Activation of Pausing RNA Polymerases by Nuclear Run-on Experiments

doi:10.1006/abio.1994.1190

Analytical Biochemistry

Volume 218, Issue 2, 1 May 1994, Pages 347-351

https://doi.org/10.1006/abio.1994.1190 Get rights and content

Abstract

The nuclear run-on transcription assay is the only approach to measure the transcriptional activity of a given gene in its genuine structural and regulatory cellular context. However, serious problems in the interpretation of results can arise from the artificial activation of paused RNA polymerases during the transcription reaction, leading to false results with regard to the level and mode of gene regulation in vivo. We have used the example of the human proto-oncogene c-myc, which has previously been reported to be regulated by premature termination of transcription, to describe the problems and pitfalls in the interpretation of nuclear run-on experiments. We show here that activation of paused, elongation-incompetent polymerases in nuclear run-on experiments produces a strong transcription signal on c-myc exon 1 in cells which do not express c-myc steady-state RNA.

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Cited by (24)

Evidence that phosphorylation of the RNA polymerase II carboxyl-terminal repeats is similar in yeast and humans
2005, Journal of Biological Chemistry
Citation Excerpt :
The phenomenon of promoter proximal pausing of Pol II has been recognized for many years (58–60) and has been correlated with changes in CTD phosphorylation since O'Brien et al. (31) directly demonstrated that the elongation-competent form of Pol II was hyperphosphorylated (55). To the best of our knowledge promoter proximal pausing does not occur in yeast (22); however, the phenomenon has been established in Drosophila and human cell lines by techniques including permanganate labeling of DNA at polymerase bubbles (59, 61), nuclear run-on assays (58, 62), and ChIP (20, 55, 63). Probably because yeast does not display promoter proximal pausing, ChIP studies in yeast have shown Pol II to be distributed evenly across genes (19, 46, 57).
Using an improved chromatin immunoprecipitation assay designed to increase immunoprecipitation efficiency, we investigated changes in RNA polymerase II (Pol II) density and carboxyl-terminal domain (CTD) phosphorylation during transcription of the cyclophilin A (PPIA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and several androgen-responsive genes in LNCaP cells. As generally observed in higher eukaryotes, promoter proximal pausing of Pol II appeared to occur on the PPIA and GAPDH genes, but apparently not on the androgen-responsive genes PSA and NKX3-1. Unlike some mammalian studies, we found that the CTD of Pol II in promoter regions contains little phosphorylation at Ser-2 of the heptad repeat, suggesting that Ser-2 phosphorylation is not involved in polymerase exit from the promoter region. In contrast, Pol II near the promoter displayed high levels of Ser-5 phosphorylation, which decreased as polymerase transcribed beyond the promoter region of the PPIA and GAPDH genes. However, total Pol II levels appear to decrease as much or more, suggesting that Ser-5 phosphorylation is maintained. In support of this conclusion, a phosphoserine 5-specific antibody quantitatively immunoprecipitates native hyperphosphorylated Pol II, suggesting that all polymerase with phosphoserine 2 also contains phosphoserine 5. Given reports indicating that phosphoserine 5 is present during elongation in yeast, our data suggest that gross changes in CTD phosphorylation patterns during transcription may be more conserved in yeast and humans than recognized previously.
The poly(A) signal, without the assistance of any downstream element, directs RNA polymerase II to pause in vivo and then to release stochastically from the template
2002, Journal of Biological Chemistry
Genes encoding polyadenylated mRNAs depend on their poly(A) signals for termination of transcription. Typically, transcription downstream of the poly(A) signal gradually declines to zero, but often there is a transient increase in polymerase density immediately preceding the decline. Special elements called pause sites are traditionally invoked to account for this increase. Using run-on transcription from the nuclei of transfected cells, we show that both the pause and the gradual decline that follow a poly(A) site are generated entirely by the poly(A) signal itself in a series of model constructs. We found no other elements to be involved and argue that the elements called pause sites do not function through pausing. Both the poly(A)-dependent pause and the subsequent decline occurred earlier for a stronger poly(A) signal than for a weaker one. Because the gradual decline resembles the abortive elongation that occurs downstream of many promoters, one model has proposed that the poly(A) signal flips the polymerase from the elongation mode to the abortive mode like a binary switch. We compared abortive elongators with poly(A) terminators and found a 4-fold difference in processivity. We conclude that poly(A) terminating polymerases do not merely revert to their prior state of low processivity but rather convert to a new termination-prone condition.
