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TSA-seq mapping suggests that gene distance to nuclear speckles is more deterministic and predictive of gene expression levels than gene radial positioning. Gene expression correlates inversely with distance to nuclear speckles, with chromosome regions of unusually high expression located at the apex of chromosome loops protruding from the nuclear periphery into the interior. Genomic distances to the nearest lamina-associated domain are larger for loop apexes mapping closest to nuclear speckles, suggesting the possibility of conservation of speckle-associated regions. To facilitate comparison of genome organization by TSA-seq, we reduced required cell numbers 10- to 20-fold for TSA-seq by deliberately saturating protein-labeling while preserving distance mapping by the still unsaturated DNA-labeling. Only ∼10% of the genome shows statistically significant shifts in relative nuclear speckle distances in pair-wise comparisons between human cell lines (H1, HFF, HCT116, K562); however, these moderate shifts in nuclear speckle distances tightly correlate with changes in cell type–specific gene expression. Similarly, half of heat shock-induced gene loci already preposition very close to nuclear speckles, with the remaining positioned near or at intermediate distance (HSPH1) to nuclear speckles but shifting even closer with transcriptional induction. Speckle association together with chromatin decondensation correlates with expression amplification upon HSPH1 activation. Our results demonstrate a largely “hardwired” genome organization with specific genes moving small mean distances relative to speckles during cell differentiation or a physiological transition, suggesting an important role of nuclear speckles in gene expression regulation.

Chen et al. present TSA-Seq, a new mapping method that measures cytological distances relative to spatially distinct nuclear subcompartments. From novel nuclear organization maps of human cells, they identify transcription hot zones of high gene density that are near nuclear speckles and enriched in highly expressed genes, housekeeping genes, and genes with low transcriptional pausing., While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. In this study, we describe TSA-Seq, a new mapping method capable of providing a “cytological ruler” for estimating mean chromosomal distances from nuclear speckles genome-wide and for predicting several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results in K562 cells reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple spatially separated nuclear domains including two types of transcription “hot zones.” Transcription hot zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, housekeeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for spatial organization of transcription and gene expression., Graphical Abstract

Genome-wide mapping of chromosomal distances relative to nuclear compartments using TSA-Seq suggests a more deterministic relationship between intranuclear gene position and expression as a function of nuclear speckle distance than radial position. Gene activity increases overall with decreasing distance to nuclear speckles, with active chromosomal regions forming the apex of chromosome loops protruding from the nuclear periphery into the interior. Interestingly, genomic distances to the nearest lamina-associated domain are larger for loop apexes mapping very close to nuclear speckles, suggesting the possibility of genomic “hardwiring” and conservation of speckle-associated regions. To facilitate comparison of genome organization relative to nuclear speckles in human K562, HCT116, HFFc6, and H1 cell lines, here we describe reducing the required cell number 10-20-fold for TSA-Seq by deliberately saturating protein-labeling while preserving distance mapping by the still unsaturated DNA-labeling. Surprisingly, in pair-wise cell line comparisons, only ∼10% of the genome shows a statistically significant shift in relative nuclear speckle distances. These modest shifts in nuclear speckle distance, however, tightly correlate with changes in cell-type specific gene expression. Similarly, half of all loci that contain induced heat-shock protein genes appear pre-positioned close to nuclear speckles, with the remaining showing small shifts towards speckles with transcriptional induction. Speckle association together with chromatin decondensation correlates with expression amplification upon HSPH1 activation. Our results demonstrate a largely “hardwired” genome organization and specific genes moving small mean distances relative to speckles during cell differentiation or physiological transition, suggesting an important role of nuclear speckles in gene expression regulation.

SummaryWhile nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. Here we describe TSA-Seq, a new mapping method able to estimate mean chromosomal distances from nuclear speckles genome-wide and predict several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple, spatially separated nuclear domains, including two types of transcription “hot-zones”. Transcription hot-zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, house-keeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for nuclear spatial organization of transcription.

A paper appearing in late 2008 attracted considerable attention with its description of a dramatic juxtaposition of two estrogen responsive genes on different chromosomes within 15-60 minutes of adding estradiol. These results challenged a growing consensus of limited chromosome mobility within interphase nuclei, while raising questions of whether a hitherto unknown molecular mechanism might exist to move chromosomes long distances within the nucleus. These results also raised the fascinating question of how two genes on widely separated chromosomes might find each other over such a short time span. Now, a more recent paper reports no such long-range interaction or chromosome movements in the same cell types under what appear to be well replicated conditions, forcing a reexamination of the prior results.

