30-nm Fiber Research Articles

Engineered chromosome regions with altered sequence composition demonstrate hierarchical large-scale folding within metaphase chromosomes.

Mitotic chromosome structure and DNA sequence requirements for normal chromosomal condensation remain unknown. We engineered labeled chromosome regions with altered scaffold-associated region (SAR) sequence composition as a formal test of the radial loop and other chromosome models. Chinese hamster ovary cells were isolated containing high density insertions of a transgene containing lac operator repeats and a dihydrofolate reductase gene, with or without flanking SAR sequences. Lac repressor staining provided high resolution labeling with good preservation of chromosome ultrastructure. No evidence emerged for differential targeting of SAR sequences to a chromosome axis within native chromosomes. SAR sequences distributed uniformly throughout the native chromosome cross section and chromosome regions containing a high density of SAR transgene insertions showed normal diameter and folding. Ultrastructural analysis of two different transgene insertion sites, both spanning less than the full chromatin width, clearly contradicted predictions of simple radial loop models while providing strong support for hierarchical models of chromosome architecture. Specifically, an ∼250-nm-diam folding subunit was visualized directly within fully condensed metaphase chromosomes. Our results contradict predictions of simple radial loop models and provide the first unambiguous demonstration of a hierarchical folding subunit above the level of the 30-nm fiber within normally condensed metaphase chromosomes.

Differences in the nuclear chromatin among various stages of the life cycle of Trypanosoma cruzi.

Trypanosoma cruzi is the etiological agent of Chagas. Although the nuclear chromatin of this parasite is organized in the form of nucleosome filaments, its chromatin is physically and enzymatically fragile, and no condensation into chromosomes occurs during mitosis. All previous investigations have been carried out with epimastigote form in its proliferate stage. It is not known whether these differences in chromatin structure are also found in the non-proliferate stationary epimastigote forms and in tissue derived trypomastigotes. Our results confirm that chromatin of logarithmic epimastigotes presents limited compaction when increasing salt concentrations from 1 to 100 mM NaCl, and no 30-nm fibers were formed. Contrary to these results, non-proliferative forms of the parasites showed a pattern of compactation similar to that observed in rat liver chromatin, where solenoids of 30-nm fibers are formed at 100-mM NaCl. In accordance with these results, digestion of the nuclear chromatin with DNase I revealed that the chromatin of logarithmic phase epimastigotes was more accessible to the enzyme. We conclude from these results that structural differences in the chromatin exist not only between T. cruzi and higher eukaryotes but also among various forms of the parasite. The functional significance of these differences are currently under investigation.

Individual somatic H1 subtypes are dispensable for mouse development even in mice lacking the H1(0) replacement subtype.

H1 linker histones are involved in facilitating the folding of chromatin into a 30-nm fiber. Mice contain eight H1 subtypes that differ in amino acid sequence and expression during development. Previous work showed that mice lacking H1(0), the most divergent subtype, develop normally. Examination of chromatin in H1(0-/-) mice showed that other H1s, especially H1c, H1d, and H1e, compensate for the loss of H1(0) to maintain a normal H1-to-nucleosome stoichiometry, even in tissues that normally contain abundant amounts of H1(0) (A. M. Sirotkin et al., Proc. Natl. Acad. Sci. USA 92:6434-6438, 1995). To further investigate the in vivo role of individual mammalian H1s in development, we generated mice lacking H1c, H1d, or H1e by homologous recombination in mouse embryonic stem cells. Mice lacking any one of these H1 subtypes grew and reproduced normally and did not exhibit any obvious phenotype. To determine whether one of these H1s, in particular, was responsible for the compensation present in H1(0-/-) mice, each of the three H1 knockout mouse lines was bred with H1(0) knockout mice to generate H1c/H1(0), H1d/H1(0), or H1e/H1(0) double-knockout mice. Each of these doubly H1-deficient mice also was fertile and exhibited no anatomic or histological abnormalities. Chromatin from the three double-knockout strains showed no significant change in the ratio of total H1 to nucleosomes. These results suggest that any individual H1 subtype is dispensable for mouse development and that loss of even two subtypes is tolerated if a normal H1-to-nucleosome stoichiometry is maintained. Multiple compound H1 knockouts will probably be needed to disrupt the compensation within this multigene family.