MAP Kinase Phosphatase-1 Gene Transcription in Rat Neuroendocrine Cells Is Modulated by a Calcium-sensitive Block to Elongation in the First Exon
2001, Journal of Biological Chemistry
Citation Excerpt :
Taken together, these results show that the block of elongation in MKP-1 is located in the exon 1 possibly in the first 300–400 nt of the 5′-end of the gene. Further fine mapping by KMnO4 footprinting (18) and nuclear run-on performed with antisense oligonucleotide probes (38) will permit us to determine if the block to transcriptional elongation correlates with promoter-proximal pauses sites and/or intrinsic sites of premature termination in the exon 1 of MKP-1. This study identified a calcium-sensitive block to elongation within the first exon of the rat MKP-1 gene as an important element for transcription regulation.
Transcriptional elongation of many eukaryotic, prokaryotic, and viral genes is tightly controlled, which contributes to gene regulation. Here we describe this phenomenon for the MAP kinase phosphatase 1 (MKP-1) immediate early gene. In rat GH4C1 pituitary cells, MKP-1 mRNA is rapidly and transiently induced by the thyrotropin-releasing hormone (TRH) and the epidermal growth factor EGF via transcriptional activation of the gene. Ca²⁺signals are necessary for the induction of MKP-1 in response to TRH but not to EGF. Reporter gene analysis with the newly cloned rat promoter sequence shows only limited induction in response to various stimuli, including TRH or EGF. By nuclear run-on assays we demonstrate that in basal conditions, a strong block to elongation in the first exon regulates the MKP-1 gene and that stimulation with either TRH or EGF overcomes the block. Ca²⁺ signals are important to release the MKP-1 elongation block in a manner similar to the c-fosoncogene. These results suggest that a common mechanism of intragenic regulation may be conserved between MKP-1 and c-fos in mammalian cells.
Influence of polyamines on DNA binding of heat shock and activator protein 1 transcription factors induced by heat shock
1999, FEBS Letters
Polyamine depletion, obtained in FAO cells with specific inhibitors of biosynthetic enzymes, prevents or decreases the accumulation of hsp 70 mRNA following heat shock [Desiderio et al., Hepatology 24 (1996) 150–156]. The present study shows that under conditions of spermidine depletion caused by α-difluoromethylornithine, the DNA binding capacity of the transcription factor HSF induced by heat shock undergoes a severe and prompt deactivation. Replenishment of the spermidine pool before heat shock re-establishes the DNA binding activity of HSF and the inducibility of hsp 70 mRNA. Similar to HSF, but with a different time-course, the DNA binding of the transcription factor AP-1 activated by heat shock is also impaired in spermidine-depleted cells and reversed by exogenous spermidine. STAT3 provides an example of a transcription factor slightly activated by heat shock but insensitive to polyamine decrease.
Identifying regulators of transcript elongation in eukaryotes
1996, Methods in Enzymology
The elongation step of transcription is a dynamic process that provides many opportunities for regulation. Biochemical analysis of this reaction has allowed transcript elongation to be staged into several distinct mechanistic steps, each of which can be assayed. The assays themselves are simple and technically straightforward, but their design and interpretation require some understanding of the transcript-elongation process. Many eukaryotic genes are subject to some form of regulation during the elongation process. The regulation is influenced by trans-acting factors that can stimulate or inhibit the activity of the transcribing RNA polymerase. This chapter highlights the various methods and the types of assays that can be used to identify such factors in eukaryotic cells. The chapter illustrates the elongation assays of one such factor, TFIIS. The process of transcript elongation is discussed, with a particular emphasis on the definition of some of the commonly used terms and the steps where transcription regulation might occur.
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Regular ArticleActivation of Pausing RNA Polymerases by Nuclear Run-on Experiments

Abstract

Regular Article
Activation of Pausing RNA Polymerases by Nuclear Run-on Experiments