Mitotic chromosome structure and pathways of mitotic condensation remain unknown. The limited amount of structural data on mitotic chromosome structure makes it impossible to distinguish between several mutually conflicting models. Here we used a Chinese hamster ovary cell line with three different lac operator-tagged vector insertions distributed over an approximately 1 microm chromosome arm region to determine positioning reproducibility, long-range correlation in large-scale chromatin folding, and sister chromatid symmetry in minimally perturbed, metaphase chromosomes. The three-dimensional positions of these lac operator-tagged spots, stained with lac repressor, were measured in isolated metaphase chromosomes relative to the central chromatid axes labeled with antibodies to topoisomerase II. Longitudinal, but not axial, positioning of spots was reproducible but showed intrinsic variability, up to approximately 300 nm, between sister chromatids. Spot positions on the same chromatid were uncorrelated, and no correlation or symmetry between the positions of corresponding spots on sister chromatids was detectable, showing the absence of highly ordered, long-range chromatin folding over tens of mega-basepairs. Our observations are in agreement with the absence of any regular, reproducible helical, last level of chromosome folding, but remain consistent with any hierarchical folding model in which irregularity in folding exists at one or multiple levels.

Summary Increasing evidence suggests functional compartmentalization of interphase nuclei [1]. This includes preferential interior localization of gene-rich and early replicating chromosome regions versus peripheral localization of gene-poor and late replicating chromosome regions [2, 3], association of some active genes with nuclear speckles [4, 5] or transcription "factories" [6], and association of transcriptionally repressed genes with heterochromatic regions [7]. Dynamic changes in chromosome compartmentalization [7–9] imply mechanisms for long-range interphase chromatin movements. However, live cell imaging in mammalian cells has revealed limited chromatin mobility [10], described as "constrained diffusion" [11]. None of these studies, though, have examined a chromosome locus undergoing an inducible repositioning between two different nuclear compartments. Here we demonstrate migration of an interphase chromosome site from the nuclear periphery to the interior 1–2 hr after targeting a transcriptional activator to this site. Spot redistribution is perturbed by specific actin or nuclear myosin I mutants. Extended periods of chromosome immobility are interspersed with several minute periods in which chromosomes move unidirectionally along curvilinear paths oriented roughly perpendicular to the nuclear envelope at velocities of 0.1–0.9 μm/min over distances of 1–5 μm. Our results suggest an active mechanism for fast and directed long-range interphase chromosome movements dependent directly or indirectly on actin/myosin.

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIα and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150–200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200–300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial “glue.”

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.

Background: Increasing evidence indicates specific changes in the three-dimensional organization of chromosomes in the cell nucleus during the cell cycle and development. These changes may be linked to changes in both the coordinated regulation of gene transcription and the timing of chromosome replication. While there is cytological evidence for short-range diffusive motion of chromosomes during interphase, the mechanisms for large-scale chromosome remodeling inside the nucleus remain unknown. Results: Chromosome motion was tracked in Drosophila spermatocyte nuclei by 3D fluorescence microscopy. The Lac repressor/ lac operator system was used to label specific chromosomal sites in live tissues, allowing extended observation of chromatin motion in different cell cycle stages. Our results reveal a highly dynamic chromosome organization governed by two types of motion: a fast, short-range component over a 1–2 s time scale and a slower component related to long-range chromosome motion within the nucleus. The motion patterns are consistent with a random walk. In early G2, short-range motion occurs within a small, approximately 0.5 μm radius domain, while long-range motion is confined to a much larger, chromosome-sized domain. Progression through G2 as cells approach meiotic prophase is accompanied by a complete arrest of long-range chromosome motion. Conclusions: Our analysis provides direct evidence for cell cycle-regulated changes in interphase chromatin motion. These changes are consistent with changes in local and long-range constraints on chromosome motility. We propose that dynamic interactions between chromosomes and internal nuclear structures modulate the range and rate of interphase chromatin diffusion and thereby regulate large-scale nuclear chromosome organization.