Sir3-dependent assembly of supramolecular chromatin structures in vitro.

Baculovirus-expressed recombinant Sir3p (rSir3p) has been purified to near homogeneity, and its binding to naked DNA, mononucleosomes, and nucleosomal arrays has been characterized in vitro. At stoichiometric levels rSir3p interacts with intact nucleosomal arrays, mononucleosomes, and naked DNA, as evidenced by formation of supershifted species on native agarose gels. Proteolytic removal of the core histone tail domains inhibits but does not completely abolish rSir3p binding to nucleosomal arrays. The linker DNA in the supershifted complexes remains freely accessible to restriction endonuclease digestion, suggesting that both the tail domains and nucleosomal DNA contribute to rSir3p--chromatin interactions. Together these data indicate that rSir3p cross-links individual nucleosomal arrays into supramolecular assemblies whose physical properties transcend those of typical 10-nm and 30-nm fibers. Based on these data we hypothesize that Sir3p functions, at least in part, by mediating reorganization of the canonical chromatin fiber into functionally specialized higher order chromosomal domains.

Structural analysis of heavy ion radiation-induced chromosome aberrations by atomic force microscopy

Heavy ion radiation (high linear energy transfer, LET, radiation) induces various types of chromosome aberration. In this report, we describe a new method employing an atomic force microscope (AFM) for nanometer-level structural analysis of chromosome damage induced by heavy ion irradiation. Metaphase mouse chromosomes with chromatid gap or chromatid breaks induced by heavy ion irradiation were marked under a light microscope. Then the detailed structure of chromosomes of Giemsa-stained or unstained samples was visualized by the AFM. The height data of chromosomes obtained by AFM provided useful information to distinguish chromatid gaps and breaks. A fibrous structure was observed on the unstained chromosome, the average width of which was about 45.8 nm in the image of AFM. These structures were considered to be 30-nm fibers on the chromosome. The structure of the break point regions induced by neon- or carbon-ion irradiation was imaged by AFM. In some cases, the fibrous structure of break points was detected by AFM imaging after carbon ion irradiation. These observations indicated that AFM is a useful tool for analysis of chromosome aberrations induced by heavy ion radiation.

Quantitative DNA fiber mapping

High resolution physical maps are indispensable for large-scale, cost-effective gene discovery. The construction of such maps of the human genome and model organisms is one of the major goals of the human genome project. The precise localization of cloned DNA fragments within much larger genomic fragments and knowledge of the extent of overlap between two clones are needed to assemble high resolution physical maps. The fluorescence in situ hybridization (FISH) provides this critical information. Isolation of DNA from cell nuclei and preparation of some sort of DNA “fibers,” that is, chromatin or 30-nm fibers representing bundles of DNA improves accessibility of the DNA target for probes and detection reagents and thus increases the hybridization efficiency. FISH applied to most preparations of decondensed nuclear or isolated cloned DNA allows visualization of probe overlap and provides some information of the existence and size of gaps between two clones. In quantitative DNA fiber mapping (QDFM), a solution of purified DNA molecules is placed on a flat surface prepared so that the DNA molecules slowly attach at one or both ends. Because of the high hybridization efficiency obtained with DNA fibers, QDFM is also the method of choice to optically map expressed sequences in limited genomic intervals.

Nuclear matrix-like filaments and fibrogranular complexes form through the rearrangement of specific nuclear ribonucleoproteins.

