Biology subjects

Pancholi, A.

Publications and source records attributed to Pancholi, A..

3 recordsLinked to original sources

bioRxiv2023

Transcriptional clusters follow a conserved condensation-dispersal sequence during stem cell differentiation

Spatiotemporal organization of transcription is essential for organism development. Most eukaryotic genes are transcribed by RNA polymerase II (Pol II). In stem cells, Pol II forms prominent clusters, which gradually disappear during differentiation, such that only smaller clusters remain. Here, we ask whether the formation and loss of large Pol II clusters is a stereotypical process explicable by changes in the Pol II transcriptional state during differentiation. We assess clusters by super-resolution microscopy in differentiating mouse embryonic stem cells, sperm precursor formation in fruit flies, and germ layer induction in zebrafish. In all cases, Pol II clusters first become larger and rounder, then unfold, and finally disperse into small clusters. These shape changes are accompanied by initial increase in recruited Pol II, subsequent transition into transcript elongation, and finally reduction of active enhancers. We reproduce these observations using a biophysical surface condensation model, where enhancers support Pol II cluster formation, and transcriptional activity unfolds clusters. Our work indicates that changes in enhancer marks and transcriptional activity during differentiation define a stereotyped trajectory through a generally applicable space of cluster shapes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/547621v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1fb769borg.highwire.dtl.DTLVardef@27f6a0org.highwire.dtl.DTLVardef@1b0ab7corg.highwire.dtl.DTLVardef@1ca932d_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2021

Deep-learning microscopy image reconstruction with quality control reveals second-scale rearrangements in RNA polymerase II clusters

Fluorescence microscopy, a central tool of biological research, is subject to inherent trade-offs in experiment design. For instance, image acquisition speed can only be increased in exchange for a lowered signal quality, or for an increased rate of photo-damage to the specimen. Computational denoising can recover some loss of signal, extending the trade-off margin for high-speed imaging. Recently proposed denoising on the basis of neural networks shows exceptional performance but raises concerns of errors typical of neural networks. Here, we present a work-flow that supports an empirically optimized reduction of exposure times, as well as per-image quality control to exclude images with reconstruction errors. We implement this work-flow on the basis of the denoising tool Noise2Void and assess the molecular state and three-dimensional shape of RNA Polymerase II (Pol II) clusters in live zebrafish embryos. Image acquisition speed could be tripled, achieving 2-second time resolution and 350-nanometer lateral image resolution. The obtained data reveal stereotyped events of approximately 10 seconds duration: initially, the molecular mark for initiated Pol II increases, then the mark for active Pol II increases, and finally Pol II clusters take on a stretched and unfolded shape. An independent analysis based on fixed sample images reproduces this sequence of events, and suggests that they are related to the transient association of genes with Pol II clusters. Our work-flow consists of procedures that can be implemented on commercial fluorescence microscopes without any hardware or software modification, and should therefore be transferable to many other applications.

systems biology↗

bioRxiv2021

RNA polymerase II clusters form in line with liquid phase wetting of chromatin

It is essential for cells to control which genes are transcribed into RNA. In eukaryotes, two major control points are recruitment of RNA polymerase II (Pol II) into a paused state and subsequent pause release to begin transcript elongation. Pol II associates with macromolecular clusters during recruitment, but it remains unclear how Pol II recruitment and pause release might affect these clusters. Here, we show that clusters exhibit morphologies that are in line with wetting of chromatin by a liquid phase enriched in recruited Pol II. Applying instantaneous structured illumination microscopy and stimulated emission double depletion microscopy to pluripotent zebrafish embryos, we find recruited Pol II associated with large clusters, and elongating Pol II with dispersed clusters. A lattice kinetic Monte Carlo model representing recruited Pol II as a liquid phase reproduced the observed cluster morphologies. In this model, chromatin is a copolymer chain containing regions that attract or repel recruited Pol II, supporting droplet formation by wetting or droplet dispersal, respectively.

biophysics↗