1. Volume 21, Issue 5, Pages 629-639 (March 2006)
Growth Factor Signaling Regulates Elongation of RNA Polymerase I Transcription in Mammals via UBF Phosphorylation and r-Chromatin Remodeling 
Victor Stefanovsky, Frédéric Langlois, Thérèse Gagnon-Kugler, Larry I. Rothblum, Tom Moss 
Molecular Cell 
Volume 21, Issue 5, Pages (March 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Growth Factor Enhancement of rRNA Gene Expression Does Not Increase in RNAPI Loading as Determined by Nuclear Run On
(A) Experimental plan. At zero time, mouse NIH3T3 cells were stimulated by the addition of EGF or serum (S), the ERK pathway was blocked with the MEK1/2 inhibitors PD98059 (PD) or U0126 (U), or cells were untreated (Ctl). The 45S rRNA was determined by pulse labeling, and RNAPI loading was determined at midpulse either by nuclear run on or chromatin immunoprecipitation (ChIP). Maximal/minimal transcription rates occur at 10–30 min poststimulation/repression (Stefanovsky et al., 2001b).
(B) 45S rRNA transcription rates are indicated relative to the level in PD98059-treated cells. Panels above show incorporation of [3H]-uridine into the 45S rRNA precursor (fluorogram) and 18S levels (EtBr staining) in one analysis.
(C) Top panels, nuclear run on hybridizations to 5′-ETS probes +1 to +293 bp (Probe 2) and +1 to +3101bp (Probe 1) compared with −167 to −1bp (Probe 3) and vector. Histogram shows the [32P] nuclear run on signals relative to those for the PD98059 for Probes 1 and 2 and the mean of these two probes.
(B) and (C) represent the average of seven independent experiments each performed in triplicate, error bars represent ± one standard error of the mean (SEM).
(D) NIH3T3 cells were treated as in (A) but then chased and labeled RNA analyzed, see the Experimental Procedures. The right panel shows the 45S to 32S rRNA ratio.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
ChIP Assays of RNAPI Loading Also Show that Growth Factor Enhancement of Ribosomal RNA Gene Expression in NIH3T3 Cells Does Not Result from an Increase in RNAPI Loading
(A) Position of the five mouse ribosomal gene segments, (PCR probes), 45S-5′, ETS, ITS-2, 45S-3′, and IGS1, used to analyze yield of immunoprecipitated DNA by quantitative PCR (Q-PCR).
(B) Comparison of 45S transcription rates determined by pulse labeling, as in Figure 1, with RNAPI loading determined as the yield of various gene segments after immunoprecipitation with an antibody raised against the β′ (194 kDa) subunit of RNAPI. The yield of 45S-5′ gene segment in control immunoprecipitations with a preimmune serum (Pre-Imm) and a serum against the phosphorylated CTD of the elongating form of RNA polymerase II (RNAPII) (Vincent et al., 1996) are also shown. Data have been normalized to the results for PD98059-treated cells. Cell treatments, labeling, and harvesting were as indicated in Figure 1A.
(C) 45S production rates and RNAPI loading were determined in parallel, respectively, by pulse labeling and ChIP in rapidly proliferating, unstarved NIH3T3 cells before (0 min) or after inhibition for 15 or 30 min with 50 ng/ml actinomycin D (ActD). Data have been normalized to the results for 0 min.
In (B) and (C), the insets show examples of 45S labelings and the 18S levels on corresponding electrophoretic analyses. Both the data in (B) and in (C) are the mean of three independent experiments, each performed in triplicate, error bars represent ± one SEM.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Direct Determination of Elongation Rates in Growth-Stimulated and Inhibited NIH3T3 Cells
(A) Diagram describing the expected time course of incorporation of radioactive label into 45S rRNA during and just after the time taken to complete the synthesis of a full transcript. RNAPI molecules are indicated as solid ellipses transcribing along the rRNA gene. Specific RNAPI molecules are shown by different open symbols to indicate the progression of transcription. When labeling is started at time t0 (top), all initiated transcripts are as yet unlabeled (vertical gray lines). As the early phase of labeling proceeds (center panels), nascent transcripts initiated before labeling began will become partially labeled (vertical black lines), dependent on their extent of synthesis at the start of labeling, t0. During this early labeling phase, the rate of incorporation into full-length transcripts (indicated as vertical lines after the plus sign [+]) will be proportional to ɛ × t2 (labeled [shaded] area of full-length transcripts in center panels), where ɛ is the RNAPI elongation rate. The RNAPI molecule indicated by an open cross will complete a full-length transcript when labeling has been continued for a time t, where t = lg × ɛ, lg being the gene length and ɛ the RNAPI elongation rate. Subsequent to this, all transcripts will be labeled from end to end, and incorporation of label into 45S rRNA will increase linearly with time (bottom).
(B) The expected curves are indicated for 45S rRNA labeling under (i) variable elongation rates and (ii) constant elongation rate. Normalizing the steady-state 45 rRNA levels permits visual comparison of the incorporation curves, dashed lines.
