UVB Induces a Genome-Wide Acting Negative Regulatory Mechanism That Operates at the Level of Transcription Initiation in Human Cells

Transcription of many genes in metazoans is subject to polymerase pausing, which corresponds to the transient arrest of transcriptionally engaged polymerase. It occurs mainly at promoter proximal regions and is not well understood. In particular, a genome-wide measurement of pausing times at high resolution has been lacking.We present here an extension of PRO-seq, time variant PRO-seq (TV-PRO-seq), that allowed us to estimate genome-wide pausing times at single base resolution. Its application to human cells reveals that promoter proximal pausing is surprisingly short compared to other regions and displays an intricate pattern. We also find precisely conserved pausing profiles at tRNA and rRNA genes and identified DNA motifs associated with pausing time. Finally, we show how chromatin states reflect differences in pausing times.

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A genome‐wide screen identifies IRF2 as a key regulator of caspase‐4 in human cells

EMBO Reports ◽

10.15252/embr.201948235 ◽

2019 ◽

Vol 20 (9) ◽

Cited By ~ 14

Author(s):

Sacha Benaoudia ◽

Amandine Martin ◽

Marta Puig Gamez ◽

Gabrielle Gay ◽

Brice Lagrange ◽

...

Keyword(s):

Human Cells ◽

Genome Wide ◽

A Genome

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The Antisense Transcriptomes of Human Cells

Science ◽

10.1126/science.1163853 ◽

2008 ◽

Vol 322 (5909) ◽

pp. 1855-1857 ◽

Cited By ~ 345

Author(s):

Yiping He ◽

Bert Vogelstein ◽

Victor E. Velculescu ◽

Nickolas Papadopoulos ◽

Kenneth W. Kinzler

Keyword(s):

Mammalian Cells ◽

Cell Types ◽

Human Cells ◽

Antisense Transcripts ◽

Genome Wide ◽

Human Genes ◽

A Genome ◽

Dna Strand ◽

The Relationship ◽

Rna Transcript

Transcription in mammalian cells can be assessed at a genome-wide level, but it has been difficult to reliably determine whether individual transcripts are derived from the plus or minus strands of chromosomes. This distinction can be critical for understanding the relationship between known transcripts (sense) and the complementary antisense transcripts that may regulate them. Here, we describe a technique that can be used to (i) identify the DNA strand of origin for any particular RNA transcript, and (ii) quantify the number of sense and antisense transcripts from expressed genes at a global level. We examined five different human cell types and in each case found evidence for antisense transcripts in 2900 to 6400 human genes. The distribution of antisense transcripts was distinct from that of sense transcripts, was nonrandom across the genome, and differed among cell types. Antisense transcripts thus appear to be a pervasive feature of human cells, which suggests that they are a fundamental component of gene regulation.

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Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

eLife ◽

10.7554/elife.55667 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 5

Author(s):

Anand Ranjan ◽

Vu Q Nguyen ◽

Sheng Liu ◽

Jan Wisniewski ◽

Jee Min Kim ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Single Molecule ◽

Transcription Initiation ◽

General Mechanism ◽

Pol Ii ◽

Promoter Escape ◽

Genome Wide ◽

A Genome ◽

Particle Imaging

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

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Hda1C restricts the transcription initiation frequency to limit divergent non-coding RNA transcription

10.1101/2021.04.06.438606 ◽

2021 ◽

Author(s):

Uthra Gowthaman ◽

Maxim Ivanov ◽

Isabel Schwarz ◽

Heta P. Patel ◽

Niels A. Müller ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Transcription Initiation ◽

Histone Deacetylation ◽

Gene Promoters ◽

Genetic Screens ◽

Fluorescence Measurements ◽

Non Coding Rna ◽

Genome Wide ◽

A Genome ◽

Initiation Frequency

ABSTRACTNucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA Polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA, and a divergent non-coding (DNC) transcript with an unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast. We identified the Hda1 histone deacetylase complex (Hda1C) as a repressor of DNC in high-throughput reverse genetic screens based on quantitative single-cell fluorescence measurements. Nascent transcription profiling showed a genome-wide role of Hda1C in DNC repression. Live-cell imaging of transcription revealed that Hda1C reduced the frequency of DNC transcription. Hda1C contributed to decreased acetylation of histone H3 in DNC regions, supporting DNC repression by histone deacetylation. Our data support the interpretation that DNC results as a consequence of the NDR-based architecture of eukaryotic promoters, but that it is governed by locus-specific repression to maintain genome fidelity.

