scholarly journals Order and stochasticity in the folding of individual Drosophila genomes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sergey V. Ulianov ◽  
Vlada V. Zakharova ◽  
Aleksandra A. Galitsyna ◽  
Pavel I. Kos ◽  
Kirill E. Polovnikov ◽  
...  
Keyword(s):  
Random Walk ◽  
High Resolution ◽  
3D Genome ◽  
Topologically Associating Domains ◽  
Chromatin Folding ◽  
A Cell ◽  
Single Nucleus ◽  
High Level ◽  
Cell To Cell Variability

AbstractMammalian and Drosophila genomes are partitioned into topologically associating domains (TADs). Although this partitioning has been reported to be functionally relevant, it is unclear whether TADs represent true physical units located at the same genomic positions in each cell nucleus or emerge as an average of numerous alternative chromatin folding patterns in a cell population. Here, we use a single-nucleus Hi-C technique to construct high-resolution Hi-C maps in individual Drosophila genomes. These maps demonstrate chromatin compartmentalization at the megabase scale and partitioning of the genome into non-hierarchical TADs at the scale of 100 kb, which closely resembles the TAD profile in the bulk in situ Hi-C data. Over 40% of TAD boundaries are conserved between individual nuclei and possess a high level of active epigenetic marks. Polymer simulations demonstrate that chromatin folding is best described by the random walk model within TADs and is most suitably approximated by a crumpled globule build of Gaussian blobs at longer distances. We observe prominent cell-to-cell variability in the long-range contacts between either active genome loci or between Polycomb-bound regions, suggesting an important contribution of stochastic processes to the formation of the Drosophila 3D genome.

scholarly journals Sci-Hi-C: a single-cell Hi-C method for mapping 3D genome organization in large number of single cells

2019 ◽  
Author(s):  
Vijay Ramani ◽  
Xinxian Deng ◽  
Ruolan Qiu ◽  
Choli Lee ◽  
Christine M Disteche ◽  
...  
Keyword(s):  
Single Cell ◽  
Genome Organization ◽  
Single Cells ◽  
Three Dimensional ◽  
Population Based ◽  
Order Structure ◽  
Chromosome Conformation ◽  
3D Genome ◽  
A Cell ◽  
Cell To Cell Variability

AbstractThe highly dynamic nature of chromosome conformation and three-dimensional (3D) genome organization leads to cell-to-cell variability in chromatin interactions within a cell population, even if the cells of the population appear to be functionally homogeneous. Hence, although Hi-C is a powerful tool for mapping 3D genome organization, this heterogeneity of chromosome higher order structure among individual cells limits the interpretive power of population based bulk Hi-C assays. Moreover, single-cell studies have the potential to enable the identification and characterization of rare cell populations or cell subtypes in a heterogeneous population. However, it may require surveying relatively large numbers of single cells to achieve statistically meaningful observations in single-cell studies. By applying combinatorial cellular indexing to chromosome conformation capture, we developed single-cell combinatorial indexed Hi-C (sci-Hi-C), a high throughput method that enables mapping chromatin interactomes in large number of single cells. We demonstrated the use of sci-Hi-C data to separate cells by karytoypic and cell-cycle state differences and to identify cellular variability in mammalian chromosomal conformation. Here, we provide a detailed description of method design and step-by-step working protocols for sci-Hi-C.

scholarly journals Resolving the 3D landscape of transcription-linked mammalian chromatin folding

2019 ◽  
Author(s):  
Tsung-Han S. Hsieh ◽  
Elena Slobodyanyuk ◽  
Anders S. Hansen ◽  
Claudia Cattoglio ◽  
Oliver J. Rando ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Regulation ◽  
Genome Organization ◽  
Regulatory Elements ◽  
Genome Architecture ◽  
Transcriptional Regulatory Elements ◽  
3D Genome ◽  
Topologically Associating Domains ◽  
Transcriptional Regulatory ◽  
Chromatin Folding

ABSTRACTChromatin folding below the scale of topologically associating domains (TADs) remains largely unexplored in mammals. Here, we used a high-resolution 3C-based method, Micro-C, to probe links between 3D-genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregate TAD regions into “microTADs” with distinct regulatory features. Enhancer-promoter and promoter-promoter interactions extending from the edge of these domains predominantly link co-regulated loci, often independently of CTCF/Cohesin. Acute inhibition of transcription disrupts the gene-related folding features without altering higher-order chromatin structures. Intriguingly, we detect “two-start” zig-zag 30-nanometer chromatin fibers. Our work uncovers the finer-scale genome organization that establishes novel functional links between chromatin folding and gene regulation.ONE SENTENCE SUMMARYTranscriptional regulatory elements shape 3D genome architecture of microTADs.

scholarly journals 5C-ID: Increased resolution Chromosome-Conformation-Capture-Carbon-Copy with in situ 3C and double alternating primer design

2018 ◽  
Author(s):  
Ji Hun Kim ◽  
Katelyn R. Titus ◽  
Wanfeng Gong ◽  
Jonathan A. Beagan ◽  
Zhendong Cao ◽  
...  
Keyword(s):  
High Resolution ◽  
High Throughput Screening ◽  
Chromosome Conformation Capture ◽  
High Coverage ◽  
Chromosome Conformation ◽  
3D Genome ◽  
Loop Detection ◽  
Need To Evaluate ◽  
Carbon Copy

