scholarly journals Histone modification dynamics as revealed by a multicolor immunofluorescence-based single-cell analysis

2020 ◽  
Author(s):  
Yoko Hayashi-Takanaka ◽  
Yuto Kina ◽  
Fumiaki Nakamura ◽  
Leontine E. Becking ◽  
Yoichi Nakao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Histone Modifications ◽  
Histone Deacetylases ◽  
Single Cell Analysis ◽  
Marine Organism ◽  
Single Cells ◽  
Cell Types ◽  
Transient Overexpression ◽  
Overexpression System ◽  
External Stimuli

AbstractPost-translational modifications on histones can be stable epigenetic marks and transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We first analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during the G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. We also screened chemical compounds in marine organism extracts that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.

scholarly journals Histone modification dynamics as revealed by multicolor immunofluorescence-based single-cell analysis

2020 ◽  
Vol 133 (14) ◽  
pp. jcs243444 ◽  
Author(s):  
Yoko Hayashi-Takanaka ◽  
Yuto Kina ◽  
Fumiaki Nakamura ◽  
Leontine E. Becking ◽  
Yoichi Nakao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Histone Modifications ◽  
Histone Deacetylases ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cell Types ◽  
Transient Overexpression ◽  
Overexpression System ◽  
Different Cell Types ◽  
External Stimuli

ABSTRACTPost-translational modifications on histones can be stable epigenetic marks or transient signals that can occur in response to internal and external stimuli. Levels of histone modifications fluctuate during the cell cycle and vary among different cell types. Here, we describe a simple system to monitor the levels of multiple histone modifications in single cells by multicolor immunofluorescence using directly labeled modification-specific antibodies. We analyzed histone H3 and H4 modifications during the cell cycle. Levels of active marks, such as acetylation and H3K4 methylation, were increased during the S phase, in association with chromatin duplication. By contrast, levels of some repressive modifications gradually increased during G2 and the next G1 phases. We applied this method to validate the target modifications of various histone demethylases in cells using a transient overexpression system. In extracts of marine organisms, we also screened chemical compounds that affect histone modifications and identified psammaplin A, which was previously reported to inhibit histone deacetylases. Thus, the method presented here is a powerful and convenient tool for analyzing the changes in histone modifications.

scholarly journals Methods and sensors for functional genomic studies of cell-cycle transitions in single cells

2020 ◽  
Vol 52 (10) ◽  
pp. 468-477
Author(s):  
Alexander C. Zambon ◽  
Tom Hsu ◽  
Seunghee Erin Kim ◽  
Miranda Klinck ◽  
Jennifer Stowe ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Imaging System ◽  
Single Cells ◽  
Cell Types ◽  
Cell Analysis ◽  
Mcf7 Cells ◽  
Genomic Studies ◽  
Cell Subpopulations

Much of our understanding of the regulatory mechanisms governing the cell cycle in mammals has relied heavily on methods that measure the aggregate state of a population of cells. While instrumental in shaping our current understanding of cell proliferation, these approaches mask the genetic signatures of rare subpopulations such as quiescent (G0) and very slowly dividing (SD) cells. Results described in this study and those of others using single-cell analysis reveal that even in clonally derived immortalized cancer cells, ∼1–5% of cells can exhibit G0 and SD phenotypes. Therefore to enable the study of these rare cell phenotypes we established an integrated molecular, computational, and imaging approach to track, isolate, and genetically perturb single cells as they proliferate. A genetically encoded cell-cycle reporter (K67p-FUCCI) was used to track single cells as they traversed the cell cycle. A set of R-scripts were written to quantify K67p-FUCCI over time. To enable the further study G0 and SD phenotypes, we retrofitted a live cell imaging system with a micromanipulator to enable single-cell targeting for functional validation studies. Single-cell analysis revealed HT1080 and MCF7 cells had a doubling time of ∼24 and ∼48 h, respectively, with high duration variability in G1 and G2 phases. Direct single-cell microinjection of mRNA encoding (GFP) achieves detectable GFP fluorescence within ∼5 h in both cell types. These findings coupled with the possibility of targeting several hundreds of single cells improves throughput and sensitivity over conventional methods to study rare cell subpopulations.