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Genome-wide CRISPR-Cas9 screen reveals the importance of the heparan sulfate pathway and the conserved oligomeric golgi complex for synthetic dsRNA uptake and Sindbis virus infection

10.1101/2020.05.20.105528 ◽

2020 ◽

Author(s):

Olivier Petitjean ◽

Erika Girardi ◽

Richard Patryk Ngondo ◽

Vladimir Lupashin ◽

Sébastien Pfeffer

Keyword(s):

Cell Death ◽

Heparan Sulfate ◽

Cell Survival ◽

Sindbis Virus ◽

Human Cells ◽

Innate Immune ◽

Genome Wide ◽

A Genome ◽

Conserved Oligomeric Golgi Complex ◽

Conserved Oligomeric Golgi

AbstractDouble stranded RNA (dsRNA) is the hallmark of many viral infections. dsRNA is produced either by RNA viruses during replication or by DNA viruses upon convergent transcription. Synthetic dsRNA is also able to mimic viral-induced activation of innate immune response and cell death. In this study, we employed a genome-wide CRISPR-Cas9 loss of function screen based on cell survival in order to identify genes implicated in the host response to dsRNA. By challenging HCT116 human cells with either synthetic dsRNA or Sindbis virus (SINV), we identified the heparan sulfate (HS) pathway as a crucial factor for dsRNA entry and we validated SINV dependency on HS. Interestingly, we uncovered a novel role for COG4, a component of the Conserved Oligomeric Golgi (COG) complex, as a factor involved in cell survival to both dsRNA and SINV in human cells. We showed that COG4 knock-out led to a decrease of extracellular HS, specifically affected dsRNA transfection efficiency and reduced viral production, explaining the increased cell survival of these mutants.ImportanceWhen facing a viral infection, the organism has to put in place a number of defense mechanisms in order to clear the pathogen from the cell. At the early phase of this preparation for fighting against the invader, the innate immune response is triggered by the sensing of danger signals. Among those molecular cues, double-stranded (dsRNA) is a very potent inducer of different reactions at the cellular level that can ultimately lead to cell death. Using a genome-wide screening approach, we set to identify genes involved in dsRNA entry, sensing and apoptosis induction in human cells. This allowed us to determine that the heparan sulfate pathway and the Conserved Oligomeric Golgi complex are key determinants allowing entry of both dsRNA and viral nucleic acid leading to cell death.

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Faculty Opinions recommendation of A genome-wide RNAi screen for modifiers of the circadian clock in human cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.2062956.1646054 ◽

2010 ◽

Author(s):

Jean Clairambault ◽

Pasquale Innominato

Keyword(s):

Circadian Clock ◽

Rnai Screen ◽

Human Cells ◽

Genome Wide ◽

A Genome

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Genome-wide RNA interference screening reveals a COPI-MAP2K3 pathway required for YAP regulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915387117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 19994-20003 ◽

Cited By ~ 1

Author(s):

Yong Joon Kim ◽

Eunji Jung ◽

Eunbie Shin ◽

Sin-Hyoung Hong ◽

Hui Su Jeong ◽

...

Keyword(s):

Rna Interference ◽

Regulatory Mechanism ◽

Inflammatory Diseases ◽

Transcriptional Regulator ◽

Endoplasmic Reticulum Stress Response ◽

Loss Of Function ◽

Genome Wide ◽

A Genome ◽

Stress Response Pathway ◽

Kinase Cascade

The transcriptional regulator YAP, which plays important roles in the development, regeneration, and tumorigenesis, is activated when released from inhibition by the Hippo kinase cascade. The regulatory mechanism of YAP in Hippo-low contexts is poorly understood. Here, we performed a genome-wide RNA interference screen to identify genes whose loss of function in a Hippo-null background affects YAP activity. We discovered that the coatomer protein complex I (COPI) is required for YAP nuclear enrichment and that COPI dependency of YAP confers an intrinsic vulnerability to COPI disruption in YAP-driven cancer cells. We identified MAP2K3 as a YAP regulator involved in inhibitory YAP phosphorylation induced by COPI subunit depletion. The endoplasmic reticulum stress response pathway activated by COPI malfunction appears to connect COPI and MAP2K3. In addition, we provide evidence that YAP inhibition by COPI disruption may contribute to transcriptional up-regulation of PTGS2 and proinflammatory cytokines. Our study offers a resource for investigating Hippo-independent YAP regulation as a therapeutic target for cancers and suggests a link between YAP and COPI-associated inflammatory diseases.

