1. Crosstalk between mRNA 3′ End Processing and Transcription Initiation
Christophe K. Mapendano, Søren Lykke-Andersen, Jørgen Kjems, Edouard Bertrand, Torben Heick Jensen 
Molecular Cell 
Volume 40, Issue 3, Pages (November 2010)
DOI: /j.molcel
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1
Adverse Effects on RNA Levels and Transcription Factor Recruitment by a pA Site Point Mutation
(A) Schematic drawing of the β-globin genes under investigation. The sequences of the β pA+ and β pA− genes differ at a single nucleotide position in the SV40 LpA hexamer (shown above the gene). The drawing is in scale, but interrupted at various positions. When relevant, the distance of the interrupted sequence is given. pc5 5′ indicates sequences from the pcDNA5 FRT/TO vector that is included in the transcript. FRT is the site of recombination—upstream sequences are from the pcDNA5-β-globin vector and downstream sequences (including the promoter-less lacZ-Zeocin gene) are from the integration site of the HEK293 Flp-In T-rex cell line (Invitrogen). ex, exon; int, intron; 6xMS2, six repeats of bacteriophage MS2 sites for RNA FISH. Positions of probes and amplicons used for northern blotting, RNaseH, RNA FISH, RT-PCR, and ChIP (with distance from center of amplicon to TATA box indicated) are shown in scale as black bars below the gene. The primer used for complementary DNA (cDNA) synthesis is indicated with an arrowhead. dT, oligo-dT oligo (shown in brackets since the sequence does not match that of the gene); rt, readthrough.
(B) Northern blotting analysis of total RNA purified from uninduced (−) or tetracycline-induced (+) β pA+ or β pA− cell lines (24 hr induction). The blots were hybridized with probes directed against β-globin RNA (top) and GAPDH RNA as an internal loading standard (bottom). Positions of the β-globin mRNA and readthrough RNA are indicated to the right of the top panel.
(C) RNaseH/Northern blotting analysis of the β-globin RNA shown in (B). RNA samples were treated with RNaseH in combination with no (−), dT, or rt oligo. mRNA-A0 denotes the position of mRNA with no pA tail (present in lanes 2 and 5); mRNA-pA/rt72 denotes the coinciding position of mRNA with a pA tail (present in lanes 1, 3, 4, and 6) or readthrough RNA (with ∼72 readthrough nucleotides relative to the site of the pA tail; present in lanes 4 and 6). The blots were probed for GAPDH RNA as an internal loading standard (not shown).
(D) Quantification of the β-globin mRNA-A0 and mRNA-pA/rt72 “focused” bands shown in (C) normalized to GAPDH levels. The histogram represents data from three independent experiments (n = 3). Error bars depict standard deviations. The levels of β-globin mRNA-pA/rt72 in lane 3 were arbitrarily set to 1. The RNA readthrough fraction was calculated as follows: 100% · (N(lane ‘rt’) −N(lane ‘dT’))/N(lane ‘rt’), where N is the normalized intensity of the “focused” band.
(E) Half-lives of spliced polyadenylated (pA-RNA) and spliced readthrough (rt-RNA) species produced from the β pA+ and β pA− genes. The values including standard deviations were obtained from three independent pulse-chase assays (n = 3), of which one is shown in Figures S1B and S1C.
(F) ChIP analysis of RNAPII (RPB1) occupancy at the indicated positions of the β-globin genes after 24 hr of induction. Levels were normalized to RPB1 occupancy at the GAPDH promoter (see Figure 2, panel vi). Histograms represent data from two independent biological experiments (n = 2), each with three technical repeats (qPCR). Error bars depict standard deviations. The same trends were observed in several additional independent experiments. m.p.i., mean percent of input.
(G) ChIP analysis of TBP occupancy performed as in (F).
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
RNAPII and GTF Levels at the β pA− Promoter Decrease between 1 hr and 2 hr after Transcription Induction
(A) ChIP analysis of RNAPII (RPB1) occupancy at the promoter (i), exon2 (ii), the 3′ end (iii), and the readthrough regions A (rt A, [iv]) and B (rt B, [v]) at times 0, 1, 2, and 24 hr after induction. RPB1 ccupancy at the GAPDH promoter, which was used for normalization, is shown in (vi).
(B and C) As in (A), but measuring TBP and TFIIB occupancies, respectively.
(A) and (B) include the data presented in Figures 1F and 1G and represent data from two independent biological experiments (n = 2) each with three technical repeats. (C) shows data from a single representative biological experiment (n = 1) with three technical repeats. Error bars depict standard deviations.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

