Chromatin In Interphase Nuclei Research Articles

Transcription Locally Disperses Chromatin and Thereby Organizes the Global Architecture of Interphase Nuclei

Interphase nuclei are segregated into chromatin domains, which are rich in DNA, and the interchromatin compartment1. Transcribing RNA polymerase II is found at chromatin domain margins1. It can be assumed that polymerase II is not just passively recruited to chromatin domain margins, but actively organizes chromatin and its interface with the interchromatin compartment. Here, we investigated how polymerase II activity impacts the bulk organization of chromatin in interphase nuclei. We used blastula stage zebrafish embryos as a model system. Blastula stage cell cycles last 1 hour or less, so we could readily follow the onset of transcription and chromatin organization after cell division. First, we imaged transcription and chromatin organization in vivo. To this end, we injected embryos with fluorescently labeled antibody fragments against transcribing polymerase II and the SiR-DNA live stain. We found that after cell division, no transcription occurred and chromatin was uniformly distributed throughout the nucleus. Within 2-3 minutes, individual foci of polymerase II activity occurred, which locally displaced chromatin from a surrounding pocket of 100-500 nm diameter. The number of polymerase II foci and chromatin-depleted pockets increased continuously and formed a nucleus-spanning network of transcription activity, which was embedded in a mirror image network of chromatin-dense domains. STED super-resolution microscopy of fixed embryonic cells showed the same intercalation of transcription and chromatin down to ∼50 nm length scales. To test the apparent requirement of transcription for this chromatin organization, we disrupted transcription with flavopiridol. This indeed abolished the intercalated nanostructure and led to precipitation of chromatin into compacted domains. Our findings suggest that foci of polymerase II activity locally manipulate bulk chromatin organization and integrate into a nucleus-spanning network required to maintain global nuclear architecture. 1Albiez et al. Chromosome Research 2006.

Chromatin structure revealed by X-ray scattering analysis and computational modeling

It remains unclear how the 2m of human genomic DNA is organized in each cell. The textbook model has long assumed that the 11-nm-diameter nucleosome fiber (beads-on-a-string), in which DNA is wrapped around core histones, is folded into a 30-nm chromatin fiber. One of the classical models assumes that the 30-nm chromatin fiber is further folded helically to form a larger fiber. Small-angle X-ray scattering (SAXS) is a powerful method for investigating the bulk structure of interphase chromatin and mitotic chromosomes. SAXS can detect periodic structures in biological materials in solution. In our SAXS results, no structural feature larger than 11nm was detected. Combining this with a computational analysis of “in silico condensed chromatin” made it possible to understand more about the X-ray scattering profiles and suggested that the chromatin in interphase nuclei and mitotic chromosomes essentially consists of irregularly folded nucleosome fibers lacking the 30-nm chromatin structure. In this article, we describe the experimental details of our SAXS and modeling systems. We also discuss other methods for investigating the chromatin structure in cells.

A male-killing Wolbachia carries a feminizing factor and is associated with degradation of the sex-determining system of its host

Endosymbiotic bacteria of the genus Wolbachia induce diverse reproductive alterations in their insect hosts. Wolbachia (wSca) infecting the moth Ostrinia scapulalis causes unusual male killing, in which males (genotype: ZZ) selectively die during embryonic and larval development, whereas females (genotype: ZW), in turn, selectively die when cured of infection. To gain insight into the interaction between wSca and the host, we analysed phenotypic and genetic sexes of the embryos and larvae of normal, wSca-infected, and infected-and-cured O. scapulalis by diagnosing the sex-specifically spliced transcripts of Osdsx—a homologue of the sex-determining gene doublesex—and sex chromatin in interphase nuclei, respectively. It was observed that the female-type Osdsx was expressed in the infected male (ZZ) progenies destined to die, whereas the male-type Osdsx was expressed in the cured female (ZW) progenies destined to die. These findings suggest that (i) wSca, a male killer, carries a genetic factor that feminizes the male host, (ii) the sex-determining system of the host is degraded, and (iii) a mismatch between the genetic and phenotypic sexes underlies the sex-specific death.

Linker histone H1 is present in centromeric chromatin of living human cells next to inner kinetochore proteins.

