scholarly journals 3 ′-5 ′ crosstalk contributes to transcriptional bursting

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Massimo Cavallaro ◽  
Mark D. Walsh ◽  
Matt Jones ◽  
James Teahan ◽  
Simone Tiberi ◽  
...  
Keyword(s):  
Stochastic Process ◽  
Mammalian Cells ◽  
Burst Size ◽  
Noise Analysis ◽  
Cell Populations ◽  
Burst Frequency ◽  
Transcriptional Noise ◽  
Mrna Species ◽  
Genome Wide ◽  
Transcriptional Bursting

Abstract Background Transcription in mammalian cells is a complex stochastic process involving shuttling of polymerase between genes and phase-separated liquid condensates. It occurs in bursts, which results in vastly different numbers of an mRNA species in isogenic cell populations. Several factors contributing to transcriptional bursting have been identified, usually classified as intrinsic, in other words local to single genes, or extrinsic, relating to the macroscopic state of the cell. However, some possible contributors have not been explored yet. Here, we focus on processes at the 3 ′ and 5 ′ ends of a gene that enable reinitiation of transcription upon termination. Results Using Bayesian methodology, we measure the transcriptional bursting in inducible transgenes, showing that perturbation of polymerase shuttling typically reduces burst size, increases burst frequency, and thus limits transcriptional noise. Analysis based on paired-end tag sequencing (PolII ChIA-PET) suggests that this effect is genome wide. The observed noise patterns are also reproduced by a generative model that captures major characteristics of the polymerase flux between the ends of a gene and a phase-separated compartment. Conclusions Interactions between the 3 ′ and 5 ′ ends of a gene, which facilitate polymerase recycling, are major contributors to transcriptional noise.

scholarly journals 3’-5’ crosstalk contributes to transcriptional bursting

2019 ◽  
Author(s):  
Massimo Cavallaro ◽  
Mark D. Walsh ◽  
Matt Jones ◽  
James Teahan ◽  
Simone Tiberi ◽  
...  
Keyword(s):  
Stochastic Process ◽  
Mammalian Cells ◽  
Burst Size ◽  
Noise Analysis ◽  
Cell Populations ◽  
Burst Frequency ◽  
Transcriptional Noise ◽  
Mrna Species ◽  
Genome Wide ◽  
Transcriptional Bursting

AbstractBackgroundTranscription in mammalian cells is a complex stochastic process involving shuttling of polymerase between genes and phase-separated liquid condensates. It occurs in bursts, which results in vastly different numbers of an mRNA species in isogenic cell populations. Several factors contributing to transcriptional bursting have been identified, usually classified as intrinsic, in other words local to single genes, or extrinsic, relating to the macroscopic state of the cell. However, some possible contributors have not been explored yet. Here, we focus on processes at the 3’ and 5’ ends of a gene that enable reinitiation of transcription upon termination.ResultsUsing Bayesian methodology, we measure the transcriptional bursting in inducible transgenes, showing that perturbation of polymerase shuttling typically reduces burst size, increases burst frequency, and thus limits transcriptional noise. Analysis based on paired-end tag sequencing (PolII ChIA-PET) suggests that this effect is genome wide. The observed noise patterns are also reproduced by a generative model that captures major characteristics of the polymerase flux between the ends of a gene and a phase-separated compartment.ConclusionsInteractions between the 3’ and 5’ ends of a gene, which facilitate polymerase recycling, are major contributors to transcriptional noise.

scholarly journals Transcriptional bursting explains the noise-versus-mean relationship in mRNA and protein levels

2016 ◽  
Author(s):  
Roy D. Dar ◽  
Sydney M. Schaffer ◽  
Siddarth S. Dey ◽  
Jonathan E. Foley ◽  
Abhyudai Singh ◽  
...  
Keyword(s):  
Conflict Of Interest ◽  
Burst Size ◽  
Inverse Correlation ◽  
Recent Analysis ◽  
Mrna Levels ◽  
Burst Frequency ◽  
Expression Variability ◽  
Protein Levels ◽  
Transcriptional Bursting ◽  
Cell Expression

Recent analysis (Dey et al, 2015), demonstrates that the HIV-1 Long Terminal Repeat (HIV LTR) promoter exhibits a range of possible transcriptional burst sizes and frequencies for any mean-expression level. However, these results have also been interpreted as demonstrating that cell-to-cell expression variability (noise) and mean are uncorrelated, a significant deviation from previous results. Here, we re-examine the available mRNA and protein abundance data for the HIV LTR and find that noise in mRNA and protein expression scales inversely with the mean along analytically predicted transcriptional burst-size manifolds. We then experimentally perturb transcriptional activity to test a prediction of the multiple burst-size model: that increasing burst frequency will cause mRNA noise to decrease along given burst-size lines as mRNA levels increase. The data show that mRNA and protein noise decrease as mean expression increases, supporting the canonical inverse correlation between noise and mean.Conflict of InterestThe authors declare that they have no conflict of interest.

