Transcriptional regulation in Drosophila during heat shock: A nuclear run-on analysis

Vazquez, Julio; Pauli, Daniel; Tissières, A.

doi:10.1007/bf00352397

Cited by 52 publications

(35 citation statements)

References 88 publications

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“…The maximum Pol II signal occurs at 5 min, decreasing 30 to 50% over the entire gene by 10 and 20 min. This result is in agreement with previously reported rates of hsp70 RNA synthesis, which peaked at 5 min (49). At all time points tested where Pol II has proceeded through the gene, Pol II signals remain stronger at the start site compared to that at the ORF (approximately 2.5-fold), consistent with the observation that escape of promoter-paused Pol II remains ratelimiting even during heat shock (10).…”

Section: Methodssupporting

confidence: 93%

Transcription Factor and Polymerase Recruitment, Modification, and Movement on dhsp70 In Vivo in the Minutes following Heat Shock

Boehm

Saunders

Werner

et al. 2003

Molecular and Cellular Biology

206

225

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The uninduced Drosophila hsp70 gene is poised for rapid activation. Here we examine the rapid changes upon heat shock in levels and location of heat shock factor (HSF), RNA polymerase II (Pol II) and its phosphorylated forms, and the Pol II kinase P-TEFb on hsp70 in vivo by using both real-time PCR assays of chromatin immunoprecipitates and polytene chromosome immunofluorescence. These studies capture Pol II recruitment and progression along hsp70 and reveal distinct spatial and temporal patterns of serine 2 and serine 5 phosphorylation: in uninduced cells, the promoter-paused Pol II shows Ser5 but not Ser2 phosphorylation, and in induced cells the relative level of Ser2-P Pol II is lower at the promoter than at regions downstream. An early time point of heat shock activation captures unphosphorylated Pol II recruited to the promoter prior to P-TEFb, and during the first wave of transcription Pol II and the P-TEFb kinase can be seen tracking together across hsp70 with indistinguishable kinetics. Pol II distributions on several other genes with paused Pol II show a pattern of Ser5 and Ser2 phosphorylation similar to that of hsp70. These studies of factor choreography set important limits in modeling transcription regulatory mechanisms.Recent studies of posttranslational modifications of, and factor interactions with, the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) have implicated this region of Pol II in productive transcriptional elongation and the coupling of transcription and pre-mRNA processing (for reviews see references 32 and 37). The CTD is highly conserved among eukaryotes, containing multiple repeats of a consensus heptad YSPTSPS; mammalian Pol II CTD consists of 52 repeats, yeast consists of 25 to 26 repeats, while Drosophila has 42 repeats (1, 6). This large flexible arm of Pol II appears to serve as a docking site for, or to stimulate the recruitment of, an orchestrated assembly of factors involved in pre-mRNA capping, splicing, and 3Ј polyadenylation at different stages in production of the nascent transcript (5,9,13,14,20,26). One way this coordination has been proposed to occur is through the known phosphorylation of serines 2 and 5 (Ser2-P and Ser5-P, respectively) of the heptad repeat (52, 56). Cdk7, the kinase subunit of general transcription factor TFIIH, has been shown to phosphorylate the CTD at Ser5, an event proposed to occur early in the transcription cycle (12,24,57). This in turn appears to influence the association and activity of the capping machinery (5,15,18,27,28,42,45). Positive transcription elongation factor b (P-TEFb) is able to phosphorylate the CTD at Ser2 and, under certain conditions, Ser5 (25, 38, 57). P-TEFb is a kinase composed of the proteins cyclin T (CycT) and cdk9 and is known to be recruited upon gene activation, overcoming the negative effects of factors like Spt5 and negative elongation factor (50) and aiding in the transition from transcription initiation to elongation (36). Cdk8, a component of the coactivator complex Mediator, h...

