Chromatin dynamics in living cells: identification of oscillatory motion.

Genomic DNA in mammalian cells is organized into ~1 Mbp chromatin domains (ChrD) which represent the basic structural units for DNA compaction, replication, and transcription. Remarkably, ChrD are highly dynamic and undergo both translational movement and configurational changes. In this study, we introduce an automated motion tracking analysis to measure, both in 2D and 3D, the linear displacement of early, mid and late S-phase replicated ChrD over short time periods (<1 sec). We conclude that previously identified large-scale transitions in the spatial position and configuration of chromatin, originate from asymmetric oscillations of the ChrD detectable in fractions of a second. The rapid oscillatory motion correlates with the replication timing of the ChrD with early S replicated ChrD showing the highest levels of motion and late S-phase chromatin the lowest. Virtually identical levels of oscillatory motion were detected when ChrD were measured during active DNA replication or during inhibition of transcription with DRB or α-amanitin. While this motion is energy independent, the oscillations of early S and mid S, but not late S replicated chromatin, are reduced by cell permeabilization. This suggests involvement of soluble factors in the regulation of chromatin dynamics. The DNA intercalating agent actinomycin D also significantly inhibits early S-labeled chromatin oscillation. We propose that rapid asymmetric oscillations of <1 sec are the basis for translational movements and configurational changes in ChrD previously detected over time spans of minutes-hours, and are the result of both the stochastic collisions of macromolecules and specific molecular interactions.

Organization of the amplified type I interferon gene cluster and associated chromosome regions in the interphase nucleus of human osteosarcoma cells.

The organization of the amplified type I interferon (IFN) gene cluster and surrounding chromosomal regions was studied in the interphase cell nucleus of the human osteosarcoma cell line MG63. Rather than being arranged in a linear ladder-like array as in mitotic chromosomes, a cluster of approximately 15 foci was detected that was preferentially associated along the periphery of both the cell nucleus and a chromosome territory containing components of chromosomes 4, 8, and 9. Interspersed within the IFN gene foci were corresponding foci derived from amplified centromere 4 and 9 sequences. Other copies of chromosomes 4 and 8 were frequently detected in pairs or higher-order arrays lacking discrete borders between the chromosomes. In contrast, while chromosomes 4 and 8 in normal WI38 human fibroblast and osteoblast cells were occasionally found to associate closely, discrete boundaries were always detected between the two. DNA replication timing of the IFN gene cluster in early- to mid-S phase of WI38 cells was conserved in the amplified IFN gene cluster of MG63. Quantitative RT-PCR demonstrated a approximately 3-fold increase in IFN beta transcripts in MG63 compared with WI38 and RNA/DNA FISH experiments revealed 1-5 foci of IFN beta transcripts per cell with only approximately 5% of the cells showing foci within the highly amplified IFN gene cluster.

Matrin 3: chromosomal distribution and protein interactions.

Matrin 3 (matr3), an abundant protein of the internal nuclear matrix, has been linked to a variety of functional events. As a step toward defining its multifunctional nature, we have studied the association of matr3 with chromosome territories and identified potential interacting proteins. A similar staining pattern of matr3 was observed in fixed WI38 fibroblast cells and in live HeLa cells using a matr3-GFP construct. Matr3 was detected throughout autosomal and the active X chromosome territories. Conversely, matr3 was strikingly excluded from the inactive X chromosome as well as within both the perinuclear and perinucleolar heterochromatin. Yeast two hybrid analysis identified matr3 interactions with 33 unique nuclear localized proteins and also revealed its propensity for self association. A majority of these proteins are involved in RNA metabolism and chromatin remodeling while others function in protein translation, DNA replication/repair and apoptosis. Further analysis of a selection of these proteins and scaffold attachment factor A (SAFA) by co-localization and co-immunoprecipitation experiments using HeLa cells confirmed their interactions with matr3.

Spatio-temporal dynamics of replication and transcription sites in the mammalian cell nucleus.

To study when and where active genes replicated in early S phase are transcribed, a series of pulse-chase experiments are performed to label replicating chromatin domains (RS) in early S phase and subsequently transcription sites (TS) after chase periods of 0 to 24 h. Surprisingly, transcription activity throughout these chase periods did not show significant colocalization with early RS chromatin domains. Application of novel image segmentation and proximity algorithms, however, revealed close proximity of TS with the labeled chromatin domains independent of chase time. In addition, RNA polymerase II was highly proximal and showed significant colocalization with both TS and the chromatin domains. Based on these findings, we propose that chromatin activated for transcription dynamically unfolds or "loops out" of early RS chromatin domains where it can interact with RNA polymerase II and other components of the transcriptional machinery. Our results further suggest that the early RS chromatin domains are transcribing genes throughout the cell cycle and that multiple chromatin domains are organized around the same transcription factory.