The behavior of nuclear pre-mRNA-binding proteins after their nuclease and/or salt-induced release from RNA was investigated. After RNase digestion or salt extraction, two proteins that initially exist as tetramers (A2)(3)B1 in isolated heterogeneous nuclear ribonucleoprotein (hnRNP) complexes quantitatively reassociated to form regular helical filaments ranging in length from 100 nm to >10 microm. In highly magnified preparations prepared for scanning transmission electron microscopy, single filaments have diameters near 18 nm. In conventional negatively stained preparations viewed at low magnification, the diameters of the thinnest filaments range from 7 to 10 nm. At protein concentrations of >0.1 mg/ml, the filaments rapidly aggregated to form thicker filamentous networks that look like the fibrogranular structures termed the "nuclear matrix." Like the residual material seen in nuclear matrix preparations, the hnRNP filaments were insoluble in 2 M NaCl. Filament formation is associated with, and may be dependent on, disulfide bridge formation between the hnRNP proteins. The reducing agent 2-mercaptoethanol significantly attenuates filament assembly, and the residual material that forms is ultrastructurally distinct from the 7- to 10-nm fibers. In addition to the protein rearrangement leading to filament formation, nearly one-third of the protein present in chromatin-clarified nuclear extracts was converted to salt-insoluble material within 1 min of digestion with RNase. These observations are consistent with the possibility that the residual material termed the nuclear matrix may be enriched in, if not formed by, denatured proteins that function in pre-mRNA packaging, processing, and transport.

Measurement of low-frequency DNA breaks using nucleoid flow cytometry.

The organization of eukaryotic DNA is increasingly well understood at each extreme of chromatin organization. Advances in understanding the organization of DNA at the level of nucleosome and chromosome have outstripped knowledge of the intermediate states and function of DNA. Most would, however, agree on the following general hierarchy (,). The base-repeating unit of approx 146 bp, including a core histone group, is further organized into the 30-nm fiber and constrained by contact with the nuclear matrix to form loop and superloop structures capable of maintaining physical tension or supercoiling (, , ). These supercoiled units of DNA subsequently adopt repetitive patterns based around a protein core template, which together are observed as chromosomes.

Chromatin structure revisited.

Independently of the enormous progress in our understanding of the structure of the core particle, there remain a multitude of structural questions still to be answered. The main points discussed here can be summarized as follows: (1) The meaning of the term 'core particle' should be widened to reflect the fact that the actual length of DNA wrapped around the histone octamer in the context of the chromatin fiber may vary between approximately 100 and approximately 170 bp. (2) In the chromatosome, the linker histone forms a bridge between one terminus of the chromatosomal DNA and a point close to the dyad axis. (3) The particle that contains one molecule of HMG1 may be classified as a bona fide chromatosome. (4) In the extended fiber, the partition of the nucleosomal DNA into core and linker is a dynamic feature, responding to environmental influences; fiber structure-related constraints demand that linker length be beyond a certain minimal value. (5) The compact fiber structure seems to be rather irregular; the precise nature of this structure is still to be determined. Finally, the term 30-nm fiber should be dropped as a designator of the compact or condensed chromatin fiber structure.

Interphase cell cycle dynamics of a late-replicating, heterochromatic homogeneously staining region: precise choreography of condensation/decondensation and nuclear positioning.

Recently we described a new method for in situ localization of specific DNA sequences, based on lac operator/repressor recognition (Robinett, C.C., A. Straight, G. Li, C. Willhelm, G. Sudlow, A. Murray, and A.S. Belmont. 1996. J. Cell Biol. 135:1685-1700). We have applied this methodology to visualize the cell cycle dynamics of an approximately 90 Mbp, late-replicating, heterochromatic homogeneously staining region (HSR) in CHO cells, combining immunostaining with direct in vivo observations. Between anaphase and early G1, the HSR extends approximately twofold to a linear, approximately 0.3-mum-diam chromatid, and then recondenses to a compact mass adjacent to the nuclear envelope. No further changes in HSR conformation or position are seen through mid-S phase. However, HSR DNA replication is preceded by a decondensation and movement of the HSR into the nuclear interior 4-6 h into S phase. During DNA replication the HSR resolves into linear chromatids and then recondenses into a compact mass; this is followed by a third extension of the HSR during G2/ prophase. Surprisingly, compaction of the HSR is extremely high at all stages of interphase. Preliminary ultrastructural analysis of the HSR suggests at least three levels of large-scale chromatin organization above the 30-nm fiber.

Nuclear morphology during the S phase.