scholarly journals Histone modifications form a cell-type-specific chromosomal bar code that persists through the cell cycle

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
John A. Halsall ◽  
Simon Andrews ◽  
Felix Krueger ◽  
Charlotte E. Rutledge ◽  
Gabriella Ficz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Histone Modifications ◽  
Expression Patterns ◽  
Cell Types ◽  
Cell Type ◽  
Bar Code ◽  
Genes Encoding ◽  
Cell Type Specific ◽  
Rolling Windows

AbstractChromatin configuration influences gene expression in eukaryotes at multiple levels, from individual nucleosomes to chromatin domains several Mb long. Post-translational modifications (PTM) of core histones seem to be involved in chromatin structural transitions, but how remains unclear. To explore this, we used ChIP-seq and two cell types, HeLa and lymphoblastoid (LCL), to define how changes in chromatin packaging through the cell cycle influence the distributions of three transcription-associated histone modifications, H3K9ac, H3K4me3 and H3K27me3. We show that chromosome regions (bands) of 10–50 Mb, detectable by immunofluorescence microscopy of metaphase (M) chromosomes, are also present in G1 and G2. They comprise 1–5 Mb sub-bands that differ between HeLa and LCL but remain consistent through the cell cycle. The same sub-bands are defined by H3K9ac and H3K4me3, while H3K27me3 spreads more widely. We found little change between cell cycle phases, whether compared by 5 Kb rolling windows or when analysis was restricted to functional elements such as transcription start sites and topologically associating domains. Only a small number of genes showed cell-cycle related changes: at genes encoding proteins involved in mitosis, H3K9 became highly acetylated in G2M, possibly because of ongoing transcription. In conclusion, modified histone isoforms H3K9ac, H3K4me3 and H3K27me3 exhibit a characteristic genomic distribution at resolutions of 1 Mb and below that differs between HeLa and lymphoblastoid cells but remains remarkably consistent through the cell cycle. We suggest that this cell-type-specific chromosomal bar-code is part of a homeostatic mechanism by which cells retain their characteristic gene expression patterns, and hence their identity, through multiple mitoses.

scholarly journals Symposium on single cell analysis and genomic approaches, Experimental Biology 2017 Chicago, Illinois, April 23, 2017

2017 ◽  
Vol 49 (9) ◽  
pp. 491-495
Author(s):  
Hilary A. Coller
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Experimental Biology ◽  
Intratumor Heterogeneity ◽  
Cell Analysis ◽  
Single Cell Genomics ◽  
Critical Issues ◽  
Cell To Cell Variability ◽  
External Stimuli

Emerging technologies for the analysis of genome-wide information in single cells have the potential to transform many fields of biology, including our understanding of cell states, the response of cells to external stimuli, mosaicism, and intratumor heterogeneity. At Experimental Biology 2017 in Chicago, Physiological Genomics hosted a symposium in which five leaders in the field of single cell genomics presented their recent research. The speakers discussed emerging methodologies in single cell analysis and critical issues for the analysis of single cell data. Also discussed were applications of single cell genomics to understanding the different types of cells within an organism or tissue and the basis for cell-to-cell variability in response to stimuli.

scholarly journals Novel microfluidic platform accelerates processing of tissue into single cells for molecular analysis and primary culture models

2020 ◽  
Author(s):  
Jeremy Lombardo ◽  
Marzieh Aliaghaei ◽  
Quy Nguyen ◽  
Kai Kessenbrock ◽  
Jered Haun
Keyword(s):  
Single Cell ◽  
Stress Responses ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cell Types ◽  
Tissue Preparation ◽  
Cell Analysis ◽  
Processing Technologies ◽  
Microfluidic Platform ◽  
Wide Range

