Báo cáo khoa học: Wisely chosen paths – regulation of rRNA synthesis Delivered on 30 June 2010 at the 35th FEBS Congress in Gothenburg, Sweden potx

THE FEBS ⁄ EMBO WOMEN IN SCIENCE LECTURE
Wisely chosen paths – regulation of rRNA synthesis
Delivered on 30 June 2010 at the 35th FEBS Congress in
Gothenburg, Sweden
Ingrid Grummt
Division of Molecular Biology of the Cell II, German Cancer Research Center, DKFZ-ZMBH-Alliance, Heidelberg, Germany
Introduction
Growing cells require continuous ribosome synthesis
to ensure that subsequent generations are provided
with the ribosomes necessary to support protein syn-
thesis. The more rapidly cells proliferate, the more rap-
idly ribosomes must be synthesized. The synthesis of
rRNA, the first event in ribosome synthesis, is a fun-
damental determinant of a cell’s capacity to grow and
proliferate. rRNA genes (rDNAs) are transcribed with
high efficiency, and rRNA synthesis is regulated in a
sophisticated way to be responsive to both general
metabolism and specific environmental challenges
[1–3]. Indeed, almost any perturbation that slows cell
growth or protein synthesis, such as nutrient and
growth factor starvation, senescence, toxic lesion or
viral infection, leads to a decrease in rDNA transcrip-
tion. Conversely, rDNA transcription is upregulated
upon reversal of such conditions and by agents that
stimulate growth. The number of rRNA genes varies
greatly among organisms, covering a vast range from
fewer than 100 to more than 10 000. Each rRNA gene
Keywords
chromatin; epigenetics; noncoding RNA;
rRNA genes; signaling; transcription
Correspondence

I. Grummt, Molecular Biology of the Cell II,
DKFZ-ZMBH Alliance, German Cancer
Research Center, Im Neuenheimer Feld
581, D-69120 Heidelberg, Germany
Fax: +49 6221 423404
Tel: +49 6221 423412
E-mail:
(Received 19 July 2010, revised 16 September
2010, accepted 22 September 2010)
doi:10.1111/j.1742-4658.2010.07892.x
All cells, from prokaryotes to vertebrates, synthesize enormous amounts of
rRNA to produce 1–2 million ribosomes per cell cycle, which are required
to maintain the protein synthesis capacity of the daughter cells. In recent
years, considerable progress has been made in the elucidation of the basic
principles of transcriptional regulation and the pathways that adapt cellular
rRNA synthesis to metabolic activity, a process that is essential for under-
standing the link between nucleolar activity, cell growth, proliferation, and
apoptosis. I will survey our present knowledge of the highly coordinated
networks that regulate transcription by RNA polymerase I, coordinating
rRNA gene transcription and ribosome production with environmental
cues. Moreover, I will discuss the epigenetic mechanisms that control the
chromatin structure and transcriptional activity of rRNA genes, in particu-
lar the role of noncoding RNA in DNA methylation and transcriptional
silencing.
Abbreviations
AMPK, AMP-activated protein kinase; Cdk, cyclin-dependent kinase; CK2, casein kinase 2; CSB, Cockayne syndrome group B protein;
DNMT, DNA methyltransferase; ERK, extracellular signal-regulated protein kinase; GSK, glycogen synthase kinase; HMG, high-mobility
group; H3K4me3, histone H3 trimethylated at Lys4; H3K9, histone H3 Lys9; H3K9me1, histone H3 methylated at Lys9; H3K9me2,
histone H3 dimethylated at Lys9; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of
rapamycin; NoRC, nucleolar remodeling complex; NPM, nucleophosmin; nsRNA, noncoding RNA; rDNA, gene encoding rRNA; PCAF, p300/