In order to evaluate at the ultrastructural level the chromatin arrangement during the S phase of the cell cycle, the detection of Bromodeoxyuridine (BrdU) by immunogold has been performed in synchronized 3T3 fibroblasts, regenerating liver, and Friend Leukemia Cells (FLC). After a 5-minute BrdU pulse, this label is detected in 10-nm-wide fibers, organized as lacework and assumed to be replication units. In the early part of the S phase, DNA replication units are localized exclusively in the dispersed chromatin domains far from the nuclear envelope. In the middle S, replication occurs at the border between condensed and dispersed chromatin and, finally, in late S, it mainly occurs in perinuclear heterochromatin regions. After replication, the 10-nm fibers can condense in heterochromatin without translocation. Chromatin is highly dispersed in early S and computer image analysis shows an increase in condensed chromatin areas ranging from 13 to 18% at the end of the S phase with a temporal and morphological pattern of distribution characteristic for each cell type. Scanning transmission electron microscopy demonstrates a regular and repetitive structure of dispersed chromatin, represented by a ring-like arrangement of the 10-nm fibers; assuming the same spatial distribution, gold particles that identify incorporated BrdU confirm this organization. By evaluating the organization and the distribution of DNA replication units during S phase, the results suggest that DNA replication occurs at a nucleosomal-like fiber level and that replicating enzymes machinery moves over a fixed template.

Expression of rat histone H1d in Escherichia coli and its purification.

Histone H1 is involved in the folding of linear polynucleosomal filament into a 30-nm fiber. In an effort to understand the role of different domains of histone H1 in chromatin folding, we have now expressed rat histone H1d in Escherichia coli using pTrc99A expression vector by providing a 6-His tag at the C-terminus to facilitate its purification. The expressed protein histone H1d was purified from the soluble extract of E. coli by employing Ni2+ NTA-agarose and heparin-agarose chromatography. The recombinant histone H1d was shown to be authentic by its N-terminal amino acid analysis, its secondary structural characteristics, and its ability to (a) condense DNA and (b) bind specifically to synthetic four-way junction DNA.

A 95-nm Spacing in Drosophila Polytene Chromatin

Abstract: It has long been debated whether the 30-nm fiber of chromatin is packed in an orderly array. The fiber may be condensed by supercoiling, producing structures of varying diameter. Alternatively, technical problems may have prevented the detection of higher-order structures. We developed a strategy to distinguish between these two possibilities. One potential obstacle to studying the order of packing was the effect of fixatives, dehydrating agents, heat, and embedding polymers on the native structure of chromatin prepared for viewing by electron microscopy. The known tendency of proteins to be degraded by osmium tetroxide and subsequently to be extracted in the conventional protocols for embedding might be particularly damaging. To avoid such denaturants and ensure the retention of proteins in chromatin, the embedding resin HACH was employed. Drosophila mimica polytene chromosomes were thin sectioned, stained with uranyl acetate, and viewed in the transmission electron microscope. Images were digitized and subjected to computerized image processing. Raw data files, containing boundary coordinates of all closed figures in the image, were edited to retain only those regions of interest (ROIs) that exhibited dimensions similar to those of 30-nm fibers in projection views. Euclidean distances between the centroids of such structures were calculated to obtain linear intercepts between recognizable 30-nm fibers. According to stereology theory, the dimensions of a lamellar structure can be determined from the volume distribution function of such intercepts. Therefore, intercept values were pooled for final data files from five processed images of chromatin. The resulting frequency histogram, showing the number of observations at different intercept values, had a sigmoidal inflection that was diagnostic of a major, new spacing at 95 nm. The 95-nm minimum was sandwiched between maxima in the 85 to 90 nm interval and throughout the range 105 to 120 nm. The results suggest that established stereological theory will be a useful tool for investigating the intractable problem of higher-order chromatin structure.

