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UBF levels determine the number of active ribosomal RNA genes in mammals.

Sanij E, Poortinga G, Sharkey K, Hung S, Holloway TP, Quin J, Robb E, Wong LH, Thomas WG, Stefanovsky V, Moss T, Rothblum L, Hannan KM, McArthur GA, Pearson RB, Hannan RD - J. Cell Biol. (2008)

Bottom Line: Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased.We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression.Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.

ABSTRACT
In mammals, the mechanisms regulating the number of active copies of the approximately 200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1-induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.

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UBF1/2 depletion causes a modest decrease in net rDNA transcription. (A) NIH3T3 cells transfected with siRNA-EGFP or -UBF1/2 and incubated in phosphate-free DME for 2 h and in phosphate-free DME/FBS containing 0.125 mCi/ml [32P]orthophosphate for 30 min. 32P-labeled cellular RNAs were resolved on 1.2% MOPS-formaldehyde gels and exposed on a PhosphoImaging screen. Total levels of 28S and 18S rRNAs were detected by ethidium bromide staining. (B) 45S rRNA levels in A were quantitated and normalized to corresponding total 28S levels. (C) Total RNA was extracted from siRNA-EGFP– or -UBF1/2–transfected NIH3T3 cells and normalized to an equal number of cells for each sample, and 45S rRNA precursor levels were determined by reverse transcription qRT-PCR using primers to the 5′ ETS (n = 3). (D) qChIP analysis of Pol I (A194 subunit) binding to the rDNA. Pol I enrichment was calculated as described in Fig. 1 D and normalized to the number of active rRNA genes as determined by psoralen cross-linking experiments in Fig. 2 B (n = 3). A representative ethidium bromide gel showing the amount of UCE and ETS1 products amplified after 22 PCR cycles. (E) UBF depletion does not affect Pol I elongation rates in NIH3T3 cells. NIH3T3 cells were transfected with siRNA-EGFP or -UBF1/2 and labeled with 10 μCi [3H]uridine for the indicated times. 3H-labeled cellular RNAs were extracted and resolved on 1% formaldehyde gels, transferred to membrane, and exposed to x-ray films. Total levels of 28S rRNAs were detected by ethidium bromide (EtBr) staining. (F) Duplicate analyses of 3H-labeled 45S rRNA in E were quantitated and normalized to corresponding total 28S levels (EtBr). The curves fitted to the data were calculated as previously shown (Stefanovsky et al., 2006a). The mean per gene elongation time was estimated to be 5 min by extrapolation of the linear phase of incorporation onto the time axis. ENH, enhancer; ITS, internal transcribed spacer; T, terminator region. Mean ± SEM (error bars).
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fig8: UBF1/2 depletion causes a modest decrease in net rDNA transcription. (A) NIH3T3 cells transfected with siRNA-EGFP or -UBF1/2 and incubated in phosphate-free DME for 2 h and in phosphate-free DME/FBS containing 0.125 mCi/ml [32P]orthophosphate for 30 min. 32P-labeled cellular RNAs were resolved on 1.2% MOPS-formaldehyde gels and exposed on a PhosphoImaging screen. Total levels of 28S and 18S rRNAs were detected by ethidium bromide staining. (B) 45S rRNA levels in A were quantitated and normalized to corresponding total 28S levels. (C) Total RNA was extracted from siRNA-EGFP– or -UBF1/2–transfected NIH3T3 cells and normalized to an equal number of cells for each sample, and 45S rRNA precursor levels were determined by reverse transcription qRT-PCR using primers to the 5′ ETS (n = 3). (D) qChIP analysis of Pol I (A194 subunit) binding to the rDNA. Pol I enrichment was calculated as described in Fig. 1 D and normalized to the number of active rRNA genes as determined by psoralen cross-linking experiments in Fig. 2 B (n = 3). A representative ethidium bromide gel showing the amount of UCE and ETS1 products amplified after 22 PCR cycles. (E) UBF depletion does not affect Pol I elongation rates in NIH3T3 cells. NIH3T3 cells were transfected with siRNA-EGFP or -UBF1/2 and labeled with 10 μCi [3H]uridine for the indicated times. 3H-labeled cellular RNAs were extracted and resolved on 1% formaldehyde gels, transferred to membrane, and exposed to x-ray films. Total levels of 28S rRNAs were detected by ethidium bromide (EtBr) staining. (F) Duplicate analyses of 3H-labeled 45S rRNA in E were quantitated and normalized to corresponding total 28S levels (EtBr). The curves fitted to the data were calculated as previously shown (Stefanovsky et al., 2006a). The mean per gene elongation time was estimated to be 5 min by extrapolation of the linear phase of incorporation onto the time axis. ENH, enhancer; ITS, internal transcribed spacer; T, terminator region. Mean ± SEM (error bars).