Ladder-like amplification of the type I interferon gene cluster in the human osteosarcoma cell line MG63.

The organization of the type I interferon (IFN) gene cluster (9p21.3) was studied in a human osteosarcoma cell line (MG63). Array comparative genomic hybridization (aCGH) showed an amplification of approximately 6-fold which ended at both ends of the gene cluster with a deletion that extended throughout the 9p21.3 band. Spectral karyotyping (SKY) combined with fluorescence in-situ hybridization (FISH) identified an arrangement of the gene cluster in a ladder-like array of 5-7 'bands' spanning a single chromosome termed the 'IFN chromosome'. Chromosome painting revealed that the IFN chromosome is derived from components of chromosomes 4, 8 and 9. Labelling with centromeric probes demonstrated a ladder-like amplification of centromeric 4 and 9 sequences that co-localized with each other and a similar banding pattern of chromosome 4, as well as alternating with the IFN gene clusters. In contrast, centromere 8 was not detected on the IFN chromosome. One of the amplified centromeric 9 bands was identified as the functional centromere based on its location at the chromosome constriction and immunolocalization of the CENP-C protein. A model is presented for the generation of the IFN chromosome that involves breakage-fusion-bridge events.

Identifying functional neighborhoods within the cell nucleus: proximity analysis of early S-phase replicating chromatin domains to sites of transcription, RNA polymerase II, HP1gamma, matrin 3 and SAF-A.

Higher order chromatin organization in concert with epigenetic regulation is a key process that determines gene expression at the global level. The organization of dynamic chromatin domains and their associated protein factors is intertwined with nuclear function to create higher levels of functional zones within the cell nucleus. As a step towards elucidating the organization and dynamics of these functional zones, we have investigated the spatial proximities among a constellation of functionally related sites that are found within euchromatic regions of the cell nucleus including: HP1gamma, nascent transcript sites (TS), active DNA replicating sites in early S-phase (PCNA) and RNA polymerase II sites. We report close associations among these different sites with proximity values specific for each combination. Analysis of matrin 3 and SAF-A sites demonstrates that these nuclear matrix proteins are highly proximal with the functionally related sites as well as to each other and display closely aligned and overlapping regions following application of the minimal spanning tree (MST) algorithm to visualize higher order network-like patterns. Our findings suggest that multiple factors within the nuclear microenvironment collectively form higher order combinatorial arrays of function. We propose a model for the organization of these functional neighborhoods which takes into account the proximity values of the individual sites and their spatial organization within the nuclear architecture.

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate.

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

Cytogenetic and cDNA microarray expression analysis of MCF10 human breast cancer progression cell lines.

We used a combination of spectral karyotyping, array comparative genomic hybridization, and cDNA microarrays to gain insights into the structural and functional changes of the genome in the MCF10 human breast cancer progression model cell lines. Spectral karyotyping data showed several chromosomal aberrations and array comparative genomic hybridization analysis identified numerous genomic gains and losses that might be involved in the progression toward cancer. Analysis of the expression levels of genes located within these genomic regions revealed a lack of correlation between chromosomal gains and losses and corresponding up-regulation or down-regulation for the majority of the approximately 1,000 genes analyzed in this study. We conclude that other mechanisms of gene regulation that are not directly related to chromosomal gains and losses play a major role in breast cancer progression.

Comparison of intensity based similarity measures for matching genomic structures in microscopic images of living cells.

This paper presents our comparative study of the application of intensity based similarity measures to the problem of matching genomic structures in microscopic images of living cells. As part of our ongoing research, we present here for the first time evidence from experiments and simulations that show the benefit of using an iterative matching algorithm guided by an intensity based similarity measure. Our experimental results are compared against a gold standard and suggest the measures that work best in the presence of fluorescent decay and other problems inherent to time-lapse microscopy. This makes our approach widely applicable in the study of the dynamics of living cells with time-lapse microscopic imaging.

On mobility analysis of functional sites from time lapse microscopic image sequences of living cell nucleus.

Recent research in biology has indicated correlations between the movement patterns of functional sites (such as replication sites in DNA) and zones of genetic activity within a nucleus. A detailed study and analysis of the motion dynamics of these sites can reveal an interesting insight into their role in DNA replication and function. In this paper, we propose a suite of novel techniques to determine, analyze, and interpret the mobility patterns of functional sites. Our algorithms are based on interesting ideas from theoretical computer science and database theory and provide for the first time the tools to interpret the seemingly stochastic motion patterns of the functional sites within the nucleus in terms of a set of tractable 'patterns' which can then be analyzed to understand their biological significance.