T H E J O U R N A L O F C E L L B I O L O G Y
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 183 No. 7 1259–1274
Correspondence to Ross D. Hannan: firstname.lastname@example.org
E. Robb ’ s present address is Mental Health Research Institute, Parkville, Victoria
Abbreviations used in this paper: AgNOR, argyrophilic NOR; ChIP, chromatin
immunoprecipitation; ETS, external transcribed spacer; GAPDH, glyceraldehyde
3-phosphate dehydrogenase; HMG, high mobility group; IGS, intergenic
spacer; MeDIP, methylated DNA immunoprecipitation; MEF, mouse embryonic
fi broblast; MPRO, murine promyelocytic; MSCV, murine stem cell virus; NOR,
nucleolar organizing region; NoRC, nucleolar remodeling complex; Pol I, poly-
merase I; pRT, pRevTRE; qChIP, quantitative ChIP; qRT-PCR, quantitative real-time
PCR; r-chromatin, ribosomal gene chromatin; rDNA, ribosomal DNA; rRNA, ri-
bosomal RNA; rUBF, rattus UBF; shRNA, short hairpin RNA; shRNAmir, shRNA –
micro RNA; SS, serum starvation; UBF, upstream binding factor; UCE, upstream
Ribosome biogenesis plays an essential role in growth control,
and transcription of the 45S precursor of the ribosomal RNA
(rRNA) by RNA polymerase I (Pol I) is limiting for prolifera-
tion in most cellular systems ( Jorgensen and Tyers, 2004 ). In
addition, through its control of nucleolar formation, rRNA gene
transcription indirectly regulates several other essential pro-
cesses, including titration of oncogenes and tumor suppressors,
cellular response to stress, and aging (for reviews see Moss,
2004 ; Mayer and Grummt, 2005 ).
A typical human cell contains ? 200 copies of the rRNA
genes arranged in tandemly repeated arrays located in nucleolar
organizing regions (NORs). Remarkably, despite rRNA gene
transcription being limiting for growth, > 50% of the rRNA
genes are believed to be transcriptionally silent at any one time
(for review see Grummt and Pikaard, 2003 ). The epigenetic
mechanisms controlling the activity status of individual ribo-
somal genes and the reasons why a majority is silenced in higher
eukaryotes remain unresolved questions ( Huang et al., 2006 ).
One factor whose function has been linked to regulation
of the rRNA gene locus is upstream binding factor (UBF).
UBF belongs to the sequence-nonspecifi c class of high mobil-
ity group (HMG) proteins and appears to function exclusively
in Pol I transcription (for review see Moss et al., 2007 ). UBF
consists of two polypeptides (UBF1 and 2), which form het-
ero- and homodimers and arise from alternative splicing of a
single transcript ( O ’ Mahony and Rothblum, 1991 ). Although
UBF1 supports robust rRNA gene transcription, UBF2 is fi ve-
fold less active ( Hannan et al., 1996 ), but the underlying
mechanisms that confer poor transcriptional activity to UBF2
We demonstrate that depletion of the transcription factor
upstream binding factor (UBF) leads to the stable and re-
versible methylation-independent silencing of rRNA genes
by promoting histone H1 – induced assembly of transcrip-
tionally inactive chromatin. Chromatin remodeling is abro-
gated by the mutation of an extracellular signal-regulated
n mammals, the mechanisms regulating the number of
active copies of the ? 200 ribosomal RNA (rRNA)
genes transcribed by RNA polymerase I are unclear.
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 remain-
ing active genes is increased. We also show that the ac-
tive rRNA gene pool is not static but decreases during
differentiation, correlating with diminished UBF expres-
sion. Thus, UBF1 levels regulate active rRNA gene chro-
matin during growth and differentiation.
UBF levels determine the number of active
ribosomal RNA genes in mammals
Elaine Sanij , 1 Gretchen Poortinga , 1 Kerith Sharkey , 1 Sandy Hung , 1 Timothy P. Holloway , 1 Jaclyn Quin , 1 Elysia Robb , 1
Lee H. Wong , 3 Walter G. Thomas , 4 Victor Stefanovsky , 5 Tom Moss , 5 Lawrence Rothblum , 6 Katherine M. Hannan , 1
Grant A. McArthur , 1,2,7 Richard B. Pearson , 1,8 and Ross D. Hannan 1,8
1 Research Division and 2 Division of Haematology and Medical Oncology, Peter MacCallum Cancer Centre, East Melbourne, Victoria 3002, Australia
3 Murdoch Childrens Research Institute, Royal Children ’ s Hospital, Parkville, Victoria 3010, Australia
4 School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia
5 Cancer Research Centre, Laval University, H ô tel-Dieu de Qu é bec, Qu é bec G1R 2J6, Canada
6 Department of Cell Biology, University of Oklahoma Medical College, Oklahoma City, OK 73104
7 Department of Medicine, St. Vincent ’ s Hospital and 8 Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria 3010, Australia
© 2008 Sanij et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the fi rst six months after the publica-
tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
on March 18, 2013
Published December 22, 2008
Supplemental Material can be found at:
JCB • VOLUME 183 • NUMBER 7 • 2008 1260
amined the effects of UBF knockdown on ribosomal gene
chromatin (r-chromatin). UBF knockdown reduced UBF1/2
mRNA and protein levels by 80% compared with the control
siRNA oligonucleotide duplex against EGFP (siRNA-EGFP;
Fig. 1, A and B ).
Quantitative chromatin immunoprecipitation (ChIP [qChIP])
analysis was used to examine the effect of UBF1/2 depletion
on the level of UBF associated with the rRNA genes. Consis-
tent with recent experiments ( O ’ Sullivan et al., 2002 ), we
found that UBF1/2 is enriched not only at the proximal pro-
moter but also across the transcribed portion of the 45S rRNA
gene ( Fig. 1, C and D ). Depletion of UBF1/2 reduced the lev-
els of UBF1/2 associated with the rRNA genes by a mean of
60% ( Fig. 1 D ). Immunofl uorescence microscopy of NIH3T3
fi broblasts in interphase demonstrated a signifi cant reduction
in both the number of UBF-positive nucleoli and the intensity
of UBF immunostaining ( Fig. 1, E and F ) after inducible
UBF1/2 knockdown using retroviral delivery of a tetracy-
cline-inducible UBF1/2 short hairpin RNA (shRNA; Dickins
et al., 2007 ).
Next, we directly examined the effect of UBF depletion
on the relative proportion of active and inactive (silent) ribo-
somal genes using psoralen, a DNA cross-linking agent.
