Defining the Status of RNA Polymerase at Promoters

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Cell Reports (Impact Factor: 8.36). 10/2012; 2(4). DOI: 10.1016/j.celrep.2012.08.034
Source: PubMed


Recent genome-wide studies in metazoans have shown that RNA polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of a global run-on sequencing assay and chromatin immunoprecipitation sequencing assays and demonstrate that the majority of the Pol II on Drosophila promoters is transcriptionally engaged; very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent-sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify the notion that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila.

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    • "Studies in vitro led to a model in which the role of NELF was to restrain Pol II in the paused state, while in vivo studies revealed that loss of NELF led to more downregulation of gene expression rather than upregulation (Gilchrist et al., 2008). Subsequent studies demonstrated that knockdown of NELF can lead to a global reduction of Pol II occupancy at promoter-proximal regions (Core et al., 2012), which can then lead to nucleosomes moving into the region to prevent re-initiation of Pol II (Core et al., 2012). Thus, how promoter-proximal paused Pol II is restrained 20–60 nt downstream of the promoters is currently unknown. "
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    ABSTRACT: The control of promoter-proximal pausing and the release of RNA polymerase II (Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify that Pol-II-associated factor 1 (PAF1) possesses an evolutionarily conserved function in metazoans in the regulation of promoter-proximal pausing. Reduction in PAF1 levels leads to an increased release of paused Pol II into gene bodies at thousands of genes. PAF1 depletion results in increased nascent and mature transcripts and increased levels of phosphorylation of Pol II's C-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase super elongation complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing PAF1 as a regulator of promoter-proximal pausing by Pol II. Copyright © 2015 Elsevier Inc. All rights reserved.
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    • "The RNA polymerase II (Pol II) recruitment step is followed by another critical regulatory event known as promoter-proximal Pol II pausing, and subsequent promoter escape into productive elongation [79]–[82]. Promoter-proximal Pol II pausing is typically found at transcriptionally active and rapidly induced genes and is characterized by a higher RNA polymerase II density at the 5′ end of the gene [79]–[81]. To investigate whether SFN might interfere with transcription elongation, RNA polymerase II occupancy along the open reading frame of the Cis gene was monitored by chromatin immunoprecipitation and quantitative PCR analysis of amplicons spread along the mouse Cis gene locus (Figure 5D). "
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    ABSTRACT: Signal transducer and activator of transcription STAT5 is an essential mediator of cytokine, growth factor and hormone signaling. While its activity is tightly regulated in normal cells, its constitutive activation directly contributes to oncogenesis and is associated to a number of hematological and solid tumor cancers. We previously showed that deacetylase inhibitors can inhibit STAT5 transcriptional activity. We now investigated whether the dietary chemopreventive agent sulforaphane, known for its activity as deacetylase inhibitor, might also inhibit STAT5 activity and thus could act as a chemopreventive agent in STAT5-associated cancers. We describe here sulforaphane (SFN) as a novel STAT5 inhibitor. We showed that SFN, like the deacetylase inhibitor trichostatin A (TSA), can inhibit expression of STAT5 target genes in the B cell line Ba/F3, as well as in its transformed counterpart Ba/F3-1*6 and in the human leukemic cell line K562 both of which express a constitutively active form of STAT5. Similarly to TSA, SFN does not alter STAT5 initial activation by phosphorylation or binding to the promoter of specific target genes, in favor of a downstream transcriptional inhibitory effect. Chromatin immunoprecipitation assays revealed that, in contrast to TSA however, SFN only partially impaired the recruitment of RNA polymerase II at STAT5 target genes and did not alter histone H3 and H4 acetylation, suggesting an inhibitory mechanism distinct from that of TSA. Altogether, our data revealed that the natural compound sulforaphane can inhibit STAT5 downstream activity, and as such represents an attractive cancer chemoprotective agent targeting the STAT5 signaling pathway.
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    • "Cells treated with flavopiridol, the most potent and selective P-TEFb inhibiting compound, result in inhibition of 60–70% of RNAPII transcription [10] [11]. Furthermore, genome-wide studies find that RNAPII occupies the promoters of most protein-coding genes in Drosophila and human embryonic stem cells without entering into productive elongation [12] [13] [14] [15] [16]. Therefore, P-TEFb plays a critical role in RNAPII regulation and has a broad impact on global gene expression patterns that affect almost every aspect of cellular functions. "
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    ABSTRACT: Bromodomain-containing protein 4 (Brd4) and hexamethylene bisacetamide (HMBA) inducible protein 1 (HEXIM1) are two opposing regulators of the positive transcription elongation factor b (P-TEFb), which is the master modulator of RNA polymerase II during transcriptional elongation. While Brd4 recruits P-TEFb to promoter-proximal chromatins to activate transcription, HEXIM1 sequesters P-TEFb into an inactive complex containing the 7SK small nuclear RNA. Besides regulating P-TEFb's transcriptional activity, recent evidence demonstrates that both Brd4 and HEXIM1 also play novel roles in cell cycle progression and tumorigenesis. Here we will discuss the current knowledge on Brd4 and HEXIM1 and their implication as novel therapeutic options against cancer.
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