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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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Loss of UBF1/2 expression correlates with rDNA silencing during granulocyte differentiation. (A) Phase-contrast microscopy of uninduced granulocytic MPRO cells (D0) and differentiated granulocytes (D4) stained with May-Grunwald–Giemsa. (B) Western blots of UBF1/2 and tubulin in day 0 and day 4 cells (n = 2). (C) One representative qChIP analysis of previously published data (Poortinga et al., 2004) showing UBF binding to the rDNA in day 0 and day 4 cells. UBF enrichment was determined as in Fig. 1 D. ENH, enhancer. (D) Nuclei from day 0 and day 4 cells (n = 2) were analyzed by psoralen cross-linking assay. (E) The results (n = 3) similar to D were quantitated (*, P < 0.05). (F) Genomic DNA from day 0 and day 4 cells was extracted, digested with HpaII or MspI, and analyzed by Southern blotting of rDNA. The relative amounts of methylated and unmethylated rRNA genes are calculated as in Fig. 4 C and represented graphically (n = 3). Mean ± SEM (error bars). Bar, 30 μm.
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fig10: Loss of UBF1/2 expression correlates with rDNA silencing during granulocyte differentiation. (A) Phase-contrast microscopy of uninduced granulocytic MPRO cells (D0) and differentiated granulocytes (D4) stained with May-Grunwald–Giemsa. (B) Western blots of UBF1/2 and tubulin in day 0 and day 4 cells (n = 2). (C) One representative qChIP analysis of previously published data (Poortinga et al., 2004) showing UBF binding to the rDNA in day 0 and day 4 cells. UBF enrichment was determined as in Fig. 1 D. ENH, enhancer. (D) Nuclei from day 0 and day 4 cells (n = 2) were analyzed by psoralen cross-linking assay. (E) The results (n = 3) similar to D were quantitated (*, P < 0.05). (F) Genomic DNA from day 0 and day 4 cells was extracted, digested with HpaII or MspI, and analyzed by Southern blotting of rDNA. The relative amounts of methylated and unmethylated rRNA genes are calculated as in Fig. 4 C and represented graphically (n = 3). Mean ± SEM (error bars). Bar, 30 μm.

Mentions: The prevailing model is that the relative amounts of active and inactive ribosomal genes are stably maintained and are not regulated in higher eukaryotic cells (Conconi et al., 1989; Stefanovsky and Moss, 2006). We have previously shown that down-regulation of rRNA gene transcription associated with terminal differentiation of the murine promyelocytic (MPRO) cell line correlates with decreased UBF1/2 expression and the amount of UBF1/2 associated with the rRNA genes (Poortinga et al., 2004). In light of our observations that UBF1 is necessary to maintain active r-chromatin, we examined whether myeloid differentiation might also be associated with a decrease in the number of active genes. Induction of the terminal differentiation of the MPRO cell line (Fig. 10 A; D0 = undifferentiated and D4 = differentiated) led to a 90% decrease in UBF1/2 expression and reduced UBF1/2 enrichment at the rRNA gene promoter and transcribed region (Fig. 10, B and C), as we have previously shown (Poortinga et al., 2004). Psoralen analysis demonstrated that the reduction in rRNA gene transcription during differentiation (Poortinga et al., 2004) correlated with a significant reduction in the number of active genes (43.7 ± 2.8% active in day 0 compared with 19.4 ± 6% active in day 4; Fig. 10, D and E). This occurred in the absence of changes in rRNA gene promoter methylation (Fig. 10 F). Thus, the pool of active ribosomal genes is not static but decreases during terminal differentiation of granulocytes most likely as a result of decreased UBF1 expression.


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)

Loss of UBF1/2 expression correlates with rDNA silencing during granulocyte differentiation. (A) Phase-contrast microscopy of uninduced granulocytic MPRO cells (D0) and differentiated granulocytes (D4) stained with May-Grunwald–Giemsa. (B) Western blots of UBF1/2 and tubulin in day 0 and day 4 cells (n = 2). (C) One representative qChIP analysis of previously published data (Poortinga et al., 2004) showing UBF binding to the rDNA in day 0 and day 4 cells. UBF enrichment was determined as in Fig. 1 D. ENH, enhancer. (D) Nuclei from day 0 and day 4 cells (n = 2) were analyzed by psoralen cross-linking assay. (E) The results (n = 3) similar to D were quantitated (*, P < 0.05). (F) Genomic DNA from day 0 and day 4 cells was extracted, digested with HpaII or MspI, and analyzed by Southern blotting of rDNA. The relative amounts of methylated and unmethylated rRNA genes are calculated as in Fig. 4 C and represented graphically (n = 3). Mean ± SEM (error bars). Bar, 30 μm.
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fig10: Loss of UBF1/2 expression correlates with rDNA silencing during granulocyte differentiation. (A) Phase-contrast microscopy of uninduced granulocytic MPRO cells (D0) and differentiated granulocytes (D4) stained with May-Grunwald–Giemsa. (B) Western blots of UBF1/2 and tubulin in day 0 and day 4 cells (n = 2). (C) One representative qChIP analysis of previously published data (Poortinga et al., 2004) showing UBF binding to the rDNA in day 0 and day 4 cells. UBF enrichment was determined as in Fig. 1 D. ENH, enhancer. (D) Nuclei from day 0 and day 4 cells (n = 2) were analyzed by psoralen cross-linking assay. (E) The results (n = 3) similar to D were quantitated (*, P < 0.05). (F) Genomic DNA from day 0 and day 4 cells was extracted, digested with HpaII or MspI, and analyzed by Southern blotting of rDNA. The relative amounts of methylated and unmethylated rRNA genes are calculated as in Fig. 4 C and represented graphically (n = 3). Mean ± SEM (error bars). Bar, 30 μm.
Mentions: The prevailing model is that the relative amounts of active and inactive ribosomal genes are stably maintained and are not regulated in higher eukaryotic cells (Conconi et al., 1989; Stefanovsky and Moss, 2006). We have previously shown that down-regulation of rRNA gene transcription associated with terminal differentiation of the murine promyelocytic (MPRO) cell line correlates with decreased UBF1/2 expression and the amount of UBF1/2 associated with the rRNA genes (Poortinga et al., 2004). In light of our observations that UBF1 is necessary to maintain active r-chromatin, we examined whether myeloid differentiation might also be associated with a decrease in the number of active genes. Induction of the terminal differentiation of the MPRO cell line (Fig. 10 A; D0 = undifferentiated and D4 = differentiated) led to a 90% decrease in UBF1/2 expression and reduced UBF1/2 enrichment at the rRNA gene promoter and transcribed region (Fig. 10, B and C), as we have previously shown (Poortinga et al., 2004). Psoralen analysis demonstrated that the reduction in rRNA gene transcription during differentiation (Poortinga et al., 2004) correlated with a significant reduction in the number of active genes (43.7 ± 2.8% active in day 0 compared with 19.4 ± 6% active in day 4; Fig. 10, D and E). This occurred in the absence of changes in rRNA gene promoter methylation (Fig. 10 F). Thus, the pool of active ribosomal genes is not static but decreases during terminal differentiation of granulocytes most likely as a result of decreased UBF1 expression.

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