scholarly journals CTCF-mediated Chromatin Structures Dictate the Spatio-temporal Propagation of Replication Foci

2019 ◽  
Author(s):  
Qian Peter Su ◽  
Ziqing Winston Zhao ◽  
Luming Meng ◽  
Miao Ding ◽  
Weiwei Zhang ◽  
...  
Keyword(s):  
Super Resolution ◽  
S Phase ◽  
Temporal Organization ◽  
Replication Origins ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Replication Foci ◽  
Spatio Temporal ◽  
Optical Reconstruction ◽  
Epigenetic Signatures

ABSTRACTMammalian DNA replication is initiated at numerous replication origins, which are clustered into thousands of replication domains (RDs) across the genome. However, it remains unclear whether the replication origins within each RD are activated stochastically. To understand how replication is regulated at the sub-RD level, we directly visualized the spatio-temporal organization, morphology, and in situ epigenetic signatures of individual replication foci (RFi) across S-phase using super-resolution stochastic optical reconstruction microscopy (STORM). Importantly, we revealed a hierarchical radial pattern of RFi propagation that reverses its directionality from early to late S-phase, and is diminished upon caffeine treatment or CTCF knockdown. Together with simulation and bioinformatic analyses, our findings point to a ‘CTCF-organized REplication Propagation’ (CoREP) model. The CoREP model suggests a non-random selection mechanism for replication activation mediated by CTCF at the sub-RD level, as well as the critical involvement of local chromatin environment in regulating replication in space and time.

scholarly journals Superresolution imaging reveals spatiotemporal propagation of human replication foci mediated by CTCF-organized chromatin structures

2020 ◽  
Vol 117 (26) ◽  
pp. 15036-15046 ◽  
Author(s):  
Qian Peter Su ◽  
Ziqing Winston Zhao ◽  
Luming Meng ◽  
Miao Ding ◽  
Weiwei Zhang ◽  
...  
Keyword(s):  
Dna Replication ◽  
S Phase ◽  
Replication Origins ◽  
Nuclear Dynamics ◽  
Superresolution Imaging ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Replication Foci ◽  
Optical Reconstruction ◽  
Epigenetic Signatures

Mammalian DNA replication is initiated at numerous replication origins, which are clustered into thousands of replication domains (RDs) across the genome. However, it remains unclear whether the replication origins within each RD are activated stochastically or preferentially near certain chromatin features. To understand how DNA replication in single human cells is regulated at the sub-RD level, we directly visualized and quantitatively characterized the spatiotemporal organization, morphology, and in situ epigenetic signatures of individual replication foci (RFi) across S-phase at superresolution using stochastic optical reconstruction microscopy. Importantly, we revealed a hierarchical radial pattern of RFi propagation dynamics that reverses directionality from early to late S-phase and is diminished upon caffeine treatment or CTCF knockdown. Together with simulation and bioinformatic analyses, our findings point to a “CTCF-organized REplication Propagation” (CoREP) model, which suggests a nonrandom selection mechanism for replication activation at the sub-RD level during early S-phase, mediated by CTCF-organized chromatin structures. Collectively, these findings offer critical insights into the key involvement of local epigenetic environment in coordinating DNA replication across the genome and have broad implications for our conceptualization of the role of multiscale chromatin architecture in regulating diverse cell nuclear dynamics in space and time.

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

2020 ◽  
Author(s):  
Yongzheng Li ◽  
Boxin Xue ◽  
Liwei Zhang ◽  
Qian Peter Su ◽  
Mengling Zhang ◽  
...  
Keyword(s):  
Dna Replication ◽  
Structural Dynamics ◽  
Super Resolution ◽  
S Phase ◽  
Replication Origins ◽  
Topologically Associating Domains ◽  
Chromatin Structural ◽  
Resolution Imaging ◽  
Epigenetic Signatures ◽  
Selection Of

ABSTRACTMetazoan 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. Despite various genetic and epigenetic signatures are found to be related with active origins, it remains elusive how the selection of origins is determined. The classic Rosette model proposes that the origins clustered in a chromatin domain are preferentially and simultaneously fired, but direct imaging evidence has been lacking due to insufficient spatial resolution. Here, we applied dual-color stochastic optical reconstruction microscopy (STORM) super-resolution imaging to map the spatial distribution of origins within individual topologically associating domains (TADs). We found that multiple replication origins initiate separately at the spatial boundary of a TAD at the beginning of the S phase, in contrary to the Rosette model. Intriguingly, while both active and dormant origins are distributed homogeneously in the TAD during the G1 phase, active origins relocate to the TAD periphery before entering the S phase. We proved that such origin relocalization is dependent on both transcription and CTCF-mediated chromatin structure. Further, we observed that the replication machinery protein PCNA forms immobile clusters around the TADs at the G1/S transition, which explains why origins at the TAD periphery are preferentially fired. Thus, we propose a “Chromatin Re-organization Induced Selective Initiation” (CRISI) model that the transcription-coupled chromatin structural re-organization determines the selection of replication origins, which transcends the scope of specific genetic and epigenetic signatures for origin efficiency. Our in situ super-resolution imaging unveiled coordination among DNA replication, transcription, and chromatin organization inside individual TADs, providing new insights into the biological functions of sub-domain chromatin structural dynamics.

