scholarly journals Quantitative imaging of RNA polymerase II activity in plants reveals the single-cell basis of tissue-wide transcriptional dynamics

2020 ◽  
Author(s):  
Simon Alamos ◽  
Armando Reimer ◽  
Krishna K. Niyogi ◽  
Hernan G. Garcia
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Cells ◽  
Quantitative Imaging ◽  
Cellular Heterogeneity ◽  
Transcription Rate ◽  
Animal Cells ◽  
Transcriptional Dynamics ◽  
Production Variability ◽  
Experimental Treatments

AbstractThe responses of plants to their environment often hinge on the spatiotemporal dynamics of transcriptional regulation. While live-imaging tools have been used extensively to quantitatively capture rapid transcriptional dynamics in living animal cells, lack of implementation of these technologies in plants has limited concomitant quantitative studies. Here, we applied the PP7 and MS2 RNA-labeling technologies for the quantitative imaging of RNA polymerase II activity dynamics in single cells of living plants as they respond to experimental treatments. Using this technology, we count nascent RNA transcripts in real-time in Nicotiana benthamiana (tobacco) and Arabidopsis thaliana (Arabidopsis). Examination of heat shock reporters revealed that plant tissues respond to external signals by modulating the number of cells engaged in transcription rather than the transcription rate of active cells. This switch-like behavior, combined with cell-to-cell variability in transcription rate, results in mRNA production variability spanning three orders of magnitude. We determined that cellular heterogeneity stems mainly from the stochasticity intrinsic to individual alleles. Taken together, our results demonstrate that it is now possible to quantitatively study the dynamics of transcriptional programs in single cells of living plants.

scholarly journals Simultaneous CUT&Tag profiling of the accessible and silenced regulome in single cells

2021 ◽  
Author(s):  
Derek H Janssens ◽  
Dominik J. Otto ◽  
Manu Setty ◽  
Kami Ahmad ◽  
Steven Henikoff
Keyword(s):  
High Resolution ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
High Throughput ◽  
Single Cells ◽  
Regulatory Elements ◽  
Small Samples ◽  
Signal Deconvolution ◽  
Chromatin Profiling

Cleavage Under Targets & Tagmentation (CUT&Tag) is an antibody-directed transposase tethering strategy for in situ chromatin profiling in small samples and single cells. We describe a modified CUT&Tag protocol using a mixture of an antibody to the initiation form of RNA Polymerase II (Pol2 Serine-5 phosphate) and an antibody to repressive Polycomb domains (H3K27me3) followed by computational signal deconvolution to produce high-resolution maps of both the active and repressive regulomes in single cells. The ability to seamlessly map active promoters, enhancers and repressive regulatory elements using a single workflow provides a complete regulome profiling strategy suitable for high-throughput single-cell platforms.

scholarly journals Quantitative imaging of mammalian transcriptional dynamics: from single cells to whole embryos

BMC Biology ◽  
2016 ◽  
Vol 14 (1) ◽  
Author(s):  
Ziqing W. Zhao ◽  
Melanie D. White ◽  
Stephanie Bissiere ◽  
Valeria Levi ◽  
Nicolas Plachta
Keyword(s):  
Single Cells ◽  
Quantitative Imaging ◽  
Transcriptional Dynamics

scholarly journals Quantification of single-cell nanoparticle concentrations and the distribution of these concentrations in cell population

2014 ◽  
Vol 11 (94) ◽  
pp. 20131152 ◽  
Author(s):  
Jason T. Rashkow ◽  
Sunny C. Patel ◽  
Ryan Tappero ◽  
Balaji Sitharaman
Keyword(s):  
Single Cell ◽  
Inductively Coupled Plasma ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Large Population ◽  
Cellular Heterogeneity ◽  
Physical Parameters ◽  
Animal Cells ◽  
Promising Tool ◽  
Icp Ms

Quantification of nanoparticle uptake into cells is necessary for numerous applications in cellular imaging and therapy. Herein, synchrotron X-ray fluorescence (SXRF) microscopy, a promising tool to quantify elements in plant and animal cells, was employed to quantify and characterize the distribution of titanium dioxide (TiO 2 ) nanosphere uptake in a population of single cells. These results were compared with average nanoparticle concentrations per cell obtained by widely used inductively coupled plasma mass spectrometry (ICP-MS). The results show that nanoparticle concentrations per cell quantified by SXRF were of one to two orders of magnitude greater compared with ICP-MS. The SXRF results also indicate a Gaussian distribution of the nanoparticle concentration per cell. The results suggest that issues relevant to the field of single-cell analysis, the limitation of methods to determine physical parameters from large population averages leading to potentially misleading information and the lack of any information about the cellular heterogeneity are equally relevant for quantification of nanoparticles in cell populations.

