Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons

Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ∼3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes.DOI: http://dx.doi.org/10.7554/eLife.02407.001

Defining NELF-E RNA binding in HIV-1 and promoter-proximal pause regions

The four-subunit Negative Elongation Factor (NELF) is a major regulator of RNA Polymerase II (Pol II) pausing. The subunit NELF-E contains a conserved RNA Recognition Motif (RRM) and is proposed to facilitate Poll II pausing through its association with nascent transcribed RNA. However, conflicting ideas have emerged for the function of its RNA binding activity. Here, we use in vitro selection strategies and quantitative biochemistry to identify and characterize the consensus NELF-E binding element (NBE) that is required for sequence specific RNA recognition (NBE: CUGAGGA(U) for Drosophila). An NBE-like element is present within the loop region of the transactivation-response element (TAR) of HIV-1 RNA, a known regulatory target of human NELF-E. The NBE is required for high affinity binding, as opposed to the lower stem of TAR, as previously claimed. We also identify a non-conserved region within the RRM that contributes to the RNA recognition of Drosophila NELF-E. To understand the broader functional relevance of NBEs, we analyzed promoter-proximal regions genome-wide in Drosophila and show that the NBE is enriched +20 to +30 nucleotides downstream of the transcription start site. Consistent with the role of NELF in pausing, we observe a significant increase in NBEs among paused genes compared to non-paused genes. In addition to these observations, SELEX with nuclear run-on RNA enrich for NBE-like sequences. Together, these results describe the RNA binding behavior of NELF-E and supports a biological role for NELF-E in promoter-proximal pausing of both HIV-1 and cellular genes

Dissection Of The Precise Mechanisms Of Rna Polymerase Ii Pausing And Elongation Using Nascent Transcript Analysis

Limiting RNA polymerase II (Pol II) at various stages of the transcription cycle is critical for gene regulation, which often occurs during the elongation stage at promoter proximal pause sites and in gene bodies. To determine the distribution of Pol II along genes, I used nascent transcript analysis as a general method. First, I identified the precise positions of Pol II pausing near promoters using a genome-wide nuclear run-on, called Precision Run-On sequencing (PRO-seq) in Drosophila embryonic cells. Using this, I revealed how the position of pausing is associated with initiation and promoter DNA elements. To further dissect the precise dynamics of paused Pol II, I probed the stability of paused Pol II and its termination by analyzing steady-state turn-over of the nascent transcript associated with Drosophila Hsp70 promoter. This shows that paused Pol II on Hsp70 is stable for around 5 min and can either terminate or elongate into the gene body, which is consistent with optical measurements of paused Pol II. I also examined how Pol II elongates during the time course of rapid and robust inhibition of pause escape in mouse embryonic stem cells. The analysis of the elongation rates in nearly 1,000 genes showed tight interplay between promoter proximal pausing, early elongation rates, and co-transcriptional splicing at the beginning of the genes. Finally, I demonstrate that the nascent transcriptome analysis methods can be directly extended into mammalian tissues, and show possibility of linking the study of the fundamental mechanism of Pol II into biomedical applications

eRNA co-expression network uncovers TF dependency and convergent cooperativity

Abstract Enhancer RNAs (eRNAs) are non-coding RNAs produced by transcriptional enhancers that are highly correlated with their activity. Using a capped nascent RNA sequencing (PRO-cap) dataset in human lymphoblastoid cell lines across 67 individuals, we identified inter-individual variation in the expression of over 80 thousand transcribed transcriptional regulatory elements (tTREs), in both enhancers and promoters. Co-expression analysis of eRNAs from tTREs across individuals revealed how enhancers are associated with each other and with promoters. Mid- to long-range co-expression showed a distance-dependent decay that was modified by TF occupancy. In particular, we found a class of “bivalent” TFs, including Cohesin, that both facilitate and isolate the interaction between enhancers and/or promoters, depending on their topology. At short distances, we observed strand-specific correlations between nearby eRNAs in both convergent and divergent orientations. Our results support a cooperative model of convergent eRNAs, consistent with eRNAs facilitating adjacent enhancers rather than interfering with each other. Therefore, our approach to infer functional interactions from co-expression analyses provided novel insights into the principles of enhancer interactions as a function of distance, orientation, and binding landscapes of TFs

TED-Seq Identifies the Dynamics of Poly(A) Length during ER Stress

Summary: Post-transcriptional RNA processing is a core mechanism of gene expression control in cell stress response. The poly(A) tail influences mRNA translation and stability, but it is unclear whether there are global roles of poly(A)-tail lengths in cell stress. To address this, we developed tail-end displacement sequencing (TED-seq) for an efficient transcriptome-wide profiling of poly(A) lengths and applied it to endoplasmic reticulum (ER) stress in human cells. ER stress induced increases in the poly(A) lengths of certain mRNAs, including known ER stress regulators, XBP1, DDIT3, and HSPA5. Importantly, the mRNAs with increased poly(A) lengths are both translationally de-repressed and stabilized. Furthermore, mRNAs in stress-induced RNA granules have shorter poly(A) tails than in the cytoplasm, supporting the view that RNA processing is compartmentalized. In conclusion, TED-seq reveals that poly(A) length is dynamically regulated upon ER stress, with potential consequences for both translation and mRNA turnover. : Woo et al. develop TED-seq, a method providing robust, sensitive, and cost-efficient transcriptome-wide measurements of poly(A) length in human cells. Using TED-seq, they show that mRNA poly(A) lengths are dynamic and associated with the control of both protein synthesis and mRNA stability under cell stress. Keywords: ER stress, RNA granules, TED-seq, poly(A) tail, poly(A) length, polyadenylation, translation, stabilit