4. Figure 3
Evidence for Decreased Transcription Initiation by Nascent RNA Analysis
(A) Schematic drawing of transcription from the β pA+ (top) and β pA− (bottom) genes under investigation to illustrate the complexity in interpreting NRO data. The circle, red bar, and purple bar on the RNA depict the 5′ cap, the pA site, and an arbitrarily picked region of the RNA, respectively. Asterisks indicate incorporation of BrUTP into the nascent RNA. To the right is shown a theoretical example of NRO measurements of the indicated features after immunoprecipitation of BrUTP-containing RNAs. In the given example, equal transcription initiation rates between the two genes will result in β pA−/β pA+ NRO ratios of 1.5, 2, and 3 for “capped,” “red,” and “purple” RNAs, respectively.
(B) NRO assay performed after 1 hr (left) and 24 hr (middle) of transcription induction. The right panel compares the values obtained for the β pA− gene after 1 hr and 24 hr. NRO levels were normalized to an internal control (PROMPT 40-13) and depicted relative to levels from the β pA+ gene. cDNA synthesis was performed with random primers. A representative experiment with three technical repeats is shown. Error bars depict standard deviations.
(C) RT-qPCR measurements of 5′ ends of total β-globin RNA relative to β-actin mRNA from chromatin, cytoplasmic and nuclear fractions of β pA+ and β pA− cell lines tetracycline-induced for the indicated periods of time. A representative experiment with three technical repeats including standard deviations is shown. cDNA synthesis was performed with the primer indicated in Figure 1A and a primer specific for β-actin.
(D) β-globin RNA FISH on tetracycline-induced β pA+ and β pA− cell lines (24 hr of induction). RNA was detected with a Cy3-labeled probe directed against the 6xMS2 sites present between exon3 and the SV40 LpA signal. Images show overlay of DAPI (blue) and Cy3 (red) signals. The bottom panel shows enlarged pictures of the nuclear foci framed in the top panel.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

5. Figure 4
Decreased Transcription Initiation by an Independent pA Site Point Mutation
(A) Schematic drawing of the HIV-1 genes under investigation. For details, see the legend to Figure 1A.
(B) Northern blotting analysis of total RNA purified from uninduced (−) or tetracycline-induced (+) HIV-1 pA+ or pA− cell lines (24 hr induction). The blots were hybridized with a probe directed against HIV-1 RNA (top), and 18S rRNA was visualized with ethidium bromide staining of the agarose gel before transfer as an internal loading standard (bottom). Positions of HIV-1 mRNA and pre-mRNA are indicated.
(C) ChIP analysis of RNAPII occupancy at the indicated positions of the HIV-1 genes after 24 hr of induction. A representative experiment with three technical repeats including standard deviations is shown. The mean percent of input levels of the GAPDH promoter control amplicon were 2.1% ± 0.1% and 1.3% ± 0.1% for pA+ and pA−, respectively.
(D) ChIP analysis of TBP as in (C). The mean percent of input levels of the GAPDH promoter control amplicon were 0.74% ± 0.04% and 0.66% ± 0.09% for β pA+ and β pA−, respectively. In (C) and (D), data handling were as in Figures 1G and 1H.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