Active rRNA genes have an open chromatin structure that is
accessible to psoralen and associated with nascent rRNA
transcripts. Conversely, the silent genes are inaccessible to
psoralen and associated with regularly spaced nucleosomes.
After psoralen cross-linking, the active and inactive rRNA
genes can be distinguished with Southern blotting by their
differing rates of migration ( Conconi et al., 1989 ; Dammann
et al., 1993 ). Strikingly, psoralen analysis of the rRNA genes
demonstrated a 70% reduction in the number of active genes
with a reciprocal increase in the fraction of silenced genes
(47.4 and 52.6% to 18.8 and 81.2%) after UBF knockdown
in NIH3T3 cells ( Fig. 2, A and B ). The change in the propor-
tion of active genes was not a result of off-target effects, as
silencing of UBF1/2 using siRNAs to different regions of the
UBF1/2 coding sequence (Fig. S1, A and B) also reduced the
number of active ribosomal genes ( Fig. 2 A , lanes 4 – 6),
whereas siRNA targeting EGFP or glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH) had no effect on the ratio of
active to inactive genes ( Fig. 2, A and C ). UBF1/2 knock-
down also increased the percentage of silenced rRNA genes
in cells arrested in G0 – G1 phase after serum starvation (SS;
Fig. 2 D , SS 24 h) and also in cells in early G1 ( Fig. 2 D ,
+Serum 6 h). Thus, silencing of rRNA genes (rDNA) after
depletion of UBF is not dependent on growth factors or chro-
matin remodeling during S phase. We further examined the
effect of UBF depletion using argyrophilic NOR (AgNOR)
staining of nucleoli from interphase NIH3T3 cells. Nucleo-
lar fi brillar centers contain undercondensed and thus tran-
scriptionally competent rRNA genes. Like mitotic NORs,
they can be silver stained because of their association with
the Pol I transcription machinery ( Roussel and Hernandez-
Verdun, 1994 ; Weisenberger and Scheer, 1995 ; Heliot et al.,
1997 ). AgNOR staining of the nucleoli demonstrated an
? 60 – 70% decrease in the overall intensity of silver staining
are unclear. In one model, UBF acts through its multiple HMG
boxes to induce looping of DNA. This creates the enhancesome,
a nucleosome-like structure thought to be responsible for the
ability of UBF to modulate rRNA gene transcription ( Stefan-
ovsky et al., 2001a,b ). Early studies suggested that UBF1
acts predominantly at the promoter in the recruitment of SL1
(selectivity factor 1) and Pol I and in the formation of the pre-
initiation complex ( Smith et al., 1990 ; McStay et al., 1991 ;
Jantzen et al., 1992 ). More recently, additional roles have been
ascribed to UBF1, including regulation of promoter escape
( Panov et al., 2006 ) and transcription elongation ( Moss et al.,
2006 ). Importantly, the association of UBF1 with rRNA genes
in vivo is not restricted to the promoter but extends across the
entire transcribed portion and to a lesser extent to the inter-
genic spacer (IGS; Fig. 1, C and D ; O ’ Sullivan et al., 2002 ).
Indeed, consistent with its ability to modify DNA conforma-
tion, the inclusion of rRNA gene sequences with high affi nity
for UBF into ectopic sites on human chromosomes results in
the formation of NOR-like structures indicative of “ open ”
chromatin ( Mais et al., 2005 ). In addition, targeting UBF1 to
regions of heterochromatin is suffi cient to induce large-scale
chromatin decondensation ( Chen et al., 2004 ). Together, these
data suggest that UBF1 binding throughout the rRNA gene re-
peat might contribute to the formation of the active chromatin
state of rRNA genes. However, direct experiments to demon-
strate this function on endogenous rRNA genes and informa-
tion on the relative contribution of the two UBF isoforms in
chromatin remodeling are lacking.
In this study, we provide strong evidence that UBF1, but
not UBF2, regulates the open chromatin structure found in
active rRNA genes by preventing linker histone H1 – induced
assembly of transcriptionally inactive chromatin. Long-term
rRNA gene silencing in response to UBF depletion is stably
propagated through the cell cycle and through many genera-
tions and is not associated with heterochromatic marks related
to nucleolar remodeling complex (NoRC) – dependent re-
modeling, including DNA methylation. Restoring UBF levels
rescues the number of active genes. Thus, in contrast to epi-
genetically silenced rRNA genes, which are methylated, silenc-
ing of rRNA genes through UBF depletion is reversible. We
also demonstrate that, contrary to accepted dogma, the pool of
active ribosomal genes is not static but decreases during dif-
ferentiation and that this decrease correlates with diminished
UBF levels in the absence of changes in ribosomal DNA
(rDNA) methylation. Together, these data suggest that modu-
lation of UBF levels might be an important determinant of
the relative proportion of active and silent rRNA genes dur-
UBF1 depletion silences rRNA genes
To determine whether UBF is necessary for maintenance of
the open chromatin structure found in active NORs, we trans-
fected siRNAs targeting conserved regions of both isoforms of
UBF (UBF1/2; Fig. S1, available at http://www.jcb.org/cgi/
content/full/jcb.200805146/DC1) into NIH3T3 cells and ex-
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Published December 22, 2008
1261UBF DETERMINES THE NUMBER OF ACTIVE RIBOSOMAL GENES • Sanij et al.
Figure 1. Silencing of UBF1/2 by siRNA. (A) NIH3T3 cells were transfected with siRNA-EGFP or -UBF1/2, and protein samples were harvested as indi-
cated and analyzed by Western blotting. (B) RNA samples were harvested, and UBF and GAPDH mRNA levels were examined by reverse transcription
qRT-PCR. n = 3; ***, P < 0.001. (C) Schematic of a murine rRNA gene and the positions of qRT-PCR amplicons. (D) qChIP analysis of UBF binding to the
rRNA gene. The percentage of DNA immunoprecipitated with anti-UBF or rabbit serum (RS) antibodies was calculated relative to the unprecipitated input
control. The percentage of DNA of rabbit serum controls was subtracted from corresponding UBF samples. n = 6; *, P < 0.05; **, P < 0.01. A representa-
tive ethidium bromide gel shows the amount of UCE and IGS2 products amplifi ed after 22 PCR cycles. (E) NIH3T3 cells stably transduced with tetracycline-
inducible TMP-UBF shRNAmir were cultured in the presence or absence of doxocyclin and analyzed by Western blotting. (F) Samples in E were analyzed
by immunofl uorescence for UBF1/2. ENH, enhancer; ITS, internal transcribed spacer; T, terminator region. Mean ± SEM (error bars). Bar, 2 μ m.
