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

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.

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

10.1101/2020.01.01.892299 ◽

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.

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

Physiological Genomics ◽

10.1152/physiolgenomics.00065.2020 ◽

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.

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

Scientific Reports ◽

10.1038/s41598-021-82539-z ◽

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.

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Integration, coincidence detection and resonance in networks of spiking neurons expressing Gamma oscillations and asynchronous states

PLoS Computational Biology ◽

10.1371/journal.pcbi.1009416 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009416

Author(s):

Eduarda Susin ◽

Alain Destexhe

Keyword(s):

Cerebral Cortex ◽

Pyramidal Neurons ◽

Network Models ◽

Cell Types ◽

Gamma Oscillations ◽

Coincidence Detection ◽

Biophysical Models ◽

Firing Mode ◽

Different Cell Types ◽

External Stimuli

Gamma oscillations are widely seen in the awake and sleeping cerebral cortex, but the exact role of these oscillations is still debated. Here, we used biophysical models to examine how Gamma oscillations may participate to the processing of afferent stimuli. We constructed conductance-based network models of Gamma oscillations, based on different cell types found in cerebral cortex. The models were adjusted to extracellular unit recordings in humans, where Gamma oscillations always coexist with the asynchronous firing mode. We considered three different mechanisms to generate Gamma, first a mechanism based on the interaction between pyramidal neurons and interneurons (PING), second a mechanism in which Gamma is generated by interneuron networks (ING) and third, a mechanism which relies on Gamma oscillations generated by pacemaker chattering neurons (CHING). We find that all three mechanisms generate features consistent with human recordings, but that the ING mechanism is most consistent with the firing rate change inside Gamma bursts seen in the human data. We next evaluated the responsiveness and resonant properties of these networks, contrasting Gamma oscillations with the asynchronous mode. We find that for both slowly-varying stimuli and precisely-timed stimuli, the responsiveness is generally lower during Gamma compared to asynchronous states, while resonant properties are similar around the Gamma band. We could not find conditions where Gamma oscillations were more responsive. We therefore predict that asynchronous states provide the highest responsiveness to external stimuli, while Gamma oscillations tend to overall diminish responsiveness.

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CA Mutation N57A Has Distinct Strain-Specific HIV-1 Capsid Uncoating and Infectivity Phenotypes

Journal of Virology ◽

10.1128/jvi.00214-19 ◽

2019 ◽

Vol 93 (9) ◽

Cited By ~ 2

Author(s):

Douglas K. Fischer ◽

Akatsuki Saito ◽

Christopher Kline ◽

Romy Cohen ◽

Simon C. Watkins ◽

...

Keyword(s):

Cell Cycle ◽

Amino Acid ◽

Cell Types ◽

Careful Consideration ◽

Immunodeficiency Virus ◽

Cell Cycle Dependence ◽

Cycle Dependent ◽

Multiple Strains ◽

Different Cell Types ◽

Hiv 1

ABSTRACTThe ability of human immunodeficiency virus type 1 (HIV-1) to transduce nondividing cells is key to infecting terminally differentiated macrophages, which can serve as a long-term reservoir of HIV-1 infection. The mutation N57A in the viral CA protein renders HIV-1 cell cycle dependent, allowing examination of HIV-1 infection of nondividing cells. Here, we show that the N57A mutation confers a postentry infectivity defect that significantly differs in magnitude between the common lab-adapted molecular clones HIV-1NL4-3(>10-fold) and HIV-1LAI(2- to 5-fold) in multiple human cell lines and primary CD4+T cells. Capsid permeabilization and reverse transcription are altered when N57A is incorporated into HIV-1NL4-3but not HIV-1LAI. The N57A infectivity defect is significantly exacerbated in both virus strains in the presence of cyclosporine (CsA), indicating that N57A infectivity is dependent upon CA interacting with host factor cyclophilin A (CypA). Adaptation of N57A HIV-1LAIselected for a second CA mutation, G94D, which rescued the N57A infectivity defect in HIV-1LAIbut not HIV-1NL4-3. The rescue of N57A by G94D in HIV-1LAIis abrogated by CsA treatment in some cell types, demonstrating that this rescue is CypA dependent. An examination of over 40,000 HIV-1 CA sequences revealed that the four amino acids that differ between HIV-1NL4-3and HIV-1LAICA are polymorphic, and the residues at these positions in the two strains are widely prevalent in clinical isolates. Overall, a few polymorphic amino acid differences between two closely related HIV-1 molecular clones affect the phenotype of capsid mutants in different cell types.IMPORTANCEThe specific mechanisms by which HIV-1 infects nondividing cells are unclear. A mutation in the HIV-1 capsid protein abolishes the ability of the virus to infect nondividing cells, serving as a tool to examine cell cycle dependence of HIV-1 infection. We have shown that two widely used HIV-1 molecular clones exhibit significantly different N57A infectivity phenotypes due to fewer than a handful of CA amino acid differences and that these clones are both represented in HIV-infected individuals. As such minor differences in closely related HIV-1 strains may impart significant infectivity differences, careful consideration should be given to drawing conclusions from one particular HIV-1 clone. This study highlights the potential for significant variation in results with the use of multiple strains and possible unanticipated effects of natural polymorphisms.

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Antiproliferative Effect of Dihydroxyacetone on Trypanosoma brucei Bloodstream Forms: Cell Cycle Progression, Subcellular Alterations, and Cell Death

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00423-07 ◽

2007 ◽

Vol 51 (11) ◽

pp. 3960-3968 ◽

Cited By ~ 27

Author(s):

Néstor L. Uzcátegui ◽

Didac Carmona-Gutiérrez ◽

Viola Denninger ◽

Caroline Schoenfeld ◽

Florian Lang ◽

...

