Single-allele analysis of transcription kinetics in living mammalian cells

Yunger, Sharon; Rosenfeld, Liat; Garini, Yuval; Shav‐Tal, Yaron

doi:10.1038/nmeth.1482

Cited by 160 publications

(165 citation statements)

References 21 publications

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“…1A) is supported by a comprehensive large-scale study in bacteria, demonstrating that >400 genes appear to follow constitutive (or Poisson-like) gene expression (1). Constitutive expression has also been reported for subsets of human genes (2). Conversely, several elegant studies showed that specific promoters in bacteria and yeast express gene products in an episodic process (Fig.…”

mentioning

confidence: 69%

Transcriptional burst frequency and burst size are equally modulated across the human genome

Dar

Razooky

Singh

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

499

571

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Gene expression occurs either as an episodic process, characterized by pulsatile bursts, or as a constitutive process, characterized by a Poisson-like accumulation of gene products. It is not clear which mode of gene expression (constitutive versus bursty) predominates across a genome or how transcriptional dynamics are influenced by genomic position and promoter sequence. Here, we use time-lapse fluorescence microscopy to analyze 8,000 individual human genomic loci and find that at virtually all loci, episodic bursting—as opposed to constitutive expression—is the predominant mode of expression. Quantitative analysis of the expression dynamics at these 8,000 loci indicates that both the frequency and size of the transcriptional bursts varies equally across the human genome, independent of promoter sequence. Strikingly, weaker expression loci modulate burst frequency to increase activity, whereas stronger expression loci modulate burst size to increase activity. Transcriptional activators such as trichostatin A (TSA) and tumor necrosis factor α (TNF) only modulate burst size and frequency along a constrained trend line governed by the promoter. In summary, transcriptional bursting dominates across the human genome, both burst frequency and burst size vary by chromosomal location, and transcriptional activators alter burst frequency and burst size, depending on the expression level of the locus.

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mentioning

confidence: 69%

Transcriptional burst frequency and burst size are equally modulated across the human genome

Dar

Razooky

Singh

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

499

571

View full text Add to dashboard Cite

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“…This resolution of detecting transcription and pausing cannot be obtained when examining single genes. 39 Interestingly, RNA Pol I transcription rates in the natural tandem rDNA array system of the nucleolus were also significantly higher (5.7 kb/min) than previous measurements. 40 Still, one might wonder whether the higher rate of Pol II transcription was specific for this artificial gene.…”

Section: Cell Systems For Following Transcription In Living Cellsmentioning

confidence: 54%

On the right track

Mor

Ben-Yishay

Shav‐Tal

2010

Nucleus

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“…These "transcriptional bursts" amount to "memory" between transcripts: The synthesis of one transcript is likely to be accompanied by the synthesis of another over certain time intervals. Transcriptional bursting has now been observed in yeast (Zenklusen et al 2008;Lenstra et al 2015), slime mold (Chubb et al 2006;Muramoto et al 2010;Stevense et al 2010), fly (Garcia et al 2013), mouse , and human (Yunger et al 2010) cell lines and is a significant source of expression variation between cells. In fact, subsequent steps of gene expression such as RNA export, RNA decay, translation, etc.…”

Section: Transcriptional Burstingmentioning

confidence: 99%

What have single-molecule studies taught us about gene expression?

Chen

Larson

2016

Genes Dev.

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The production of a single mRNA is the result of many sequential steps, from docking of transcription factors to polymerase initiation, elongation, splicing, and, finally, termination. Much of our knowledge about the fundamentals of RNA synthesis and processing come from ensemble in vitro biochemical measurements. Single-molecule approaches are very much in this same reductionist tradition but offer exquisite sensitivity in space and time along with the ability to observe heterogeneous behavior and actually manipulate macromolecules. These techniques can also be applied in vivo, allowing one to address questions in living cells that were previously restricted to reconstituted systems. In this review, we examine the unique insights that single-molecule techniques have yielded on the mechanisms of gene expression.Single-molecule experiments are now pervasive in biology. What started out as an experimental approach for characterizing ion channels in the 1970s (Neher and Sakmann 1976) has now become a fixture in hundreds of laboratories addressing fundamental questions in biochemistry, cell biology, genetics, and development. The methodology is nearly as diverse as the problems that are addressed and encompasses imaging, optical tweezers, atomic force microscopy, electrophysiology, and cryo-electron microscopy (cryo-EM), to list a few. The unifying principle behind these approaches is straightforward: the ability to observe the heterogeneous, rare, or fleeting behavior of macromolecules that is normally masked by ensemble techniques. In this review, we focus on the role that single-molecule approaches have played in advancing our understanding of the early steps in gene expression.In general, transcription by RNA polymerase (RNAP) in bacteria or RNA polymerase II (Pol II) in eukaryotes begins when transcription factors (TFs) are recruited to the promoter. This leads to the assembly of the preinitiation complex (PIC) that contains a semicompetent polymerase. The PIC is necessary for unwinding duplex DNA and setting the stage for processive elongation by the polymerase. After conformational changes in the PIC, the polymerase escapes the promoter region and enters into productive elongation. In eukaryotes, the nascent RNA undergoes further modifications such as addition of a 5 ′ cap, synthesis of a poly-A tail, and splicing before the mature mRNA is formed. These early steps of gene expression are uniquely suited to elucidation through singlemolecule methods. For example, single-molecule experimental approaches allow one to visualize the order of assembly for molecular complexes (i.e., the PIC) and observe the variety of pathways that can result in initiation of RNAP. The ability to observe kinetics in unperturbed systems can provide clues to the mechanisms of transcription. RNAPs can also be manipulated by singlemolecule optical trapping to reveal the inner workings of force generation by this enzyme. Furthermore, singlemolecule imaging has also revealed the heterogeneity of gene expression that exists among cells in ...

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Single-allele analysis of transcription kinetics in living mammalian cells

Cited by 160 publications

References 21 publications

Transcriptional burst frequency and burst size are equally modulated across the human genome

Transcriptional burst frequency and burst size are equally modulated across the human genome

On the right track

What have single-molecule studies taught us about gene expression?

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