The vertebrate kinetochore complex assembles at the centromere on α-satellite DNA. In humans, α-satellite DNA has a repeat length of 171 bp slightly longer than the DNA in the chromatosome containing the linker histone H1. The centromere-binding protein CENP-B binds specifically to α-satellite DNA with properties of a centromeric-linker histone. Here, we analysed if linker histone H1 is present at or excluded from centromeric chromatin by CENP-B. By immunostaining we detected the presence, but no enrichment or depletion of five different H1 subtypes at centromeric chromatin. The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome. These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap. By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1° and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.

Condensation of rye chromatin in somatic interphase nuclei of Ph1 and ph1b wheat

The Ph1 locus in hexaploid wheat (Triticum aestivum L.) enforces diploid-like behavior in the first metaphase of meiosis. To test the hypothesis that this chromosome pairing control is exercised by affecting the degree of chromatin condensation, the dispersion of rye chromatin in interphase nuclei in somatic tissues of wheat-rye chromosome translocations 1RS.1BL, 2RS.2BL, 2BS.2RL, 3RS.3DL and 5RS.5BL was compared in Ph1 and ph1b isogenic backgrounds. No significant differences in rye chromatin condensation that could be attributed to the Ph1 locus were detected. Regardless of the Ph1 status, each rye chromosome arm tested conformed to the general Rabl’s orientation and occupied portions of the nuclei proportional to their length. Earlier observations that indicated the involvement of Ph1 locus in rye chromatin condensation in wheat could have been due either to specific loci on the studied 5RL rye arm that control the chromosome condensation process or to damage to the genetic system controlling chromatin condensation in the existing ph1b stocks of wheat. That damage might have been caused by homoeologous recombination and uneven disjunction of chromosomes from multivalents.

Arabidopsis Chromatin-Associated HMGA and HMGB Use Different Nuclear Targeting Signals and Display Highly Dynamic Localization within the Nucleus

In plants, the chromatin-associated high mobility group (HMG) proteins occur in two subfamilies termed HMGA and HMGB. The HMGA proteins are characterized by the presence of four AT-hook DNA binding motifs, and the HMGB proteins contain an HMG box DNA binding domain. As architectural factors, the HMG proteins appear to be involved in the regulation of transcription and other DNA-dependent processes. We have examined the subcellular localization of Arabidopsis thaliana HMGA, HMGB1, and HMGB5, revealing that they localize to the cell nucleus. They display a speckled distribution pattern throughout the chromatin of interphase nuclei, whereas none of the proteins associate with condensed mitotic chromosomes. HMGA is targeted to the nucleus by a monopartite nuclear localization signal, while efficient nuclear accumulation of HMGB1/5 requires large portions of the basic N-terminal part of the proteins. The acidic C-terminal domain interferes with nucleolar targeting of HMGB1. Fluorescence recovery after photobleaching experiments revealed that HMGA and HMGB proteins are extremely dynamic in the nucleus, indicating that they bind chromatin only transiently before moving on to the next site, thereby continuously scanning the genome for targets. By contrast, the majority of histone H2B is basically immobile within the nucleus, while linker histone H1.2 is relatively mobile.

Proteomics analysis of the centromere complex from HeLa interphase cells: UV-damaged DNA binding protein 1 (DDB-1) is a component of the CEN-complex, while BMI-1 is transiently co-localized with the centromeric region in interphase.

CENP-A, a centromere-specific histone H3, is conserved throughout eukaryotes, and formation of CENP-A chromatin defines the active centromere region. Here, we report the isolation of CENP-A chromatin from HeLa interphase nuclei by chromatin immunoprecipitation using anti-CENP-A monoclonal antibody, and systematic identification of its components by mass spectrometric analyses. The isolated chromatin contained CENP-B, CENP-C, CENP-H, CENP-I/hMis 6 and hMis 12 as well as CENP-A, suggesting that the isolated chromatin may represent the centromere complex (CEN-complex). Mass spectrometric analyses of the CEN-complex identified approximately 40 proteins, including the previously reported centromere proteins and the proteins of unknown function. In addition, we unexpectedly identified a series of proteins previously reported to be related to functions other than chromosome segregation, such as uvDDB-1, XAP8, hSNF2H, FACTp180, FACTp80/SSRP1, polycomb group proteins (BMI-1, RING1, RNF2, HPC3 and PHP2), KNL5 and racGAP. We found that uvDDB-1 was actually localized to the centromeric region throughout cell cycle, while BMI-1 was transiently co-localized with the centromeres in interphase. These results give us new insights into the architecture, dynamics and function of centromeric chromatin in interphase nuclei, which might reflect regulation of cell proliferation and differentiation.

Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules.