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JCB • VOLUME 183 • NUMBER 7 • 2008 1262
Figure 2. UBF1/2 depletion decreases the number of active ribosomal genes. (A) NIH3T3 cells transfected with siRNA-EGFP ( n = 2) or three independent siRNAs
targeting UBF1/2. Nuclei were extracted and irradiated in the presence of ethanol (ETOH; lane 1) or psoralen (lanes 2 – 6). Genomic DNA was isolated and
analyzed by Southern blotting for rRNA genes. (B) A map of the murine rRNA gene promoter and the probe used for Southern blotting (top). The proportion of
active versus inactive rDNA from experiments in A was quantitated (bottom). ENH, enhancer. n = 5; mean ± SEM (error bars); ***, P < 0.001. (C) NIH3T3 cells
were transfected with siRNA-GAPDH, -EGFP, or -UBF1/2 and analyzed by Western blotting (top) and psoralen cross-linking experiments (bottom). (D) NIH3T3 cells
transfected with siRNA-EGFP or -UBF1/2 were serum deprived for 24 h (SS) and serum refed for 6 h (+Serum). The psoralen cross-linking assay was performed as
in A (top). Cell cycle analysis of NIH3T3 cells serum starved for 24 h and serum refed for the indicated times. Fixed cells were stained with propidium iodide and
analyzed by fl ow cytometry. The percentages of cells in G0 – G1, S, and G2 – M phases were determined using Modfi t 3.0 software. (E) NIH3T3 were transfected
with siRNA-EGFP (A and C) and -UBF1/2 (B and D), and AgNOR staining was performed. Arrows indicate stained interphase NORs. Bar, 1 μ m.
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1263 UBF DETERMINES THE NUMBER OF ACTIVE RIBOSOMAL GENES • Sanij et al.
1994 ; Hannan et al., 1999 ). However, the relative ability
of UBF1 and UBF2 to regulate r-chromatin in vivo has not
been investigated. To address this, we performed replace-
ment experiments with rat UBF1 and UBF2, which contain
nucleic acid sequence differences in the region targeted by
the murine-specific siRNA-UBF1/2 (Fig. S2, available at
mortalized mouse embryonic fibroblast (MEF) cell lines
(NIH3T3 MEFs) expressing tetracycline-inducible Flag epi-
tope – tagged rat UBF1 or rat UBF2 were generated ( Fig. 3 A ).
Western analysis demonstrated that the rat UBF1 and 2
transgenes were expressed approximately two- to threefold
above endogenous levels and were resistant to the siRNA-
UBF1/2 ( Fig. 3 B ). Knockdown of UBF1/2 reduced the per-
centage of active and inactive genes from 31.7 and 68.3%
and number of fi brillar centers after UBF1/2 knockdown
( Fig. 2 E ). The strong correlation between decreased AgNOR
staining and UBF depletion is consistent with experiments
demonstrating that UBF is responsible for recruiting a ma-
jority of the Pol I transcription machinery to the rDNA re-
peats ( Mais et al., 2005 ). Together, these data provide strong
evidence that the association of UBF with rRNA genes is
necessary for maintenance of the open chromatin structure
found in active NORs.
UBF2 does not function in r-chromatin
UBF2, the naturally occurring splice variant of UBF1, is a
poor activator of rDNA transcription compared with UBF1
( O ’ Mahony et al., 1992 ; O ’ Neill et al., 1993 ; Kuhn et al.,
Figure 3. Differential regulation of active r-chromatin by UBF1 and UBF2. (A) RevTet-Off – inducible FLAG-rUBF1 and -rUBF2 NIH3T3 MEF cell lines were
stimulated by doxocyclin for 48 h and analyzed by Western blotting. (B) RevTet-Off – inducible RNAi-resistant UBF1, UBF2, and pRT (empty vector) NIH3T3
MEF cell lines were transfected with siRNA-EGFP or -UBF1/2 and analyzed by Western blotting (top). Nuclei from corresponding samples were analyzed
by psoralen cross-linking (bottom). (C) Nuclei from pRT and RNAi-resistant wild-type UBF1, UBF1-T117E, and UBF1-T117A – overexpressing NIH3T3 MEF
cell lines were collected 48 h after transfection with siRNA-EGFP or -UBF1/2 and analyzed by psoralen cross-linking. (D) The results ( n = 3) in B and C
were quantitated. Mean ± SEM (error bars); *, P < 0.05.
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JCB • VOLUME 183 • NUMBER 7 • 2008 1264
The ability of UBF1 to regulate r-chromatin
is abrogated by mutation of an extracellular
signal-regulated kinase site within the HMG
box 1 domain
To examine in more detail the structural requirements for
UBF1 to regulate r-chromatin, we performed UBF replacement
to 5.3 and 94.7%, respectively, after 48 h ( Fig. 3, B and D ).
Expression of rat UBF1 in these cells was able to prevent
the loss of active genes (41.3 and 58.7%), whereas rat UBF2
could not (8.5 and 91.5%; Fig. 3, B and D ). Thus, UBF2
does not function in r-chromatin remodeling in vivo in the
absence of UBF1.
Figure 4. DNA methylation and other heterochromatic markers are unaffected in response to UBF1/2 depletion. (A) Schematic of the murine rRNA gene
promoter and 5 ? ETS region with the position of restriction enzyme sites indicated. (B) Southern blot analysis of rDNA using methylation-sensitive restriction
enzymes. Genomic DNA from an NIH3T3 cell transfected with siRNA-EGFP, -UBF1, or -UBF1/2 was digested with HpaII or MspI, subjected to electrophoresis,
and hybridized to the probe depicted in Fig. 2 A. The white line indicates that intervening lanes have been spliced out. (C) The results ( n = 2) in B were
quantitated, and HpaII-sensitive (unmethylated) bands were quantitated as a proportion of the total rDNA (MspII band), and the difference was designated
as methylated. (D) Analysis of DNA methylation across the rDNA by MeDIP in siRNA-EGFP or -UBF1/2 cells ( n = 3). Samples were analyzed by qRT-PCR as
described in Fig. 1 D. (E – H) Loss of UBF1/2 does not alter SNF2H binding or histone modifi cations associated with NoRC silencing of rDNA. qChIP analysis of
the rRNA genes in siRNA-EGFP – or -UBF1/2 – transfected NIH3T3 cells using antibodies against SNF2H (E), H3K9Me2 (F), H3K9Me3 (G), or hyperacetylated
H4 (H). Samples were analyzed by qRT-PCR as described in Fig. 1 D. qChIPs (F – H) were normalized to total histone H3 or H4 loading (Fig. 6, A and B) and
expressed as a ratio of the percentage of DNA ( n = 3). ENH, enhancer; ITS, internal transcribed spacer; T, terminator region. Mean ± SEM (error bars).