Abstract Tissues are composed of highly heterogeneous mixtures of cell subtypes, and this diversity is increasingly being characterized using high-throughput single cell analysis methods. However, these efforts are hindered by the fact that tissues must first be dissociated into single cell suspensions that are viable and still accurately represent phenotypes from the original tissue. Current methods for breaking down tissues are inefficient, labor-intensive, subject to high variability, and potentially biased towards cell subtypes that are easier to release. Here, we present a microfluidic platform consisting of three different tissue processing technologies that can perform the complete tissue to single cell workflow, including digestion, disaggregation, and filtration. First, we developed a new microfluidic digestion device that can be loaded with minced tissue specimens quickly and easily, and then use the combination of proteolytic enzyme activity and fluid shear forces to accelerate tissue breakdown. Next, we integrated dissociation and filter technologies into a single device, which enhanced single cell numbers and fully prepared the sample for single cell analysis. The final multi-device platform was then evaluated using a diverse array of tissue types that exhibited a wide range of properties. For murine kidney and mammary tumor, we found that microfluidic processing produced 2.5-fold more single, viable cells. Single cell RNA sequencing (scRNA-seq) further revealed that device processing enriched for endothelial cells, fibroblasts, and basal epithelium, and did not increase stress responses. For murine liver and heart, which are softer tissues containing fragile cell types, processing time could be reduced to 15 min, and even as short as 1 min. We also demonstrated that periodic recovery at defined time intervals produced substantially more hepatocytes and cardiomyocytes than continuous operation, most likely by preventing damage to fragile cell types. In future work, we will seek to integrate additional operations such as upstream tissue preparation and downstream microfluidic cell sorting and detection to create powerful point-of-care single cell diagnostic platforms.

scholarly journals HiCluster: A Robust Single-Cell Hi-C Clustering Method Based on Convolution and Random Walk

2018 ◽  
Author(s):  
Jingtian Zhou ◽  
Jianzhu Ma ◽  
Yusi Chen ◽  
Chuankai Cheng ◽  
Bokan Bao ◽  
...  
Keyword(s):  
Random Walk ◽  
Single Cell ◽  
Clustering Algorithm ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Genome Structure ◽  
Real Data ◽  
Cell Types ◽  
3D Genome ◽  
Cell Clustering

3D genome structure plays a pivotal role in gene regulation and cellular function. Single-cell analysis of genome architecture has been achieved using imaging and chromatin conformation capture methods such as Hi-C. To study variation in chromosome structure between different cell types, computational approaches are needed that can utilize sparse and heterogeneous single-cell Hi-C data. However, few methods exist that are able to accurately and efficiently cluster such data into constituent cell types. Here, we describe HiCluster, a single-cell clustering algorithm for Hi-C contact matrices that is based on imputations using linear convolution and random walk. Using both simulated and real data as benchmarks, HiCluster significantly improves clustering accuracy when applied to low coverage Hi-C datasets compared to existing methods. After imputation by HiCluster, structures similar to topologically associating domains (TADs) could be identified within single cells, and their consensus boundaries among cells were enriched at the TAD boundaries observed in bulk samples. In summary, HiCluster facilitates visualization and comparison of single-cell 3D genomes.

scholarly journals Histone modifications form a cell-type-specific chromosomal bar code that modulates and maintains patterns of gene expression through the cell cycle

2020 ◽  
Author(s):  
John A. Halsall ◽  
Simon Andrews ◽  
Felix Krueger ◽  
Charlotte E. Rutledge ◽  
Gabriella Ficz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Histone Modifications ◽  
Expression Patterns ◽  
Cell Types ◽  
Cell Type ◽  
Bar Code ◽  
Genes Encoding ◽  
Cell Type Specific ◽  
Rolling Windows

ABSTRACTBackgroundChromatin configuration influences gene expression in eukaryotes at multiple levels, from individual nucleosomes to chromatin domains several Mb long. Post-translational modifications (PTM) of core histones seem to be involved in chromatin structural transitions, but how remains unclear.To explore this, we used ChIP-seq and two cell types, HeLa and lymphoblastoid (LCL) to define how changes in chromatin packaging through the cell cycle influence the distributions of three transcription-associated histone modifications, H3K9ac, H3K4me3 and H3K27me3.ResultsChromosome regions (bands) of 10-50Mb, detectable by immunofluorescence microscopy of metaphase (M) chromosomes, are also present in G1 and G2. We show that they comprise 1-5Mb sub-bands that differ between HeLa and LCL but remain consistent through the cell cycle. The same sub-bands are defined by H3K9ac and H3K4me3, while H3K27me3 spreads more widely.We found little change between cell cycle phases, whether compared by 5Kb rolling windows or when analysis was restricted to functional elements such as transcription start sites and topologically associating domains.Only a small number of genes showed cell-cycle related changes: at genes encoding proteins involved in mitosis, H3K9 became highly acetylated in G2M, possibly because of ongoing transcription.ConclusionsModified histone isoforms H3K9ac, H3K4me3 and H3K27me3 exhibit a characteristic genomic distribution at resolutions of 1Mb and below that differs between HeLa and lymphoblastoid cells but remains remarkably consistent through the cell cycle. We suggest that this cell-type-specific chromosomal bar-code is part of a homeostatic mechanism by which cells retain their characteristic gene expression patterns, and hence their identity, through multiple mitoses.

