Endogenous Real Time Imaging Reveals Dynamic Chromosomal Mobility During Ligand-Mediated Transcriptional Burst Events

Reviewer #1 (Public review):

Summary:

This study investigates the effects of transcriptional activation on chromatin dynamics and mobility. Using a breast cancer model, the authors examine the effects of estrogen receptor-a (ERa) stimulation and the resulting transcriptional activation on chromatin behavior at ERa-dependent loci during three distinct phases: unstimulated, acute stimulation, and chronic stimulation. Through live DNA and RNA imaging, the authors claim that ERa-dependent target genes display distinct bursting dynamics during periods of acute versus chronic simulation, accompanied by an overall increase in chromatin mobility. Notably, they claim that ERa-dependent loci display increased mobility during the non-bursting phase compared to the bursting phase. The study also attempts to explore the role of condensates in mediating these transcriptional and chromatin mobility changes using a single-molecule tracking assay to identify a unique population of low diffusion-coefficient molecules that appears upon E2 stimulation and is sensitive to 1,6-hexanediol.

Strengths:

While the study develops interesting tools that have the potential to provide useful insights into the relationship between transcriptional state, genomic locus mobility, and condensate formation, several major claims lack key supportive evidence, and the methods are inadequately established and described.

Weaknesses:

(1) The use of 1,6 hexanediol experiments is not suitable for drawing conclusions in live cell experiments, as this assay is now widely recognized to be plagued with artifacts and inadequate as a test for condensate formation. 1,6 hexanediol perturbs all hydrophobic interactions and has effects ranging from perturbing kinase and phosphatase activities (Düster et al, J. Biol. Chem., 2021), immobilizing and condensing chromatin in living cells (Itoh et al., Life Sci. Alliance 2021), disrupting nuclear pore complexes (Ribbeck et al., EMBO 2002), nuclear transport (Barrientos et al., Nucleus, 2023), and does not disrupt charge-mediated phase separation (Zheng et al., EMBO, 2025). There is also a discussion on these effects in a recent article: Current practices in the study of biomolecular condensates: a community comment, Alberti, Nat. Comm., 2025.

(2) The chromatin mobility is analyzed using displacement, and the differences are typically less than 50 nm. There is no discussion on the precision of this measurement and what these small differences may mean. No control loci are assessed to see if this effect is specific to the genes of interest or global.

(3) The SMT analysis is performed using Mean Square Displacement fitting of short single trajectories, which is error-prone, and no analysis is performed on the localization precision or error in estimation of the key parameters. Potential artifacts from this analysis are reflected in the distribution of alpha and diffusion coefficients that are presented in this paper, which include physically impossible values on which major claims rest.

(4) No experiment is performed to directly connect foci/cluster/condensation formation of ER at the genes of interest. Given these points alone, it is impossible to assess whether any of the claims made in the current manuscript are correct.

Reviewer #2 (Public review):

Summary:

The authors use a combination of state-of-the-art live-cell imaging techniques to track transcriptional bursting, DNA mobility, and single-molecule tracking to discern biophysical behaviours of chromatin and condensate formation in response to ER𝛼 activation. Surprisingly, the authors find that loci in estradiol-stimulated cells display enhanced mobility during the non-bursting phase. The authors attribute the reduced mobility of the loci during transcriptional bursts to condensate formation of ER𝛼 on enhancers regulating the bursting gene. Inhibition of transcription with flavopiridol shifts the loci and ER𝛼 to a non-confined state. These findings open the door to performing more complex multi-color live-cell imaging assays to fully interrogate the role of transcription factor condensates, DNA mobility, and subnuclear localization in the regulation of transcriptional bursting kinetics, and should be of great benefit to researchers studying mechanisms of gene regulation.

Strengths:

The authors presented a series of advanced multi-color live cell imaging assays used to correlate changes in DNA mobility with transcriptional bursting of a gene. By using such a defined temporal trigger associated with the addition of estroldiol to cells, the authors were also able to elegantly characterize changes in the diffusive properties of different classes of ER𝛼 during the acute (early, <2 hours) and chronic (late, >2 hours) phases of estrogen-responsive gene activation. Interestingly, one particular class of ER𝛼 that changed between acute and chronic phases was also responsive to 1,6-hexanediol treatment, suggesting that the authors are assaying ER𝛼 behaviours related to condensate formation. The authors also examined how the proximity of the NRIP1 gene to interchromatin granules impacted transcriptional bursting kinetics. There was no correlation of DNA mobility nor transcription bursting associated with localization to interchromatin granules, suggesting that other higher-order, architectural associations are regulating these processes. The imaging data were also supported by genomic GRO-seq and ChIP-seq assays showing changes in genomic occupancy of a number of transcription factors, including ER𝛼, during the pre-acute, acute, and chronic phases.

Weaknesses:

Although there are a number of compelling strengths to support the author's interpretation of the data, the paper is written in a way that lacks clarity and detail on a number of technical components. This lack of details, in particular related to how endogenous tagging of DNA, ER𝛼, and interchromatin granules (e.g. SC35) potentially impacts transcriptional bursting, makes it difficult for the reader to sufficiently judge any potential limitations of these complex engineered cell lines. Another potential weakness is the lack of any experiments directly measuring ER𝛼 diffusive properties in close proximity to the bursting gene. It is noted that this type of experiment examining transcription factor binding on a bursting gene is very technically challenging, given the different timescales of measurement of bursting (seconds-minutes) versus ER𝛼 diffusion (sub-seconds). However, these types of experiments would go a long way to supporting the authors' conclusions regarding how changes in DNA mobility and transcription bursting may be directly related to ER𝛼 condensate formation on enhancers.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors explore dynamic chromosomal mobility and transcriptional bursting events in mammalian cells, particularly focusing on ERα-dependent gene activation. The authors investigate how the physical movement of DNA loci changes during different phases of gene transcription (bursting vs. non-bursting, acute vs. chronic stimulation). Using advanced live-cell imaging techniques, including SMT of ERα and dual DNA/RNA visualization, the study reveals a multi-state model of DNA mobility linked to the formation of transcription factor condensates. The authors conclude that differential DNA kinetics serve as a reliable indicator for detecting condensate formation during gene activation, offering new insights into the mechanisms regulating gene expression within the nucleus.

Strengths:

The authors have done substantial work, and a major strength of the manuscript is being able to image both DNA and RNA from the same gene, as well as the TF that acts on that gene. This multi-pronged approach leads to complementary insights into transcription bursting mechanisms.

Weaknesses:

A major weakness of the manuscript is the lack of appropriate controls that support the specificity of the effects observed. The exclusive focus on condensates as the underlying mechanism to explain their data is also a bit limiting.