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1265UBF DETERMINES THE NUMBER OF ACTIVE RIBOSOMAL GENES • Sanij et al.
Silencing of rDNA in response to UBF
depletion is reversible
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 chro-
matin confi guration 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 , fi rst and second lanes) and reduced loading of UBF on
the rDNA repeats ( Fig. 5 C ). This was not accompanied by in-
creased 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 , com-
pare the third, fourth, and fi fth lanes). CpG methylation at the
rDNA promoter was again unchanged ( Fig. 5 D ). We also per-
formed 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.
rRNA gene silencing leads to increased
association of linker histone H1
Next, we examined the mechanism by which the UBF – DNA
complexes might promote decondensation of the rRNA genes.
One possibility is that association of UBF with the rDNA leads
to ejection of the core histone octamers, thus promoting an open
r-chromatin structure. However, qChIP analysis failed to dem-
onstrate any signifi cant changes in the relative amount of total
H3 and H4 occupancy at the promoter and transcribed portion
of the rRNA genes after silencing induced by UBF1/2 knock-
down ( Fig. 6, A and B ).
Alternatively, experiments performed with cell-free sys-
tems demonstrate that UBF out competes linker histone H1 for
binding to a nucleosome core ( Kermekchiev et al., 1997 ), sug-
gesting that UBF might form active r-chromatin by preventing
the formation of higher order repressive chromatin structures
regulated by histone H1. This model predicts that the associa-
tion of UBF and histone H1 with r-chromatin in vivo would be
mutually exclusive and that UBF knockdown would increase
histone H1 association with the pool of rRNA genes that are
undergoing silencing. To monitor relative UBF and histone H1 oc-
cupancy at silenced (methylated) and active (unmethylated)
rRNA genes, we used the ChIP-CHOP assay ( Lawrence et al.,
2004 ). In this assay, immunoprecipitated DNA from UBF and
histone H1 ChIP experiments was digested with HpaII before
quantitative real-time PCR (qRT-PCR) using the core primers
( Fig. 1 C ) that span the ? 133 CpG dinucleotide in the rRNA
gene promoter ( Fig. 6 C ). The percentage of unmethylated ver-
sus methylated DNA was determined and used to distinguish
between active and silent rRNA genes. In wild-type cells, the
experiments as described in the previous section with struc-
ture/function mutants that affect the ability of UBF1 to bend
and loop DNA in vitro and to activate transcription
( Stefanovsky et al., 2001a ). Phosphorylation of T117 and
T201 in HMG boxes 1 and 2 of UBF1 reduces their ability to
bend DNA, leading to a cooperative unfolding of the en-
hancesome structure, as does substitution of these residues
with glutamic acid (UBF-threonin to glutamic acid muta-
tion; Stefanovsky et al., 2001b , 2006b ). The T117A UBF1
mutant that can still bend DNA and form the enhancesome
was similar to wild-type UBF in its ability to remodel chro-
matin ( Fig. 3, C and D ). However, the T117E UBF1 mutant
was unable to prevent the loss of inactive ribosomal genes in
UBF1/2-depleted fibroblasts ( Fig. 3, C and D ). Thus, muta-
tions that block the ability of UBF to bend DNA and form
the enhancesome in vitro also significantly reduce the ability
of UBF1 to remodel r-chromatin into an active configuration
rRNA gene silencing in response to UBF
depletion does not require NoRC-mediated
The chromatin-remodeling complex NoRC recruits DNA
methyltransferases and histone deacetylases to the rRNA gene
promoter – proximal terminator, contributing to the formation
of a closed nucleosomal structure ( Santoro and Grummt,
2005 ). In particular, NoRC-induced DNA methylation of a
CpG dinucleotide at ? 133 in the core region of the rRNA
gene promoter has been implicated in silencing of murine
rRNA genes ( Santoro and Grummt, 2001 ) and also has been
shown to reduce UBF binding to the rRNA gene promoter. We
examined the methylation status of the CpG dinucleotide at
? 133 in response to acute UBF1/2 depletion by Southern
analysis of genomic DNA digested with the enzymes HpaII
and MspI. These enzymes demonstrate differential sensitivity
to methylation ( Fig. 4 A ; Santoro and Grummt, 2001 ), and the
analysis revealed that ? 48% of the ribosomal genes were un-
methylated at the CpG dinucleotide ? 133 in exponentially
growing NIH3T3 cells ( Fig. 4, B and C ), which is in accor-
dance with the aforementioned psoralen data and previous ex-
periments ( Santoro and Grummt, 2001 ). This percentage was
not affected by depletion of UBF1/2 or UBF1 only ( Fig. 4, B
and C ; and Fig. S1). We extended this analysis across the en-
tire rRNA gene using methylated DNA immunoprecipitation
(MeDIP; Fig. 4 D ) but failed to observe a signifi cant change in
CpG methylation at any of the amplicons upon UBF deple-
tion. Moreover, ChIP analysis demonstrated that enrichment
of SNF2H, a component of the NoRC complex ( Strohner et al.,
2001 ; Santoro et al., 2002 ; Percipalle et al., 2006 ), was not
signifi cantly altered at the rDNA promoter and transcribed re-
gion in response to UBF depletion ( Fig. 4 E ). In addition, the
levels of histone marks associated with NoRC-dependent gene
silencing such as H3K9Me2 and H3K9Me3 ( Fig. 4, F – H )
were unchanged after UBF depletion. Thus, the loss of UBF is
suffi cient to induce a closed r-chromatin state without the need
for increased rDNA methylation and other NoRC-mediated
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JCB • VOLUME 183 • NUMBER 7 • 2008 1266
ChIP-CHOP demonstrated that this enrichment occurred on the
unmethylated fraction of genes ( Fig. 6, D and E ). These would,
presumably, correspond to the fraction of newly silenced genes
depleted of UBF. Together, these results suggest a model in
which direct association of UBF with the unmethylated rRNA
genes prevents the assembly of transcriptionally inactive higher
order chromatin structures catalyzed by linker histone H1.
UBF1 overexpression does not increase
UBF loading on the rRNA genes
Our aforementioned data and previously published experi-
ments ( Santoro and Grummt, 2001 ) demonstrate that UBF
active repeats are unmethylated and sensitive to HpaII cleavage,
whereas the silent ones are methylated and resistant to HpaII
digestion ( Santoro and Grummt, 2001 ).
