Human Interphase Nuclei Research Articles

Cell thermoregulation hypothesis: its origin, material basis, mechanisms and meaning

The accumulated knowledge about the chromosome structure, redundant DNA and an interphase nucleus inevitably raises, at least, two questions: (1) Why are there so much non-coding DNAs in chromosomes of higher eukaryotes and (2) what is the role of their organization in interphase nucleus on cellular function? These questions in cell biology are attracting increased attention as the genomes of higher eukaryotes are being sequenced. Based on investigations of chromosomal heterochromatin regions variability in human populations, condensed chromatin (CC), interphase nucleus and redundant non-coding DNAs in the genome, an attempt is made to justify the view of possible participation of CC in cell thermoregulation. CC, being the densest domains in a cell, apparently conducts heat between the cytoplasm and nucleus when there is a difference in temperature between them. Keywords: Cell Thermoregulation; Condensed Chromatin; Q-heterochromatin; C-heterochromatin; Human Body Heat Conductivity; Human Adaptation

Revealing Gene Function and Transcription Relationship by Reconstructing Gene-Level Chromatin Interaction

Chromatin is hierarchically organized in human interphase nuclei. Dynamic chromatin interactions are thought to influence gene transcription and cell fate determination. A consensus concept is that genes may form transcription factories within nucleus by spatially interaction. However, it is still not well known whether the function-related genes co-locate in three-dimensional (3D) space for co-transcription. Especially, there is a lack of visualization method that directly reflect the relationship between gene spatial interaction, gene function and co-transcription. In this study, we constructed three kinds of matrices based on gene ontology annotations, high-through chromosome conformation capture (Hi-C) data and RNA-seq data from twenty human tissues and cell lines. The comparative analysis for gene pairs revealed that 3D genome organization influences gene transcription predominantly at local scale. We found that the local genes within family clusters have similar transcription patterns. We also found that spatial reorganization of a histone gene cluster could control gene transcription. These observations suggest that function-related genes are close in space and activated or repressed together. Our work provided a framework for genome-wide studying the relationship between gene function, co-transcription and spatial interaction.

Systems-level chromosomal parameters represent a suprachromosomal basis for the non-random chromosomal arrangement in human interphase nuclei.

Forty-six chromosome territories (CTs) are positioned uniquely in human interphase nuclei, wherein each of their positions can range from the centre of the nucleus to its periphery. A non-empirical basis for their non-random arrangement remains unreported. Here, we derive a suprachromosomal basis of that overall arrangement (which we refer to as a CT constellation), and report a hierarchical nature of the same. Using matrix algebra, we unify intrinsic chromosomal parameters (e.g., chromosomal length, gene density, the number of genes per chromosome), to derive an extrinsic effective gene density matrix, the hierarchy of which is dominated largely by extrinsic mathematical coupling of HSA19, followed by HSA17 (human chromosome 19 and 17, both preferentially interior CTs) with all CTs. We corroborate predicted constellations and effective gene density hierarchy with published reports from fluorescent in situ hybridization based microscopy and Hi-C techniques, and delineate analogous hierarchy in disparate vertebrates. Our theory accurately predicts CTs localised to the nuclear interior, which interestingly share conserved synteny with HSA19 and/or HSA17. Finally, the effective gene density hierarchy dictates how permutations among CT position represents the plasticity within its constellations, based on which we suggest that a differential mix of coding with noncoding genome modulates the same.

Computational model of dose response for low-LET-induced complex chromosomal aberrations.

Experiments with full-colour mFISH chromosome painting have revealed high yield of radiation-induced complex chromosomal aberrations (CAs). The ratio of complex to simple aberrations is dependent on cell type and linear energy transfer. Theoretical analysis has demonstrated that the mechanism of CA formation as a result of interaction between lesions at a surface of chromosome territories does not explain high complexes-to-simples ratio in human lymphocytes. The possible origin of high yields of γ-induced complex CAs was investigated in the present work by computer simulation. CAs were studied on the basis of chromosome structure and dynamics modelling and the hypothesis of CA formation on nuclear centres. The spatial organisation of all chromosomes in a human interphase nucleus was predicted by simulation of mitosis-to-interphase chromosome structure transition. Two scenarios of CA formation were analysed, 'static' (existing in a nucleus prior to irradiation) centres and 'dynamic' (formed in response to irradiation) centres. The modelling results reveal that under certain conditions, both scenarios explain quantitatively the dose-response relationships for both simple and complex γ-induced interchromosomal exchanges observed by mFISH chromosome painting in the first post-irradiation mitosis in human lymphocytes.