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Genome-Wide CRISPR-Cas9 Screen Reveals the Importance of the Heparan Sulfate Pathway and the Conserved Oligomeric Golgi Complex for Synthetic Double-Stranded RNA Uptake and Sindbis Virus Infection

mSphere ◽

10.1128/msphere.00914-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Olivier Petitjean ◽

Erika Girardi ◽

Richard Patryk Ngondo ◽

Vladimir Lupashin ◽

Sébastien Pfeffer

Keyword(s):

Cell Death ◽

Heparan Sulfate ◽

Cell Survival ◽

Sindbis Virus ◽

Human Cells ◽

Double Stranded Rna ◽

Genome Wide ◽

A Genome ◽

Conserved Oligomeric Golgi Complex ◽

Conserved Oligomeric Golgi

ABSTRACT Double-stranded RNA (dsRNA) is the hallmark of many viral infections. dsRNA is produced either by RNA viruses during replication or by DNA viruses upon convergent transcription. Synthetic dsRNA is also able to mimic viral-induced activation of innate immune response and cell death. In this study, we employed a genome-wide CRISPR-Cas9 loss-of-function screen based on cell survival in order to identify genes implicated in the host response to dsRNA. By challenging HCT116 human cells with either synthetic dsRNA or Sindbis virus (SINV), we identified the heparan sulfate (HS) pathway as a crucial factor for dsRNA entry, and we validated SINV dependency on HS. Interestingly, we uncovered a novel role for COG4, a component of the conserved oligomeric Golgi (COG) complex, as a factor involved in cell survival to both dsRNA and SINV in human cells. We showed that COG4 knockout led to a decrease of extracellular HS that specifically affected dsRNA transfection efficiency and reduced viral production, which explains the increased cell survival of these mutants. IMPORTANCE When facing a viral infection, the organism has to put in place a number of defense mechanisms in order to clear the pathogen from the cell. At the early phase of this preparation for fighting against the invader, the innate immune response is triggered by the sensing of danger signals. Among those molecular cues, double-stranded RNA (dsRNA) is a very potent inducer of different reactions at the cellular level that can ultimately lead to cell death. Using a genome-wide screening approach, we set to identify genes involved in dsRNA entry, sensing, and apoptosis induction in human cells. This allowed us to determine that the heparan sulfate pathway and the conserved oligomeric Golgi complex are key determinants allowing entry of both dsRNA and viral nucleic acid leading to cell death.

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High-Throughput Fluorescence-Based Screen Identifies the Neuronal MicroRNA miR-124 as a Positive Regulator of Alphavirus Infection

Journal of Virology ◽

10.1128/jvi.02145-19 ◽

2020 ◽

Vol 94 (9) ◽

Cited By ~ 2

Author(s):

Paula López ◽

Erika Girardi ◽

Bryan C. Mounce ◽

Amélie Weiss ◽

Béatrice Chane-Woon-Ming ◽

...

Keyword(s):

Binding Site ◽

Human Cells ◽

Viral Rna ◽

Positive Regulation ◽

Cellular Mirnas ◽

Positive Regulator ◽

Genome Wide ◽

A Genome ◽

Arbovirus Infection ◽

Alphavirus Infection

ABSTRACT MicroRNAs (miRNAs) are small regulatory RNAs which act by modulating the expression of target genes. In addition to their role in maintaining essential physiological functions in the cell, miRNAs can also regulate viral infections. They can do so directly by targeting RNAs of viral origin or indirectly by targeting host mRNAs, and this can result in a positive or negative outcome for the virus. Here, we performed a fluorescence-based miRNA genome-wide screen in order to identify cellular miRNAs involved in the regulation of arbovirus infection in human cells. We identified 16 miRNAs showing a positive effect on Sindbis virus (SINV) expressing green fluorescent protein (GFP), among which were a number of neuron-specific ones such as miR-124. We confirmed that overexpression of miR-124 increases both SINV structural protein translation and viral production and that this effect is mediated by its seed sequence. We further demonstrated that the SINV genome possesses a binding site for miR-124. Both inhibition of miR-124 and silent mutations to disrupt this binding site in the viral RNA abolished positive regulation. We also proved that miR-124 inhibition reduces SINV infection in human differentiated neuronal cells. Finally, we showed that the proviral effect of miR-124 is conserved in other alphaviruses, as its inhibition reduces chikungunya virus (CHIKV) production in human cells. Altogether, our work expands the panel of positive regulation of the viral cycle by direct binding of host miRNAs to the viral RNA and provides new insights into the role of cellular miRNAs as regulators of alphavirus infection. IMPORTANCE Arthropod-borne (arbo) viruses are part of a class of pathogens that are transmitted to their final hosts by insects. Because of climate change, the habitat of some of these insects, such as mosquitoes, is shifting, thereby facilitating the emergence of viral epidemics. Among the pathologies associated with arbovirus infection, neurological diseases such as meningitis and encephalitis represent a significant health burden. Using a genome-wide miRNA screen, we identified neuronal miR-124 as a positive regulator of the Sindbis and chikungunya alphaviruses. We also showed that this effect was in part direct, thereby opening novel avenues to treat alphavirus infections.

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