INTRANUCLEAR NETWORK OF 3–5 nm AND 8–10 nm FIBERS IN EL-4 LYMPHOMA CELLS

While much evidence indicates a high degree of spatial organization in the nucleus, the underlying molecular structures that support it remain poorly characterized. By extracting with high concentrations of RNase A in a modification of the sequential extraction protocol of Penman, we have identified a novel intranuclear network in the mouse lymphoma cell line, EL-4. Micrographs of embedment-free sections of extracted cells reveal anastomosing filaments of two different diameters: 3–5nm and 8–10nm. The 3–5-nm filaments are interconnected in many junctions and appear to blend smoothly into each other. The 8–10-nm fibers frequently split into two 3–5-nm filaments. Some 3–5-nm fibers appear to be connected at 90° angles with the 8–10-nm fibers. All junctions are smooth with no apparent junction protein. Flow cytometric analysis of RNase A- (and DNase I-) extracted nuclear matrices indicates that they do not contain significant amounts of protein that react with anti-actin and anti-vimentin monoclonal antibodies. Extraction of EL-4 nuclear matrices with high salt does not reveal 8–10-nm core filaments described after similar treatment of tumor cell lines of cervical and mammary origin. The novel characteristics of the core filaments in EL-4 lymphoma cells may reflect cell-type specificity of the nuclear matrix.

Mice develop normally without the H1(0) linker histone.

H1 histones bind to the linker DNA between nucleosome core particles and facilitate the folding of chromatin into a 30-nm fiber. Mice contain at least seven nonallelic subtypes of H1, including the somatic variants H1a through H1e, the testis-specific variant H1t, and the replacement linker histone H1(0). H1(0) accumulates in terminally differentiating cells from many lineages, at about the time when the cells cease dividing. To investigate the role of H1(0) in development, we have disrupted the single-copy H1(0) gene by homologous recombination in mouse embryonic stem cells. Mice homozygous for the mutation and completely lacking H1(0) mRNA and protein grew and reproduced normally and exhibited no anatomic or histologic abnormalities. Examination of tissues in which H1(0) is normally present at high levels also failed to reveal any abnormality in cell division patterns. Chromatin from H1(0)-deficient animals showed no significant change in the relative proportions of the other H1 subtypes or in the stoichiometry between linker histones and nucleosomes, suggesting that the other H1 histones can compensate for the deficiency in H1(0) by occupying sites that normally contain H1(0). Our results indicate that despite the unique properties and expression pattern of H1(0), its function is dispensable for normal mouse development.

High-resolution FEISEM detection of DNA centromeric probes in HeLa metaphase chromosomes.

HeLa metaphase chromosome spreads were hybridized with centromeric biotinylated DNA probes and detected with gold-conjugated anti-biotin antibodies. Chromosomes were observed by an in-lens field emission scanning electron microscope (FEISEM), which permits detection of biological samples without any coating. DNA probes were well localized in the centromeric region of chromosomes and there was clear discrimination between 10 nm fibers that hybridized to DNA probes and those that did not hybridize. This approach shows that in situ hybridization can be directly visualized at the FEISEM level by evaluating only secondary electron emission, which allows physical localization of the hybridized probe with high resolution so that backscatter detection represents only a control. Because chromosomes maintain the 10-nm fiber organization after in situ hybridization procedures, our data suggest that this fiber represents the lowest order of chromatin arrangement that permits transitory DNA denaturation.

Study of DNA accessibility in the condensed chromatin structures by resonance energy transfer

The linker DNA accessibility of chicken erythrocyte chromatin was studied by diffusion-enhanced resonance energy transfer (DERET). The 4″-{9‴-[((4-carboxy-3-hydroxyphenyl)-acetatamido)-3‴,6‴,9‴-(triacetyl)-3″,6‴,9‴-triazanonamido]-2″,6″-diazanonyl}-4,5',8-trimethyl psoralen-terbium complex was photocovalently bound to linker DNA and transferred its energy to fluorescein free in solution or bound on proteins of different sizes. We observed a diminution of linker DNA accessibility in chromatin as the protein size increased. Free fluorescein and proteins (up to a molecular weight of 24,000) labeled with fluorescein isothiocyanate (FITC) showed no variation in linker accessibility as chromatin condensation from 10- to 30-nm fibers was induced by an increase in ionic strength. We can conclude from these observations that linker DNA is located on the outside of the condensed chromatin fiber or, alternatively, that small proteins are free to diffuse toward an inside-located linker DNA, even in the condensed state of chromatin, possibly through the central cavity of the solenoïd model.

