Chromosome territory organization: A study of 3-D topology and interchromosomal networks
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Recent research has led to the emerging view that the 3-D arrangement of chromosome territories (CT) is linked to genomic function and regulation. Despite this progress, the degree of non-random arrangement of CT remains unclear and no overall model has been proposed. Similarly, our understanding of the 3-D organization of chromatin within individual CT (topology) is very limited as is our knowledge of gene positioning within CT in relationship to gene expression. In an effort to address these fundamental problems in genomic research, we used fluorescence in situ hybridization (FISH), 3-D microscopy and computer imaging. In-house built computational algorithms further enabled us to explore the intrachromosomal and interchromosomal organization. At a global level, the morphology of CT was assessed in terms of their shape and regularity (Chapter 2). This study was driven by the hypothesis that gene density affects overall shape of CT. By applying both visual inspection and algorithms that measure the degree of shape ellipticity and regularity, we demonstrate a strong inverse correlation between the degree of regular CT shape and gene density for those CT that are most gene rich (19, 17, 11) and gene poor (18, 13, Y). CT more intermediate in gene density, showed a strong negative correlation with shape regularity but not with ellipticity. An even more striking correlation between gene density and CT shape was determined for the CT which contain the nucleolar organizing region (NOR-CT). Correspondingly significant differences in shape between the X active and inactive CT implies that, aside from gene density, the overall global level of gene transcription is also an important determinant of chromosome territory shape. Differences in the shapes of different CT led us to investigate how individual chromosomes are folded. 3-D organization (topology) of six different CT was then investigated and probabilistic CT models were generated (Chapter 3). We performed multi-color FISH using 6 probes extending across each chromosome in human WI38 lung fibroblasts (Chapter 3). CT were selected ranging in size and gene density (1, 4,12,17,18 and X). Our findings demonstrate a high degree of non-random arrangement of individual CT that vary from chromosome to chromosome and display distinct changes during the cell cycle. Application of a novel data mining and pattern recognition approach termed the "k-means" generated 3-D models for the most probabilistic arrangement of each chromosome. These predicted models correlated well with the detailed distance measurements and analysis and signify the non-random arrangement of the CT. In order to examine the CT positioning and alterations of interchromosomal associations in relationship to coordinated gene expression, we selected the well-established keratinocyte differentiation model and performed re-FISH and computational image analysis on a sub-set of seven chromosomes in undifferentiated (0 h) and differentiating keratinocytes (24 h, Chapter 4). We demonstrate several stage specific alterations in cellular morphology and nuclear architecture, such as, a) ~56% increase in the nuclear volume at 24 h with a corresponding increase in the CT volume, b) radial positioning of CT17 changes significantly as the cells progress towards differentiation, c) there are differences in the single and multiple interaction profiles, e) interchromosomal associations strikingly differ between the two time points, f) probabilistic models based on interchromosomal associations demonstrate major reorganization of the network. While 27 non-random associations were detected at 0 h, this number increased to 33 at 24 h. However, only 11 associations were common to both time points. This study provides a new insight into our understanding of CT organization and function. We demonstrate a high degree of complexity and non-randomness of CT arrangement and how they alter across cell cycle and differentiation. We conclude that CT morphology is reflective of its intrachromosomal organization. This 3-D CT arrangement may further affect the interchromosomal networks, which may occur as a result of co-regulated gene expression.