Mentions: Unexpectedly, rRNA synthesis rates as measured by metabolic labeling (Fig. 8, A and B) or levels of the 5′ external transcribed spacer (ETS; Fig. 8 C) were reduced by only ∼15% in response to depletion of UBF, which is fourfold less than the decrease in number of active genes (∼70% decrease; Fig. 2, A and B). This suggests that the rate of transcription on the remaining active genes was increased. Consistent with this, ChIP analysis using antibodies to the largest subunit of Pol I (RPA194) demonstrated that UBF1/2 knockdown led to a twofold increase in Pol I loading on the remaining 15% of active rRNA genes (Fig. 8 D). This would maintain a nearly constant transcriptional output. We also examined Pol I transcription elongation rates, which are regulated by UBF and are limiting for rRNA gene transcription (Stefanovsky et al., 2006a). In vivo elongation rates were determined as we previously described (Stefanovsky et al., 2006a) using [3H]uridine pulse labeling (Fig. 8, E and F). If slowing of elongation in response to UBF depletion occurs, it would lead to an observable lag before the linear phase of label incorporation is reached. However, if the elongation rates are similar, no lag should be apparent. When 45S rRNA labeling was followed with time in NIH3T3 cells, no difference in the initial incorporation curves in the control siRNA-EGFP– and -UBF1/2–transfected cells was observed (Fig. 8 F), suggesting that UBF depletion did not affect elongation rates. Thus, increased Pol I loading per gene in the absence of appreciable changes in elongation suggests that initiation rates must have increased on the genes remaining active after UBF knockdown.


UBF levels determine the number of active ribosomal RNA genes in mammals.

Sanij E, Poortinga G, Sharkey K, Hung S, Holloway TP, Quin J, Robb E, Wong LH, Thomas WG, Stefanovsky V, Moss T, Rothblum L, Hannan KM, McArthur GA, Pearson RB, Hannan RD - J. Cell Biol. (2008)