Analysis of the relationship between transcriptional activators and chromatin organization has focused largely on lower levels of chromatin structure. Here we describe striking remodeling of large-scale chromatin structure induced by a strong transcriptional activator. A VP16-lac repressor fusion protein targeted the VP16 acidic activation domain to chromosome regions containing lac operator repeats. Targeting was accompanied by increased transcription, localized histone hyperacetylation, and recruitment of at least three different histone acetyltransferases. Observed effects on large-scale chromatin structure included unfolding of a 90-Mbp heterochromatic chromosome arm into an extended 25–40-μm chromonema fiber, remodeling of this fiber into a novel subnuclear domain, and propagation of large-scale chromatin unfolding over hundreds of kilobase pairs. These changes in large-scale chromatin structure occurred even with inhibition of ongoing transcription by α-amanitin. Our results suggest a functional link between recruitment of the transcriptional machinery and changes in large-scale chromatin structure. Based on the observed long-range propagation of changes in large-scale chromatin structure, we suggest a possible rationale for the observed clustering of housekeeping genes within Mbp-sized chromosome bands.

The exceptional cytology provided by polytene chromosomes has made Drosophila melanogaster a premier model for chromosome studies, but full exploitation of polytene cytology is impeded by the difficulty in preparing high-quality chromosome spreads. Here we describe use of high pressure to produce formaldehyde-fixed chromosome spreads, which upon light-microscopy examination reveal structural detail previously observed only in electron microscopy preparations. We demonstrate applications to immunofluorescence and in situ hybridization.

Background: Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus. Results: To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture. Conclusions: We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.

We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac repressor. This system was first characterized by visualization of chromosome homogeneously staining regions (HSRs) produced by gene amplification using a dihydrofolate reductase (DHFR) expression vector with methotrexate selection. Using electron microscopy, most HSRs showed approximately 100-nm fibers, as described previously for the bulk, large-scale chromatin organization in these cells, and by light microscopy, distinct, large-scale chromatin fibers could be traced in vivo up to 5 microns in length. Subsequent experiments demonstrated the potential for more general applications of this labeling technology. Single and multiple copies of the integrated vector could be detected in living CHO cells before gene amplification, and detection of a single 256 lac operator repeat and its stability during mitosis was demonstrated by its targeted insertion into budding yeast cells by homologous recombination. In both CHO cells and yeast, use of the green fluorescent protein-lac repressor protein allowed extended, in vivo observations of the operator-tagged chromosomal DNA. Future applications of this technology should facilitate structural, functional, and genetic analysis of chromatin organization, chromosome dynamics, and nuclear architecture.

The past several years has seen increasing appreciation for plasticity of higher-level chromatin folding. Four distinct “30 nm” chromatin fiber structures have been identified, while new in situ imaging approaches have questioned the universality of 30 nm chromatin fibers as building blocks for chromosome folding in vivo. 3C-based approaches have provided a non-microscopic, genomic approach to investigating chromosome folding while uncovering a plethora of long-distance cis interactions difficult to accommodate in traditional hierarchical chromatin folding models. Recent microscopy based studies have suggested complex topologies co-existing within linear interphase chromosome structures. These results call for a reappraisal of traditional models of higher-level chromatin folding.

Folding of gene loci organized in tandem arrays reveals variation of chromatin structure as a function of cell differentiation., Interphase chromatin compaction well above the 30-nm fiber is well documented, but the structural motifs underlying this level of chromatin folding remain unknown. Taking a reductionist approach, we analyzed in mouse embryonic stem (ES) cells and ES-derived fibroblasts and erythroblasts the folding of 10–160-megabase pair engineered chromosome regions consisting of tandem repeats of bacterial artificial chromosomes (BACs) containing ∼200 kilobases of mammalian genomic DNA tagged with lac operator (LacO) arrays. Unexpectedly, linear mitotic and interphase chromatid regions formed from noncontiguously folded DNA topologies. Particularly, in ES cells, these model chromosome regions self-organized with distant sequences segregating into functionally distinct, compact domains. Transcriptionally active and histone H3K27me3-modified regions positioned toward the engineered chromosome subterritory exterior, with LacO repeats and the BAC vector backbone localizing within an H3K9me3, HP1-enriched core. Differential compaction of Dhfr and α- and β-globin transgenes was superimposed on dramatic, lineage-specific reorganization of large-scale chromatin folding, demonstrating a surprising plasticity of large-scale chromatin organization.

