Applying thiouracil tagging to mouse transcriptome analysis

Karfilis, Kate V.; Miller, Michael R.; Doe, Chris Q.; Stankunas, Kryn

doi:10.1038/nprot.2014.023

Cited by 41 publications

(50 citation statements)

References 17 publications

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“…Total RNA was extracted as outlined above. The 4sU-tagging was performed according to (Gay et al, 2014) (Synthesize cDNA), RNA and ending with part 3.C (Synthesize 3'-Tagged DNA) to 3.G.…”

Section: Tagging the Nascent Transcriptomementioning

confidence: 99%

The Spatio-Temporal Control of Zygotic Genome Activation

Gentsch

Owens

Smith

2018

Preprint

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7One of the earliest and most significant events in embryonic development is zygotic 8 genome activation (ZGA). In several species, bulk transcription begins at the mid-9 blastula transition (MBT) when, after a certain number of cleavages, the embryo attains 10 a particular nuclear-to-cytoplasmic (N/C) ratio, maternal repressors become sufficiently 11 diluted, and the cell cycle slows down. Here we resolve the frog ZGA in time and space 12 by profiling RNA polymerase II (RNAPII) engagement and its transcriptional readout. We 13 detect a gradual increase in both the quantity and the length of RNAPII elongation before 14 the MBT, revealing that >1,000 zygotic genes disregard the N/C timer for their activation, 15 and that the sizes of newly transcribed genes are not necessarily constrained by cell 16 cycle duration. We also find that Wnt, Nodal and BMP signaling together generate most 17 of the spatio-temporal dynamics of regional ZGA, directing the formation of orthogonal 18 body axes and proportionate germ layers. 102respectively. Gene activation reached its peak at the MBT (~4.5 hpf), with 1,854 newly-103 activated genes, before dropping to 724 genes at the early-to-mid gastrula stage (~7.5 hpf) and 104 increasing again to 1,214 genes towards the end of gastrulation (~10 hpf) (Figures 1B,C and 105 S1E and Table S2). The dramatic increase in transcriptional activity that occurs in the 1.5 hours 106 between the 128-cell stage and the MBT can be illustrated by Hilbert curves ( Figure 1C), which 107 Page 4 of 43 provide a genome-wide overview of RNAPII enrichment by folding chromosomes into two-108 dimensional space-filling and position-preserving plots (Gu et al., 2016). While most zygotic 109 genes remain active beyond the mid-gastrula stage, 197 (including siamois2 [sia2], nodal5 and 110 znf470) of the 4,836 zygotic genes (~4%) are deactivated within ~6 h of development (Figures 1111C and S1F,G). Slightly less than one third of the activated genes were differentially expressed 112 along either or both of the animal-vegetal and dorso-ventral axes ( Figures 1D and S1F). 113The temporal order of enriched biological processes supported by ZGA matched the regulatory 114 flow of gene expression, starting with nucleosome assembly, nucleic acid synthesis, mRNA 115 metabolism and production, post-translational modification and degradation of proteins (Figure 116 1E). The earliest transcriptional engagement, beginning at the 32 to 128-cell stages, was 117 detected in gene clusters of tens to hundreds of kilobases (Figures 1A,C and S1H-J). These 118 clusters featured close relatives of the same genes, some of which are critical to Nodal signaling 119 (Nodal ligands), the formation of the Spemann organiser (Siamois homeobox transcription 120 factors), nucleosome assembly (histones), mRNA decay (MIR-427), and ongoing gene 121 regulation (zinc finger [ZF] transcription factors with on average 10 Cys2-His2 [C2H2] domains; 122 Figure S1J). These earliest activated genes were shorter and encoded smaller proteins than 123 those wi...

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“…Total RNA was extracted as outlined above. The 4sU-tagging was performed according to (Gay et al, 2014) (Synthesize cDNA), RNA and ending with part 3.C (Synthesize 3'-Tagged DNA) to 3.G.…”

Section: Tagging the Nascent Transcriptomementioning

confidence: 99%

The Spatio-Temporal Control of Zygotic Genome Activation

Gentsch

Owens

Smith

2018

Preprint

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“…More recently, metabolic labeling with 4sU has been used to isolate RNA from yeast [17] and metazoan cells [19] for transcriptome studies. To our knowledge, this represents the first use of 4sU to isolate newly synthesized RNA in bacteria.…”

Section: Introductionmentioning

confidence: 99%

Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting

Hulscher

Bohon

Rappé

et al. 2016

Methods

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The assembly of the E. coli ribosome has been widely studied and characterized in vitro. Despite this, ribosome biogenesis in living cells is only partly understood because assembly is coupled with transcription, modification and processing of the pre-ribosomal RNA. We present a method for footprinting and isolating pre-rRNA as it is synthesized in E. coli cells. Pre-rRNA synthesis is synchronized by starvation, followed by nutrient upshift. RNA synthesized during outgrowth is metabolically labeled to facilitate isolation of recent transcripts. Combining this technique with two in vivo RNA probing methods, hydroxyl radical and DMS footprinting, allows the structure of nascent RNA to be probed over time. Together, these can be used to determine changes in the structures of ribosome assembly intermediates as they fold in vivo.

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“…Current efforts include optimizing cell sorting by magnetic (MACS) or fluorescent (FACS) labeling and combining this with RNA-seq, DNase-seq, or ChIP-seq to get expression and chromatin profiles of pure cell populations (Thurman et al 2012;Zhu et al 2013a;Harzer et al 2013). In order to overcome issues of cell sorting procedures and more directly assess genome-wide aspects of transcriptional regulation in vivo, it may be necessary to use transgenic approaches that allow for direct labeling and isolation of cell typespecific nuclei from tissues (Bonn et al 2012;Deal and Henikoff 2011) or cell typespecific RNA species through "TU tagging," i.e., 4-thiouracil labeling and biotinylation of RNA from a particular cell type (Gay et al 2014). Further development of these labeling technologies and their integration with genome-wide technologies will be an important step for the ability to investigate transcriptional regulation in vivo.…”

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confidence: 99%

A Genome-Wide Perspective on Metabolism

Rauch

Mandrup

2015

Metabolic Control

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Mammals have at least 210 histologically diverse cell types (Alberts, Molecular biology of the cell. Garland Science, New York, 2008) and the number would be even higher if functional differences are taken into account. The genome in each of these cell types is differentially programmed to express the specific set of genes needed to fulfill the phenotypical requirements of the cell. Furthermore, in each of these cell types, the gene program can be differentially modulated by exposure to external signals such as hormones or nutrients. The basis for the distinct gene programs relies on cell type-selective activation of transcriptional enhancers, which in turn are particularly sensitive to modulation. Until recently we had only fragmented insight into the regulation of a few of these enhancers; however, the recent advances in high-throughput sequencing technologies have enabled the development of a large number of technologies that can be used to obtain genome-wide insight into how genomes are reprogrammed during development and in response to specific external signals. By applying such technologies, we have begun to reveal the cross-talk between metabolism and the genome, i.e., how genomes are reprogrammed in response to metabolites, and how the regulation of metabolic networks is coordinated at the genomic level.

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Applying thiouracil tagging to mouse transcriptome analysis

Cited by 41 publications

References 17 publications

The Spatio-Temporal Control of Zygotic Genome Activation

The Spatio-Temporal Control of Zygotic Genome Activation

Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting

A Genome-Wide Perspective on Metabolism

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