scholarly journals Global architecture of the nucleus in single cells by DNA seqFISH+ and multiplexed immunofluorescence

2020 ◽  
Author(s):  
Yodai Takei ◽  
Jina Yun ◽  
Noah Ollikainen ◽  
Shiwei Zheng ◽  
Nico Pierson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cells ◽  
Hidden Variables ◽  
Embryonic Stem ◽  
Super Resolution ◽  
Molecular Heterogeneity ◽  
Organization Studies ◽  
Chromatin States ◽  
Resolution Imaging ◽  
Nascent Transcripts

AbstractIdentifying the relationships between chromosome structures, chromatin states, and gene expression is an overarching goal of nuclear organization studies. Because individual cells are highly variable at all three levels, it is essential to map all three modalities in the same single cell, a task that has been difficult to accomplish with existing tools. Here, we report the direct super-resolution imaging of over 3,660 chromosomal loci in single mouse embryonic stem cells (mESCs) by DNA seqFISH+, along with 17 chromatin marks by sequential immunofluorescence (IF) and the expression profile of 70 RNAs, in the same cells. We discovered that the nucleus is separated into zones defined by distinct combinatorial chromatin marks. DNA loci and nascent transcripts are enriched at the interfaces between specific nuclear zones, and the level of gene expression correlates with an association between active or nuclear speckle zones. Our analysis also uncovered several distinct mESCs subpopulations with characteristic combinatorial chromatin states that extend beyond known transcriptional states, suggesting that the metastable states of mESCs are more complex than previously appreciated. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3K27me3 and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. The long-lived chromatin states may represent “hidden variables” that explain the observed functional heterogeneity in differentiation decisions in single mESCs. Our integrated spatial genomics approach can be used to further explore the existence and biological relevance of molecular heterogeneity within cell populations in diverse biological systems.

scholarly journals Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-17 ◽  
Author(s):  
Sabine Conrad ◽  
Hossein Azizi ◽  
Maryam Hatami ◽  
Mikael Kubista ◽  
Michael Bonin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Adult Stem Cells ◽  
Single Cells ◽  
Human Fibroblasts ◽  
Embryonic Stem ◽  
Human Adult

The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen−/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profilein vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers.

scholarly journals Quantitative Transcription Factor Analysis of Undifferentiated Single Human Embryonic Stem Cells

2009 ◽  
Vol 55 (12) ◽  
pp. 2162-2170 ◽  
Author(s):  
Anders Ståhlberg ◽  
Martin Bengtsson ◽  
Martin Hemberg ◽  
Henrik Semb
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Cell Line ◽  
Human Embryonic Stem Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Measurement Noise ◽  
Sox2 Expression

Abstract Background: Human embryonic stem cells (hESCs) require expression of transcription factor genes POU5F1 (POU class 5 homeobox 1), NANOG (Nanog homeobox), and SOX2 [SRY (sex determining region Y)-box 2] to maintain their capacity for self-renewal and pluripotency. Because of the heterogeneous nature of cell populations, it is desirable to study the gene regulation in single cells. Large and potentially important fluctuations in a few cells cannot be detected at the population scale with microarrays or sequencing technologies. We used single-cell gene expression profiling to study cell heterogeneity in hESCs. Methods: We collected 47 single hESCs from cell line SA121 manually by glass capillaries and 57 single hESCs from cell line HUES3 by flow cytometry. Single hESCs were lysed and reverse-transcribed. Reverse-transcription quantitative real-time PCR was then used to measure the expression POU5F1, NANOG, SOX2, and the inhibitor of DNA binding genes ID1, ID2, and ID3. A quantitative noise model was used to remove measurement noise when pairwise correlations were estimated. Results: The numbers of transcripts per cell varied >100-fold between cells and showed lognormal features. POU5F1 expression positively correlated with ID1 and ID3 expression (P < 0.05) but not with NANOG or SOX2 expression. When we accounted for measurement noise, SOX2 expression was also correlated with ID1, ID2, and NANOG expression (P < 0.05). Conclusions: We demonstrate an accurate method for transcription profiling of individual hESCs. Cell-to-cell variability is large and is at least partly nonrandom because we observed correlations between core transcription factors. High fluctuations in gene expression may explain why individual cells in a seemingly undifferentiated cell population have different susceptibilities for inductive cues.

scholarly journals A dual role for H2A.Z.1 in modulating the dynamics of RNA Polymerase II initiation and elongation

2020 ◽  
Author(s):  
Constantine Mylonas ◽  
Alexander L. Auld ◽  
Choongman Lee ◽  
Ibrahim I. Cisse ◽  
Laurie A. Boyer
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Super Resolution ◽  
Fine Tuning ◽  
Transcriptional Initiation ◽  
Single Molecule Level