DNA, the hereditary material of the cell, is folded into protein-containing fibers in the nucleus of eukaryotes. The structure and location of these fibers is thought to be important in determining whether molecular machines can access the DNA for important cellular processes such as gene activation. Keywords: chromosome; chromatin; nucleus; transcription; structure; topology; organization; territory

How DNA is folded into chromosomes is unknown. Mitotic chromosome banding shows reproducibility in longitudinal compaction at a resolution of several megabase pairs, but it is less clear whether DNA sequences are targeted laterally to specific locations. The in vitro chromosome assembly of prokaryotic DNA1 suggests that there is a lack of sequence requirements for chromosome condensation, implying an absence of DNA targeting. Protein extraction experiments indicate, however, that specific DNA sequences may bind to a chromosome scaffold2,3. Chromosome banding patterns, using dyes with differential sequence specificity, have been interpreted to result from the alignment of AT-rich sequences in a partially helically folded chromosome scaffold4. But fluorescence in situ hybridization experiments, perhaps owing to technical limitations, have shown at best only slight deviation from a random, lateral sequence distribution5. Here we show that there is highly reproducible targeting of specific chromosome segments to the metaphase chromatid axis, but that these segments localize to the periphery of prophase and telophase chromosomes. Unfolding intermediates during anaphase and telophase suggest that sequence repositioning occurs through the global uncoiling of an underlying chromatid structure.

We examined changes in intranuclear chromosome positioning induced by a transcriptional activator in a simple experimental system. Targeting the VP16 acidic activation domain (AAD) to an engineered chromosome site resulted in its transcriptional activation and redistribution from a predominantly peripheral to a more interior nuclear localization. Direct visualization in vivo revealed that the chromosome site normally moves into the nuclear interior transiently in early G1 and again in early S phase. In contrast, VP16 AAD targeting induced this site's permanent interior localization in early G1. A single transcriptional activator therefore can modify the cell-cycle-dependent programme of intranuclear positioning of chromosome loci.

Publisher Summary In situ hybridization methods have been pivotal in allowing the exploration of chromosome and gene arrangements within interphase nuclei. However, the requirement of DNA denaturation for in situ hybridization is incompatible with direct in vivo observations. Moreover, the harsh conditions required for DNA denaturation are antagonistic to ultrastructural preservation. This has led to a large gap between primarily light microscopy studies, using in situ hybridization to examine the locations of specific chromosomal regions or loci, and ultrastructural analysis of higher order, “generic” chromatin structure, primarily by electron microscopy. In an attempt to bridge these two structural approaches, this chapter explores the use of protein–DNA recognition as a tag for localizing specific chromosome regions to provide an alternative to in situ hybridization. The chapter uses the lac operator/repressor system for an initial test case for several reasons. The lac repressor had been demonstrated as having the capability of binding in vitro to an operator sequence packaged within nucleosomes with only a small decrease in binding affinity. Later in vivo experiments confirmed the specific binding of lac repressor to lac operator within eukaryotic cells.

To investigate chromosome segregation in B. subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus. We then visualized the position of the operator cassettes with green fluorescent protein fused to the LacI repressor. In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle. In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle. In contrast, termini showed little preference for the poles. These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell.

Analysis of cellular ultrastructure currently is seriously handicapped by the lack of structural tools capable of visualizing macromolecular assemblies and organelles which extend over large subcellular volumes. This problem is particularly severe with regard to structural dissection of chromosome architecture in which a hierarchy of folding motifs give rise to linear compaction ratios tens of thousands to one for mitotic chromosomes and hundreds or thousands to one for interphase chromatids. Whereas analysis of the highest levels of chromosome folding requires tracing individual chromatids over at least several um distances, analysis of the lowest levels of chromatin folding requires a resolution of at least several nm. In practice, because of the extremely high DNA packing ratios, these different levels of chromosome folding are not easily isolated for separate, sequential structural determination. This is particularly true for mitotic chromosomes, in which the several nm spatial separation between larger units of chromatin folding is comparable to the spacing between lower levels of chromatin folding contained within these larger domains. Therefore direct identification within native chromosomes of structural motifs underlying the highest level of chromosome folding may first require the unambiguous tracing of the lower levels of chromatin folding.