AbstractMammalian genomes are folded in a hierarchy of compartments, topologically associating domains (TADs), subTADs and looping interactions. Currently, there is a great need to evaluate the link between chromatin topology and genome function across many biological conditions and genetic perturbations. Hi-C generates high quality, high resolution maps of looping interactions genome-wide, but is intractable for high-throughput screening of loops across conditions due to the requirement of an enormous number of reads (>6 Billion) per library. Here, we describe 5C-ID, an updated version of Chromosome-Conformation-Capture-Carbon-Copy (5C) with restriction digest and ligation performed in the nucleus (in situ Chromosome-Conformation-Capture (3C)) and ligation-mediated amplification performed with a new double alternating design. 5C-ID reduces spatial noise and enables higher resolution 3D genome folding maps than canonical 5C, allowing for a marked improvement in sensitivity and specificity of loop detection. 5C-ID enables the creation of high-resolution, high-coverage maps of chromatin loops in up to a 30 Megabase subset of the genome at a fraction of the cost of Hi-C.

scholarly journals Multiscale and integrative single-cell Hi-C analysis with Higashi

2021 ◽  
Author(s):  
Ruochi Zhang ◽  
Tianming Zhou ◽  
Jian Ma
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Three Dimensional ◽  
Representation Learning ◽  
Specific Gene ◽  
Separate Analysis ◽  
3D Genome ◽  
Genome Features ◽  
Single Nucleus ◽  
Cell To Cell Variability

AbstractSingle-cell Hi-C (scHi-C) can identify cell-to-cell variability of three-dimensional (3D) chromatin organization, but the sparseness of measured interactions poses an analysis challenge. Here we report Higashi, an algorithm based on hypergraph representation learning that can incorporate the latent correlations among single cells to enhance overall imputation of contact maps. Higashi outperforms existing methods for embedding and imputation of scHi-C data and is able to identify multiscale 3D genome features in single cells, such as compartmentalization and TAD-like domain boundaries, allowing refined delineation of their cell-to-cell variability. Moreover, Higashi can incorporate epigenomic signals jointly profiled in the same cell into the hypergraph representation learning framework, as compared to separate analysis of two modalities, leading to improved embeddings for single-nucleus methyl-3C data. In an scHi-C dataset from human prefrontal cortex, Higashi identifies connections between 3D genome features and cell-type-specific gene regulation. Higashi can also potentially be extended to analyze single-cell multiway chromatin interactions and other multimodal single-cell omics data.

scholarly journals Ultrastructural Visualization of 3D Chromatin Folding Using Serial Block-Face Scanning Electron Microscopy and In Situ Hybridization (3D-EMISH)

2020 ◽  
Author(s):  
Paweł Trzaskoma ◽  
Błażej Ruszczycki ◽  
Byoungkoo Lee ◽  
Katarzyna K. Pels ◽  
Katarzyna Krawczyk ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
In Situ Hybridization ◽  
3 Dimensional ◽  
Face Scanning ◽  
Chromatin Folding ◽  
Block Face ◽  
High Level ◽  
Scanning Electron

AbstractThe human genome is extensively folded into 3-dimensional organization, yet the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra- resolution imaging capability. Here, we report the development of a novel electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5×5×30 nm in xyz dimensions, respectively). We applied 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualized a large number of distinctive 3D chromatin folding structures in high ultra-resolution. We further quantitatively characterized the reconstituted chromatin folding structures by identifying sub-domains, and uncovered a high level of heterogeneity in chromatin folding ultrastructures, suggestive of extensive dynamic fluidity in 3D chromatin states.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

Ultrastructural analysis of the spatial distribution of MRNA

1992 ◽  
Vol 50 (1) ◽  
pp. 552-553
Author(s):  
Gary Bassell ◽  
Robert H. Singer
Keyword(s):  
Electron Microscopy ◽  
Spatial Distribution ◽  
In Situ Hybridization ◽  
High Resolution ◽  
Nucleic Acids ◽  
Colloidal Gold ◽  
Dna Probe ◽  
Nucleotide Analogs ◽  
Gold Particles

We have been investigating the spatial distribution of nucleic acids intracellularly using in situ hybridization. The use of non-isotopic nucleotide analogs incorporated into the DNA probe allows the detection of the probe at its site of hybridization within the cell. This approach therefore is compatible with the high resolution available by electron microscopy. Biotinated or digoxigenated probe can be detected by antibodies conjugated to colloidal gold. Because mRNA serves as a template for the probe fragments, the colloidal gold particles are detected as arrays which allow it to be unequivocally distinguished from background.

High-Resolution Resistance Mapping in SEM

2018 ◽  
Author(s):  
Grigore Moldovan ◽  
Wolfgang Joachimi ◽  
Guillaume Boetsch ◽  
Jörg Jatzkowski ◽  
Frank Altman
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Reference Structure ◽  
Total Resistance ◽  
Embedded Electronics ◽  
Improved Performance ◽  
Mapping Techniques ◽  
Scanning Electron

Abstract This work presents advanced resistance mapping techniques based on Scanning Electron Microscopy (SEM) with nanoprobing systems and the related embedded electronics. Focus is placed on recent advances to reduce noise and increase speed, such as integration of dedicated in situ electronics into the nanoprobing platform, as well as an important transition from current-sensitive to voltagesensitive amplification. We show that it is now possible to record resistance maps with a resistance sensitivity in the 10W range, even when the total resistance of the mapped structures is in the range of 100W. A reference structure is used to illustrate the improved performance, and a lowresistance failure case is presented as an example of analysis made possible by these developments.

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