CBP-associated factor; PFH8, PHD finger protein 8; PIC, preinitiation complex; pre-rRNA, ribosomal precursor RNA; Pol I, DNA-dependent
RNA polymerase I; pRNA, promoter-associated RNA; PTEN, phosphatase and tensin homolog deleted on chromosome 10; RSK, ribosomal
S6 kinase, 90 kDa; S6K, ribosomal S6 kinase, 60 kDa; TAF
I
, Pol I-specific TBP-associated factor; TBP, TATA-binding protein; TFIIH,
transcription factor IIH; TTF-I, transcription termination factor I; UBF, upstream binding factor; UCE, upstream control element.
4626 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS
encodes a long precursor RNA (45S pre-rRNA) that is
processed and post-transcriptionally modified to gener-
ate one molecule each of 18S, 5.8S and 28S rRNA.
Actually, almost all signaling pathways that affect cell
growth and proliferation directly regulate rRNA
synthesis, their downstream effectors converging at the
DNA-dependent RNA polymerase I (Pol I) transcrip-
tion machinery.
Given the repetitive nature of rRNA genes, two
strategies for regulating rRNA synthesis are conceiv-
able. Pol I transcription may be controlled either by
changing the rate of transcription from active genes or
by adjusting the number of genes that are involved in
transcription (Fig. 1). There is evidence for both
options. In most cases, short-term regulation is
brought about by reversible modification of Pol I tran-
scription factors that affect the efficiency of transcrip-
tion initiation and ⁄ or the rate of transcription from
active rRNA genes, whereas long-term regulation dur-
ing development and differentiation is achieved by epi-
genetic mechanisms that alter the ratio of active to
silent copies of rRNA genes, thereby regulating the
number of genes transcribed.

This article discusses and summarizes work on the
mechanisms that mammalian cells use to regulate
rRNA synthesis, and hence ribosome production, in
response to external signals. Although the emerging
picture of transcriptional regulation is one of unex-
pected variety and complexity, we are beginning to
understand the functions of individual components of
the Pol I transcription apparatus, the pathways that
link rDNA transcription to cell growth, and the role
of epigenetic mechanisms that establish the active and
inactive states of rRNA genes. As both transcription
of rDNA and maturation of rRNA play central roles
in the complex network that controls cell growth and
proliferation, the elucidation of the molecular path-
ways that transmit information on the growth state of
a cell population to the Pol I transcription apparatus
represents a challenging and rewarding subject of
research.
The Pol I transcription machinery
Ribosome biogenesis is a major cellular process that
occurs in distinct nuclear compartments, the nucleoli.
Nucleoli form around the multiple tandem arrayed
copies of rRNA genes, known as nucleolus organizer
regions, which are located at one or several acrocentric
chromosomes. Nucleoli disappear if rRNA synthesis is
curtailed, indicating that the nucleolar structure is
dependent on rDNA transcription. Actually, nucleolar
morphology is diagnostic for the general metabolism
of the cell, and morphological changes in the number
and size of nucleoli constitute a reliable marker of the

proliferative state of cancer cells. Mammalian rDNA
clusters are characterized by multiple alternating mod-
ules of a long intergenic spacer of approximately 30 kb
and a pre-rRNA coding region of approximately
14 kb. Each active rRNA gene is transcribed by Pol I
to generate 45S pre-rRNA. After synthesis, pre-rRNA
is processed and modified to generate one molecule
each of mature 18S, 5.8S and 28S rRNA, which,
together with 5S rRNA, which is transcribed by DNA-
dependent RNA polymerase III, form the RNA back-
bone of the ribosome.
Altered ratio of
Altered rate of
active versus silent genes
transcription initiation
Active copies
Silent copies
Fig. 1. Two methods of rDNA transcription regulation. Cells regulate rRNA synthesis by modulating the rate of transcription initiation,
thereby controlling the number of nascent pre-rRNA molecules (green lines) that are generated from active genes (left panel). Alternatively,
as there are hundreds of rRNA genes, subsets of rDNA repeats are turned either ‘on’ or ‘off’ as required. The gray ellipses indicate the more
compact, heterochromatic conformation of silent rRNA genes; the red boxes represent transcription terminator elements located upstream
and downstream of the rDNA transcription units.
I. Grummt et al. Regulation of rRNA synthesis
FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS 4627
Transcription initiation is a complex process that
requires the assembly of a specific multiprotein com-
plex at the rDNA promoter, containing Pol I and a
surprising number of associated proteins that promote
Pol I transcription (Fig. 2). In mammals, the assembly
of the preinitiation complex (PIC) is mediated by the

