Reversible phase separation of HSF1 is required for an acute transcriptional response during heat shock.

Heat-shock transcription factor 1 (HSF1) orchestrates the fast and vast cellular response to heat shock through increased expression of heat-shock proteins. However, how HSF1 rapidly and reversibly regulates transcriptional reprogramming remains poorly defined. Here by combining super-resolution imaging, in vitro reconstitution and high-throughput sequencing, we reveal that HSF1 forms small nuclear condensates via liquid-liquid phase separation at heat-shock-protein gene loci and enriches multiple transcription apparatuses through co-phase separation to promote the transcription of target genes. Furthermore, the phase-separation capability of HSF1 is fine-tuned through phosphorylation at specific sites within the regulatory domain. Last, we discovered that HSP70 disperses HSF1 condensates to attenuate transcription following the cessation of heat shock and further prevents the gel-like phase transition of HSF1 under extended heat-shock stress. Our work reveals an inducible and reversible phase-separation feedback mechanism for dynamic regulation of HSF1 activity to drive the transcriptional response and maintain protein homeostasis during acute stress.

Transcription-coupled structural dynamics of topologically associating domains regulate replication origin efficiency.

BACKGROUND: Metazoan cells only utilize a small subset of the potential DNA replication origins to duplicate the whole genome in each cell cycle. Origin choice is linked to cell growth, differentiation, and replication stress. Although various genetic and epigenetic signatures have been linked to the replication efficiency of origins, there is no consensus on how the selection of origins is determined. RESULTS: We apply dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We find that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase. Intriguingly, while both high-efficiency and low-efficiency origins are distributed homogeneously in the TAD during the G1 phase, high-efficiency origins relocate to the TAD periphery before the S phase. Origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observe that the replication machinery protein PCNA forms immobile clusters around TADs at the G1/S transition, explaining why origins at the TAD periphery are preferentially fired. CONCLUSION: Our work reveals a new origin selection mechanism that the replication efficiency of origins is determined by their physical distribution in the chromatin domain, which undergoes a transcription-dependent structural re-organization process. Our model explains the complex links between replication origin efficiency and many genetic and epigenetic signatures that mark active transcription. The coordination between DNA replication, transcription, and chromatin organization inside individual TADs also provides new insights into the biological functions of sub-domain chromatin structural dynamics.

PN-ImTLSM facilitates high-throughput low background single-molecule localization microscopy deep in the cell.

When imaging the nucleus structure of a cell, the out-of-focus fluorescence acts as background and hinders the detection of weak signals. Light-sheet fluorescence microscopy (LSFM) is a wide-field imaging approach which has the best of both background removal and imaging speed. However, the commonly adopted orthogonal excitation/detection scheme is hard to be applied to single-cell imaging due to steric hindrance. For LSFMs capable of high spatiotemporal single-cell imaging, the complex instrument design and operation largely limit their throughput of data collection. Here, we propose an approach for high-throughput background-free fluorescence imaging of single cells facilitated by the Immersion Tilted Light Sheet Microscopy (ImTLSM). ImTLSM is based on a light-sheet projected off the optical axis of a water immersion objective. With the illumination objective and the detection objective placed opposingly, ImTLSM can rapidly patrol and optically section multiple individual cells while maintaining single-molecule detection sensitivity and resolution. Further, the simplicity and robustness of ImTLSM in operation and maintenance enables high-throughput image collection to establish background removal datasets for deep learning. Using a deep learning model to train the mapping from epi-illumination images to ImTLSM illumination images, namely PN-ImTLSM, we demonstrated cross-modality fluorescence imaging, transforming the epi-illumination image to approach the background removal performance obtained with ImTLSM. We demonstrated that PN-ImTLSM can be generalized to large-field homogeneous illumination imaging, thereby further improving the imaging throughput. In addition, compared to commonly used background removal methods, PN-ImTLSM showed much better performance for areas where the background intensity changes sharply in space, facilitating high-density single-molecule localization microscopy. In summary, PN-ImTLSM paves the way for background-free fluorescence imaging on ordinary inverted microscopes.