Using ChIP-CHOP, we found that immunoprecipitated
UBF was almost exclusively associated with active, unmethyl-
ated rRNA gene promoters ( > 90%; Fig. 6 D ). In contrast,
immunoprecipitated histone H1 was almost entirely associated
with the silenced, methylated rRNA gene promoters ( > 90%;
Fig. 6 D ). Thus, the rRNA genes are either associated with UBF
or histone H1; they are mutually exclusive. UBF1/2 knockdown
led to a twofold increase in the amount of H1 at the promoter
and transcribed portion of the total pool of rRNA genes ( Fig. 6 E ).
Figure 5. 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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1267UBF DETERMINES THE NUMBER OF ACTIVE RIBOSOMAL GENES • Sanij et al.
Increased rRNA gene silencing leads to
increased transcription from the remaining
active rRNA genes
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 load-
ing 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 deter-
mined as we previously described ( Stefanovsky et al., 2006a )
using [ 3 H]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 incorpora-
tion is reached. However, if the elongation rates are similar, no
does not bind to methylated rRNA genes. When considered
with the UBF rescue experiments in Fig. 5 , the data suggest a
model whereby methylated rRNA genes are permanently si-
lenced because of their inability to load UBF, which is re-
quired for rDNA activation. Consistent with this model,
three- to fourfold overexpression of Flag-tagged UBF1 ( Fig. 7 A )
failed to signifi cantly increase the amount of UBF1 at the pro-
moter, transcribed, and IGS regions of the rDNA ( Fig. 7 B ).
In addition, ChIP-CHOP experiments revealed that in control
as well as UBF1-overexpressing cells, immunoprecipitated
UBF was almost exclusively associated with unmethylated
rDNA promoters ( Fig. 7 C ). Thus, UBF cannot be loaded onto
the methylated pool of rRNA genes. Surprisingly, despite the
inability to increase the net loading of UBF onto the rRNA
genes, overexpression of UBF1 induced a modest but statis-
tically signifi cant increase in the proportion of active genes
from 45.8 to 60.4% ( Fig. 7 D ). One explanation for the in-
crease in active gene number without more UBF loading is
that overexpression of UBF1 increases the preponderance of
UBF1 – UBF1 homodimers, which our data ( Fig. 3 ) indicate
would be more active in r-chromatin remodeling than UBF1 –
UBF2 or UBF2 – UBF2 dimers.
Figure 6. Loss of UBF1/2 leads to an increase in total levels of histone H1 associated with rRNA genes. (A and B) Depletion of UBF does not alter the total
levels of core histones associated with rDNA. qChIP analysis of the rRNA genes in siRNA-EGFP – or -UBF1/2 – transfected NIH3T3 cells using antibodies
against total histone H4 (A) or total histone H3 (B). Samples were analyzed by qRT-PCR as described in Fig. 1 D ( n = 3). (C) Schematic of the ChIP-CHOP
assay representing the murine rDNA promoter and the 5 ? ETS region as in Fig. 4 A with the position of restriction enzyme sites and primers used for qRT-PCR
indicated. (D) ChIP-CHOP assay of UBF or histone H1 ChIPs from siRNA-EGFP – or -UBF1/2 – transfected NIH3T3 cells. Samples were either mock digested
or digested with HpaII. The relative level of HpaII-resistant methylated rDNA was determined by qRT-PCR using the core primers, and the difference was
designated as unmethylated rDNA ( n = 4). (E) qChIP analysis of the rRNA genes in siRNA-EGFP – or -UBF1/2 – transfected NIH3T3 cells using antibodies to
total histone H1. Chromatin samples were analyzed by qRT-PCR as described in Fig. 1 D ( n = 3; *, P < 0.05). Mean ± SEM (error bars).
on March 18, 2013
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JCB • VOLUME 183 • NUMBER 7 • 2008 1268
of rRNA gene transcription associated with terminal differentia-
tion 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 en-
richment 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 signifi cant reduction in the number of active
genes (43.7 ± 2.8% active in day 0 compared with 19.4 ± 6% ac-
tive 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 dur-
ing terminal differentiation of granulocytes most likely as a result
of decreased UBF1 expression.
Despite previous investigation, the specifi c mechanisms that re-
model the r-chromatin from an inactive condensed state into an
open structure and the ensuing steps that result in the initiation of
lag should be apparent. When 45S rRNA labeling was followed
with time in NIH3T3 cells, no difference in the initial incorpo-
ration 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 elon-
gation suggests that initiation rates must have increased on the
genes remaining active after UBF knockdown.
Intriguingly, the increased rate of transcription on the sur-
viving active genes correlated with a 2.5 – 4-fold and a 1.5 – 2.5-
fold increase in the euchromatic marks H3K4Me2 and H3K4Me3,
respectively, across the promoter ( Fig. 9, A and B ; left). Simi-
larly, we observed a twofold increase in acetylated H3K9, a
marker of gene activation, at the enhancer and upstream control
element (UCE; Fig. 9 C , left) of the rRNA genes. ChIP-CHOP
demonstrated that in each case the increase in active chromatin
marks occurred on an unmethylated fraction of rRNA genes
( Fig. 9, A – C ; right).
Granulocyte differentiation is characterized
by increased rRNA gene silencing, which
correlates with decreased UBF levels but
not rDNA methylation
The prevailing model is that the relative amounts of active and in-
active 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
Figure 7. Overexpression of UBF1 is not suffi cient to activate silenced rDNA. (A) Western blot analysis of NIH3T3 cell lines expressing rUBF1-MSCV or
empty vector MSCV. (B) qChIP analysis of UBF binding to rDNA in cell lines in A. Samples were analyzed by qRT-PCR as described in Fig. 1 D ( n = 3).
(C) ChIP-CHOP assay of ChIP samples in B was performed as in Fig. 6 D ( n = 3). (D) Nuclei from cells listed in A analyzed by psoralen cross-linking (left).
The proportion of active versus inactive rDNA was quantitated (right; n = 5; **, P < 0.01). ENH, enhancer; ITS, internal transcribed spacer. Mean ± SEM
on March 18, 2013
Published December 22, 2008
1269 UBF DETERMINES THE NUMBER OF ACTIVE RIBOSOMAL GENES • Sanij et al.
Figure 8. 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 [ 32 P]orthophosphate for 30 min. 32 P-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 amplifi ed 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 μ Ci [ 3 H]uridine for the
indicated times. 3 H-labeled cellular RNAs were extracted and resolved on 1% formaldehyde gels, transferred to membrane, and exposed to x-ray fi lms.