UBF1/2 depletion causes a modest decrease in net rDNA transcription. (A) NIH3T3 cells transfected with siRNA-EGFP or -UBF1/2 and incubated in phosphate-free DME for 2 h and in phosphate-free DME/FBS containing 0.125 mCi/ml [32P]orthophosphate for 30 min. 32P-labeled cellular RNAs were resolved on 1.2% MOPS-formaldehyde gels and exposed on a PhosphoImaging screen. Total levels of 28S and 18S rRNAs were detected by ethidium bromide staining. (B) 45S rRNA levels in A were quantitated and normalized to corresponding total 28S levels. (C) Total RNA was extracted from siRNA-EGFP– or -UBF1/2–transfected NIH3T3 cells and normalized to an equal number of cells for each sample, and 45S rRNA precursor levels were determined by reverse transcription qRT-PCR using primers to the 5′ ETS (n = 3). (D) qChIP analysis of Pol I (A194 subunit) binding to the rDNA. Pol I enrichment was calculated as described in Fig. 1 D and normalized to the number of active rRNA genes as determined by psoralen cross-linking experiments in Fig. 2 B (n = 3). A representative ethidium bromide gel showing the amount of UCE and ETS1 products amplified after 22 PCR cycles. (E) UBF depletion does not affect Pol I elongation rates in NIH3T3 cells. NIH3T3 cells were transfected with siRNA-EGFP or -UBF1/2 and labeled with 10 μCi [3H]uridine for the indicated times. 3H-labeled cellular RNAs were extracted and resolved on 1% formaldehyde gels, transferred to membrane, and exposed to x-ray films. Total levels of 28S rRNAs were detected by ethidium bromide (EtBr) staining. (F) Duplicate analyses of 3H-labeled 45S rRNA in E were quantitated and normalized to corresponding total 28S levels (EtBr). The curves fitted to the data were calculated as previously shown (Stefanovsky et al., 2006a). The mean per gene elongation time was estimated to be 5 min by extrapolation of the linear phase of incorporation onto the time axis. ENH, enhancer; ITS, internal transcribed spacer; T, terminator region. Mean ± SEM (error bars).
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fig8: UBF1/2 depletion causes a modest decrease in net rDNA transcription. (A) NIH3T3 cells transfected with siRNA-EGFP or -UBF1/2 and incubated in phosphate-free DME for 2 h and in phosphate-free DME/FBS containing 0.125 mCi/ml [32P]orthophosphate for 30 min. 32P-labeled cellular RNAs were resolved on 1.2% MOPS-formaldehyde gels and exposed on a PhosphoImaging screen. Total levels of 28S and 18S rRNAs were detected by ethidium bromide staining. (B) 45S rRNA levels in A were quantitated and normalized to corresponding total 28S levels. (C) Total RNA was extracted from siRNA-EGFP– or -UBF1/2–transfected NIH3T3 cells and normalized to an equal number of cells for each sample, and 45S rRNA precursor levels were determined by reverse transcription qRT-PCR using primers to the 5′ ETS (n = 3). (D) qChIP analysis of Pol I (A194 subunit) binding to the rDNA. Pol I enrichment was calculated as described in Fig. 1 D and normalized to the number of active rRNA genes as determined by psoralen cross-linking experiments in Fig. 2 B (n = 3). A representative ethidium bromide gel showing the amount of UCE and ETS1 products amplified after 22 PCR cycles. (E) UBF depletion does not affect Pol I elongation rates in NIH3T3 cells. NIH3T3 cells were transfected with siRNA-EGFP or -UBF1/2 and labeled with 10 μCi [3H]uridine for the indicated times. 3H-labeled cellular RNAs were extracted and resolved on 1% formaldehyde gels, transferred to membrane, and exposed to x-ray films. Total levels of 28S rRNAs were detected by ethidium bromide (EtBr) staining. (F) Duplicate analyses of 3H-labeled 45S rRNA in E were quantitated and normalized to corresponding total 28S levels (EtBr). The curves fitted to the data were calculated as previously shown (Stefanovsky et al., 2006a). The mean per gene elongation time was estimated to be 5 min by extrapolation of the linear phase of incorporation onto the time axis. ENH, enhancer; ITS, internal transcribed spacer; T, terminator region. Mean ± SEM (error bars).
Mentions: Unexpectedly, rRNA synthesis rates as measured by metabolic labeling (Fig. 8, A and B) or levels of the 5′ external transcribed spacer (ETS; Fig. 8 C) were reduced by only ∼15% in response to depletion of UBF, which is fourfold less than the decrease in number of active genes (∼70% decrease; Fig. 2, A and B). This suggests that the rate of transcription on the remaining active genes was increased. Consistent with this, ChIP analysis using antibodies to the largest subunit of Pol I (RPA194) demonstrated that UBF1/2 knockdown led to a twofold increase in Pol I loading on the remaining 15% of active rRNA genes (Fig. 8 D). This would maintain a nearly constant transcriptional output. We also examined Pol I transcription elongation rates, which are regulated by UBF and are limiting for rRNA gene transcription (Stefanovsky et al., 2006a). In vivo elongation rates were determined as we previously described (Stefanovsky et al., 2006a) using [3H]uridine pulse labeling (Fig. 8, E and F). If slowing of elongation in response to UBF depletion occurs, it would lead to an observable lag before the linear phase of label incorporation is reached. However, if the elongation rates are similar, no lag should be apparent. When 45S rRNA labeling was followed with time in NIH3T3 cells, no difference in the initial incorporation curves in the control siRNA-EGFP– and -UBF1/2–transfected cells was observed (Fig. 8 F), suggesting that UBF depletion did not affect elongation rates. Thus, increased Pol I loading per gene in the absence of appreciable changes in elongation suggests that initiation rates must have increased on the genes remaining active after UBF knockdown.

Bottom Line: Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased.We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression.Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Research Division, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia.

ABSTRACT
In mammals, the mechanisms regulating the number of active copies of the approximately 200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1-induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.

Show MeSH
Related in: MedlinePlus