synergistic action of two basal Pol I-specific transcrip-
tion factors that bind to the rDNA promoter, i.e. the
upstream binding factor (UBF) and the promoter
selectivity factor, termed SL1 in humans and TIF-IB
in mice [4,5]. UBF is a member of the high-mobility
group (HMG) protein family, which contains five
HMG boxes. The multiple HMG boxes enable UBF
to loop approximately 140 bp of DNA into a single
turn, thereby inducing a nucleosome-like structure [6].
UBF activates rRNA gene transcription by recruiting
Pol I to the rDNA promoter [7] and through displace-
ment of nonspecific DNA-binding proteins, such as
histone H1, from rDNA [8]. Depletion of UBF leads
to stable and reversible repression of rDNA transcrip-
tion by promoting histone H1-induced assembly of
compact, transcriptionally inactive chromatin [9]. Addi-
tionally, UBF regulates promoter escape of Pol I [10]
and transcription elongation [11]. UBF expression is
reduced in differentiated cells, indicating that UBF levels
regulate rDNA transcription during growth and differ-
entiation [9].
UBF acts synergistically with SL1, a complex contain-
ing the TATA-binding protein (TBP) and four Pol I-
specific TBP-associated factors (TAF
I
s), which nucleates
transcription complex assembly and confers promoter
selectivity on Pol I [12,13]. The TAF
I
subunits mediate

specific interactions between the rDNA promoter and
Pol I, thus playing an important role in recruiting
Pol I – together with a collection of Pol I-associated
factors – to the rDNA promoter. In addition, the asso-
ciation of Pol I with the preinitiation complex involves
interactions with UBF and PAF53 (53 kDa Pol I-asso-
ciated factor) [14], and with a Pol I-associated factor,
termed PAF49. PAF49 is a homolog of the yeast Pol I
subunit A34.5, previously identified as a subunit of the
T-cell receptor complex (CAST) [15].
Pol I exists in two distinct forms, Pol Ia and Pol Ib,
the latter being capable of assembling into productive
T
0
T
1–10
18S RNA
Topo I
SIRT7
CK2
UCE
CORE
AcƟn
Pol I
TTF-I
SL1
NM1
28S RNA
UBF
TIF-IA

T
0
Fig. 2. Cartoon depicting the structural organization of mammalian rDNA repeats and the basal factors required for transcription initiation.
The sites of transcription initiation of 47S pre-rRNA (black arrow) and transcripts from the spacer promoter (red arrow) are indicated. Binding
sites for the transcription termination factors located downstream of the transcription unit (T
1–10
) and upstream of the gene promoter (T
o
)
are indicated by red boxes. Repetitive enhancer elements located between the spacer promoter and major gene promoter are indicated by
blue boxes. The factors that are associated with the rDNA promoter and Pol I, respectively, are depicted by ellipsoids. Synergistic binding of
UBF and SL1 to the rDNA promoter is required for the recruitment of Pol I and multiple Pol I-associated factors to the transcription start site
to initiate pre-rRNA synthesis. An electron microscopic image visualizing active amphibian rRNA genes is shown above. It reveals the tan-
dem head-to-tail arrangement of rRNA genes that are separated by ‘nontranscribed spacers’ and the characteristic Christmas tree appear-
ance of active transcription units (from Miller and Beatty [75]).
Regulation of rRNA synthesis I. Grummt et al.
4628 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS
transcription initiation complexes [16]. Pol Ib is associ-
ated with numerous proteins, including the basal tran-
scription factors, protein kinase CK2, nuclear actin,
nuclear myosin 1 (NM1), chromatin modifiers, such as
G9a and SIRT7, and proteins involved in replication
and DNA repair, such as topoisomerases I and IIa,
Ku70 ⁄ 80, proliferating cell nuclear antigen, transcrip-
tion factor IIH (TFIIH) and CSB [17]. These findings
are compatible with a mechanism in which a Pol I
‘holoenzyme’ is recruited to the rDNA promoter to
coordinate rRNA synthesis and maturation as well as
chromatin modification and DNA repair. However,
the concept of the Pol I transcription machinery as a

massive multiprotein complex that assembles in a sto-
chastic manner from freely diffusible subunits has been
eclipsed by measurements of the movement of fluores-
cently tagged subunits of Pol I and basal transcription
factors. These studies revealed that the Pol I transcrip-
tion machinery is highly dynamic, assembling in a sto-
chastic fashion, sometimes individually and sometimes
in subcomplexes [18]. Quantitative single-cell imaging
combined with computational modeling and biochemi-
cal analysis revealed that upregulation of transcription
is accompanied by prolonged retention of Pol I factors
at the rDNA promoter [19], demonstrating that
modulation of the efficiency of transcription initiation
complex assembly is a decisive step in the regulation of
rDNA transcription.
Basal Pol I transcription factors are
targeted by multiple signaling pathways
Transcription of rRNA genes is efficiently regulated to
be responsive to both general metabolism and specific
environmental challenges. Conditions that impair cellu-
lar metabolism, such as nutrient starvation, oxidative
stress, inhibition of protein synthesis and cell conflu-
ence, will downregulate rDNA transcription, whereas
growth factors and agents that stimulate growth and
proliferation will upregulate Pol I transcription
(Fig. 3). There is evidence that almost all proteins
required for Pol I transcription can serve as targets for
regulatory pathways. For example, Cdk (cyclin-depen-
dent kinase)1–cyclin B-dependent phosphorylation of
TAF

I
110, a subunit of SL1 that nucleates PIC assem-
bly, causes shutdown of rDNA transcription during
mitosis. Mitotic phosphorylation of TAF
I
110 at
Thr852 impairs the ability of SL1 to interact with
UBF, thereby abrogating transcription complex forma-
tion [20,21]. Thus, reversible phosphorylation of SL1 is
used as a molecular switch to shut down rDNA tran-
scription during mitosis. Resetting of the Pol I tran-
scription machinery at the end of mitosis is brought
about by Cdc14B, a phosphatase that is sequestered
within the nucleolus during interphase and activated
upon release from rDNA at prometaphase [22].
hCdc14B dephosphorylates Thr852 at the exit from
mitosis [23], thereby allowing transcription complex
assembly and resumption of rRNA synthesis in early
G
1
-phase (Fig. 4).
In early G
1
-phase, rDNA transcription remains low,
although the activity of SL1 has been fully recovered.
To achieve optimal transcriptional activity, UBF has
to be phosphorylated at Ser484 by Cdk4–cyclin D1
and at Ser388 by Cdk2–cyclin E ⁄ A [23,24]. Mutations
that prevent phosphorylation of Ser388 impair the
interaction of UBF with Pol I and abrogate rDNA

transcription. The finding that specific Cdk–cyclin
complexes modulate the activity of SL1 and UBF in a
cell cycle-dependent manner links the control of cell
cycle progression with regulation of Pol I transcrip-
tion. In quiescent cells, UBF is hypophosphorylated
[25,26], and phosphorylation of the two N-terminal
HMG boxes of UBF by extracellular signal-regulated
protein kinase (ERK) is essential for activation of
rDNA transcription by growth factors [27]. Moreover,
the mammalian target of rapamycin (mTOR), a key
regulator of cell growth and proliferation, stimulates
Pol I transcription in part through phosphorylation of
the C-terminal activation domain of UBF [28], under-
scoring the importance of UBF phosphorylation in the
control of rRNA synthesis. In addition to transcription
initiation, phosphorylation of UBF plays an important
role in transcription elongation. UBF is bound along
the pre-rRNA coding region through which Pol I must
pass [29]. UBF phosphorylated by ERK permits Pol I
elongation, whereas hypophosphorylated UBF inhibits
elongation, demonstrating that transcription elongation
Growth factors
Nutrients
Oncogenes
Nutrients
Oncogenes
Genotoxic
Tumor
stress suppressors
Starvation

