1. Volume 46, Issue 3, Pages 311-324 (May 2012)
mRNA Decapping Factors and the Exonuclease Xrn2 Function in Widespread Premature Termination of RNA Polymerase II Transcription 
Kris Brannan, Hyunmin Kim, Benjamin Erickson, Kira Glover-Cutter, Soojin Kim, Nova Fong, Lauren Kiemele, Kirk Hansen, Richard Davis, Jens Lykke-Andersen, David L. Bentley 
Molecular Cell 
Volume 46, Issue 3, Pages (May 2012)
DOI: /j.molcel
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2. Molecular Cell 2012 46, 311-324DOI: (10.1016/j.molcel.2012.03.006)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1
Xrn2 Associates with Termination and mRNA Decapping Factors, and These Factors Colocalize at Promoter-Proximal Regions
(A) Selected proteins enriched in the Xrn2 IP from RNaseA-treated HeLa nuclear extract analyzed by MS on LTQ and FT platforms (see Experimental Procedures and Table S1). Anti-GFP is a negative control. Total assigned spectra were and for the anti-GFP and and for the anti-Xrn2 LTQ and FT analyses respectively.
(B) Western blots of Xrn2 and GFP IPs probed with anti-TTF2, -Edc3, -Dcp1a, -Dcp2, and Aly as a negative control. Inputs are 7.5% of the IP.
(C) ChIP-seq of pol II, Xrn2 (see also Figure S2A), TTF2, and Dcp1a in HeLa cells on RBM39 and MAT2A. The significance values for co-occurrence of peaks determined by a one-tailed hypergeometic test are: TTF2 versus Xrn e-26; TTF2 versus Dcp e-15; XRN2 versus Dcp e-53 (see Table S4). In all figures genes are oriented 5′–3′ left to right (black arrows) and maps indicate exons and introns.
(D) ChIP-seq heatmap profiles (Experimental Procedures) for pol II, Xrn2, TTF2, and Dcp1a in HeLa cells for 10,034 genes > 2 kb long in the region −1 kb to +2 kb relative to the TSS ranked by pol II signal.
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4. Figure 2
Knockdown of Xrn2 and TTF2 Increases Relative Pol II Occupancy Upstream and Downstream of Start Sites
(A) Pol II ChIP-seq normalized to total read counts on indicated genes in HEK293 cells stably expressing shScramble (scr) and shXrn2+shTTF2 shRNAs. Note reduced pol II density near the TSS and increased density within the gene and 3′ flank with knockdown of Xrn2+TTF2 (see Figure S2A for individual knockdowns). Blue arrow indicates divergent transcription at RBM39 (see Figure S5D) that is slightly elevated by Xrn2+TTF2 knockdown. Images were made using the IGB6.4 Browser (Nicol et al., 2009).
(B) Pol II densities expressed as log2 RPKM (reads per kb per million) in the region from −30 to the poly(A) site (see diagram) in the shScrambled (scr) control relative to uninfected parent and double knockdown lines (see also Figure S2C). Each plot represents a set of 5507 genes > 2 kb long and > 2 kb from neighbors enriched for pol II (FDR < .05) within 500 bases of a TSS that are shared in common between the uninfected parent, scrambled control, and knockdown datasets. We noted some decrease in RPKM in knockdown lines among more highly expressed genes but the number of genes that significantly diverge from the scrambled control (p < .01, two-tailed t test) is a small fraction of the total.
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5. Figure 3
Knockdown of Xrn2 and TTF2 Shifts the Distribution of Pol II Away from the TSS
(A) ChIP-seq relative frequency profiles (reads per 50 bp bin divided by total reads in all bins, see Experimental Procedures) in shScramble (scr) control and shXrn2+shTTF2 expressing HEK293 cells across 5507 genes used in Figure 2B.
(B) Log2 ratio of pol II ChIP-seq read frequencies in uninfected parent and knockdown cell lines relative to the shScramble (scr) control. Note the additive effect with Xrn2+TTF2 knockdown.
(C) Definition of Escape Index (EI).