6. Figure 5
PCF11 Depletion Causes Decreased Levels of RNAPII and TFIIB at the β pA+ and Endogenous Promoters
(A) Western blot analysis demonstrating efficient depletion of PCF11. The blot was probed with antibodies against PCF11 and TFIIB. The lower panel shows an additional loading control, which is an unspecific cross-reactive species detected by the PCF11 antibody.
(B) Levels of spliced, 3′ end extended (cont.) or readthrough region (rt A) β pA+ RNA normalized to the RNAPIII transcript Y5 in the control and PCF11-depleted samples measured by RT-qPCR. For visualization purposes, the signal for each amplicon is shown relative to the control level. Standard deviations are shown.
(C) ChIP analysis of RNAPII (RBP1) occupancy at the indicated positions of the β pA+ gene after 24 hr of induction in PCF11-depleted or control conditions. Levels are not normalized to GAPDH promoter occupancy, since these levels are significantly altered upon PCF11 depletion (see G). A representative experiment with three technical repeats including standard deviations is shown. The relative enrichment of readthrough RNAPII in PCF11-depleted cells compared to the control is estimated by calculating the ratio of the ratios between readthrough and gene body amplicon signals as indicated below the histogram (e.g., readthrough at amplicon rt A compared to “body” amplicon ex2 is calculated with the formula NrtAPCF11/Nex2PCF11/NrtAcontrol/Nex2control), where N is the measured ChIP signal.
(D) ChIP analysis of TFIIB occupancy at the β pA+ promoter performed as in (C).
(E) Top: Levels of spliced, 3′ end extended (cont.) or readthrough region (rt) cMYC RNA normalized to Y5 RNA and to levels from control samples. Bottom: ChIP analysis of RNAPII (RBP1) and TFIIB occupancy at the cMYC promoter. Error bars depict standard deviations. As in (C), the relative enrichment of readthrough (rt) RNAPII in the PCF11-depleted samples is calculated relative to either promoter (prom) signal or the signal from an amplicon positioned approximately in the middle of the gene (body); ChIP signals from these positions are shown in Figure S3.
(F–G) As (E), but for the p21 and GAPDH genes, respectively.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

7. Figure 6
The RNAPII CTD-Ser2 Phosphorylation Profile Is Altered upon pA Site Mutation
(A) ChIP analysis of RNAPII CTD-Ser5P relative to RNAPII (RPB1) occupancy at the indicated positions of the β-globin genes after 24 hr of induction. Levels were normalized to occupancy at the GAPDH promoter. A representative experiment with three technical repeats is shown. Error bars depict standard deviations. The ratios of mean percent of input levels of the GAPDH promoter control amplicon were 0.17 ± 0.05 and 0.17 ± 0.02 for β pA+ and β pA−, respectively.
(B) ChIP analysis of RNAPII CTD-Ser2P per RNAPII occupancy performed as in (A). GAPDH control ratios were 0.10 ± 0.03 and 0.13 ± 0.01 for β pA+ and β pA−, respectively.
(C) ChIP analysis of CDK9 occupancy performed as in (A).
(D) ChIP analysis of CDK9 relative to RNAPII (RPB1) performed as in (A). The data in (C) and (D) are also shown in the context of the CDK9 induction time course ChIP experiment presented in Figure S5, where the GAPDH control levels are also shown.
See also Figures S4 and S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions

8. Figure 7
Model
Working model illustrating RNAPII, GTF (TBP, TFIIB) and CDK9 dynamics in the transcription of genes with a WT (A) or a mutated (B) polyadenylation site. (i) and (ii) depict two nonmutually exclusive scenarios explaining the depletion of RNAPII from the pA− gene. (i) suggests inefficient recycling of CDK9 and RNAPII due to the extended residence time on the pA− transcription unit. (ii) suggests displacement of GTFs from promoter to readthrough region followed by displacement of nonreset RNAPII back to the promoter and GTFs into the gene body. Dashed lines designate recycling of factors in the indicated direction. Black solid lines indicate putative transient interactions. RNAPII(O) and RNAPII(A) indicate hyper- (elongation mode) and hypophosphorylated RNAPII (inititiation mode), respectively. Additionally, white circles with a 5 (=Ser5P) or a 2 (=Ser2P) indicate the predominant phosphorylation status of the CTD. The red bar on the RNA indicates the pA site. See the main text for details.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2010 Elsevier Inc. Terms and Conditions