scholarly journals Transcriptional bursting shape autosomal dynamic random monoallelic expression in pre-gastrulation embryos

2020 ◽  
Author(s):  
C H Naik ◽  
D Chandel ◽  
S Mandal ◽  
S Gayen
Keyword(s):  
Monoallelic Expression ◽  
Wide Spread ◽  
Mammalian Development ◽  
Burst Frequency ◽  
Genome Wide ◽  
Allele Specific ◽  
Transcriptional Bursting ◽  
Burst Kinetics ◽  
Cell Expression ◽  
Early Mammalian Development

AbstractRecent years, allele-specific single cell RNA-seq (scRNA-seq) analysis have demonstrated wide-spread dynamic random monoallelic expression of autosomal genes (aRME). However, the origin of dynamic aRME remains poorly understood. It is believed that dynamic aRME is originated from discrete transcriptional burst of two alleles. Here, for the first time, we have profiled genome-wide pattern of dynamic aRME and allele-specific burst kinetics in mouse pre-gastrulation embryos. We found wide-spread dynamic aRME across the different lineages of pre-gastrulation embryos and which is linked to the allelic burst kinetics. Specially, we found that expression level and burst frequency are the key determinants of dynamic aRME. Altogether, our study provides significant insight about the origin of prevalent dynamic aRME and cell to cell expression heterogeneity during the early mammalian development.

scholarly journals Transcriptional bursts explain autosomal random monoallelic expression and affect allelic imbalance

2021 ◽  
Vol 17 (3) ◽  
pp. e1008772
Author(s):  
Anton J. M. Larsson ◽  
Christoph Ziegenhain ◽  
Michael Hagemann-Jensen ◽  
Björn Reinius ◽  
Tina Jacob ◽  
...  
Keyword(s):  
Allelic Imbalance ◽  
Burst Size ◽  
Single Cells ◽  
Expression Patterns ◽  
Allelic Expression ◽  
Monoallelic Expression ◽  
Burst Frequency ◽  
Biallelic Expression ◽  
High Consistency ◽  
Transcriptional Bursting

Transcriptional bursts render substantial biological noise in cellular transcriptomes. Here, we investigated the theoretical extent of allelic expression resulting from transcriptional bursting and how it compared to the amount biallelic, monoallelic and allele-biased expression observed in single-cell RNA-sequencing (scRNA-seq) data. We found that transcriptional bursting can explain the allelic expression patterns observed in single cells, including the frequent observations of autosomal monoallelic gene expression. Importantly, we identified that the burst frequency largely determined the fraction of cells with monoallelic expression, whereas the burst size had little effect on monoallelic observations. The high consistency between the bursting model predictions and scRNA-seq observations made it possible to assess the heterogeneity of a group of cells as their deviation in allelic observations from the expected. Finally, both burst frequency and size contributed to allelic imbalance observations and reinforced that studies of allelic imbalance can be confounded from the inherent noise in transcriptional bursting. Altogether, we demonstrate that allele-level transcriptional bursting renders widespread, although predictable, amounts of monoallelic and biallelic expression in single cells and cell populations.

scholarly journals Genome-wide analysis of transcriptional bursting-induced noise in mammalian cells

2019 ◽  
Author(s):  
Hiroshi Ochiai ◽  
Tetsutaro Hayashi ◽  
Mana Umeda ◽  
Mika Yoshimura ◽  
Akihito Harada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Transcription Elongation ◽  
Mapk Signaling ◽  
Kinetic Properties ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
A Genome ◽  
Transcriptional Bursting

AbstractTranscriptional bursting is stochastic activation and inactivation of promoters, leading to discontinuous production of mRNA, and is considered to be a contributing factor to cell-to-cell heterogeneity in gene expression. However, it remains elusive how the kinetic properties of transcriptional bursting (e.g., burst size, burst frequency, and noise induced by transcriptional bursting) are regulated in mammalian cells. In this study, we performed a genome-wide analysis of transcriptional bursting in mouse embryonic stem cells (mESCs) using single-cell RNA-sequencing. We found that the kinetics of transcriptional bursting was determined by a combination of promoter and gene body binding proteins, including polycomb repressive complex 2 and transcription elongation-related factors. Furthermore, large-scale CRISPR-Cas9-based screening and functional analysis revealed that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncover key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

scholarly journals Position Effects Are Influenced by the Orientation of a Transgene with Respect to Flanking Chromatin

2001 ◽  
Vol 21 (1) ◽  
pp. 298-309 ◽  
Author(s):  
Yong-Qing Feng ◽  
Matthew C. Lorincz ◽  
Steve Fiering ◽  
John M. Greally ◽  
Eric E. Bouhassira
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Methylation Status ◽  
Regulatory Elements ◽  
Cell Populations ◽  
Methylation Analysis ◽  
Position Effects ◽  
Methylation State ◽  
Cassette Exchange

ABSTRACT We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

Controlling quality and amount of mitochondria by mitophagy: insights into the role of ubiquitination and deubiquitination

2016 ◽  
Vol 397 (7) ◽  
pp. 637-647 ◽  
Author(s):  
Tao Tan ◽  
Marcel Zimmermann ◽  
Andreas S. Reichert
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Fission ◽  
Baker’S Yeast ◽  
Negative Regulator ◽  
Mitochondrial Outer Membrane ◽  
Baker's Yeast ◽  
Genome Wide ◽  
A Genome ◽  
Autophagy Pathway ◽  
Quantity Control

Abstract Mitophagy is a selective autophagy pathway conserved in eukaryotes and plays an essential role in mitochondrial quality and quantity control. Mitochondrial fission and fusion cycles maintain a certain amount of healthy mitochondria and allow the isolation of damaged mitochondria for their elimination by mitophagy. Mitophagy can be classified into receptor-dependent and ubiquitin-dependent pathways. The mitochondrial outer membrane protein Atg32 is identified as the only known receptor for mitophagy in baker’s yeast, whereas mitochondrial proteins FUNDC1, NIX/BNIP3L, BNIP3 and Bcl2L13 are recognized as mitophagy receptors in mammalian cells. Earlier studies showed that ubiquitination and deubiquitination occurs in yeast, yet there is no direct evidence for an ubiquitin-dependent mitophagy pathway in this organism. In contrast, a ubiquitin-/PINK1-/Parkin-dependent mitophagy pathway was unraveled and was extensively characterized in mammals in recent years. Recently, a quantitative method termed synthetic quantitative array (SQA) technology was developed to identify modulators of mitophagy in baker’s yeast on a genome-wide level. The Ubp3-Bre5 deubiquitination complex was found as a negative regulator of mitophagy while promoting other autophagic pathways. Here we discuss how ubiquitination and deubiquitination regulates mitophagy and other selective forms of autophagy and what argues for using baker’s yeast as a model to study the ubiquitin-dependent mitophagy pathway.

Mapping replication timing domains genome wide in single mammalian cells with single-cell DNA replication sequencing

2020 ◽  
Vol 15 (12) ◽  
pp. 4058-4100
Author(s):  
Hisashi Miura ◽  
Saori Takahashi ◽  
Takahiro Shibata ◽  
Koji Nagao ◽  
Chikashi Obuse ◽  
...  
Keyword(s):  
Dna Replication ◽  
Single Cell ◽  
Mammalian Cells ◽  
Replication Timing ◽  
Genome Wide

scholarly journals A Systematic Pan-Cancer Analysis of Genetic Heterogeneity Reveals Associations with Epigenetic Modifiers

Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 391 ◽  
Author(s):  
Mafalda de Matos ◽  
Ioana Posa ◽  
Filipa Carvalho ◽  
Vanessa Morais ◽  
Ana Grosso ◽  
...  
Keyword(s):  
Cancer Treatment ◽  
Genetic Heterogeneity ◽  
Dna Methyltransferase ◽  
Tumor Development ◽  
Predictive Biomarkers ◽  
Cell Populations ◽  
Major Mechanism ◽  
Molecular Features ◽  
Genome Wide ◽  
Cancer Types

Intratumor genetic heterogeneity (ITH) is the main obstacle to effective cancer treatment and a major mechanism of drug resistance. It results from the continuous evolution of different clones of a tumor over time. However, the molecular features underlying the emergence of genetically-distinct subclonal cell populations remain elusive. Here, we conducted an exhaustive characterization of ITH across 2807 tumor samples from 16 cancer types. Integration of ITH scores and somatic variants detected in each tumor sample revealed that mutations in epigenetic modifier genes are associated with higher ITH levels. In particular, genes that regulate genome-wide histone and DNA methylation emerged as being determinant of high ITH. Indeed, the knockout of histone methyltransferase SETD2 or DNA methyltransferase DNMT3A using the CRISPR/Cas9 system on cancer cells led to significant expansion of genetically-distinct clones and culminated in highly heterogeneous cell populations. The ITH scores observed in knockout cells recapitulated the heterogeneity levels observed in patient tumor samples and correlated with a better mitochondrial bioenergetic performance under stress conditions. Our work provides new insights into tumor development, and discloses new drivers of ITH, which may be useful as either predictive biomarkers or therapeutic targets to improve cancer treatment.

scholarly journals VPS37A directs ESCRT recruitment for phagophore closure

2019 ◽  
Vol 218 (10) ◽  
pp. 3336-3354 ◽  
Author(s):  
Yoshinori Takahashi ◽  
Xinwen Liang ◽  
Tatsuya Hattori ◽  
Zhenyuan Tang ◽  
Haiyan He ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Growth Factor Receptor ◽  
Multivesicular Body ◽  
Regulatory Mechanisms ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Escrt Machinery ◽  
Genome Wide ◽  
A Genome ◽  
Epidermal Growth

The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

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