(C) Experimental determination of the time course of [3H]-uridine incorporation into 45S rRNA (left) and of [3H]-uridine uptake (right) in NIH3T3 cells treated with EGF or PD98059 (PD).
(D) The 45S data in (C) were normalized to the steady-state levels of labeling and the early time points analyzed by curve fit. Upper panels show an example electrophoretic analysis of 45S and 32S rRNA labeling in EGF-treated cells. 45S labeling and [3H]-uridine uptake data are the mean respectively of five and three independent experiments in duplicate, error bars represent ± one SEM.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
High Template Occupancy by UBF Inhibits RNAPI Transcription In Vitro
(A) Coomassie-stained SDS-PAGE analysis of baculovirus-expressed mammalian UBF1.
(B) In vitro run-off transcription from the mouse RNAPI promoter with addition of increasing amounts of UBF1. The DNA template was transcribed in a reaction containing nuclear extract supplemented with UBF1, see the Experimental Procedures.
(C) A transcription reaction as in (B) was also performed, but UBF1 was replaced by Nbox123 or Nbox123-T117/201E. Template occupancy was estimated by using a binding constant (Kd) of 20 × 10−9 M (Leblanc et al., 1993; Stefanovsky et al., 2006).
(D) Experimental determination of template occupancy by Nbox123. Characteristic changes in the UBF DNase I cleavage pattern around the initiation site were used to estimate template occupancy under the conditions of in vitro transcription (Leblanc and Moss, 2001; Leblanc et al., 1993), the left panel shows representative DNase I cleavage patterns and the right panel the corresponding cleavage profiles from phosphoimager analyses at increasing Nbox123 amounts, see the Experimental Procedures.
(E) In vitro transcription as in (B) and (D) but in the presence of 300 ng Nbox123 or Nbox123-T117/203 phosphorylated with ERK2 for increasing times.
(F) Right, top, Coomassie-stained SDS-PAGE gel of a time course of ERK2 phosphorylation of Nbox123; bottom, PhosphoImager analysis of the same gel. Left, PhosphoImager quantitation of transcription levels in (E) plotted against time of Nbox123 reaction with ERK2.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
UBF Inhibits RNAPI Transcription Downstream of +34 bp Dependent on Its Phosphorylation State
(A) Order of addition of reaction components on the 34 bp G-less cassette template.
(B) In vitro transcription reactions using the 34 bp G-less cassette linked to the mouse RNAPI promoter. Full-length UBF1 (FL-UBF1), see (Figure 4A), was added in increasing amounts to reactions in which the RNAPI transcription complexes had previously been paused at +34 bp by withdrawal of GTP.
(C) Nbox123 was ERK2 or mock phosphorylated for increasing times, e.g., see Figure 4F, and 600 ng added to reactions in which the RNAPI transcription complexes had been paused at +34 bp.
In both (B) and (C), transcription was continued by the addition of GTP and 32P incorporation suppressed by the addition of excess UTP (GTP chase). “5S” refers to end labeling of endogenous 5S RNA during the transcription reaction. In (B) and (C), the gel analyses are shown above the corresponding graphic of the phosphoimager quantification, error bars give an estimated error of ± In (C), phosphorylation levels of Nbox123, determined by γ[32P]-ATP tracing, exceeded 1 mole per mole Nbox123 in a 30 min reaction. Thus, greater than 50% of the ERK sites of Nbox123 were phosphorylated.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
UBF Reversibly Inhibits the Full Elongation Phase of RNAPI Transcription
(A) Order of addition of reaction components on the 64 bp G-less cassette template.
(B) Elongation of preformed transcription complexes paused at +64 was inhibited by 600 ng of Nbox123. heparin, 80 μg/ml, released Nbox123 and relieved this inhibition.
(C) Increasing amounts of full-length UBF1 or double mutant −T117/201E were added to preformed transcription complexes paused at +64 bp before continuing elongation by GTP addition.
(D) Alternatively, increasing amounts of Nbox123 or the −T117/201 mutant were added to complexes paused at +64 bp before continuing elongation. In the experiment shown, a +155 bp template was added in 10-fold excess (Comp. Template) along with the chase.
(E) ERK2- or mock-phosphorylated NBox123 was added to complexes paused at +64 bp before continuing elongation. Phosphorylation levels of Nbox123 exceeded 1 mole per mole Nbox123 in a 30 min reaction.
The graphic plots in (D) and (E) display the mean of three independent experiments, error bars represent ± one SEM.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
Engagement of UBF throughout the 45S rRNA Coding Region Is Not Significantly Affected by Growth Factor Stimulation
(A) UBF ChIP and Q-PCR assays, data are the average of five independent experiments. Error bars represent ± one SEM.
(B) Model for elongation regulation by UBF phosphorylation. In the top panel, multiple enhancesomes are shown impeding the passage of the polymerase, whereas in the bottom panel, local unfolding of the enhancesomes by ERK phosphorylation of UBF (∗) permits elongation. The DNA template is shown in light gray, dimers of core UBF in darker gray shading, and the transcript as a black wavy line.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions