To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. Optical reconstruction of chromatin architecture and multiplex RNA labelling traces the DNA path in single cells and its relationship to transcription.Īlthough chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. Together, these results illustrate an approach for high-resolution, single-cell DNA domain analysis in vivo, identify domain structures that change with cell identity, and show that border elements contribute to the formation of physical domains in Drosophila. Deletion of Polycomb-independent borders led to ectopic enhancer–promoter contacts, aberrant gene expression, and developmental defects.
We identified cell-type-specific physical borders between active and Polycomb-repressed DNA, and unexpected Polycomb-independent borders. We used ORCA to study a Hox gene cluster in cryosectioned Drosophila embryos and labelled around 30 RNA species in parallel. Here we describe optical reconstruction of chromatin architecture (ORCA), a method that can trace the DNA path in single cells with nanoscale accuracy and genomic resolution reaching two kilobases. We have a limited understanding of how these interactions look in three dimensions, vary across cell types in complex tissue, and relate to transcription. The establishment of cell types during development requires precise interactions between genes and distal regulatory sequences. We will mainly focus on state-of-the-art developments in the yeast model but also cover higher eukaryotic systems.
#Tritag michigan update
Finally, this review will give an update on the mechanisms of heterogeneous gene expression among genetically identical individual cells. Furthermore, we will dissect how cell physiology or age impacts on dynamic gene regulation and especially discuss molecular insights into acquired transcriptional memory. Intrinsic factors modulating gene expression dynamics will then be discussed, focusing on chromatin modifications.
We will start by comparing recent in vivo procedures to capture gene expression in real time. In this review, we highlight the dynamic nature of transient gene expression changes to better understand cell physiology and development in general. Changes in gene expression are highly dynamic and depend on many intrinsic and extrinsic factors. The regulation of gene expression is a fundamental process enabling cells to respond to internal and external stimuli or to execute developmental programs. Three representative cells were analyzed, including the one from (H). (I) Quantifications were performed as those in (G) for the condition in (H). (H) Live-cell imaging snapshots of a cell illustrating chromatin dynamics and transcription activation with the addition of triptolide under the same stress condition as (F). Three representative cells were measured, including the one from (F). (G) Quantifications of the transcription activity (top) defined by the total intensity of stdMCP-tdTomato spots, and chromatin dynamics (bottom) by measuring the size of dCas9-GFP 14X spots, respectively. (F) Live-cell imaging snapshots of a cell showing chromatin dynamics and transcriptional activation of the HSPA1A gene under the stress condition (42 Red line at the center of each box denotes the median value, top and bottom edges indicate the 25th and 75th percentiles, respectively. (D, E) Quantifications of the confinement area (D) and the microscopic diffusion coefficient (E) estimated from the MSD curves in (C). Each pair was from the same cell, with one active and the other one silent. The data are displayed as means ± SEM for 44 pairs of alleles. (C) The averaged mean square displacement (MSD) curves of LMNA loci with (TF on) or without (TF off) transcription. The trajectory lengths are 400 frames with 0.25 s per frame. (B) Movement trajectories of dCas9-GFP 14X spots representing the two indicated loci in (A). BFP-LMNA was captured to highlight the nuclear membrane. (A) Colocalization of dCas9-GFP 14X and stdMCP-tdTomato demonstrates the transcription status of LMNA alleles. Transcription-coupled chromatin dynamics revealed by the TriTag system.
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