Most chromatin in interphase nuclei is part of condensed chromatin domains. Previous work has indicated that transcription takes place primarily at the surface of chromatin domains, that is, in the perichromatin region. It is possible that genes inside chromatin domains are silenced due to inaccessibility to macromolecular components of the transcription machinery. We have tested the accessibility of chromatin domains in nuclei of living cells with proteins and dextrans of different molecular sizes. Our results show that chromatin domains are readily accessible to large macromolecules, including proteins with a molecular weight of several hundred kilodaltons. Therefore, the silencing of genes that are incorporated into such domains is not due to the physical inaccessibility of condensed chromatin domains to transcription factors.

Two means of transcriptional reactivation within heterochromatin

DNA methylation levels and specific histone modifications of chromatin in interphase nuclei are taken as an indicator of transcriptional activity or silencing. Arabidopsis mutants impaired in maintenance of transcriptional gene silencing (TGS) alleviate TGS with or without affecting DNA methylation. Mutant ddm1, representing the first type, lacks a chromatin remodeling factor that regulates histone and DNA methylation. Mutant mom1, representing the second type, is affected in a different but still unknown silencing mechanism. Both classes of mutation have been studied mainly for their effects on specific loci. Here, we describe the cytological analysis of chromatin in ddm1 and mom1 mutants. The ddm1 mutation causes a striking decondensation of centromeric heterochromatin, a re-distribution of the remaining methylation of DNA, and a drastic change in the pattern of histone modification. A complex transgenic locus, which underwent stable inactivation and became heterochromatin-like, follows similar structural alterations. In contrast, nuclear organization in mom1 appears unaltered, demonstrating an involvement of MOM1 in transcriptional regulation within a heterochromatic environment.

Changes of Nuclear Structure Induced by Increasing Temperatures

Despite the recent improvement in understanding the higher-order structure of chromatin fibers, the organization of interphase chromosomes in specific nuclear domains emerged only recently and it is still controversial. This study took advantage of an integrated approach using complementary techniques in order to investigate the structure and organization of chromatin in interphase nucleus. Native CHO-K1 cells were progressively heated from 310 K to 410 K and the effects of increasing temperatures on nuclear chromatin were analyzed in situ by means of cytometric and calorimetric techniques. Distribution and organization of chromatin domains were analyzed by Fluorescence microscopy, while the mean condensation of nuclear chromatin was measured by Differential scanning calorimetry. The results show as changes of nuclear structures (envelope and matrix, namely) affect significantly organization and condensation of in situ chromatin. Moreover when volume is modified by an external force (the temperature gradient in our case) we observe significant alterations of chromatin structure. These data are in accordance with the hypothesis of an inverse relationship between nuclear volume and chromatin condensation.

Distinct cytoplasmic and nuclear fractions of Drosophila heterochromatin protein 1: their phosphorylation levels and associations with origin recognition complex proteins.

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.

Restricted expression of Epstein-Barr virus (EBV)-encoded, growth transformation-associated antigens in an EBV- and human herpesvirus type 8-carrying body cavity lymphoma line.

A body cavity lymphoma-derived cell line (BC1), known to carry both Epstein-Barr virus (EBV) and human herpes virus type 8 (HHV-8; or Kaposi's sarcoma-associated herpesvirus, KSHV), was analysed for the expression of EBV-encoded, growth transformation-associated antigens and cellular phenotype by immunofluorescence staining, Western blotting, RT-PCR and flow cytometry. A similar phenotypic analysis was also performed on another body cavity lymphoma line, BCBL1, that is singly infected with HHV-8. Phenotypically, the two lines were closely similar. Although both lines are known to carry rearranged immunoglobulin genes, they were mostly negative for B-cell surface markers. Both expressed the HHV-8-encoded nuclear antigen (LNA1). Similarly to Epstein-Barr nuclear antigen type 1 (EBNA1), LNA1 was associated with the chromatin in interphase nuclei and the mitotic chromosomes in metaphase. It accumulated in a few well-circumscribed nuclear bodies that did not co-localize with EBNA1. BC1 cells expressed EBNA1, LMP2A and EBV-encoded small RNAs but not EBNA2-6, LMP1 and LMP2B. They were thus similar to type I Burkitt's lymphoma cells and latently infected peripheral B-cells. Analysis of the splicing pattern of the EBNA1-encoding message by RT-PCR showed that BC1 cells used the QUK but not the YUK splice, indicating that the mRNA was initiated from Qp and not from Cp or Wp.

