scholarly journals Super-resolution microscopy of chromatin fibers and quantitative DNA methylation analysis of DNA fiber preparations

2021 ◽  
Author(s):  
Michal Franek ◽  
Agata Kilar ◽  
Petr Fojtík ◽  
Marie Olšinová ◽  
Aleš Benda ◽  
...  
Keyword(s):  
Single Cell Analysis ◽  
Super Resolution ◽  
Single Copy ◽  
Histone Variants ◽  
Dna Repeats ◽  
Genome Wide ◽  
Custom Script ◽  
Chromatin Immunoprecipitation Sequencing ◽  
Super Resolution Microscopy ◽  
Dna Methylation Analysis

Analysis of histone variants and epigenetic marks is dominated by genome-wide approaches in the form of chromatin immunoprecipitation-sequencing (ChIP-seq) and related methods. While uncontested in their value for single-copy genes, mapping the chromatin of DNA repeats is problematic for biochemical techniques based on averaging cell populations or high number of repeats in a single cell analysis. Extending chromatin and DNA fibers allows us to study the epigenetics of individual repeats in their specific chromosomal context and thus constitutes an important tool for a wholesome understanding of the epigenetic organization of genomes. We present that using an optimized fiber extension protocol is essential to obtain more reproducible data, where the clustering of fibers is minimized. We also demonstrate that applying super-resolution microscopy is important to reliably evaluate the distribution of histone modifications on individual fibers. Furthermore, we introduce a custom script to analyse methylation levels on DNA fibers and apply it to map the methylation of telomeres, ribosomal genes and centromeres.

scholarly journals Spatial sequestration and detoxification of Huntingtin by the ribosome quality control complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Junsheng Yang ◽  
Xinxin Hao ◽  
Xiuling Cao ◽  
Beidong Liu ◽  
Thomas Nyström
Keyword(s):  
Quality Control ◽  
Regulatory System ◽  
Super Resolution ◽  
Age Related ◽  
Genome Wide ◽  
A Genome ◽  
Polyglutamine Expansions ◽  
Key Factor ◽  
Super Resolution Microscopy ◽  
Control Complex

Huntington disease (HD) is a neurological disorder caused by polyglutamine expansions in mutated Huntingtin (mHtt) proteins, rendering them prone to form inclusion bodies (IB). We report that in yeast, such IB formation is a factor-dependent process subjected to age-related decline. A genome-wide, high-content imaging approach, identified the E3 ubiquitin ligase, Ltn1 of the ribosome quality control complex (RQC) as a key factor required for IB formation, ubiquitination, and detoxification of model mHtt. The failure of ltn1∆ cells to manage mHtt was traced to another RQC component, Tae2, and inappropriate control of heat shock transcription factor, Hsf1, activity. Moreover, super-resolution microscopy revealed that mHtt toxicity in RQC-deficient cells was accompanied by multiple mHtt aggregates altering actin cytoskeletal structures and retarding endocytosis. The data demonstrates that spatial sequestration of mHtt into IBs is policed by the RQC-Hsf1 regulatory system and that such compartmentalization, rather than ubiquitination, is key to mHtt detoxification.

scholarly journals CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication

2019 ◽  
Vol 159 (1) ◽  
pp. 48-53 ◽  
Author(s):  
Alžběta Němečková ◽  
Christina Wäsch ◽  
Veit Schubert ◽  
Takayoshi Ishii ◽  
Eva Hřibová ◽  
...  
Keyword(s):  
Dna Replication ◽  
Chromatin Structure ◽  
Super Resolution ◽  
Dna Repeats ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Replication Sites ◽  
And Function ◽  
Super Resolution Microscopy

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.

scholarly journals Stochastic association of neighboring replicons creates replication factories in budding yeast