Laser confocal microscopy analysis of human interphase nuclei by three-dimensional FISH reveals dynamic perinucleolar clustering of telomeres

Nuclear functions are strongly dependent on the three-dimensional organization of the interphase nucleus. Given the importance of telomeres in the behaviour and stability of chromosomes, we have investigated the architecture of the human nucleus from the telomere perspective by 3D-FISH and laser confocal microscopy. We observed a randomly scattered telomere distribution in all confocal sections of the interphase nuclear volume with various levels of telomere clustering in different cell types. This distribution is independent of H2AX presence or phosphorylation status. We also observed that telomeres usually cluster at the periphery of the nucleolus following its cell cycle dependent dynamic formation but are never present in the interior of the nucleolus. These perinucleolar telomeric clusters contain the telomeres of the short arms of acrocentric chromosomes, explaining the p-arm association of acrocentric chromosomes frequently found in metaphase. Thus, chromosome positioning in metaphase spreads is tightly connected to the three-dimensional architecture of the interphase nucleus.

Fluorescence intensity profiles of in situ hybridization signals depict genome architecture within human interphase nuclei

An approach towards construction of two-dimensional (2D) and three-dimensional (3D) profiles of interphase chromatin architecture by quantification of fluorescence in situ hybridization (FISH) signal intensity is proposed. The technique was applied for analysis of signal intensity and distribution within interphase nuclei of somatic cells in different human tissues. Whole genomic DNA, fraction of repeated DNA sequences (Cot 1) and cloned satellite DNA were used as probes for FISH. The 2D and 3D fluorescence intensity profiles were able to depict FISH signal associations and somatic chromosome pairing. Furthermore, it allowed the detection of replicating signal patterns, the assessment of hybridization efficiency, and comparative analysis of DNA content variation of specific heterochromatic chromosomal regions. The 3D fluorescence intensity profiles allowed the analysis of intensity gradient within the signal volume. An approach was found applicable for determination of assembly of different types of DNA sequences, including classical satellite and alphoid DNA, gene-rich (G-negative bands) and gene-poor (G-positive bands) chromosomal regions as well as for assessment of chromatin architecture and targeted DNA sequence distribution within interphase nuclei. We conclude the approach to be a powerful additional tool for analysis of interphase genome architecture and chromosome behavior in the nucleus of human somatic cells.

The Three-Dimensional Structure of Human Interphase Chromosomes Is Related to the Transcriptome Map

The three-dimensional (3D) organization of the chromosomal fiber in the human interphase nucleus is an important but poorly understood aspect of gene regulation. Here we quantitatively analyze and compare the 3D structures of two types of genomic domains as defined by the human transcriptome map. While ridges are gene dense and show high expression levels, antiridges, on the other hand, are gene poor and carry genes that are expressed at low levels. We show that ridges are in general less condensed, more irregularly shaped, and located more closely to the nuclear center than antiridges. Six human cell lines that display different gene expression patterns and karyotypes share these structural parameters of chromatin. This shows that the chromatin structures of these two types of genomic domains are largely independent of tissue-specific variations in gene expression and differentiation state. Moreover, we show that there is remarkably little intermingling of chromatin from different parts of the same chromosome in a chromosome territory, neither from adjacent nor from distant parts. This suggests that the chromosomal fiber has a compact structure that sterically suppresses intermingling. Together, our results reveal novel general aspects of 3D chromosome architecture that are related to genome structure and function.