Flavonoids inhibit the AU-rich element binding of HuC

Post-transcriptional regulation of mRNA stability by Hu proteins is an important mechanism for tumorigenesis. We focused on the molecular interactions between the HuC protein and AU-rich elements (AREs) to find chemical inhibitors of RNA-protein interactions using RNA electrophoretic mobility shift assay with non-radioactive probes. Screening of 52 natural compounds identified 14 candidate compounds that displayed potent inhibitory activity. Six (quercetin, myricetin, (-)-epigallocatechin gallate, ellagic acid, (-)-epicatechin gallate, and rhamnetin) were categorized as phytochemicals, and their IC(50) values were low (0.2-1.8 mu M).This work was supported by the BK21 program of the Korea Ministry of Education and Human Resources Development and by grant No. 03-2008-017 from the SNUH Research fund.Gong YH, 2008, BMB REP, V41, P287Han KY, 2008, BMB REP, V41, P328Dormoy-Raclet V, 2007, MOL CELL BIOL, V27, P5365, DOI 10.1128/MCB.00113-07Eberhardt W, 2007, PHARMACOL THERAPEUT, V114, P56, DOI 10.1016/j.pharmthera.2007.01.002Jung KC, 2006, LEUKEMIA, V20, P122, DOI 10.1038/sj.leu.2404022Akamatsu W, 2005, P NATL ACAD SCI USA, V102, P4625, DOI 10.1073/pnas.0407523102Li Y, 2004, J BIOCHEM BIOPH METH, V60, P85, DOI 10.1016/j.jbbm.2004.03.008Park-Lee S, 2003, J BIOL CHEM, V278, P39801, DOI 10.1074/jbc.M307105200Bandyopadhyay S, 2003, BIOCHEM PHARMACOL, V66, P1151, DOI 10.1016/S0006-2952(03)00453-2Rodgers JT, 2000, ANAL BIOCHEM, V277, P254, DOI 10.1006/abio.1999.4394King P H, 2000, Nucleic Acids Res, V28, pE20, DOI 10.1093/nar/28.7.e20Akamatsu W, 1999, P NATL ACAD SCI USA, V96, P9885, DOI 10.1073/pnas.96.17.9885Lazarova DL, 1999, ONCOGENE, V18, P2703, DOI 10.1038/sj.onc.1202621Sakai K, 1999, FEBS LETT, V446, P157, DOI 10.1016/S0014-5793(99)00206-9Peng SSY, 1998, EMBO J, V17, P3461, DOI 10.1093/emboj/17.12.3461Dobashi Y, 1998, BIOCHEM BIOPH RES CO, V244, P226, DOI 10.1006/bbrc.1998.8247Okano HJ, 1997, J NEUROSCI, V17, P3024Myer VE, 1997, EMBO J, V16, P2130, DOI 10.1093/emboj/16.8.2130Chagnovich D, 1996, J BIOL CHEM, V271, P33587Chung SM, 1996, J BIOL CHEM, V271, P11518Ma WJ, 1996, J BIOL CHEM, V271, P8144LIU J, 1995, NEUROLOGY, V45, P544CAMPOS AR, 1985, J NEUROGENET, V2, P197, DOI 10.3109/01677068509100150

Defining the Status of RNA Polymerase at Promoters

SummaryRecent 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

Chemical inhibitors destabilize HuR binding to the AU-rich element of TNF-α mRNA

Hu protein R (HuR) binds to the AU-rich element (ARE) in the 3'UTR to stabilize TNF-α mRNA. Here, we identified chemical inhibitors of the interaction between HuR and the ARE of TNF-α mRNA using RNA electrophoretic mobility gel shift assay (EMSA) and filter binding assay. Of 179 chemicals screened, we identified three with a half-maximal inhibitory concentration (IC50) below 10 µM. The IC50 of quercetin, b-40, and b-41 were 1.4, 0.38, and 6.21 µM, respectively, for binding of HuR protein to TNF-α mRNA. Quercetin and b-40 did not inhibit binding of tristetraprolin to the ARE of TNF-α mRNA. When LPS-treated RAW264.7 cells were treated with quercetin and b-40, we observed decreased stability of TNF-α mRNA and decreased levels of secreted TNF-α. From these results, we could find inhibitors for the TNF-α mRNA stability, which might be used advantageously for both the study for post-transcriptional regulation and the discovery of new anti-inflammation drugs