to chromatin in a dose-dependent manner. Consistent with these findings, H1-containing chromatin assembled in the presence of ParaT has reduced nucleosome spacing. These observations suggest that interaction of the two proteins might result in a conformational change of H1. Fluorescence spectroscopy and circular dichroism-based measurements on mixtures of H1 and ParaT confirm this hypothesis. Human sperm nuclei challenged with ParaT become highly decondensed, whereas overexpression of green fluorescent protein- or FLAG-tagged protein in HeLa cells induces global chromatin decondensation and increases the accessibility of chromatin to micrococcal nuclease digestion. Our data suggest a role of parathymosin in the remodeling of higher order chromatin structure through modulation of H1 interaction with nucleosomes and point to its involvement in chromatin-dependent functions.     

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.     

Structure and dynamics of interphase chromosomes

During interphase chromosomes decondense, but fluorescent in situ hybridization experiments reveal the existence of distinct territories occupied by individual chromosomes inside the nuclei of most eukaryotic cells. We use computer simulations to show that the existence and stability of territories is a kinetic effect that can be explained without invoking an underlying nuclear scaffold or protein-mediated interactions between DNA sequences. In particular, we show that the experimentally observed territory shapes and spatial distances between marked chromosome sites for human, Drosophila, and budding yeast chromosomes can be reproduced by a parameter-free minimal model of decondensing chromosomes. Our results suggest that the observed interphase structure and dynamics are due to generic polymer effects: confined Brownian motion conserving the local topological state of long chain molecules and segregation of mutually unentangled chains due to topological constraints.