Sambit Bhattacharya

Cell type specific chromosome territory organization in the interphase nucleus of normal and cancer cells.

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 G0 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 ∼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. J. Cell. Physiol. 221: 130–138, 2009.

Identifying functional neighborhoods within the cell nucleus: Proximity analysis of early S-phase replicating chromatin domains to sites of transcription, RNA polymerase II, HP1γ, 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: HP1γ, 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. J. Cell. Biochem. 105: 391–403, 2008.

A probabilistic model for the arrangement of a subset of human chromosome territories in WI38 Human fibroblasts

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 G0‐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. J. Cell. Physiol. 221: 120–129, 2009.

Gesture classification with machine learning using Kinect sensor data

We present approaches for gesture classification and gesture segmentation by using machine learning on the Kinect sensors data stream. Our work involved three phases. Firstly we developed gesture classification from a known vocabulary of gestures in an edited data stream. Secondly we extended those techniques to detect and classify a gesture in an unedited stream which also captures random movements. Thirdly, we apply rules to filter out movements that were not intentional gestures and yet resembled certain gestures in our vocabulary.

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. J. Cell. Physiol. 228: 609–616, 2013.

The architecture of functional neighborhoods within the mammalian cell nucleus

There is growing realization that the spatio-temporal positioning of functional sites and factors within the cell nucleus are critical determinants of genomic function and regulation within the interphase cell nucleus. Previous studies from our group identified higher order functional zones of replication and transcriptional activity in chromatin domains replicated in early S phase where actively transcribed genes are enriched. It was proposed that these higher order zones represent the modular basis for the coordinate regulation of genomic replication and transcription in the cell. In this article we review our recent findings concerning the spatio-temporal dynamics of early S phase replicated chromatin domains, sites of gene transcription and associated protein factors. Our findings led us to propose that an important step in activation for transcription in the cell nucleus is the unfolding of regions within the discrete chromatin domains so that transcriptionally active chromatin can interact with the machinery for transcription such as RNA polymerase II and the multitude of additional factors involved in transcription and its regulation. Regulation of transcription from this global perspective likely involves temporal programming for the coordinate and differential unfolding of chromatin regions in accordance with the transcriptional programming of the cell. For example, active genes from different chromatin domains could undergo transcription at common sites or transcription factories. In addition, genes regions from within the same chromatin domains could be active at different transcription factories. All this is likely made possible by the spatial arrangement of the protein factors involved in the replicational and transcriptional processes which form combinatorial arrays that define the functional landscape of the cell nucleus. Our results demonstrate a degree of precision for the higher order spatial arrangement of these combinatorial arrays of functional components. It is thus conceivable that alterations in the spatial organization of these arrays could lead to dysfunction or altered regulation of replicational or transcriptional processes at the global level. Studies of the nuclear matrix associated proteins matrin 3 and SAF-A further suggest that anchoring to the nuclear matrix architecture may be a critical determining factor in the combinatorial properties and for the higher order arrangement of these genomic functional complexes into 3-D network-like arrays.

Cell Type Specific Alterations in Interchromosomal Networks across the Cell Cycle

The interchromosomal organization of a subset of human chromosomes (#1, 4, 11, 12, 16, 17, and 18) was examined in G1 and S phase of human WI38 lung fibroblast and MCF10A breast epithelial cells. Radial positioning of the chromosome territories (CTs) was independent of gene density, but size dependent. While no changes in radial positioning during the cell cycle were detected, there were stage-specific differences between cell types. Each CT was in close proximity (interaction) with a similar number of other CT except the gene rich CT17 which had significantly more interactions. Furthermore, CT17 was a member of the highest pairwise CT combinations with multiple interactions. Major differences were detected in the pairwise interaction profiles of MCF10A versus WI38 including cell cycle alterations from G1 to S. These alterations in interaction profiles were subdivided into five types: overall increase, overall decrease, switching from 1 to ≥2 interactions, vice versa, or no change. A global data mining program termed the chromatic median determined the most probable overall association network for the entire subset of CT. This probabilistic interchromosomal network was nearly completely different between the two cell lines. It was also strikingly altered across the cell cycle in MCF10A, but only slightly in WI38. We conclude that CT undergo multiple and preferred interactions with other CT in the nucleus and form preferred -albeit probabilistic- interchromosomal networks. This network of interactions is altered across the cell cycle and between cell types. It is intriguing to consider the relationship of these alterations to the corresponding changes in the gene expression program across the cell cycle and in different cell types.