2013 ◽  
Vol 202 (7) ◽  
pp. 1001-1012 ◽  
Author(s):  
Nazan Saner ◽  
Jens Karschau ◽  
Toyoaki Natsume ◽  
Marek Gierliński ◽  
Renata Retkute ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Cell Imaging ◽  
Budding Yeast ◽  
Super Resolution ◽  
Replication Proteins ◽  
Replication Factory ◽  
Genome Wide ◽  
Nuclear Structures ◽  
Super Resolution Microscopy ◽  
Insight Into

Inside the nucleus, DNA replication is organized at discrete sites called replication factories, consisting of DNA polymerases and other replication proteins. Replication factories play important roles in coordinating replication and in responding to replication stress. However, it remains unknown how replicons are organized for processing at each replication factory. Here we address this question using budding yeast. We analyze how individual replicons dynamically organized a replication factory using live-cell imaging and investigate how replication factories were structured using super-resolution microscopy. Surprisingly, we show that the grouping of replicons within factories is highly variable from cell to cell. Once associated, however, replicons stay together relatively stably to maintain replication factories. We derive a coherent genome-wide mathematical model showing how neighboring replicons became associated stochastically to form replication factories, which was validated by independent microscopy-based analyses. This study not only reveals the fundamental principles promoting replication factory organization in budding yeast, but also provides insight into general mechanisms by which chromosomes organize sub-nuclear structures.

scholarly journals Second Harmonic Super-resolution Microscopy for Quantification of mRNA at Single Copy Sensitivity

ACS Nano ◽  
2014 ◽  
Vol 8 (12) ◽  
pp. 12418-12427 ◽  
Author(s):  
Jing Liu ◽  
Il-Hoon Cho ◽  
Yi Cui ◽  
Joseph Irudayaraj
Keyword(s):  
Second Harmonic ◽  
Super Resolution ◽  
Single Copy ◽  
Super Resolution Microscopy

scholarly journals Analysis of novel hyperosmotic shock response suggests “beads in liquid” cytosol structure

2019 ◽  
Author(s):  
A.I. Alexandrov ◽  
E.V. Grosfeld ◽  
A.A. Dergalev ◽  
V.V. Kushnirov ◽  
R.N. Chuprov-Netochin ◽  
...  
Keyword(s):  
Super Resolution ◽  
Close Correlation ◽  
Shock Onset ◽  
Hyperosmotic Shock ◽  
Amyloidogenic Proteins ◽  
Genome Wide ◽  
A Genome ◽  
Targeted Testing ◽  
Body Components ◽  
Super Resolution Microscopy

AbstractProteins can aggregate in response to stresses, including hyperosmotic shock. Formation and disassembly of aggregates is a relatively slow process. We describe a novel instant response of the cell to hyperosmosis, during which chaperones and other proteins form numerous foci with properties uncharacteristic of classical aggregates. These foci appeared/disappeared seconds after shock onset/removal, in close correlation with cell volume changes. Genome-wide and targeted testing revealed chaperones, metabolic enzymes, P-body components and amyloidogenic proteins in the foci. Most of these proteins can form large assemblies and for some, the assembled state was pre-requisite for participation in foci. A genome-wide screen failed to identify genes whose absence prevented foci participation by Hsp70. Shapes of and interconnections between foci revealed by super-resolution microscopy indicated that the foci were compressed between other entities. Based on our findings, we propose a new model of the cytosol architecture as a collection of numerous of gel-like regions suspended in a liquid network. This network is reduced in volume in response to hyperosmosis and forms small pockets between the gel-like regions.

Super-Resolution Microscopy in Studying the Structure and Function of the Cell Nucleus

Acta Naturae ◽  
2017 ◽  
Vol 9 (4) ◽  
pp. 42-51
Author(s):  
S. S. Ryabichko ◽  
◽  
A. N. Ibragimov ◽  
L. A. Lebedeva ◽  
E. N. Kozlov ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Super Resolution ◽  
Structure And Function ◽  
And Function ◽  
Super Resolution Microscopy

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

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