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Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.

ABSTRACT: Mammalian genomes are organized into three-dimensional DNA structures called A/B compartments that are associated with transcriptional activity/inactivity. However, whether these structures are simply correlated with gene expression or are permissive/impermissible to transcription has remained largely unknown because we lack methods to measure DNA organization and transcription simultaneously. Recently, we developed RNA & DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial organization of RNA and DNA. Here we show that RD-SPRITE measures genomic structure surrounding nascent pre-mRNAs and maps their spatial contacts. We find that transcription occurs within B compartments-with multiple active genes simultaneously colocalizing within the same B compartment-and at genes proximal to nucleoli. These results suggest that localization near or within nuclear structures thought to be inactive does not preclude transcription and that active transcription can occur throughout the nucleus. In general, we anticipate RD-SPRITE will be a powerful tool for exploring relationships between genome structure and transcription.

SUBMITTER: Goronzy IN

PROVIDER: S-EPMC9793828 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.

Goronzy Isabel N IN Quinodoz Sofia A SA Jachowicz Joanna W JW Ollikainen Noah N Bhat Prashant P Guttman Mitchell M

Cell reports 20221101 9

Mammalian genomes are organized into three-dimensional DNA structures called A/B compartments that are associated with transcriptional activity/inactivity. However, whether these structures are simply correlated with gene expression or are permissive/impermissible to transcription has remained largely unknown because we lack methods to measure DNA organization and transcription simultaneously. Recently, we developed RNA & DNA (RD)-SPRITE, which enables genome-wide measurements of the spatial org ...[more]

PMID: 36450242

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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

Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.

Publications

Simultaneous mapping of 3D structure and nascent RNAs argues against nuclear compartments that preclude transcription.

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