Wide-scale Alterations in Interchromosomal Organization in Breast Cancer Cells: Defining a Network of Interacting Chromosomes

The interchromosomal spatial positionings of a subset of human chromosomes was examined in the human breast cell line MCF10A (10A) and its malignant counterpart MCF10CA1a (CA1a). The nine chromosomes selected (#1, 4, 11, 12, 15, 16, 18, 21 and X) cover a wide range in size and gene density and compose ∼40% of the total human genome. Radial positioning of the chromosome territories (CT) was size dependent with certain of the CT more peripheral in CA1a. Each CT was in close proximity (interaction) with a similar number of other CT except the inactive CTXi. It had lower levels of interchromosomal partners in 10A which increased strikingly in CA1a. Major alterations from 10A to CA1a were detected in the pairwise interaction profiles which were subdivided into five types of altered interaction profiles: overall increase, overall decrease, switching from 1 to ≥2, vice versa or no change. A global data mining program termed the chromatic median calculated the most probable overall association network for the entire subset of CT. This interchromosomal network was drastically altered in CA1a with only 1 of 20 shared connections. We conclude that CT undergo multiple and preferred interactions with other CT in the cell nucleus and form preferred-albeit probabilistic-interchromosomal networks. This network of interactions is highly altered in malignant human breast cells. It is intriguing to consider the relationship of these alterations to the corresponding changes in the gene expression program of these malignant cancer cells.

Non‐Random Patterns in the Distribution of NOR‐Bearing Chromosome Territories in Human Fibroblasts: A Network Model of Interactions

We present a 3‐D mapping in WI38 human diploid fibroblast cells of chromosome territories (CT) 13,14,15,21, and 22, which contain the nucleolar organizing regions (NOR) and participate in the formation of nucleoli. The nuclear radial positioning of NOR‐CT correlated with the size of chromosomes with smaller CT more interior. A high frequency of pairwise associations between NOR‐CT ranging from 52% (CT13–21) to 82% (CT15–21) was detected as well as a triplet arrangement of CT15–21‐22 (72%). The associations of homologous CT were significantly lower (24–36%). Moreover, singular contacts between CT13–14 or CT13–22 were found in the majority of cells, while CT13–15 or CT13–21 predominantly exhibited multiple interactions. In cells with multiple nucleoli, one of the nucleoli (termed “dominant”) always associated with a higher number of CT. Moreover, certain CT pairs more frequently contributed to the same nucleolus than to others. This nonrandom pattern suggests that a large number of the NOR‐chromosomes are poised in close proximity during the postmitotic nucleolar recovery and through their NORs may contribute to the formation of the same nucleolus. A global data mining program termed the chromatic median determined the most probable interchromosomal arrangement of the entire NOR‐CT population. This interactive network model was significantly above randomized simulation and was composed of 13 connections among the NOR‐CT. We conclude that the NOR‐CT form a global interactive network in the cell nucleus that may be a fundamental feature for the regulation of nucleolar and other genomic functions. J. Cell. Physiol. 230: 427–439, 2015.

Automated Matching of Genomic Structures in Microscopic Images of Living Cells Using an Information Theoretic Approach

Advances in microscopic imaging technology have revolutionized biology in recent years by enabling the study of dynamic processes inside living cells. Time-lapse microscopy produces large numbers of sequential images of living cells taken over time. In this paper we describe the novel approaches we have developed to automate and introduce high accuracy to the process of identifying genomic structures in living cells and matching them between consecutive time-points. We derive control points from landmarks within the structures and use the Kulback-Leibler divergence as an information-theoretic approach to correctly resolve potential close matches within the iterative closest point (ICP) algorithm. We also describe the steps needed to extend our techniques to analyze three dimensional voxel images. The approaches we describe are widely applicable in the analysis of time-lapse microscopy data