Keyword(s):

Energy Source ◽

Cell Cycle ◽

Trypanosoma Brucei ◽

Rational Design ◽

Cell Cycle Progression ◽

Cell Types ◽

Cycle Arrest ◽

Starting Point ◽

M Phase ◽

Different Cell Types

ABSTRACT We evaluated the effects of dihydroxyacetone (DHA) on Trypanosoma brucei bloodstream forms. DHA is considered an energy source for many different cell types. T. brucei takes up DHA readily due to the presence of aquaglyceroporins. However, the parasite is unable to use it as a carbon source because of the absence of DHA kinase (DHAK). We could not find a homolog of the relevant gene in the genomic database of T. brucei and have been unable to detect DHAK activity in cell lysates of the parasite, and the parasite died quickly if DHA was the sole energy source in the medium. In addition, during trypanosome cultivation, DHA induced growth inhibition with a 50% inhibitory concentration of about 1 mM, a concentration that is completely innocuous to mammals. DHA caused cell cycle arrest in the G2/M phase of up to 70% at a concentration of 2 mM. Also, DHA-treated parasites showed profound ultrastructural alterations, including an increase of vesicular structures within the cytosol and the presence of multivesicular bodies, myelin-like structures, and autophagy-like vacuoles, as well as a marked disorder of the characteristic mitochondrion structure. Based on the toxicity of DHA for trypanosomes compared with mammals, we consider DHA a starting point for a rational design of new trypanocidal drugs.

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The Histone Modification Code in the Pathogenesis of Autoimmune Diseases

Mediators of Inflammation ◽

10.1155/2017/2608605 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 27

Author(s):

Yasuto Araki ◽

Toshihide Mimura

Keyword(s):

Autoimmune Diseases ◽

Histone Modifications ◽

Lupus Erythematosus ◽

Cell Types ◽

Biliary Cirrhosis ◽

Loss Of Self ◽

Self Tolerance ◽

Different Cell Types

Autoimmune diseases are chronic inflammatory disorders caused by a loss of self-tolerance, which is characterized by the appearance of autoantibodies and/or autoreactive lymphocytes and the impaired suppressive function of regulatory T cells. The pathogenesis of autoimmune diseases is extremely complex and remains largely unknown. Recent advances indicate that environmental factors trigger autoimmune diseases in genetically predisposed individuals. In addition, accumulating results have indicated a potential role of epigenetic mechanisms, such as histone modifications, in the development of autoimmune diseases. Histone modifications regulate the chromatin states and gene transcription without any change in the DNA sequence, possibly resulting in phenotype alteration in several different cell types. In this paper, we discuss the significant roles of histone modifications involved in the pathogenesis of autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, primary biliary cirrhosis, and type 1 diabetes.

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

Physiological Genomics ◽

10.1152/physiolgenomics.00049.2017 ◽

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.

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Computational approaches towards reducing contamination in single-cell RNA-seq data

10.1101/2020.07.15.205062 ◽

2020 ◽

Author(s):

Siamak Yousefi ◽

Hao Chen ◽

Jesse F. Ingels ◽

Melinda S. McCarty ◽

Arthur G. Centeno ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Real Life ◽

Cell Types ◽

Cell Capture ◽

Rna Seq ◽

Sequence Analyses ◽

Cell Functions ◽

Biological Interpretation ◽

Different Cell Types

SUMMARYSingle cell RNA sequencing has enabled quantification of single cells and identification of different cell types and subtypes as well as cell functions in different tissues. Single cell RNA sequence analyses assume acquired RNAs correspond to cells, however, RNAs from contamination within the input data are also captured by these assays. The sequencing of background contamination as well as unwanted cells making their way to the final assay Potentially confound the correct biological interpretation of single cell transcriptomic data. Here we demonstrate two approaches to deal with background contamination as well as profiling of unwanted cells in the assays. We use three real-life datasets of whole-cell capture and nucleotide single-cell captures generated by Fluidigm and 10x technologies and show that these methods reduce the effect of contamination, strengthen clustering of cells and improves biological interpretation.

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Mapping multicellular programs from single-cell profiles

10.1101/2020.08.11.245472 ◽

2020 ◽

Author(s):

Livnat Jerby-Arnon ◽

Aviv Regev

Keyword(s):

Single Cell ◽

Spatial Data ◽

Spatial Information ◽

Single Cells ◽

Cell Types ◽

Risk Genes ◽

Health And Disease ◽

Different Cell Types ◽

Cellular Components ◽

Acting In Concert

ABSTRACTTissue homeostasis relies on orchestrated multicellular circuits, where interactions between different cell types dynamically balance tissue function. While single-cell genomics identifies tissues’ cellular components, deciphering their coordinated action remains a major challenge. Here, we tackle this problem through a new framework of multicellular programs: combinations of distinct cellular programs in different cell types that are coordinated together in the tissue, thus forming a higher order functional unit at the tissue, rather than only cell, level. We develop the open-access DIALOGUE algorithm to systematically uncover such multi-cellular programs not only from spatial data, but even from tissue dissociated and profiled as single cells, e.g., by single-cell RNA-Seq. Tested on spatial transcriptomes from the mouse hypothalamus, DIALOGUE recovered spatial information, predicted the properties of a cell’s environment only based on its transcriptome, and identified multicellular programs that mark animal behavior. Applied to brain samples and colon biopsies profiled by scRNA-Seq, DIALOGUE identified multicellular configurations that mark Alzheimer’s disease and ulcerative colitis (UC), including a program spanning five cell types that is predictive of response to anti-TNF therapy in UC patients and enriched for UC risk genes from GWAS, each acting in different cell types, but all cells acting in concert. Taken together, our study provides a novel conceptual and methodological framework to unravel multicellular regulation in health and disease.

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