Distinct patterns of histone methylation and acetylation in human interphase nuclei

To study 3D nuclear distributions of epigenetic histone modifications such as H3(K9) acetylation, H3(K4) dimethylation, H3(K9) dimethylation, and H3(K27) trimethylation, and of histone methyltransferase Suv39H1, we used advanced image analysis methods, combined with Nipkow disk confocal microscopy. Total fluorescence intensity and distributions of fluorescently labelled proteins were analyzed in formaldehyde-fixed interphase nuclei. Our data showed reduced fluorescent signals of H3(K9) acetylation and H3(K4) dimethylation (di-me) at the nuclear periphery, while di-meH3(K9) was also abundant in chromatin regions closely associated with the nuclear envelope. Little overlapping (intermingling) was observed for di-meH3(K4) and H3(K27) trimethylation (tri-me), and for di-meH3(K9) and Suv39H1. The histone modifications studied were absent in the nucleolar compartment with the exception of H3(K9) dimethylation that was closely associated with perinucleolar regions which are formed by centromeres of acrocentric chromosomes. Using immunocytochemistry, no di-meH3(K4) but only dense di-meH3(K9) was found for the human acrocentric chromosomes 14 and 22. The active X chromosome was observed to be partially acetylated, while the inactive X was more condensed, located in a very peripheral part of the interphase nuclei, and lacked H3(K9) acetylation. Our results confirmed specific interphase patterns of histone modifications within the interphase nuclei as well as within their chromosome territories.

Topology of genes and nontranscribed sequences in human interphase nuclei

Knowledge about the functional impact of the topological organization of DNA sequences within interphase chromosome territories is still sparse. Of the few analyzed single copy genomic DNA sequences, the majority had been found to localize preferentially at the chromosome periphery or to loop out from chromosome territories. By means of dual-color fluorescence in situ hybridization (FISH), immunolabeling, confocal microscopy, and three-dimensional (3D) image analysis, we analyzed the intraterritorial and nuclear localization of 10 genomic fragments of different sequence classes in four different human cell types. The localization of three muscle-specific genes FLNA, NEB, and TTN, the oncogene BCL2, the tumor suppressor gene MADH4, and five putatively nontranscribed genomic sequences was predominantly in the periphery of the respective chromosome territories, independent from transcriptional status and from GC content. In interphase nuclei, the noncoding sequences were only rarely found associated with heterochromatic sites marked by the satellite III DNA D1Z1 or clusters of mammalian heterochromatin proteins (HP1α, HP1β, HP1γ). However, the nontranscribed sequences were found predominantly at the nuclear periphery or at the nucleoli, whereas genes tended to localize on chromosome surfaces exposed to the nuclear interior.

Suspension (S)-FISH, a new technique for interphase nuclei.

We describe a versatile method for performing fluorescence in situ hybridization (FISH) in suspension instead of on a slide as usually done. This so-called suspension-FISH (S-FISH) opens new possibilities for the analysis of shape and functions of the human interphase nucleus. The procedure is described and the first results using this approach are presented.

Chromosomes are predominantly located randomly with respect to each other in interphase human cells.

To test quantitatively whether there are systematic chromosome–chromosome associations within human interphase nuclei, interchanges between all possible heterologous pairs of chromosomes were measured with 24-color whole-chromosome painting (multiplex FISH), after damage to interphase lymphocytes by sparsely ionizing radiation in vitro. An excess of interchanges for a specific chromosome pair would indicate spatial proximity between the chromosomes comprising that pair. The experimental design was such that quite small deviations from randomness (extra pairwise interchanges within a group of chromosomes) would be detectable. The only statistically significant chromosome cluster was a group of five chromosomes previously observed to be preferentially located near the center of the nucleus. However, quantitatively, the overall deviation from randomness within the whole genome was small. Thus, whereas some chromosome–chromosome associations are clearly present, at the whole-chromosomal level, the predominant overall pattern appears to be spatially random.

Triplex-forming DNAs in the human interphase nucleus visualized in situ by polypurine/polypyrimidine DNA probes and antitriplex antibodies.