High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq)

2021 ◽  
Vol 220 (12) ◽  
Author(s):  
Daniel A. Bartlett ◽  
Vishnu Dileep ◽  
Tetsuya Handa ◽  
Yasuyuki Ohkawa ◽  
Hiroshi Kimura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Cell ◽  
Rna Polymerase Ii ◽  
High Throughput ◽  
Single Cells ◽  
Mapping Method ◽  
Ctcf Binding ◽  
Linear Amplification ◽  
Site Mapping ◽  
Chromatin Profiling

Chromatin profiling in single cells has been extremely challenging and almost exclusively limited to histone proteins. In cases where single-cell methods have shown promise, many require highly specialized equipment or cell type–specific protocols and are relatively low throughput. Here, we combine the advantages of tagmentation, linear amplification, and combinatorial indexing to produce a high-throughput single-cell DNA binding site mapping method that is simple, inexpensive, and capable of multiplexing several independent samples per experiment. Targeted insertion of promoters sequencing (TIP-seq) uses Tn5 fused to proteinA to insert a T7 RNA polymerase promoter adjacent to a chromatin protein of interest. Linear amplification of flanking DNA with T7 polymerase before sequencing library preparation provides ∼10-fold higher unique reads per single cell compared with other methods. We applied TIP-seq to map histone modifications, RNA polymerase II (RNAPII), and transcription factor CTCF binding sites in single human and mouse cells.

Darkness Affects Differentially the Expression of Plastid-Encoded Genes and Delays the Senescence-Induced Down-Regulation of Chloroplast Transcription in Cotyledons of Cucurbita pepo L. (Zucchini)

2011 ◽  
Vol 66 (3-4) ◽  
pp. 159-166 ◽  
Author(s):  
Kiril Mishev ◽  
Anna Dimitrova ◽  
Evguéni D. Ananiev
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Rate ◽  
Dot Blot ◽  
Chloroplast Gene Expression ◽  
Chloroplast Transcription ◽  
Nuclear Rna ◽  
Dark Stress ◽  
Nuclear Rna Polymerase

In contrast to differentiated leaves, the regulatory mechanisms of chloroplast gene expression in darkened cotyledons have not been elucidated. Although some results have been reported indicating accelerated senescence in Arabidopsis upon reillumination, the capacity of cotyledons to recover after dark stress remains unclear. We analysed the effect of twodays dark stress, applied locally or at the whole-plant level, on plastid gene expression in zucchini cotyledons. Our results showed that in the dark the overall chloroplast transcription rate was much more inhibited than the nuclear run-on transcription. While the activities of the plastid-encoded RNA polymerase (PEP) and nuclear RNA polymerase II were strongly reduced, the activities of the nuclear-encoded plastid RNA polymerase (NEP) and nuclear RNA polymerase I were less affected. During recovery upon reillumination, chloroplast transcription in the cotyledons was strongly stimulated (3-fold) compared with the naturally senescing controls, suggesting delayed senescence. Northern blot and dot blot analyses of the expression of key chloroplast-encoded photosynthetic genes showed that in contrast to psbA, which remained almost unaffected, both the transcription rate and mRNA content of psaB and rbcL were substantially decreased

scholarly journals Activation of the ATF3 gene through a co-ordinated amino acid-sensing response programme that controls transcriptional regulation of responsive genes following amino acid limitation

2006 ◽  
Vol 401 (1) ◽  
pp. 299-307 ◽  
Author(s):  
Yuan-Xiang Pan ◽  
Hong Chen ◽  
Michelle M. Thiaville ◽  
Michael S. Kilberg
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Initial Increase ◽  
Transcription Rate ◽  
Activating Transcription Factor 3 ◽  
Histone H4 ◽  
Histone H4 Acetylation

Expression of ATF3 (activating transcription factor 3) is induced by a variety of environmental stress conditions, including nutrient limitation. In the present study, we demonstrate that the increase in ATF3 mRNA content following amino acid limitation of human HepG2 hepatoma cells is dependent on transcriptional activation of the ATF3 gene, through a highly co-ordinated amino acid-responsive programme of transcription factor synthesis and action. Studies using transient over-expression and knockout fibroblasts showed that several ATF and C/EBP (CCAAT/enhancer-binding protein) family members contribute to ATF3 regulation. Promoter analysis showed that a C/EBP-ATF composite site at −23 to −15 bp relative to the transcription start site of the ATF3 gene functions as an AARE (amino acid response element). Chromatin immunoprecipitation demonstrated that amino acid limitation increased ATF4, ATF3, and C/EBPβ binding to the ATF3 promoter, but the kinetics of each was markedly different. Immediately following histidine removal, there was a rapid increase in histone H3 acetylation prior to an enhancement in ATF4 binding and in histone H4 acetylation. These latter changes closely paralleled the initial increase in RNA pol II (RNA polymerase II) binding to the promoter and in the transcription rate from the ATF3 gene. The increase in ATF3 and C/EBPβ binding was considerably slower and more closely correlated with a decline in transcription rate. A comparison of the recruitment patterns between ATF and C/EBP transcription factors and RNA polymerase II at the AARE of several amino acid-responsive genes revealed that a highly co-ordinated response programme controls the transcriptional activation of these genes following amino acid limitation.

scholarly journals Integrative analysis of RNA polymerase II and transcriptional dynamics upon MYC activation

2017 ◽  
Vol 27 (10) ◽  
pp. 1658-1664 ◽  
Author(s):  
Stefano de Pretis ◽  
Theresia R. Kress ◽  
Marco J. Morelli ◽  
Arianna Sabò ◽  
Chiara Locarno ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Integrative Analysis ◽  
Transcriptional Dynamics
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