Functional architecture of the mammalian cell nucleus: An analysis of proximity relationships between genomic functions
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The results of simultaneous replication (RS) and transcription site (TS) labeling in HeLa cells not only revealed that these fundamental genomic processes exist in two spatially separate domains or zones, but also raised important questions about their spatial and temporal coordination. A series of pulse-chase-pulse experiments using nucleotide analogs, their subsequent detection and high resolution imaging followed by computer analysis revealed that for various chase periods (0 to 24 hours) the transcriptional activity was highly association but spatially separated from replicated chromatin domains. A thorough investigation of labeled RS, TS and RNA polymerase II (pol II) sites using high resolution imaging and subsequent segmentation and proximity analyses lead us to propose a new model whereby chromatin activated for transcription, dynamically unfolds or 'loops out' of densely packed discrete chromatin domains. These results further lead us to characterize the nuclear micro-environments that surround the early S phase replicating RS, TS and pol II sites. Based on the proximity relationships between key genomic functions (RS & TS), proteins factor sites such as RNA polymerase II and HP1γ, and nuclear matrix proteins such as matrin 3 and Saf A, we propose a model for the organization of the dynamic chromatin domains and their associated protein factors that further create higher levels of functional neighborhoods. The model presented here, takes into account the stochastic ratios of available sites and their spatial organization within the nuclear architecture. In this study, this emerging view of the dynamic mammalian cell nucleus is also extended to living cells. It is known that the transcriptional activity is preferentially associated with the early S replicating chromatin domains. Proliferating cell nuclear antigen (PCNA) a marker of actively replicating chromatin domains was fused to GFP to study their dynamics in living cells. The configurational changes of these domains in living cells may be indicative of dynamic transitions between transcriptionally active and inactive states of the chromatin. Consistent with the above hypothesis, time lapse imaging of PCNA-GFP sites in living cells followed by the application of algorithms for minimal spanning tree (MST), mobility based clustering and motion analysis revealed that, mid or late S phase replicating PCNA-GFP sites demonstrated lower levels of dynamics as compared to those replicating in early S phase. In this thesis, high resolution imaging of nuclei labeled for multiple functions and factors is combined with computer image analysis to explore the spatial relationships of key genomic functions and factors that may represent the structural underpinning for the functional programming and coordination of the mammalian cell nucleus.