Chromosome position effects combined with transgene silencing of multi-copy plasmid insertions lead to highly variable and usually quite low expression levels of mini-genes integrated into mammalian chromosomes. Together, these effects greatly complicate obtaining high-level expression of therapeutic proteins in mammalian cells or reproducible expression of individual or multiple transgenes. Here, we report a simple, one-step procedure for obtaining high-level, reproducible mini-gene expression in mammalian cells. By inserting mini-genes at different locations within a BAC containing the DHFR housekeeping gene locus, we obtain copy-number-dependent, position-independent expression with chromosomal insertions of one to several hundred BAC copies. These multi-copy DHFR BAC insertions adopt similar large-scale chromatin conformations independent of their chromosome integration site, including insertions within centromeric heterochromatin. Prevention of chromosome position effects, therefore, may be the result of embedding the mini-gene within the BAC-specific large-scale chromatin structure. The expression of reporter mini-genes can be stably maintained during continuous, long-term culture in the presence of drug selection. Finally, we show that this method is extendable to reproducible, high-level expression of multiple mini-genes, providing improved expression of both single and multiple transgenes.

A recent paper demonstrates that coregulated genes on different chromosomes show surprisingly high frequencies of colocalization within the nucleus; this complements similar results found previously for genes localized tens of megabases apart on a single chromosome. Colocalization could be related to the earlier observation of active genes associating with foci where RNA polymerase II is concentrated.

We describe findings on the architecture of Drosophila melanogaster mitotic chromosomes, made using a three-dimensional-oriented structural approach. Using high-voltage and conventional transmission electron microscopy combined with axial tomography and digital contrast-enhancement techniques, we have for the first time visualized significant structural detail within minimally perturbed mitotic chromosomes. Chromosomes prepared by several different preparative procedures showed a consistent size hierarchy of discrete chromatin structural domains with cross-sectional diameters of 120, 240, 400-500, and 800-1,000 A. In fully condensed, metaphase-arrested chromosomes, there is evidence for even larger-scale structural organization in the range of 1,300-3,000-A size. The observed intrachromosomal arrangements of these higher-order structural domains show that both the radial loop and sequential helical coiling models of chromosome structure are over-simplifications of the true situation. Finally, our results suggest that the pathway of chromatin condensation through mitosis consists of concurrent changes occurring at several levels of chromatin organization, rather than a strictly sequential folding process.

Higher-order chromatin structural domains approximately 130 nm in width are observed as prominent components of both Drosophila melanogaster and human mitotic chromosomes using buffer conditions which preserve chromosome morphology as determined by light microscopic comparison with chromosomes within living cells. Spatially discrete chromatin structural domains of similar size also exist as prominent components within interphase nuclei prepared under equivalent conditions. Examination of chromosomes during the anaphase-telophase transition suggests that chromosomes decondense largely through the progressive straightening or uncoiling of these large-scale chromatin domains. A quantitative analysis of the size distribution of these higher-order domains in telophase nuclei indicated a mean width of 126 +/- 36 nm. Three-dimensional views using stereopairs of chromosomes and interphase nuclei from 0.5 micron thick sections suggest that these large-scale chromatin domains consist of 30 nm fibers packed by tight folding into larger, linear, fiber-like elements. Reduction in vitro of either polyamine or divalent cation concentrations within two different buffer systems results in a loss of these large-scale domains, with no higher-order chromatin organization evident above the 20-30 nm fiber. Under these conditions the DNA distribution within mitotic chromosomes and interphase nuclei appears significantly diffuse relative to the appearance by light microscopy within living cells, or, by electron microscopy, within cells fixed directly without permeabilization in buffer. These results suggest that these large-scale chromatin structural domains are fundamental elements of chromosome architecture in vivo.

A recent review (1,2) indicates that significant correlation exist between the structure of chromatin and the extent of cell proliferation (aging, serum stimulation, virus transformation, cell-cycle phases, etc.). Chromatin, of course, refers to the diffuse interphase form of chromosome isolated from eukaryotic cells and the question remains open as to whether observed chromatin changes are reflected in the intact cell.

In previous papers the existence of two cycles of chromosome condensation-decondensation per cell cycle was suggested based on experiments involving nuclear morphometry measurements of Feulgen-stained nuclei. This conclusion can be criticized since its assumption of a relationship between nuclear morphology and chromatin structure is derived from indirect evidence. In this paper, we report simultaneous measurements of nuclear area and nuclear fluorescence intensity on individual cells stained with the intercalating dye, acridine orange (AO). Using cells in various stages of G1 and synchronized by two different methods, our results demonstrate a linear correlation between nuclear area and fluorescence intensity. They also indicate two cycles of chromatin condensation-decondensation during the G1 period, as assayed by the number of chromatin primary, intercalating AO binding sites. Finally, they show that the first of these cycles involves a transition in early G1 from a very small condensed nucleus (immediately after telephase) to a relatively large, dispersed nucleus that occurs abruptly.