A specific conformation of the territory of chromosome 17 locates ERBB - 2 sequences to a DNase-hypersensitive domain at the nuclear periphery

Chromatin in interphase nuclei exhibits a topology that is associated with the transcriptional state of cells. We examined the spatial, intranuclear distribution of chromosome 17 and the ERBB-2 (HER2/neu) sequence thereon, relative to that of DNase-hypersensitive chromatin (DHC), in breast tumour cells exhibiting different levels of expression of ERBB-2. These sequences were specifically associated with the nuclear periphery, within a band of DHC. The remainder of the chromosome 17 mass showed no preferential position within the nucleus. The peripheral placement of ERBB-2 sequences is associated with a specific conformation of chromosome 17. We propose that the conformational organization of chromosome territories might represent a fundamental control mechanism in gene expression.

Cytology ofViciaspecies. VI. Nuclear chromatin organization, karyomorphological analysis and DNA amount inVicia serratifoliaJacq.

SUMMARYKaryomorphological features of Vicia serratifolia Jacq. were studied: the nuclear DNA content was determined in meristematic root cells, karyotype and morphometric data were generated by an automated image analysis system and the distribution and staining properties of heterochromatin were assessed in metaphase chromosomes. Moreover, Feulgen absorption at different thresholds of optical density provided evidence on the organization of the chromatin in interphase nucleus. The collected data were compared with those previously gathered in V. faba used as internal standard and V. narbonensis considered the reference species of the Narbonensis complex.

Chromatin dynamics in interphase nuclei and its implications for nuclear structure.

Translational dynamics of chromatin in interphase nuclei of living Swiss 3T3 and HeLa cells was studied using fluorescence microscopy and fluorescence recovery after photobleaching. Chromatin was fluorescently labeled using dihydroethidium, a membrane-permeant derivative of ethidium bromide. After labeling, a laser was used to bleach small (approximately 0.4 microm radius) spots in the heterochromatin and euchromatin of cells of both types. These spots were observed to persist for >1 h, implying that interphase chromatin is immobile over distance scales >/=0.4 microm. Over very short times (<1 s), a partial fluorescence recovery within the spots was observed. This partial recovery is attributed to independent dye motion, based on comparison with results obtained using ethidium homodimer-1, which binds essentially irreversibly to nucleic acids. The immobility observed here is consistent with chromosome confinement to domains in interphase nuclei. This immobility may reflect motion-impeding steric interactions that arise in the highly concentrated nuclear milieu or outright attachment of the chromatin to underlying nuclear substructures, such as nucleoli, the nuclear lamina, or the nuclear matrix.

Oligonucleotide PRINS DNA synthesis on extended chromatin preparations.

AbstractOne of the inherent problems with conventional fluorescent in situ hybridization (FISH) on metaphase chromosomes has been the difficulty in resolving closely associated markers. Any targets separated by less than about 1 Mb (1 × 106 bp) tend to appear as a single locus on metaphase chromosomes. The first approach to improving this resolution was hybridization to interphase nuclei (1,2), in which, because the chromatin is decondensed, the markers are further apart and, therefore, more easily resolved. However, the chromatin in interphase nuclei is not identifiable as discrete strands, so it is both difficult to see whether two markers are definitely linked and impossible to tell whether, if they are physically linked, the chromatin between them follows a straight course or a convoluted one. Thus, it is necessary to measure the distance between a large number of signal pairs to establish modal and average figures for the distance between them. By this means, it has been possible to establish physical distances between markers as little as 50–100 kb (5 × 104–105 kbp) apart.KeywordsWash BufferMetaphase ChromosomeInterphase NucleusSkim Milk PowderChromatin FiberThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Interphase fluorescence in situ hybridization mapping: a physical mapping strategy for plant species with large complex genomes.