Analysis of electron-specimen interactions of thick biological specimens in Transmission Electron Microscopy at 200 KEV

The primary project of our laboratory is the investigation of chromatin structure by three dimensional electron microscope tomography. The goal is to understand how 30nm fibers fold into higher order chromatin structures. Three dimensional tomography involves the reconstruction of an object by combining multiple projection views of the object at different tilt angles. Due to the electronspecimen interaction and the characteristics of lens aberration in the electron microscope, however, the image is not always an accurate representation of the projected object mass density. In this abstract, we analyze the various types of electron-specimen interaction for thick biological specimens up to 0.7 microns thickness.Electron-specimen interactions include single elastic and inelastic, and multiple elastic and inelastic scattering. Of the imaging electrons, the single elastic and the plasmon electrons give rise to image intensities that can be linearly related to the projected object mass density. Multiply scattered elastic electrons contribute to an increase in background intensity. In addition, due to the chromatic aberration of the TEM’s objective lens, multiply scattered inelastic electrons cause a blurring of the image because of an effective broadening of the focus spread.

Automated data collection for electron tomography

Three-dimensional imaging has become an important addition to the variety of methods available for research on biological structures. Non-crystalline samples can be examined by high resolution electron tomography which requires that projection data be collected over a large range of specimen tilts. Practical limitations of tomography are set by the large number of micrographs to be processed, and by the required (and tedious) recentering and refocusing of the object during data collection; especially for dose sensitive specimens. With automated electron tomography a number of these problems can be overcome. First, the images are recorded directly in digital format, using a cooled slow scan CCD camera, and, with automatic tracking and correction for image shift and focus variation, a pre-aligned dataset is obtained, with every image recorded under well defined imaging conditions.At UCSF, we use intermediate voltage electron tomography to study higher-order chromatin structure. Of central interest is elucidating the higher-order arrangement of the 30nm chromatin fiber within condensed chromosomes through several phases of the cell cycle and, in collaboration with Chris Woodcock, the structure of the 30 nm fiber.

Chromatin Compaction and the Efficiency of Formation of DNA-Protein Crosslinks in γ-Irradiated Mammalian Cells

Chromatin has been prepared from Chinese hamster V79 cell nuclei by successive suspension and sedimentation in buffers of decreasing ionic strength. For buffer concentrations from 50 to 1 mM, the resultant chromatin maintained a normal histone content, nucleosomal organization, and attachment to the nuclear matrix; however, as the buffer concentration was reduced from 50 to 10 and 1 mM, the higher-order chromatin structures became increasingly relaxed. Fully expanded chromatin is 5- to 10-fold more susceptible to the induction of DNA-protein crosslinks (DPCs) by gamma radiation than is chromatin residing in living interphase cells. As much as 60-70% of expanded chromatin can be induced to form DPCs as compared to a maximum of about 20% of cellular DNA. For expanded chromatin, the maximum level of induced DPCs is two to three times higher than would be expected if only matrix-associated DNA were induced to form DPCs. Therefore, DNA in distal regions of chromatin loops must also be induced to form DPCs with histones or other nonhistone chromosomal proteins. The hypersensitivity of isolated chromatin to radiation-induced production of DPCs appears to be related to the expansion of chromatin conformation rather than to the removal of intracellular radical scavengers for the following reasons: (a) there is an inverse relationship between the buffer concentration in which the chromatin is suspended and DPC formation, and (b) the induction of a more compact 30-nm chromatin fiber from the expanded 10-nm chromatin fiber in the presence of a low concentration of MgCl2 results in a marked reduction in DPC formation. The formation of radiation-induced DPC seems to occur at maximum efficiency in fully expanded chromatin, since DPC formation cannot be further stimulated by the addition of Cu2+, which can catalyze the production of OH by Fenton chemistry. It is concluded that radiation-induced DNA damage production is greatly influenced by chromatin conformation, and that chromatin as it exists in the cell is a relatively poor substrate for DNA-protein crosslinking in comparison to completely expanded chromatin.