Total levels of 28S rRNAs were detected by ethidium bromide (EtBr) staining. (F) Duplicate analyses of 3 H-labeled 45S rRNA in E were quantitated and
normalized to corresponding total 28S levels (EtBr). The curves fi tted 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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JCB • VOLUME 183 • NUMBER 7 • 2008 1270
UBF to regulate Pol I transcription at the level of elongation
( Stefanovsky et al., 2006a ). Our data demonstrate that muta-
tions that block the ability of UBF1 to bend DNA and form the
enhancesome in vitro also severely reduce the ability of UBF
to defi ne a unique psoralen-accessible chromatin structure
across the rRNA gene in vivo, suggesting that these two pro-
cesses are linked. Specifi cally, a UBF1 mutant (UBF-T117E)
that mimics extracellular signal-regulated kinase phosphoryla-
tion at threonine 117 and is thus defective in the ability to form
a functional enhancesome ( Stefanovsky et al., 2001a,b , 2006b )
was unable to prevent the loss of active genes. In contrast, the
equivalent nonphosphorylatable mutant (UBF-T117A) that re-
tains its ability to form the enhancesome in vitro was able to
remodel r-chromatin to the same extent as recombinant wild-
Interestingly, although the T117A mutant is able to func-
tion in enhancesome formation, it, like the T117E mutant, is se-
verely compromised in its ability to regulate transcription by
Pol I compared with wild-type UBF1 ( Stefanovsky et al.,
2001b ). However, we do not think this demonstrates that tran-
scription and chromatin remodeling can be separated. Rather,
we conclude that psoralen cross-linking measures the number
of actively transcribed genes but does not differentiate between
highly transcribed genes (i.e., rescue with wild-type UBF1) and
poorly transcribed genes (i.e., rescue with T117A). This is con-
sistent with the observation that the number of active genes, as
determined by psoralen cross-linking, does not vary between
Pol I transcription remain unclear. Studies in higher eukaryotes
have implicated rRNA gene promoter methylation and the post-
translational modifi cation of histones in regulating the epigenetic
silencing switch of rRNA gene clusters ( Hirschler-Laszkiewicz
et al., 2001 ; Santoro and Grummt, 2001 ; Lawrence et al., 2004 ).
However, these fi ndings are diffi cult to reconcile with two recent
studies that indirectly implicate UBF in regulating the open
chromatin state of active ribosomal genes in mammalian cells
( Chen et al., 2004 ; Mais et al., 2005 ). In this study, we show that
depletion of UBF1/2 leads to an increase in the number of rRNA
genes in an inactive condensed state. rRNA genes inactivated in
response to UBF depletion have characteristics of epigenetically
silenced rDNA repeats, as they are psoralen inaccessible and sta-
bly propagated though the cell cycle and through many cell gen-
erations. However, in contrast to classical epigenetic silencing of
NORs, rRNA gene inactivation in response to UBF depletion is
not associated with CpG methylation and thus is reversible; re-
storing UBF levels restores the wild-type ratio of active to inac-
tive genes. Thus, we term this form of rRNA gene inactivation
methylation-independent silencing or pseudosilencing to distin-
guish it from epigenetic silencing characteristic of imprinting.
These data provide strong evidence that UBF binding to rRNA
repeats is necessary for maintenance of the open chromatin
structure found in active NORs.
Through its HMG boxes, UBF has been shown to bend
? 140 bp of DNA into a near 360 ° loop. This protein DNA
structure, coined the enhancesome, is central to the ability of
Figure 9. UBF1/2 depletion correlates with an increase in euchromatic histone modifi cations at rDNA. (A – C) qChIP analysis of the rDNA in siRNA-EGFP – or
-UBF1/2 – transfected NIH3T3 cells using antibodies against H3K4Me2 (A), H3K4Me3 (B), and acetylated H3K9 (C). ChIP samples were analyzed by qRT-PCR
as described in Fig. 1 D, normalized to total H3 loading (Fig. 6 A), and expressed as a ratio of the percentage of DNA ( n = 3; *, P < 0.05; **, P < 0.01).
ChIP-CHOP assays performed as in Fig. 6 D are represented graphically on the right of corresponding ChIPs ( n = 3). Mean ± SEM (error bars).
on March 18, 2013
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1271UBF DETERMINES THE NUMBER OF ACTIVE RIBOSOMAL GENES • Sanij et al.
Our data also demonstrate that UBF1 and its natural splice
variant UBF2 possess distinct abilities to remodel r-chromatin.
UBF1 was able to effi ciently replace endogenous UBF1/2 to
exponentially growing cells and serum-starved cells, although
clearly the rate of transcription on the latter pool of rRNA genes
is considerably repressed ( Stefanovsky and Moss, 2006 ).
Figure 10. 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 μ m.
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Published December 22, 2008
JCB • VOLUME 183 • NUMBER 7 • 2008 1272
Regulation of rRNA synthesis at the level
of rate per gene rather than number of
Another important fi nding of our study is that the reduction in
the number of inactive genes through the depletion of UBF does
not lead to a commensurate decrease in net cellular rRNA gene
transcription rates. This was because the density of Pol I load-
ing on the remaining active genes was increased, thus maintain-
ing rRNA synthesis rates per cell. Such a phenomenon has been
observed in yeast where artifi cially reducing the rRNA gene
copy number from 143 to 42 does not affect growth rates be-
cause the mean number of polymerases per rRNA gene in-
creases to maintain transcription output ( French et al., 2003 ).
Interestingly, upon UBF knockdown, a pool of unmethylated
rRNA genes exhibited increases in markers of gene activation
(acetylated H3K9 and di- and trimethylated H3K4) at their pro-
moter regions ( Fig. 9, A – C ). It is probable that these changes
are functionally associated with the increased Pol I loading.
UBF regulates the active ribosomal gene
pool during differentiation
The prevailing view is that the relative amounts of active and
inactive ribosomal genes are stably maintained as a result of
CpG methylation of a fi xed number of rRNA repeats ( Con-
coni et al., 1989 ; Stefanovsky and Moss, 2006 ). In contrast,
our experiments demonstrate that the number of silenced
genes increases markedly during granulocyte differentiation.