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Section: Methodssupporting

confidence: 93%

Transcription Factor and Polymerase Recruitment, Modification, and Movement on dhsp70 In Vivo in the Minutes following Heat Shock

Boehm

Saunders

Werner

et al. 2003

Molecular and Cellular Biology

206

225

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“…Transcript imaging detected increases above 3-fold in relative expression levels for 9 genes encoding Drosophila heatshock proteins: Hsp22, Hsp26, Hsp27, Hsp23, DnaJ-1, Hsp67Bc, Hsp83, Hsp70Ab, and Hsp70Bb (17,18). The largest changes (Ͼ10-fold) were observed for Hsp22, Hsp26, Hsp27, and Hsp23, in accordance with several studies that report that these four small Hsps are expressed during normal fly development and are up-regulated under heat shock (19,20). For five other genes known to encode heat-shock proteins, DnaJ-1, Hsp67Bc, Hsp83, Hsp70Ab, and Hsp70Bb, we detect an increase in expression in the 3-to 6-fold range.…”

Section: Quantitative Transcript Imaging Of Heat-shocked Compared Withsupporting

confidence: 88%

“…For five other genes known to encode heat-shock proteins, DnaJ-1, Hsp67Bc, Hsp83, Hsp70Ab, and Hsp70Bb, we detect an increase in expression in the 3-to 6-fold range. All of these genes are known to be responsive to heat shock (20). The heat-shock cognate genes (Hsc) have been reported to be expressed at normal temperatures but are not further induced by heat shock (21,22).…”

Section: Quantitative Transcript Imaging Of Heat-shocked Compared Withmentioning

confidence: 99%

Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays

Leemans

Egger

Loop

et al. 2000

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

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Embryonic development in Drosophila is characterized by an early phase during which a cellular blastoderm is formed and gastrulation takes place, and by a later postgastrulation phase in which key morphogenetic processes such as segmentation and organogenesis occur. We have focused on this later phase in embryogenesis with the goal of obtaining a comprehensive analysis of the zygotic gene expression that occurs during development under normal and altered environmental conditions. For this, a functional genomic approach to embryogenesis has been developed that uses highdensity oligonucleotide arrays for large-scale detection and quantification of gene expression. These oligonucleotide arrays were used for quantitative transcript imaging of embryonically expressed genes under standard conditions and in response to heat shock. In embryos raised under standard conditions, transcripts were detected for 37% of the 1,519 identified genes represented on the arrays, and highly reproducible quantification of gene expression was achieved in all cases. Analysis of differential gene expression after heat shock revealed substantial expression level changes for known heat-shock genes and identified numerous heat shock-inducible genes. These results demonstrate that highdensity oligonucleotide arrays are sensitive, efficient, and quantitative instruments for the analysis of large scale gene expression in Drosophila embryos. R ecently the genome of the first multicellular eukaryote Caenorhabditis elegans was completely elucidated (1). Sequencing of the Drosophila melanogaster genome has now also been carried out, and currently the corresponding putative open reading frames are being defined and verified (2). On the basis of this complete genomic information, it will now be important to determine the complex expression of all encoded genes and to analyze physiological as well as pathological phenomena from a global genetic perspective. Large-scale transcript analysis is made possible by DNA micro-or oligonucleotide arrays (3, 4), both of which allow the simultaneous monitoring of hundreds of mRNA expression profiles (5, 6). In this study, we used Drosophila high-density oligonucleotide arrays to monitor the simultaneous expression of zygotically active genes during the later postgastrulation stages of embryonic development (7-9). We analyzed the relative abundance levels of hundreds of embryonically expressed genes under normal physiological conditions and in response to heat shock (10). In embryos raised under normal conditions, we obtained highly reproducible quantification for 563 expressed genes corresponding to different functional classes. After a 36°C heat shock, we detected increases in expression levels for known heat-shock genes and identified numerous heat-shock-inducible genes. Materials and MethodsEmbryos. D. melanogaster Oregon R stocks were kept on standard cornmeal͞yeast͞agar medium at 25°C. Embryos were collected overnight on grape-juice plates for 12 h and were kept for a further 5 h at 25°C before RNA isolation...

