scholarly journals Rad21 is the core subunit of the cohesin complex involved in directing genome organization

2021 ◽  
Author(s):  
Yujie Sun ◽  
Yuao Sun ◽  
Xin Xu ◽  
Wenxue Zhao ◽  
Yu Zhang ◽  
...  
Keyword(s):  
Important Determinant ◽  
Genome Structure ◽  
Super Resolution ◽  
Loop Model ◽  
Cohesin Complex ◽  
Aberrant Expression ◽  
The Core ◽  
Topologically Associating Domains ◽  
Resolution Imaging ◽  
Cohesin Subunit

Abstract The ring-shaped cohesin complex is an important factor regulating genome structure. It is thought to mediate the formation of chromatin loops and topologically associating domains (TADs) by loop extrusion. However, the regulation of association between cohesin and chromatin is poorly understood. In this study, we directly visualized cohesin loading after up-regulation of cohesin subunit Rad21 by identifying the formation of vermicelli-like structures via live cell super-resolution imaging. We also reveal that cohesin loading can be promoted by Rad21-loader interactions and accumulated contacts were shown at TAD corners while inter-TAD interactions increased after vermicelli formation, indicating that Rad21 is an important determinant of chromatin structure. Moreover, we find that cohesin saddle on topologically associating domains by FISH assay, which is consistent with the CTCF/cohesin-anchored chromatin loop model. Importantly, expression of Rad21 is strictly controlled, and aberrant expression of Rad21 leads to the formation of Rad21 “beads” in the nucleus. In summary, our observations provided important new biological insights into the mechanism of cohesin loading and its functions.

scholarly journals Genetically encoded photo-switchable molecular sensors for optoacoustic and super-resolution imaging

2021 ◽  
Author(s):  
Kanuj Mishra ◽  
Juan Pablo Fuenzalida-Werner ◽  
Francesca Pennacchietti ◽  
Robert Janowski ◽  
Andriy Chmyrov ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Super Resolution ◽  
Structural Level ◽  
Optoacoustic Imaging ◽  
Molecular Sensors ◽  
The Core ◽  
Resolution Imaging ◽  
G Protein Coupled

AbstractReversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca2+ receptor moiety. We demonstrate super-resolution imaging of Ca2+ concentration in cultured cells and optoacoustic Ca2+ imaging in implanted tumor cells in mice under controlled Ca2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

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 Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin

2019 ◽  
Author(s):  
Ezequiel Miron ◽  
Roel Oldenkamp ◽  
Jill M. Brown ◽  
David M. S. Pinto ◽  
C. Shan Xu ◽  
...  
Keyword(s):  
Nuclear Organization ◽  
Three Dimensional ◽  
Super Resolution ◽  
Mammalian Genome ◽  
Functional Modules ◽  
Cell Level ◽  
The Core ◽  
Topologically Associating Domains ◽  
Functional Topography ◽  
Physical Features

ABSTRACTThree-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function, however many of its physical features at the single-cell level remain underexplored. Here we use 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify linked chromatin domains (CDs) composed of irregular ∼200-300-nm-wide aggregates of nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin region. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications towards the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in post-replicative chromatin, but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization.

scholarly journals Chromatin arranges in chains of mesoscale domains with nanoscale functional topography independent of cohesin

2020 ◽  
Vol 6 (39) ◽  
pp. eaba8811 ◽  
Author(s):  
Ezequiel Miron ◽  
Roel Oldenkamp ◽  
Jill M. Brown ◽  
David M. S. Pinto ◽  
C. Shan Xu ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Super Resolution ◽  
Functional Modules ◽  
Cell Level ◽  
The Core ◽  
Topologically Associating Domains ◽  
Interchromatin Compartment ◽  
Functional Topography ◽  
Fixed Cell ◽  
Physical Features

Three-dimensional (3D) chromatin organization plays a key role in regulating mammalian genome function; however, many of its physical features at the single-cell level remain underexplored. Here, we use live- and fixed-cell 3D super-resolution and scanning electron microscopy to analyze structural and functional nuclear organization in somatic cells. We identify chains of interlinked ~200- to 300-nm-wide chromatin domains (CDs) composed of aggregated nucleosomes that can overlap with individual topologically associating domains and are distinct from a surrounding RNA-populated interchromatin compartment. High-content mapping uncovers confinement of cohesin and active histone modifications to surfaces and enrichment of repressive modifications toward the core of CDs in both hetero- and euchromatic regions. This nanoscale functional topography is temporarily relaxed in postreplicative chromatin but remarkably persists after ablation of cohesin. Our findings establish CDs as physical and functional modules of mesoscale genome organization.

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