The polypurine/polypyrimidine (PuPy) tracts present in the human genome are known to be scattered among and within chromosomes. In PuPy tract sequences, triplex formation occurs readily under physiological conditions, leaving single-stranded DNAs capable of hybridization with complementary single-stranded DNAs and RNAs. The formation of single-strands and transmolecular triplexes is thought to enable sequences spaced distantly along the genome to associate with each other and organize nuclear DNA into ordered configurations. Triplex-forming DNAs in the human interphase nucleus were analyzed by combining fluorescence in situ "nondenaturing" hybridization employing PuPy tract probes and immunodetection by antitriplex antibodies. The nondenaturing hybridization technique, which has been used to detect RNA, may detect single-stranded DNAs in nondenatured nuclei, if present. Probes such as (GA/TC)(n) and (GAA/TTC)(n) sequences gave sequence-specific signals that overlapped with or were closely associated with triplexes immunolocalized by using known antitriplex antibodies. Pretreatment of nuclei with antitriplex antibodies blocked probe signal formation. Signal formation was resistant to pretreatment of nuclei with RNases but sensitive to single strand-specific nucleases. Triplexes visualized differentially with distinct PuPy tract probes were associated spatially with centromeric sequences in the interphase nucleus in a sequence-specific manner.

Two-Photon Multicolor FISH: A Versatile Technique to Detect Specific Sequences with in Single DNA Molecules in Cells and Tissues

Two-photon multicolor fluorescence in situ hybridisation (FISH) is presented as an advanced single molecule detection technique. Based on a two-photon excitation process it enables pinhole-free 3D laser scanning microscopy. Nonresonant two-photon absorption in a sub-femtoliter excitation volume was realized with a femtosecond laser scanning microscope equipped with a high numerical aperture objective. Nonlinear near infrared laser excitation and multicolor FISH has been used to image a variety of specific DNA regions. This innovative single molecule technique was applied to visualize genomic regions in fiber-DNA, human metaphase chromosomes, interphase nuclei and histological sections. Intense 170 fs laser pulses at 800 nm and GW/cm2 intensities have been employed to induce visible fluorescence of a variety of FISH fluorophores coupled to DNA probes. In particular, two-photon excited FITC-labeled 40 kb probes and multicolor labeled centromeric probes that bind to repetitive sequences of 0.340-2.000 kb have been used to visualize subtelomeric and different centromeric regions in metaphase chromosome spreads. Spectrum Orange and Spectrum Green labeled bcr and abl gene probes that target a stretch of 300 kb and 650 kb, respectively, have been imaged in a single fiber-DNA molecule with an estimated number of fluorescent molecules of 75 - 225 μm-1. Using the advantages of pinhole-free optical sectioning with submicron spatial resolution and multi-fluorophore single-wavelength-excitation, 3D images of multiple labeled centromeric regions of amniotic fluid cells in interphase have been obtained and used in diagnosis of trisomy 18. In addition, first 3D two-photon studies on centromer distribution in human kidney biopsies by labeling chromosomes X, Y and 4 with the FISH-fluorophores Spectrum Blue, Spectrum Green and Spectrum Orange have been performed. As demonstrated, two-photon Multicolor FISH has the potential to perform Multi-Gene-Imaging with high spatial resolution also in turbid tissue layers.

Reliability and significance of DNA measurements in interphase nuclei and division figures in histological sections

DNA contents from single cells at interphase and division were analysed in histological sections and in imprints from 73 breast cancer specimens. Fetal livers from 18 terminations of normal pregnancies provided the standard for truly mitotic prophases, metaphases and telophases. The reliability of DNA quantities from image microphotometry was improved using paraffin-embedded tissue samples from which 4, 8 and 15 μm slices were Feulgen stained. Imprinted replicas from the mirror surface of each freshly cut specimen provided matching domains and represent the crucial approach in this project. A close positive relationship was observed between interphase nuclei in 8 μm sections and their imprinted counterparts ( r=0.992; n=73). Interphase nuclei in 4 μm sections yielded insufficient DNA contents when compared with the imprints ( r=0.815; n=21) and with endogenous lymphocyte nuclei. This 2 c DNA standard also calibrated 232 mitotic figures to 3.91±0.01 c in 15 μm sections from fetal liver. Prophases, metaphases and telophases were slightly scattered (coefficient of variation=0.04 each). The 0.09 c deficiency to plain 4.0 c was read as an artifact from sectioning. However, the methodical bias did not challenge the most irregular DNA distribution profiles recorded from chromosome division figures (CDFs) in 15 μm sections of breast cancers. Poorly differentiated and aggressive breast cancer (Auer type IV, Zetterberg type A) exhibited a 4.5 c exceeding rate of 82.24% from a total of 752 CDFs in 10 randomly selected cases. Well differentiated, slowly growing cancer with diploid interphase nuclei (Auer I, Zetterberg D) surprisingly showed a 4.5 c exceeding rate of 29.26% from a total of 173 mitoses and CDFs in 10 randomly selected cases. The bulk of data beyond the mitotic 4.0 c level discriminates biological bias from methodical impairment. We concluded that 8 μm sections are sufficient for human interphase nuclei, whereas a depth of 15 μm preserves intact mitoses and CDFs.

