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Probing the dynamic organization of transcription compartments and gene loci within the nucleus of living cells.

ABSTRACT: The three-dimensional organization of nuclear compartments within living cells determines genome function and yet their underlying self-organizing principles are unclear. We visualize in real-time transcriptionally active compartments (TCs) by the transient enrichment of fluorescently-labeled uridine 5'-triphosphate molecules within living cells. These TCs partially colocalize with active RNA-Pol II in the cell nucleus. Fluorescence anisotropy maps of chromatin compaction evidences a more open chromatin structure at the TCs. Using live-cell timelapse imaging, heterogeneity in the dynamic behavior of TCs has been revealed which falls into three distinct classes: subdiffusive, super-diffusive, and normal diffusive behavior. In contrast, the mobility of a candidate gene locus, either in the repressed or activated state, undergoes a differential restricted motion that is coupled to TC movement. Further TC dynamics is directly affected by small molecule chromatin structure modulators and adenosine triphosphate depletion. This heterogeneous behavior in TC dynamics within living cells could provide an interesting paradigm to explore the spatiotemporal dimension to gene transcription control.

SUBMITTER: Sinha DK

PROVIDER: S-EPMC2586563 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Probing the dynamic organization of transcription compartments and gene loci within the nucleus of living cells.

Sinha Deepak Kumar DK Banerjee Bidisha B Maharana Shovamayee S Shivashankar G V GV

Biophysical journal 20080919 11

The three-dimensional organization of nuclear compartments within living cells determines genome function and yet their underlying self-organizing principles are unclear. We visualize in real-time transcriptionally active compartments (TCs) by the transient enrichment of fluorescently-labeled uridine 5'-triphosphate molecules within living cells. These TCs partially colocalize with active RNA-Pol II in the cell nucleus. Fluorescence anisotropy maps of chromatin compaction evidences a more open c ...[more]

PMID: 18805931

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Project description:Transcription in higher organisms requires spatiotemporal coordination of transcription machinery and the transcription factors at promoter sites. Toward this, recent evidence suggests that both static compartmentalization and dynamic self-organization of transcriptional apparatus are in effect at sites of transcription. Although the dynamics of transcription machinery is essential to genome regulation, the principles underlying this organization and its functional coupling to nuclear architecture is unclear. In a recent study we revealed that Uridine-5'-triphosphate (UTP) uptake in living cells labeled transcription-related compartments. In this article, we quantitatively establish multicolor labeling strategies for UTP-enriched transcription compartments (TCs) and probe their dynamic organization. UTP-enriched TCs were found to be in two distinct fractions: one colocalized with phosphorylated RNA pol II and the other as nascent aggregates. The fraction colocalized with the phosphorylated RNA pol II decreased with the inhibition of transcription initiation or elongation. Fluorescence anisotropy imaging and photobleaching experiments suggest that TCs are functional aggregates of nascent transcripts that are assembled in a transcription-dependent manner. Fluorescence correlation spectroscopy analysis revealed the relative fraction and sizes of fluorescent UTP-labeled transcripts in the nucleoplasm. Time-lapse imaging experiments of TCs exhibited pause and a mobile nature of these compartments within interchromosome territories. Perturbation of either nucleoskeletal protein or the cytoskeleton resulted in reduced active mobility of TCs, whereas inhibitors of transcription enhanced the mobile fraction of TCs. Further, high temporal resolution imaging showed evidence of stepping dynamics of TCs regulated by nucleoskeleton and chromatin modifications. Taken together, our experiments suggest the transient compartmentalization of UTP-enriched aggregates and their dynamic reorganization in a transcription-dependent manner. These results may have important implications for understanding spatiotemporal control of eukaryotic transcription.

Dataset Information

Probing the dynamic organization of transcription compartments and gene loci within the nucleus of living cells.

Publications

Probing the dynamic organization of transcription compartments and gene loci within the nucleus of living cells.

Similar Datasets

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