AbstractRNAPII pausing immediately downstream of the transcription start site (TSS) is a critical rate limiting step at most metazoan genes that allows fine-tuning of gene expression in response to diverse signals1–5. During pause-release, RNA Polymerase II (RNAPII) encounters an H2A.Z.1 nucleosome6–8, yet how this variant contributes to transcription is poorly understood. Here, we use high resolution genomic approaches2,9 (NET-seq and ChIP-nexus) along with live cell super-resolution microscopy (tcPALM)10 to investigate the role of H2A.Z.1 on RNAPII dynamics in embryonic stem cells (ESCs). Using a rapid, inducible protein degron system11 combined with transcriptional initiation and elongation inhibitors, our quantitative analysis shows that H2A.Z.1 slows the release of RNAPII, impacting both RNAPII and NELF dynamics at a single molecule level. We also find that H2A.Z.1 loss has a dramatic impact on nascent transcription at stably paused, signal-dependent genes. Furthermore, we demonstrate that H2A.Z.1 inhibits re-assembly and re-initiation of the PIC to reinforce the paused state and acts as a strong additional pause signal at stably paused genes. Together, our study suggests that H2A.Z.1 fine-tunes gene expression by regulating RNAPII kinetics in mammalian cells.

scholarly journals Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probes

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yanxiang Ni ◽  
Bo Cao ◽  
Tszshan Ma ◽  
Gang Niu ◽  
Yingdong Huo ◽  
...  
Keyword(s):  
Repetitive Dna ◽  
Molecular Beacon ◽  
Genomic Sequence ◽  
Single Cells ◽  
Nuclear Genome ◽  
Super Resolution ◽  
Resolution Limit ◽  
Fish Method ◽  
Resolution Imaging

High-resolution visualization of short non-repetitive DNA in situ in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence in situ hybridization (FISH) using Oligopaint probes have enabled super-resolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used three-dimensional stochastic optical reconstruction microscopy (3D-STORM) with optimized imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and only 29–34 individual MB probes, we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome, respectively. We demonstrated our MB-based FISH method was capable of visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution.

scholarly journals Microtubules Regulate Spatial Localization and Availability of Insulin Granules in Pancreatic Beta Cells

2019 ◽  
Author(s):  
Kai M. Bracey ◽  
Kung-Hsien Ho ◽  
Dmitry Yampolsky ◽  
Guoqiang Gu ◽  
Irina Kaverina ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Single Cells ◽  
Critical Role ◽  
Super Resolution ◽  
Transport Dynamics ◽  
Insulin Granules ◽  
Resolution Imaging ◽  
Glucose Stimulated Insulin Secretion

AbstractTwo key prerequisites for glucose stimulated insulin secretion (GSIS) in Beta cells are the proximity of insulin granules to the plasma membrane and their anchoring or docking to the plasma membrane (PM). While recent evidence has indicated that both of these factors are altered in the context of diabetes, it is unclear what regulates localization of insulin and its interactions with the PM within single cells. Here we demonstrate that microtubule (MT) motor mediated transport dynamics have a critical role in regulating both factors. Super-resolution imaging shows that while the MT cytoskeleton resembles a random meshwork in the cells’ interior, MTs near the cells surface are preferentially aligned with the PM. Computational modeling demonstrates two consequences of this alignment. First, this structured MT network preferentially withdraws granules from the PM. Second, the binding and transport of insulin granules by MT motors prevents their stable anchoring to the PM. The MT cytoskeleton thus negatively regulates GSIS by both limiting the amount of insulin proximal to the PM and preventing/breaking interactions between the PM and the remaining nearby insulin. These results predict that altering MT structure in beta cells can be used to tune GSIS. Thus, our study points to a potential of an alternative therapeutic strategy for diabetes by targeting specific MT regulators.

scholarly journals Single cell, super-resolution imaging reveals an acid pH-dependent conformational switch in SsrB regulates SPI-2

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrew Tze Fui Liew ◽  
Yong Hwee Foo ◽  
Yunfeng Gao ◽  
Parisa Zangoui ◽  
Moirangthem Kiran Singh ◽  
...  
Keyword(s):  
Dna Binding ◽  
Single Molecule ◽  
Single Cells ◽  
Super Resolution ◽  
Single Particle Tracking ◽  
Live Cells ◽  
Bacterial Cells ◽  
Acid Ph ◽  
Resolution Imaging ◽  
Ph Dependent

After Salmonella is phagocytosed, it resides in an acidic vacuole. Its cytoplasm acidifies to pH 5.6; acidification activates pathogenicity island 2 (SPI-2). SPI-2 encodes a type three secretion system whose effectors modify the vacuole, driving endosomal tubulation. Using super-resolution imaging in single bacterial cells, we show that low pH induces expression of the SPI-2 SsrA/B signaling system. Single particle tracking, atomic force microscopy, and single molecule unzipping assays identified pH-dependent stimulation of DNA binding by SsrB. A so-called phosphomimetic form (D56E) was unable to bind to DNA in live cells. Acid-dependent DNA binding was not intrinsic to regulators, as PhoP and OmpR binding was not pH-sensitive. The low level of SPI-2 injectisomes observed in single cells is not due to fluctuating SsrB levels. This work highlights the surprising role that acid pH plays in virulence and intracellular lifestyles of Salmonella; modifying acid survival pathways represents a target for inhibiting Salmonella.