Adaptation of a preembedding electron microscopic in situ hybridization for detection of the telomere region in human interphase nuclei

The in situ hybridization (ISH) method is generally used in light microscopy but not in electron microscopy. We investigated whether the ISH method can be applied to electron microscopy (EM) by a preembedding method using human lymphocyte nuclei as a material. In addition, we also present some data from fluorescence in situ hybridization (FISH) using the same DNA probe. We indicate the suitability of preembedding EM-ISH to isolated cells.

The localization of chromosome domains in human interphase nuclei. Three-dimensional distance determinations of fluorescence in situ hybridization signals from confocal laser scanning microscopy

The localization of chromosome domains in human interphase nuclei. Three-dimensional distance determinations of fluorescence in situ hybridization signals from confocal laser scanning microscopy

The localization of chromosome domains in human interphase nuclei. Three‐dimensional distance determinations of fluorescence in situ hybridization signals from confocal laser scanning microscopy

The localization of chromosome domains in human interphase nuclei. Three‐dimensional distance determinations of fluorescence in situ hybridization signals from confocal laser scanning microscopy

The localization of chromosome domains in human interphase nuclei. Semi‐automated two‐dimensional image acquisition and analysis of fluorescence in situ hybridization signals

The localization of chromosome domains in human interphase nuclei. Semi‐automated two‐dimensional image acquisition and analysis of fluorescence in situ hybridization signals

Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84.

The localization of chromosome 18 in human interphase nuclei is demonstrated by use of radioactive and non-radioactive in situ hybridization techniques with a DNA clone designated L1.84. This clone represents a distinct subpopulation of the repetitive human alphoid DNA family, located in the centric region of chromosome 18. Under stringent hybridization conditions hybridization of L1.84 is restricted to chromosome 18 and reflects the number of these chromosomes present in the nuclei, namely, two in normal diploid human cells and three in nuclei from cells with trisomy 18. Under conditions of low stringency, cross-hybridization with other subpopulations of the alphoid DNA family occurs in the centromeric regions of the whole chromosome complement, and numerous hybridization sites are detected over interphase nuclei. Detection of chromosome-specific target DNAs by non-radioactive in situ hybridization with appropriate DNA probes cloned from individual chromosomal subregions presents a rapid means of identifying directly numerical or even structural chromosome aberrations in the interphase nucleus. Present limitations and future applications of interphase cytogenetics are discussed.

Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes.

Two cloned repetitive DNA probes, pXBR and CY1, which bind preferentially to specific regions of the human X and Y chromosome, respectively, were used to study the distribution of the sex chromosomes in human lymphocyte nuclei by in situ hybridization experiments. Our data indicate a large variability of the distances between the sex chromosomes in male and female interphase nuclei. However, the mean distance observed between the X and Y chromosome was significantly smaller than the mean distance observed between the two X-chromosomes. The distribution of distances determined experimentally is compared with three model distributions of distances, and the question of a non-random distribution of sex chromosomes is discussed. Mathematical details of these model distributions are provided in an Appendix to this paper. In the case of a human translocation chromosome (Xqter----Xp22.2::Yq11----Yqter) contained in the Chinese hamster X human hybrid cell line 445 X 393, the binding sites of pXBR and CY1 were found close to each other in most interphase nuclei. These data demonstrate the potential use of chromosome-specific repetitive DNA probes to study the problem of interphase chromosome topography.