scholarly journals What you see is what you get: activity-based probes in single-cell analysis of enzymatic activities

2020 ◽  
Vol 401 (2) ◽  
pp. 233-248
Author(s):  
Christian S. Lentz
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Subcellular Location ◽  
Data Interpretation ◽  
Enzymatic Activities ◽  
Biological Processes ◽  
Chemical Probes ◽  
Spatio Temporal ◽  
External Stimuli

AbstractMolecular imaging methods can provide spatio-temporal information about the distribution of biomolecules or biological processes, such as certain enzymatic activities, in single cells. Within a cell, it is possible to define the subcellular location of a target, its trafficking through the cell, colocalization with other biomolecules of interest and involvement in certain cell biological processes. On the other hand, single-cell imaging promises to distinguish cells that are phenotypically different from each other. The corresponding cellular diversity comprises the presence of functionally distinct cells in a population (‘phenotypic heterogeneity’), as well as dynamic cellular responses to external stimuli (‘phenotypic plasticity’), which is highly relevant, e.g. during cell differentiation, activation (of immune cells), or cell death. This review focuses on applications of a certain class of chemical probes, the so-called activity-based probes (ABPs), for visualization of enzymatic activities in the single-cell context. It discusses the structure of ABPs and other chemical probes, exemplary applications of ABPs in single-cell studies in human, mouse and bacterial systems and considerations to be made with regard to data interpretation.

Statistical single-cell analysis of cell cycle-dependent quantum dot cytotoxicity and cellular uptake using a microfluidic system

RSC Advances ◽  
2014 ◽  
Vol 4 (47) ◽  
pp. 24929-24934 ◽  
Author(s):  
Jing Wu ◽  
Haifang Li ◽  
Qiushui Chen ◽  
Xuexia Lin ◽  
Wu Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Quantum Dot ◽  
Single Cell ◽  
Cellular Uptake ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Microfluidic System ◽  
Cell Analysis ◽  
System P ◽  
Cycle Dependent

The response of single cells in different cell cycle phases to QD cytotoxicity studied on a microfluidic device.

scholarly journals Deconvolving multiplexed histone modifications in single cells

2021 ◽  
Author(s):  
Jake Yeung ◽  
Maria Florescu ◽  
Peter Zeller ◽  
Buys Anton de Barbanson ◽  
Alexander van Oudenaarden
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Histone Modification ◽  
Histone Modifications ◽  
Blood Cells ◽  
Single Cells ◽  
Cell Types ◽  
Mouse Bone Marrow ◽  
Immunoglobulin Genes ◽  
Computational Framework

Recent advances have enabled mapping of histone modifications in single cells, but current methods are constrained to profile only one histone modification per cell. Here we present an integrated experimental and computational framework, scChIX (single-cell chromatin immunocleavage and unmixing), to map multiple histone modifications in single cells. We first validate this method using purified blood cells and show that although the two repressive marks, H3K27me3 and H3K9me3, are generally mutually exclusive, the transitions between the two regions can vary between cell types. Next we apply scChIX to a heterogenous cell population from mouse bone marrow to generate linked maps of active (H3K4me1) and repressive (H3K27me3) chromatin landscapes in single cells, where coordinates in the active modification map correspond to coordinates in the repressive map. Linked analysis reveals that immunoglobulin genes in the region are in a repressed chromatin state in pro-B cells, but become activated in B cells. Overall, scChIX unlocks systematic interrogation of the interplay between histone modifications in single cells.

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