scholarly journals Multiview super-resolution microscopy

2021 ◽  
Author(s):  
Yicong Wu ◽  
Xiaofei Han ◽  
Yijun Su ◽  
Melissa Glidewell ◽  
Jonathan S. Daniels ◽  
...  
Keyword(s):  
Single Cells ◽  
Super Resolution ◽  
Structured Illumination ◽  
Reconstruction Algorithms ◽  
Structured Illumination Microscopy ◽  
Drosophila Wing ◽  
Resolution Imaging ◽  
Imaginal Disks ◽  
Developing Neurons ◽  
Super Resolution Microscopy

We enhance the performance of confocal microscopy over imaging scales spanning tens of nanometers to millimeters in space and milliseconds to hours in time, improving volumetric resolution more than 10-fold while simultaneously reducing phototoxicity. We achieve these gains via an integrated, four-pronged approach: 1) developing compact line-scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; 2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; 3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labeled, thick samples; 4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than twenty distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae, and adults; myoblasts in Drosophila wing imaginal disks; and mouse renal, esophageal, cardiac, and brain tissues.

scholarly journals Manifold alignment reveals correspondence between single cell transcriptome and epigenome dynamics

2017 ◽  
Author(s):  
Joshua D. Welch ◽  
Alexander J. Hartemink ◽  
Jan F. Prins
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Epigenetic Modification ◽  
Single Cells ◽  
Embryonic Stem ◽  
Genomic Data ◽  
Chromatin Accessibility ◽  
Data Sets ◽  
Cell Transcriptome ◽  
Dynamic Interplay

AbstractSingle cell genomic techniques promise to yield key insights into the dynamic interplay between gene expression and epigenetic modification. However, the experimental difficulty of performing multiple measurements on the same cell currently limits efforts to combine multiple genomic data sets into a united picture of single cell variation. We show that it is possible to construct cell trajectories, reflecting the changes that occur in a sequential biological process, from single cell ATAC-seq, bisulfite sequencing, and ChIP-seq data. In addition, we present an approach called MATCHER that computationally circumvents the experimental difficulties inherent in performing multiple genomic measurements on a single cell by inferring correspondence between single cell transcriptomic and epigenetic measurements performed on different cells of the same type. MATCHER works by first learning a separate manifold for the trajectory of each kind of genomic data, then aligning the manifolds to infer a shared trajectory in which cells measured using different techniques are directly comparable. Using scM&T-seq data, we confirm that MATCHER accurately predicts true single cell correlations between DNA methylation and gene expression without using known cell correspondence information. We also used MATCHER to infer correlations among gene expression, chromatin accessibility, and histone modifications in single mouse embryonic stem cells. These results reveal the dynamic interplay between epigenetic changes and gene expression underlying the transition from pluripotency to differentiation priming. Our work is a first step toward a united picture of heterogeneous transcriptomic and epigenetic states in single cells.

scholarly journals Single-cell analysis of Hodgkin and Reed-Sternberg cells: molecular heterogeneity of gene expression and p53 mutations

Blood ◽  
1993 ◽  
Vol 81 (11) ◽  
pp. 3097-3115 ◽  
Author(s):  
LH Trumper ◽  
G Brady ◽  
A Bagg ◽  
D Gray ◽  
SL Loke ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Hodgkin's Disease ◽  
Single Cell Analysis ◽  
Hodgkin’S Disease ◽  
Single Cells ◽  
Gene Expression Pattern ◽  
Pcr Amplification ◽  
Lymphoid Cells ◽  
Molecular Heterogeneity

Abstract We have used a single-cell based polymerase chain reaction (PCR) amplification technique to examine the gene expression pattern in single Hodgkin's and Reed-Sternberg (H&RS) cells from seven patients with Hodgkin's disease. Single cells were isolated from lymph nodes obtained at diagnosis (5 of 7 patients) or in first or second relapse (2 of 7 patients). Gene expression was examined by hybridization to a panel of 22 cDNA probes. Forty-nine H&RS cells (and 23 CD3+ or CD20+ lymphocytes as controls) from four patients with nodular sclerosing Hodgkin's disease (HD) and one patient each with lymphocyte predominant and mixed-cellularity HD were successfully analyzed by PCR. This analysis provides evidence that single H&RS cells can coexpress genes characteristic of several hematopoietic lineages (monocytes and lymphocytes). Genes characteristic of activated lymphoid cells are expressed in most H&RS cells. Heterogeneity of expression for certain genes between different cases was found and may eventually define molecular subgroups of HD. These findings indicate that H&RS cells of HD resemble activated hematopoietic cells. Phenotypically similar cells from different cases exhibit characteristic molecular differences. In one patient, 5 of 7 single RS cells showed identical p53 cDNA mutations at codon 246 on specific reverse transcriptase [RT]-PCR and sequencing of exons 5 through 8. The novel experimental approach may provide a valuable tool for understanding the molecular events in newly diagnosed Hodgkin's disease and progression of the disease.

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