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“…Heat shock is another cellular stress that causes global changes in Pol II transcription in eukaryotic cells. Although some genes, such as those encoding heat shock proteins, are significantly activated in response to heat shock, most mRNA genes are thought to be transcriptionally repressed (36)(37)(38)(39). We were interested in learning whether the genes repressed during HSV-1 infection also showed a profound loss in Pol II occupancy in response to heat shock, which would suggest similarities in how Pol II transcription is downregulated in response to the two different stresses.…”

Section: Resultsmentioning

confidence: 99%

Infection by Herpes Simplex Virus 1 Causes Near-Complete Loss of RNA Polymerase II Occupancy on the Host Cell Genome

Abrisch

Eidem

Yakovchuk

et al. 2016

J Virol

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Lytic infection by herpes simplex virus 1 (HSV-1) triggers a change in many host cell programs as the virus strives to express its own genes and replicate. Part of this process is repression of host cell transcription by RNA polymerase II (Pol II), which also transcribes the viral genome. Here, we describe a global characterization of Pol II occupancy on the viral and host genomes in response to HSV-1 infection using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). The data reveal near-complete loss of Pol II occupancy throughout host cell mRNA genes, in both their bodies and promoter-proximal regions. Increases in Pol II occupancy of host cell genes, which would be consistent with robust transcriptional activation, were not observed. HSV-1 infection induced a more potent and widespread repression of Pol II occupancy than did heat shock, another cellular stress that widely represses transcription. Concomitant with the loss of host genome Pol II occupancy, we observed Pol II covering the HSV-1 genome, reflecting a high level of viral gene transcription. Interestingly, the positions of the peaks of Pol II occupancy at HSV-1 and host cell promoters were different. The primary peak of Pol II occupancy at HSV-1 genes is ϳ170 bp upstream of where it is positioned at host cell genes, suggesting that specific steps in transcription are regulated differently at HSV-1 genes than at host cell mRNA genes. IMPORTANCE We investigated the effect of herpes simplex virus 1 (HSV-1) infection on transcription of host cell and viral genes by RNA polymerase II (Pol II). The approach we used was to determine how levels of genome-bound Pol II changed after HSV-1 infection.We found that HSV-1 caused a profound loss of Pol II occupancy across the host cell genome. Increases in Pol II occupancy were not observed, showing that no host genes were activated after infection. In contrast, Pol II occupied the entire HSV-1 genome. Moreover, the pattern of Pol II at HSV-1 genes differed from that on host cell genes, suggesting a unique mode of viral gene transcription. These studies provide new insight into how HSV-1 causes changes in the cellular program of gene expression and how the virus coopts host Pol II for its own use. Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that proliferates in the nuclei of host cells during lytic infection (reviewed in reference 1). HSV-1 can cause lifelong infection by establishing asymptomatic latency in host sensory neurons, where it remains in a transcriptionally silenced state until it is stimulated by stress to reactivate and replicate in epithelial cells (2). The majority of the population are infected with HSV-1, which is largely responsible for oral cold sores; however, in rarer cases, it can cause severe conditions, such as blindness and encephalitis (3, 4). Recent experimental and epidemiological evidence also suggests a role for recurrent HSV-1 infection in Alzheimer's disease (5). Despite its clear medical significance, the relationship between the virus ...

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Transcriptional regulation in Drosophila during heat shock: A nuclear run-on analysis

Cited by 52 publications

References 88 publications

Transcription Factor and Polymerase Recruitment, Modification, and Movement on dhsp70 In Vivo in the Minutes following Heat Shock

Transcription Factor and Polymerase Recruitment, Modification, and Movement on dhsp70 In Vivo in the Minutes following Heat Shock

Quantitative transcript imaging in normal and heat-shocked Drosophila embryos by using high-density oligonucleotide arrays

Infection by Herpes Simplex Virus 1 Causes Near-Complete Loss of RNA Polymerase II Occupancy on the Host Cell Genome

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