The chromatin in interphase nuclei is much less condensed than are metaphase chromosomes, making the resolving power of fluorescence in situ hybridization (FISH) two orders of magnitude higher in interphase nuclei than on metaphase chromosomes. In mammalian species it has been demonstrated that within a certain range the interphase distance between two FISH sites can be used to estimate the linear DNA distance between the two probes. The interphase mapping strategy has never been applied in plant species, mainly because of the low sensitivity of the FISH technique on plant chromosomes. Using a CCD (charge-coupled device) camera system, we demonstrate that DNA probes in the 4 to 8 kb range can be detected on both metaphase and interphase chromosomes in maize. DNA probes pA1-Lc and pSh2.5.SstISalI, which contain the maize loci a1 and sh2, respectively, and are separated by 140 kb, completely overlapped on metaphase chromosomes. However, when the two probes were mapped in interphase nuclei, the FISH signals were well separated from each other in 86% of the FISH sites analyzed. The average interphase distance between the two probes was 0.50 micron. This result suggests that the resolving power of interphase FISH mapping in plant species can be as little as 100 kb. We also mapped the interphase locations of another pair of probes, ksu3/4 and ksu16, which span the Rp1 complex controlling rust resistance of maize. Probes ksu3/4 and ksu16 were mapped genetically approximately 4 cM apart and their FISH signals were also overlapped on metaphase chromosomes. These two probes were separated by an average of 2.32 microns in interphase nuclei. The possibility of estimating the linear DNA distance between ksu3/4 and ksu16 is discussed.

Nuclear Localization of Nucleoside Diphosphate Kinase Type B (nm23-H2) in Cultured Cells

Nucleoside diphosphate (NDP) kinases are metabolic enzymes found ubiquitously in cells. Recently, two known human isoforms of NDP kinase (A and B), identical to the protein products of the genesnm23-H1andnm23-H2,respectively, have been implicated in cancer metastasis and transcriptional regulation. To date, NDP kinase has been studied extensively in tissue sections and its cellular localization was described as being cytoplasmic. However, the recently discovered role of thenm23-H2gene product in transcriptional activation of thec-mycproto-oncogene also suggests a nuclear localization of the protein. In this study, we used isoform-specific antibodies against NDPK-B to examine the subcellular localization of thenm23-H2gene product. The cytoplasmic fluorescence is intense and homogeneous with pronounced labeling in the centromere region. The distribution of NDPK-B in interphase nuclei exhibits a pattern of numerous uniformly dispersed fine dots with reduced staining of the nucleoli. To further characterize the nuclear localization of NDPK-B,in situsequential extraction of nuclear components was performed. Brief exposure to Triton X-100 and subsequent treatment with RNase A do not change the nuclear staining pattern of NDPK-B. In contrast, treatment of Triton X-100-permeabilized nuclei with DNase I results in a significant loss of fluorescence. In mitotic prophase cells, the protein segregates from forming chromosomes and reappears in newly formed daughter nuclei after cell division. Taken together, the results indicate an association of NDPK-B with chromatin in interphase nuclei, supporting its proposed role in transcription.

High-resolution fluorescencein situ hybridization ofRBM- andTSPY-related cosmids on released Y chromatin in humans and pygmy chimpanzees

Applying two-colour fluorescence in situ hybridization (FISH) we simultaneously hybridized RBM- and TSPY-related cosmids to Y chromosomes in prophase and to released Y chromatin in interphase nuclei of man and pygmy chimpanzee. Whereas, even on prophasic Y chromosomes, no resolution of the overlapping RBM and TSPY signal clusters could be achieved, the RBM and TSPY signals are completely separated from each other in our maximum released Y chromatin stretches in interphase nuclei. These results unequivocally lend support to the view that the RBM and TSPY families have an interspersed organization on the Y chromosomes of man and higher apes. Thus, the distribution of RBM and TSPY signals might well go back to a common organization of these genes next to each other on an ancient Y chromosome.

Centromeric DNA cloned from functional kinetochore fragments in mitotic cells with unreplicated genomes

Treatment of cells arrested in the cell cycle at the G1/S-phase boundary with 5 mM caffeine induces premature mitosis, resulting in chromosomal fragmentation and detachment of centromere-kinetochore fragments, which are subsequently attached to the mitotic spindle and segregated in anaphase. Taking advantage of this in vivo separation of the centromere, we have developed a procedure for isolation of a centromere-enriched fraction of mitotic chromatin. Using this method, we have isolated and cloned DNA from the centromere-enriched material of Chinese hamster cells. One of the clones thus obtained was characterized in detail. It contains 6 kb of centromere-associated sequence that exhibits no recognizable homology with other mammalian centromeric sequences and is devoid of any extensive repetitive structure. This sequence is present in a single copy on chromosome 1 and is species-specific. Distinctive features of the clone include the presence of several A+T-rich regions and clusters of multiple topoisomerase II consensus cleavage sites and other sequence motifs characteristic of nuclear matrix-associated regions. We hypothesize that these features might be related to the more compact packaging of centromeric chromatin in interphase nuclei and mitotic chromosomes.