Furthermore, the silencing correlates with a reduction in UBF
levels and UBF loading on the rDNA repeat but not promoter
methylation of rRNA genes, suggesting that the loss of UBF
is causative in the rRNA gene inactivation. Furthermore, as
we and others have shown that reduced UBF expression is
common during the terminal differentiation of many cell
types ( Larson et al., 1993 ; Datta et al., 1997 ; Alzuherri and
White, 1999 ; Poortinga et al., 2004 ; Li et al., 2006 ; Liu et al.,
2007 ), it is likely that down-regulation of UBF is a wide-
spread mechanism for the silencing of active rRNA genes
The level of rRNA gene silencing during granulocyte dif-
ferentiation is similar to that observed during UBF depletion in
NIH3T3 cells. However, UBF depletion did not change rRNA
gene transcription rates signifi cantly. Thus, although rRNA
gene silencing may be required for the down-regulation of Pol I
transcription during differentiation, it is not suffi cient to regu-
late this process. Interestingly, our preliminary experiments
show that during granulocyte differentiation, in addition to
UBF, the expression of a majority of the other components of
the Pol I complex is down-regulated (unpublished data). We
propose that this results in limiting amounts of the Pol I com-
plex, which prevents increased loading of Pol I on the remain-
ing active rRNA genes after UBF depletion. This would ensure
that rRNA gene silencing during differentiation leads to de-
creased rRNA synthesis rates. Future studies are needed to de-
termine whether coordinate regulation of Pol I transcription
factors and modulation of the number of active rRNA genes is a
general mechanism to effect long-term changes in rRNA gene
transcription rates during differentiation.
maintain rRNA genes in an open chromatin state. In marked
contrast, UBF2 exhibited little, if any, chromatin remodeling
capability. UBF2 contains a 27 – amino acid deletion in HMG
box 2, which reduces the DNA-binding capacity of this domain
( Stefanovsky and Moss, 2008 ). Thus, reminiscent of the afore-
mentioned UBF1 mutants, UBF2 may function poorly in re-
modeling r-chromatin as a result of an inability to bend and loop
DNA as effi ciently as UBF1 ( Stefanovsky and Moss, 2008 ).
Moreover, these data also suggest that in vivo variations in
UBF2 levels might function to fi ne tune the number of active
rRNA genes by forming less active UBF2 – UBF2 homodimers
or UBF1 – UBF2 heterodimers.
NoRC activity is not required for sustained
silencing of ribosomal genes in response to
Importantly, our data demonstrate that ribosomal genes pseudo-
silenced in response to UBF depletion do not require in-
creased CpG methylation or histone deacetylation across the
rDNA repeats. These data are consistent with a model in which
rRNA genes loaded with UBF are open; rRNA genes devoid of
UBF are closed regardless of their CpG methylation status. In
this model, the chromatin-remodeling complex NoRC lies up-
stream of UBF in the rRNA gene – silencing program, most
likely by controlling the local r-chromatin landscape, including
CpG methylation, to regulate the overall level of UBF binding
to the rRNA genes ( Santoro and Grummt, 2001 ).
Our data also demonstrate that rRNA genes pseudo-
silenced by UBF depletion do not become CpG methylated even
after many generations, and, thus, the silencing is stable and can
be reversed by restoring UBF levels. This suggests that UBF
does not normally function to prevent “ constitutive ” NoRC-
mediated methylation of rRNA genes, which is consistent with
a model in which epigenetic silencing of genes through CpG
methylation and UBF exclusion only occurs during defi ned
stages during development.
Our data also allow us to make some conclusions about
the mechanism by which UBF facilitates decondensation and
formation of an active chromatin environment at rRNA genes.
First, it is apparent that the UBF – DNA complexes and the core
histones can coexist on rRNA genes. We found no evidence that
UBF depletion was associated with increases in the total level
of the core histones associated with the rRNA genes, which
would be expected if UBF chromatin remodeling functioned to
eject nucleosomes from DNA. These fi ndings do not necessar-
ily imply that the histones associated with active rRNA genes
are nucleosomal. Indeed, much earlier experiments suggested
that actively transcribed ribosomal genes remain associated
with core histones, but in an unfolded “ half-histone ” state ( Prior
et al., 1983 ).
Second, rRNA gene silencing through loss of UBF leads
to signifi cant increases in the level of linker histone H1 associ-
ated with the new fraction of silenced genes. Thus, the com-
bined data argue strongly that UBF contributes to the open
r-chromatin structure by preventing the assembly of transcrip-
tionally inactive higher order chromatin structures catalyzed by
linker histone H1 binding.
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Published December 22, 2008
1273UBF DETERMINES THE NUMBER OF ACTIVE RIBOSOMAL GENES • Sanij et al.
tracted according to standard methods. To estimate RNA recovery rate, a
32 P-labeled RNA probe was mixed with RNA lysates before extraction.
RNA amounts were quantitated and normalized to equal numbers of cells.
First-strand cDNA was synthesized using a random hexamer primer and
avian myeloblastosis virus reverse transcription (Promega) according to the
manufacturer ’ s protocol. qRT-PCR was performed as described in the ChIP
section. Mouse 5 ? ETS primer sequences were previously published
( Poortinga et al. 2004 ).
AgNOR staining was performed as described previously ( Ploton et al.,
1984 ), and nucleoli were visualized using transmission EM.
Psoralen cross-linking assay
Cells were lysed in 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl 2 ,
and 0.5% NP-40, and nuclei were pelleted, resuspended in 50 mM Tris-
HCl, pH 8.3, 40% glycerol, 5 mM MgCl 2 , and 0.1 mM EDTA, and irradi-
ated in the presence of 4,5,8 ? -trimethylpsoralen (Sigma-Aldrich) with a
366-nm UV light box at a distance of 6 cm ( Conconi et al., 1989 ). 200
μ g/ml psoralen was added at 1:20 dilution every 4 min for a total irradia-
tion time of 20 min. Genomic DNA was isolated, digested with SalI, and
separated on a 0.9% agarose gel, and alkaline Southern blotting was per-
formed. To reverse psoralen cross-linking, fi lters were treated with 254-nm
UV rays at 1,875 × 100 μ J/cm 2 using a UV cross-linker (Stratalinker 2400;
Agilent Technologies). The membrane was then hybridized to a purifi ed 32 P
(Amersham)-labeled rDNA as depicted in Fig. 2 A , visualized by scanning
on a PhosphoImager (GE Healthcare), and quantitated using ImageQuant
(TLv2005.04; GE Healthcare).
DNA methylation and MeDIP
Genomic DNA was digested with the methylation-sensitive enzyme HpaII
or MspI ( Santoro and Grummt, 2001 ). Southern blotting was performed
using the aforementioned probe. MeDIP was performed by incubating
4 μ g of heat-denatured sonicated, genomic DNA with 4 μ g anti-5mC anti-
body for 2 h at 4 ° C in 0.14 M NaCl, 16.7 mM Tris, pH 8.0, and 0.05%
Triton X-100. DNA – antibody complexes were incubated with protein A –
Sepharose beads (Millipore) for 2 h at 4 ° C, and the precipitates were
eluted in 50 mM Tris, pH 8.0, 10 mM EDTA, and 0.5% SDS. DNA was pu-
rifi ed and analyzed by qRT-PCR, and the percentage of bound DNA was
calculated after normalization to 20 ng of input DNA.