scholarly journals Single-molecule Photoswitching and Localization

2011 ◽  
Vol 64 (5) ◽  
pp. 503 ◽  
Author(s):  
Sebastian van de Linde ◽  
Steve Wolter ◽  
Markus Sauer
Keyword(s):  
Single Molecule ◽  
Optical Resolution ◽  
Super Resolution ◽  
Image Plane ◽  
Localization Microscopy ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Spatio Temporal ◽  
Optical Reconstruction ◽  
20 Nm

Within only a few years super-resolution fluorescence imaging based on single-molecule localization and image reconstruction has attracted considerable interest because it offers a comparatively simple way to achieve a substantially improved optical resolution down to ∼20 nm in the image plane. Since super-resolution imaging methods such as photoactivated localization microscopy, fluorescence photoactivation localization microscopy, stochastic optical reconstruction microscopy, and direct stochastic optical reconstruction microscopy rely critically on exact fitting of the centre of mass and the shape of the point-spread-function of isolated emitters unaffected by neighbouring fluorophores, controlled photoswitching or photoactivation of fluorophores is the key parameter for resolution improvement. This review will explain the principles and requirements of single-molecule based localization microscopy, and compare different super-resolution imaging concepts and highlight their strengths and limitations with respect to applications in fixed and living cells with high spatio-temporal resolution.

scholarly journals Label-retention expansion microscopy

2019 ◽  
Author(s):  
Xiaoyu Shi ◽  
Qi Li ◽  
Zhipeng Dai ◽  
Arthur A. Tran ◽  
Siyu Feng ◽  
...  
Keyword(s):  
High Efficiency ◽  
Super Resolution ◽  
Fluorescence Signal ◽  
Signal Loss ◽  
Coated Pits ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Cell Tissue ◽  
Wide Range ◽  
Optical Reconstruction

ABSTRACTExpansion microscopy (ExM) improves the resolution of fluorescence microscopy by physically expanding the sample embedded in a hydrogel1–4. Since its invention, ExM has been successfully applied to a wide range of cell, tissue and animal samples 2–9. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency protein labeling using enzymatic tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with super-resolution Stochastic Optical Reconstruction Microscopy (STORM), we have achieved 5 nm resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

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

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yongzheng Li ◽  
Boxin Xue ◽  
Mengling Zhang ◽  
Liwei Zhang ◽  
Yingping Hou ◽  
...  
Keyword(s):  
Dna Replication ◽  
Structural Dynamics ◽  
Replication Origin ◽  
High Efficiency ◽  
S Phase ◽  
Chromatin Domain ◽  
Replication Origins ◽  
Replication Efficiency ◽  
Topologically Associating Domains ◽  
Epigenetic Signatures

Abstract 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.

scholarly journals Focus on Super-Resolution Imaging with Direct Stochastic Optical Reconstruction Microscopy (dSTORM)

2014 ◽  
Vol 67 (2) ◽  
pp. 179 ◽  
Author(s):  
Donna R. Whelan ◽  
Thorge Holm ◽  
Markus Sauer ◽  
Toby D. M. Bell
Keyword(s):  
Cell Biology ◽  
Super Resolution ◽  
Diffraction Limit ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Resolution Imaging ◽  
Optical Reconstruction ◽  
Set Up ◽  
Microscopy Techniques ◽  
Super Resolution Microscopy ◽  
Microscopic Techniques

The last decade has seen the development of several microscopic techniques capable of achieving spatial resolutions that are well below the diffraction limit of light. These techniques, collectively referred to as ‘super-resolution’ microscopy, are now finding wide use, particularly in cell biology, routinely generating fluorescence images with resolutions in the order of tens of nanometres. In this highlight, we focus on direct Stochastic Optical Reconstruction Microscopy or dSTORM, one of the localisation super-resolution fluorescence microscopy techniques that are founded on the detection of fluorescence emissions from single molecules. We detail how, with minimal assemblage, a highly functional and versatile dSTORM set-up can be built from ‘off-the-shelf’ components at quite a modest budget, especially when compared with the current cost of commercial systems. We also present some typical super-resolution images of microtubules and actin filaments within cells and discuss sample preparation and labelling methods.

scholarly journals G1/S transcription factors assemble in discrete clusters that increase in number as cells grow

2019 ◽  
Author(s):  
Labe Black ◽  
Sylvain Tollis ◽  
Guo Fu ◽  
Jean-Bernard Fiche ◽  
Savanna Dorsey ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Super Resolution ◽  
Temporal Organization ◽  
Stochastic Simulations ◽  
Live Cells ◽  
Number Of Clusters ◽  
Localization Microscopy ◽  
Spatio Temporal ◽  
Cluster Behavior ◽  
Target Promoters

AbstractThe spatio-temporal organization of transcription factor (TF)-promoter interactions is critical for the coordination of transcriptional programs. In budding yeast, the main G1/S transcription factors, SBF and MBF, are limiting with respect to target promoters in small G1 phase cells and accumulate as cells grow, raising the question of how SBF/MBF are dynamically distributed across the G1/S regulon. Super-resolution Photo-Activatable Localization Microscopy (PALM) mapping of the static positions of SBF/MBF subunits revealed that 85% were organized into discrete clusters containing ∼8 copies regardless of cell size, while the number of clusters increased with growth. Stochastic simulations with a mathematical model based on co-localization of promoters in clusters recapitulated observed cluster behavior. A prediction of the model that SBF/MBF should exhibit both fast and slow dynamics was confirmed in PALM experiments on live cells. This spatio-temporal organization of the TFs that activate the G1/S regulon may help coordinate commitment to division.

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