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Genome-wide CRISPR library screening to identify genes involved in the regulation of the kinetic properties of transcriptional bursting

ABSTRACT: Genome-wide CRISPR library screening to identify genes involved in the regulation of the kinetic properties of transcriptional bursting

PROVIDER: PRJNA548636 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9278140.fastq.gz	Fastqsanger.gz
	SRR9278141.fastq.gz	Fastqsanger.gz
	SRR9278132.fastq.gz	Fastqsanger.gz
	SRR9278133.fastq.gz	Fastqsanger.gz
	SRR9278134.fastq.gz	Fastqsanger.gz

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Genome-wide CRISPR library screening to identify genes involved in the regulation of the kinetic properties of transcriptional bursting

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Regulation of stochastic gene bursting by chromatin-independent acetylation

Project description:Stochastic transcriptional bursting is now recognized as a universal property of gene expression. While different genes exhibit distinct bursting patterns, the molecular basis for differences in bursting are largely unknown. We have developed and applied a high-throughput-imaging based screening strategy to identify cellular factors and mechanisms that determine the bursting behavior of human genes. Focusing on epigenetic regulators, we find that global acetylation is the strongest acute modulator of burst frequency, size and heterogeneity of bursting. Acetylation similarly affected the Off-time but On-time changes were gene-specific. These effects were not strongly linked to promoter acetylation, which did not correlate with bursting properties and forced promoter acetylation altered bursting for only some genes. Instead, we identified acetylation of the Integrator complex as a key determinant of gene bursting with elevated Integrator acetylation leading to faster bursting. Taken together our results suggest a dominant role of non-histone proteins in determining gene bursting properties and they identify non-epigenetic acetylation of a transcription cofactor as an allosteric modulator of bursting via a pause-release related bursting checkpoint

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RNA-seq analysis of mouse embryonic stem cells cultured in medium containing MEK and Akt inhibitors

Project description:Cell-to-cell heterogeneity in gene expression can even be observed in the same type of cells, present in a similar environment. Transcriptional bursting is thought to be one of contributing factors to the heterogeneity, but 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. To unbiasedly identify genes regulating the kinetic properties of transcriptional bursting, we performed large-scale CRISPR/-Cas9 based screening and functional analysis, and found that Akt/MAPK signaling pathways are involved in the regulation of the kinetic properties of transcriptional bursting. To further characterize how these pathways affect mESC gene expression programs, we performed RNA-Seq analysis of mESCs cultured in standard (Std), 2i and Akt/MAPK signal-inhibitor containing medium (PD-MK). We found that expression levels of some genes encoding transcription elongation related factors were upregulated in PD-MK condition.

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Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells

Project description:This SuperSeries is composed of the SubSeries listed below.

2020-06-04 | GSE132593 | GEO

Allele-specific mRNA expression analysis in 129/CAST hybrid mESCs by single-cell full-length total RNA-sequencing

Project description:Cell-to-cell heterogeneity in gene expression can even be observed in the same type of cells, present in a similar environment. Transcriptional bursting is thought to be one of contributing factors to the heterogeneity, but 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. Here, by using single-cell, full-length total RNA-sequencing, we performed genome-wide analysis of transcriptional bursting in mouse embryonic stem cells (mESCs). We found that the kinetics of transcriptional bursting is gene-specific and is determined by a combination of promoter and gene body binding proteins, including polycomb repressive complex 2 and transcription elongation-related factors.

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Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells.

Project description:Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

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