(D) Escape index (EI) for the 5507 genes analyzed in Figure 2B (Table S2). The numbers of genes that differ from the scr control (FDR < 0.01, one-sample t test) are given. Note widespread elevation of EI in the Xrn2:TTF2 knockdown line. Best fit lines (orange) were generated by loess fitting (Local Polynomial Regression Fitting). Black dots correspond to genes with significant (FDR < .05) 5′ peaks of Dcp1a ChIP signal in HeLa (Table S2).
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6. Figure 4
Knockdown of Decapping Factors Increases Relative Pol II Occupancy Upstream and Downstream of Start Sites
(A) Pol II ChIP-seq normalized to total read counts in HEK293 cells stably expressing shScramble (scr) and shDcp2 shRNA. Note reduced pol II density near the TSS and increased density within the gene and 3′ flank with knockdown of Dcp2 (see Figure S2B for Edc3 and Dcp1a knockdowns). Blue arrow indicates divergent transcription at RBM39 (see Figure S5D) that is slightly elevated by Dcp2 knockdown.
(B) Pol II densities expressed as log2 RPKM (reads per kb per million) in the region from −30 to the poly(A) site in the scr control relative to uninfected parent and shDcp2 as in Figure 2B (see also Figure S2C). Note knockdown of Dcp2 does not have a major effect on total pol II occupancy within genes.
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7. Figure 5
Knockdown of Decapping Factors and Spt5 Cause Similar Repositioning of Pol II Away from the TSS
(A) ChIP-seq relative frequency profiles (5507 genes) in shScramble (scr) and shDcp2 expressing HEK293 cells as in Figure 3A.
(B) Log2 ratio of pol II ChIP-seq read frequencies in uninfected parent and knockdown cell lines relative to the scr control as in Figure 3B.
(C) Escape index (EI, see Figure 3C) in knockdown lines and uninfected parent compared to scr control as in Figure 3D. The numbers of genes that differ from the scr control (FDR < 0.01, one-sample t test) are given. Note widespread elevation of EI particularly in the Dcp2 knockdown line. Black dots correspond to genes with significant 5′ peaks of Dcp1a ChIP signal as in Figure 3D.
(D) ChIP-seq relative frequency profiles as in (A) in mouse ES cells expressing shScramble (scr) (6813 genes) shNelfA (6467 genes) and shSpt5 (5604 genes) shRNAs from (Rahl et al., 2010).
(E) Log2 ratios of pol II ChIP-seq read frequencies in shNelfA and shSpt5 expressing ES cells relative to the scr control.
(F) Escape index (EI) in shNelfA and shSpt5 ES cells relative to the scr control. FDR was not calculated because only one control, the scrambled shRNA, was analyzed. Note elevation of EI (F) and pol II relative frequency in the gene body (D) in Spt5 knockdown cells is comparable to decapping factor knockdown cells (A–C).
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8. Figure 6
Knockdown of Termination and Decapping Factors Enhances Pol II Density within “Pausing-Regulated” Genes
(A) Pol II ChIP-seq profiles on the c-Myc and HSF targets HSP90AA1, PIM1, CCNB1, and UBA52 in HEK293 cells knocked down for Xrn2+TTF2 and Dcp2 and in mouse ES cells knocked down for NelfA and Spt5 (Rahl et al., 2010) with shScramble (scr) controls.
(B) Escape index (EI) plots in knockdown lines and uninfected parent versus scr control as in Figure 3D for 173 genes (Table S2) likely to be regulated at the level of elongation including targets of Myc (Li et al., 2003) and HSF. Note widespread elevation of EI in the knockdown lines.
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9. Figure 7
The Promoter-Proximal “Torpedo” Model for Premature Termination of Pol II Transcription
Cotranscriptional decapping by Dcp2 at promoter-proximal pause sites is proposed to expose a 5′ PO4 that is attacked by the exonuclease Xrn2 leading to termination facilitated by TTF2 and possibly other factors. Polymerases that escape decapping are bound by cap binding complex (CBC) and may pause, resume productive elongation, or terminate by an alternative mechanism. We speculate that promoter-proximal pausing serves as a decision point for regulated decapping.
Molecular Cell  , DOI: ( /j.molcel )
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