Online supplemental material
Figs. S1 and S2 describe siRNA oligonucleotide sequences and positions
within the murine UBF1/2 coding regions. Table S1 includes ChIP RT-PCR
primer sequences. Online supplemental material is available at http://
We thank Sarah Ellis for performing the EM experiments and Anna Jenkins for
This work was supported by grants from the National Health and Medi-
cal Research Council (NHMRC) of Australia and the Cancer Council Victoria
to R.D. Hannan, R.B. Pearson, and G.A. McArthur and by a National Institutes
of Health grant (5R01HL077814 USA) to L. Rothblum. R.D. Hannan, R.B.
Pearson, and G.A. McArthur were supported by NHMRC fellowships.
Submitted: 23 May 2008
Accepted: 25 November 2008
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scription in response to F9 embryonal carcinoma stem cell differentia-
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Chen , D. , A.S. Belmont , and S. Huang . 2004 . Upstream binding factor associa-
tion induces large-scale chromatin decondensation. Proc. Natl. Acad. Sci.
USA . 101 : 15106 – 15111 .
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matin structures coexist in ribosomal RNA genes throughout the cell
cycle. Cell . 57 : 753 – 761 .
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Materials and methods
Anti – trimethyl H3K9, anti – dimethyl H3K9, anti – trimethyl H3K4, anti –
dimethyl H3K4, anti-H4, and anti-SNF2H antibodies were obtained from
Abcam. Anti – hyperacetylated H4, anti – acetyl H3K9, anti-H3, anti-H2A,
and anti-H1 (AE-4) antibodies were obtained from Millipore. Anti – 5-methyl
cytosine antibody (anti-5mC) was obtained from Megabase Research Prod-
ucts. Antibodies to ? -tubulin and GAPDH were obtained from Sigma-Aldrich
and Abcam, respectively. In-house rabbit anti – Pol I, -UBF1/2, and – rabbit
sera were used for Western and ChIP assays ( Poortinga et al., 2004 ).
siRNAs encoding the sequences of EGFP or UBF were synthesized by
Sigma-Aldrich. A complementary sequence of each oligonucleotide was
designed to produce a two-nucleotide overhang at both of the 3 ? ends of
the duplex. The oligonucleotide RNA sequences and positions on UBF1/2
are marked in Fig. S1. ON-TARGETplus siCONTROL GAPDH siRNA was
purchased from Thermo Fisher Scientifi c. Short hairpin – targeting UBF1/2
sequences (Fig. S1) were subcloned into the tetracycline-regulated retro-
viral vector TMP containing the entire micro-RNA cassette.
Cell culture, transfection, and retroviral infection
NIH3T3 cells were cultured in DME with 10% FBS at 37 ° C. Lipofectamine
reagent (Invitrogen) was used to transfect siRNA at 25 nM according to the
manufacturer ’ s protocol, and cells were harvested 48 h after transfection
unless otherwise specifi ed. For inducible RNAi targeting UBF, NIH3T3 cells
were stably cotransduced with the pRevTet-On tetracycline transactiva-
tor (Clontech Laboratories, Inc.) and TMP-UBF shRNA – micro RNA#1
(shRNAmir#1) retroviral vectors. Single clones were isolated and selected
with 1 μ g/ml doxocyclin to induce UBF1/2 knockdown. To establish cell
lines overexpressing rattus UBF 1 (rUBF1), NIH3T3 cells were stably trans-
duced with rUBF1 – murine stem cell virus (MSCV) – GFP and empty MSCV-
GFP retroviral vectors. Cells were sorted for high GFP expression using
fl ow cytometry. The tetracycline-regulated rat UBF1, UBF2, rUBF1-T117E,
and rUBF1-T117A NIH3T3 MEF cell lines were established using a RevTet-
Off expression system (Clontech Laboratories, Inc.). NIH3T3 MEF cells ex-
pressing the RevTet-Off – responsive elements were infected with pRevTRE
(pRT)-UBF recombinant viruses. The MPRO granulocyte cell line was gener-
ated from whole bone marrow of mice and maintained in culture as de-
scribed previously ( Walkley et al., 2004 ). Differentiation of MPRO cells
into mature granulocytes was induced by stimulation with 10 ? 6 M of the
retinoid agonist AGN 194204 for 4 d ( McArthur et al., 2002 ).
ChIP was performed as described previously ( Poortinga et al., 2004 ;
Walkley et al., 2004 ). Cross-linking was achieved with 0.6% formalde-
hyde and assays performed using 10 6 cells per immunoprecipitation. For
all ChIPs, 4 μ g of purifi ed antibody or 8 μ l of sera was used per immuno-
precipitation. Samples were analyzed in triplicate using the SYBR green
dye on the ABI Prism 7000 (Applied Biosystems). To calculate the percent-
age of total DNA bound, unprecipitated input samples from each condition
were used as reference for all qRT-PCR reactions. Primer sequences are
listed in Table S1 (available at http://www.jcb.org/cgi/content/full/
jcb.200805146/DC1). ChIP-CHOP assays were performed by digesting
DNA with HpaII before qRT-PCR. The relative level of HpaII resistance was
calculated after normalization to mock-digested DNA.
Immunofl uorescence and microscopy
Cells were fi xed in 3% paraformaldehyde for 10 min at room temperature,
permeabilized with ice-cold 0.05% Triton X-100 in PBS for 15 min, and
blocked with 5% skim milk powder and 0.5% chicken serum in PBS for 30
min. Anti-UBF sera was used at 1:500 dilution and detected with Alexa Fluor
594 anti – rabbit secondary antibody (Invitrogen) at 1:1,000 dilution. DNA
was counterstained with DAPI in ProLong Gold antifade reagent (Invitrogen).
Images were acquired on a microscope (BX-51; Olympus) equipped with a
camera (RT model 25.4; SPOT) using the UPlanAPO 60 × NA 1.2 water im-
mersion objective. Images were acquired using Advanced software (version
188.8.131.52; SPOT). All UBF images were taken at a 1-s exposure with a gain
(excitation power) of two. Settings for adjusting the image after acquisition
(i.e., ? adjust and background subtract settings) were identical for all images.
RNA extraction and expression analysis
Cells were lysed in 4 M guanidine thiocyanate, 25 mM sodium citrate, pH
7.0, 0.5% sarcosyl, and 0.1 M ? -mercaptoethanol, and RNA was ex-
on March 18, 2013
Published December 22, 2008
JCB • VOLUME 183 • NUMBER 7 • 2008 1274 Download full-text
polymerase I transcription factors UBF1 and UBF2. Nucleic Acids Res.
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some cores by phage T7 RNA polymerase in vitro. Proc. Natl. Acad. Sci.
USA . 90 : 6203 – 6207 .
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on March 18, 2013
Published December 22, 2008