Abstract
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.