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ABSTRACT: 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 s). 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 <1s are the basis for translational movements and configurational changes in ChrD previously detected over time spans of minutes to hours, and are the result of both the stochastic collisions of macromolecules and specific molecular interactions. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.
Journal of Cellular Physiology 08/2012; · 3.87 Impact Factor
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Advances in enzyme regulation 11/2009; 50(1):126-34.
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ABSTRACT: There is growing evidence that chromosome territories have a probabilistic non-random arrangement within the cell nucleus of mammalian cells. Other than their radial positioning, however, our knowledge of the degree and specificity of chromosome territory associations is predominantly limited to studies of pair-wise associations. In this study we have investigated the association profiles of eight human chromosome pairs (numbers 1, 2, 3, 4, 6, 7, 8, 9) in the cell nuclei of G(0)-arrested WI38 diploid lung fibroblasts. Associations between heterologous chromosome combinations ranged from 52% to 78% while the homologous chromosome pairs had much lower levels of association (3-25%). A geometric computational method termed the Generalized Median Graph enabled identification of the most probable arrangement of these eight chromosome pairs. Approximately 41% of the predicted associations are present in any given nucleus. The association levels of several chromosome pairs were very similar in a series of lung fibroblast cell lines but strikingly different in skin and colon derived fibroblast cells. We conclude that a large subset of human chromosomes has a preferred probabilistic arrangement in WI38 cells and that the resulting chromosomal associations show tissue origin specificity.
Journal of Cellular Physiology 07/2009; 221(1):120-9. · 3.87 Impact Factor
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ABSTRACT: Numerous studies indicate that the genome of higher eukaryotes is organized into distinct chromosome territories and that the 3-D arrangement of these territories may be closely connected to genomic function and the global regulation of gene expression. Despite this progress, the degree of non-random arrangement remains unclear and no overall model has been proposed for chromosome territory associations. To address this issue, a re-FISH approach was combined with computational analysis to analysis the pair-wise associations for six pairs of human chromosomes (chr #1, 4, 11, 12, 16, 18) in the G(0) state of normal human WI38 lung fibroblast and MCF10A epithelial breast cells. Similar levels of associations were found in WI38 and MCF10A for several of the chromosomes whereas others showed striking differences. A novel computational geometric approach, the generalized median graph (GMG), revealed a preferred probabilistic arrangement distinct for each cell line. Statistical analysis demonstrated that approximately 50% of the associations depicted in the GMG models are present in each individual nucleus. A nearly twofold increase of chromosome 4/16 associations in a malignant breast cancer cell line (MCFCA1a) compared to the related normal epithelial cell line (MCF10A) further demonstrates cancer related changes in chromosome arrangements. Our findings of highly preferred chromosome association profiles that are cell type specific and undergo alterations in cancer cells, lead us to propose a probabilistic chromosome code whereby the 3-D association profile of chromosomes contributes to the functional landscape of the cell nucleus, the global regulation of gene expression and the epigenetic state of chromatin.
Journal of Cellular Physiology 07/2009; 221(1):130-8. · 3.87 Impact Factor
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ABSTRACT: Discrete chromatin domains (ChrD), containing an average of approximately 1 Mbp DNA, represent the basic structural units for the regulation of DNA organization and replication in situ. In this study, a bio-computational approach is employed to simultaneously measure the translational motion of large populations of ChrD in the cell nucleus of living cells. Both movement and configurational changes are strikingly higher in early S-phase replicating ChrD compared to those that replicate in mid and late S-phase. The chromatin dynamics was not sensitive to transcription inhibition by alpha-amanitin but was significantly reduced by actinomycin D treatment. Since a majority of active genes replicate in early S-phase, our results suggest a correlation between levels of chromatin dynamics and chromatin poised for active transcription. Analysis of ChrD colocalization with transcription sites and cDNA with ChrD and transcription sites further supports this proposal.
Chromosoma 04/2009; 118(4):459-70. · 3.85 Impact Factor
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ABSTRACT: 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.
Chromosoma 08/2008; 117(6):553-67. · 3.85 Impact Factor
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ABSTRACT: 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.
Journal of Cellular Biochemistry 08/2008; 105(2):391-403. · 2.87 Impact Factor
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Proceedings of the 2008 International Conference on Image Processing, Computer Vision, & Pattern Recognition, IPCV 2008, July 14-17, 2008, Las Vegas Nevada, USA, 2 Volumes; 01/2008
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ABSTRACT: 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.
Conference proceedings: ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference 02/2006; 1:3057-61.
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Advances in Enzyme Regulation 02/2005; 45:17-26.
