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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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Restoration of UBF levels after short- or long-term depletion rescues the number of active rRNA genes. (A) Schematic of experimental timeline. NIH3T3 cells stably transduced with tetracycline-inducible TMP-UBF shRNAmir were cultured in the presence or absence of doxocyclin (DOX) for 12 d. Doxocyclin-treated cells were either maintained in doxocyclin-supplemented media or grown without doxocyclin for a further 8 d. (B) Cells in A were analyzed by Western blotting (top) and psoralen cross-linking experiments (bottom). The white line indicates that intervening lanes have been spliced out. (C) qChIP analysis of UBF binding to the rDNA in cells in A. UBF enrichment was determined as in Fig. 1 D. Mean ± SEM (error bars) of samples in duplicates. (D) MeDIP analysis of rDNA promoter methylation in cells in A. Samples were analyzed by qRT-PCR using the core primers as described in Fig. 1 D. Mean ± SEM (error bars; n = 2). (E) siRNA-EGFP– or -UBF1/2–transfected NIH3T3 cells from Fig. 2 C were maintained in culture for a further 7 d to allow restoration of UBF1/2 protein levels and were harvested for Western blotting (top) and psoralen cross-linking (bottom). ENH, enhancer.
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fig5: Restoration of UBF levels after short- or long-term depletion rescues the number of active rRNA genes. (A) Schematic of experimental timeline. NIH3T3 cells stably transduced with tetracycline-inducible TMP-UBF shRNAmir were cultured in the presence or absence of doxocyclin (DOX) for 12 d. Doxocyclin-treated cells were either maintained in doxocyclin-supplemented media or grown without doxocyclin for a further 8 d. (B) Cells in A were analyzed by Western blotting (top) and psoralen cross-linking experiments (bottom). The white line indicates that intervening lanes have been spliced out. (C) qChIP analysis of UBF binding to the rDNA in cells in A. UBF enrichment was determined as in Fig. 1 D. Mean ± SEM (error bars) of samples in duplicates. (D) MeDIP analysis of rDNA promoter methylation in cells in A. Samples were analyzed by qRT-PCR using the core primers as described in Fig. 1 D. Mean ± SEM (error bars; n = 2). (E) siRNA-EGFP– or -UBF1/2–transfected NIH3T3 cells from Fig. 2 C were maintained in culture for a further 7 d to allow restoration of UBF1/2 protein levels and were harvested for Western blotting (top) and psoralen cross-linking (bottom). ENH, enhancer.

Mentions: Next, we examined whether the newly silenced repeats would eventually become methylated and permanently silenced after long-term UBF depletion or, alternatively, whether they would remain unmethylated and thus could be returned to an open chromatin configuration by restoration of UBF levels. Long-term tetracycline-inducible knockdown of UBF (12 d; Fig. 5, A and B) led to a sustained reduction in the number of active rRNA genes (Fig. 5 B, first and second lanes) and reduced loading of UBF on the rDNA repeats (Fig. 5 C). This was not accompanied by increased methylation at the rRNA gene promoter (Fig. 5 D). Removal of tetracycline after 12 d of knockdown led to a recovery of UBF expression (Fig. 5 B, top) and its occupancy of the rRNA genes (Fig. 5 C), which correlated with the restoration of the number of active genes back to wild-type levels (Fig. 5 B, compare the third, fourth, and fifth lanes). CpG methylation at the rDNA promoter was again unchanged (Fig. 5 D). We also performed recovery experiments by transient knockdown of UBF using siRNA oligonucleotides (Fig. 1, A and B) and followed the recovery of UBF expression and active rRNA genes with time as the siRNA oligonucleotides were depleted. Loss of UBF siRNA led to a recovery of UBF to control levels and restoration of the active gene number (Fig. 5 E). Thus, silencing of rRNA genes in response to UBF depletion is stable and reversible.


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)

Restoration of UBF levels after short- or long-term depletion rescues the number of active rRNA genes. (A) Schematic of experimental timeline. NIH3T3 cells stably transduced with tetracycline-inducible TMP-UBF shRNAmir were cultured in the presence or absence of doxocyclin (DOX) for 12 d. Doxocyclin-treated cells were either maintained in doxocyclin-supplemented media or grown without doxocyclin for a further 8 d. (B) Cells in A were analyzed by Western blotting (top) and psoralen cross-linking experiments (bottom). The white line indicates that intervening lanes have been spliced out. (C) qChIP analysis of UBF binding to the rDNA in cells in A. UBF enrichment was determined as in Fig. 1 D. Mean ± SEM (error bars) of samples in duplicates. (D) MeDIP analysis of rDNA promoter methylation in cells in A. Samples were analyzed by qRT-PCR using the core primers as described in Fig. 1 D. Mean ± SEM (error bars; n = 2). (E) siRNA-EGFP– or -UBF1/2–transfected NIH3T3 cells from Fig. 2 C were maintained in culture for a further 7 d to allow restoration of UBF1/2 protein levels and were harvested for Western blotting (top) and psoralen cross-linking (bottom). ENH, enhancer.
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Related In: Results  -  Collection

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fig5: Restoration of UBF levels after short- or long-term depletion rescues the number of active rRNA genes. (A) Schematic of experimental timeline. NIH3T3 cells stably transduced with tetracycline-inducible TMP-UBF shRNAmir were cultured in the presence or absence of doxocyclin (DOX) for 12 d. Doxocyclin-treated cells were either maintained in doxocyclin-supplemented media or grown without doxocyclin for a further 8 d. (B) Cells in A were analyzed by Western blotting (top) and psoralen cross-linking experiments (bottom). The white line indicates that intervening lanes have been spliced out. (C) qChIP analysis of UBF binding to the rDNA in cells in A. UBF enrichment was determined as in Fig. 1 D. Mean ± SEM (error bars) of samples in duplicates. (D) MeDIP analysis of rDNA promoter methylation in cells in A. Samples were analyzed by qRT-PCR using the core primers as described in Fig. 1 D. Mean ± SEM (error bars; n = 2). (E) siRNA-EGFP– or -UBF1/2–transfected NIH3T3 cells from Fig. 2 C were maintained in culture for a further 7 d to allow restoration of UBF1/2 protein levels and were harvested for Western blotting (top) and psoralen cross-linking (bottom). ENH, enhancer.
Mentions: Next, we examined whether the newly silenced repeats would eventually become methylated and permanently silenced after long-term UBF depletion or, alternatively, whether they would remain unmethylated and thus could be returned to an open chromatin configuration by restoration of UBF levels. Long-term tetracycline-inducible knockdown of UBF (12 d; Fig. 5, A and B) led to a sustained reduction in the number of active rRNA genes (Fig. 5 B, first and second lanes) and reduced loading of UBF on the rDNA repeats (Fig. 5 C). This was not accompanied by increased methylation at the rRNA gene promoter (Fig. 5 D). Removal of tetracycline after 12 d of knockdown led to a recovery of UBF expression (Fig. 5 B, top) and its occupancy of the rRNA genes (Fig. 5 C), which correlated with the restoration of the number of active genes back to wild-type levels (Fig. 5 B, compare the third, fourth, and fifth lanes). CpG methylation at the rDNA promoter was again unchanged (Fig. 5 D). We also performed recovery experiments by transient knockdown of UBF using siRNA oligonucleotides (Fig. 1, A and B) and followed the recovery of UBF expression and active rRNA genes with time as the siRNA oligonucleotides were depleted. Loss of UBF siRNA led to a recovery of UBF to control levels and restoration of the active gene number (Fig. 5 E). Thus, silencing of rRNA genes in response to UBF depletion is stable and reversible.

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