Viral infection
Metabolic stress
Fig. 3. Extracellular signals impinge on transcription of rRNA
genes. The cartoon illustrates the signaling pathways that upregu-
late (green arrows) or downregulate (red arrows) nucleolar tran-
scription, converging at Pol I transcription.
I. Grummt et al. Regulation of rRNA synthesis
FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS 4629
is a major rate-limiting step for growth factor-depen-
dent regulation of rDNA transcription [11].
Acetylation is another post-translational modifica-
tion that regulates the activity of UBF and SL1. Acet-
ylation of TAF
I
68 by the the histone acetyltransferase
PCAF augments SL1 activity and stimulates transcrip-
tion initiation [30,31]. PCAF-dependent acetylation of
TAF
I
68 is counteracted by SIRT1, the founding mem-
ber of a family of highly conserved NAD
+
-dependent
histone deacetylases, termed sirtuins. SIRT1 is con-
served from bacteria to humans, and regulates a wide
range of biological processes, such as gene silencing,
aging, differentiation, and cell metabolism [32]. SIRT1
deacetylates TAF
I
68, leading to impaired binding of

SL1 to the rDNA promoter and inhibition of transcrip-
tion initiation. In contrast, SIRT7, another member of
the sirtuin family, exerts a positive effect on Pol I tran-
scription. SIRT7 localizes to nucleoli, is associated with
active rDNA repeats, interacts with Pol I, and stimu-
lates rDNA transcription by enhancing Pol I occu-
pancy at rDNA [33]. Knockdown of SIRT7 leads to
cell cycle arrest and apoptosis, underscoring the piv-
otal role of SIRT7 in cell survival. As the activity of
sirtuins depends on the level of cellular NAD
+
,
changes in the cellular energy status are translated into
changes in rRNA synthesis and ribosome production.
Thus, sirtuins are central players in the regulation of
rDNA transcription, SIRT1 repressing and SIRT7
activating rRNA genes, thereby linking Pol I transcrip-
tion to the metabolic activity of the cell.
In a recent study, Murayama et al. uncovered an
additional interrelationship between the cellular energy
status and rDNA transcription [34]. They identified a
novel protein complex, termed energy-dependent nucle-
olar silencing complex, which contains the NAD
+
-
dependent histone deacetylase SIRT1, the histone
methyltransferase SUV39H1, and a nucleolar protein,
termed nucleomethylin, which binds to histone H3
dimethylated at Lys9 (H3K9me2). If the intracellular
energy supply is limited, the deacetylase activity of

SIRT1 is enhanced, leading to elevated levels of his-
tone H3 Lys9 (H3K9) methylation and an increased
number of silent rDNA repeats. These results suggest
the existence of a mechanism that links cell physiology
to rDNA silencing, which in turn is a prerequisite for
nucleolar integrity and cell survival.
TIF-IA – a transcription factor that is
targeted by multiple signaling
pathways
Conditions that negatively affect cell growth, including
stress, nutrient starvation, and toxic lesions, down-
regulate transcription of rDNA, whereas agents that
Cdk2–cyclin E
Cdk4–cyclin D
S6K
Cdk2–cyclin A
ERK
RSK
G
0
Cdk1–cyclin B
PP
P
TIF-IA
UBF
P
P
UBF
PP
UBF

P
P
P
TIF-IA
PP
SL1
P
SG
1
G
2
M
Fig. 4. Regulation of Pol I transcription during the cell cycle. During progression through the G
1
-phase and S-phase, UBF is activated by
phosphorylation of Ser484 by Cdk4–cyclin D and Ser388 and Cdk2–cyclin E ⁄ A, respectively. In addition, mTOR-dependent and ERK-depen-
dent pathways activate TIF-IA by phosphorylation of Ser44, Ser633 and Ser649. At entry into mitosis, Cdk1–cyclin B phosphorylates TAF
I
110,
a subunit of the TAF
I
–TBP complex SL1, at Thr852. Phosphorylation at Thr852 inactivates SL1, leading to repression of Pol I transcription
during mitosis. At the exit from mitosis, Cdc14B dephosphorylates Thr852, leading to recovery of SL1–TIF-IB activity. Activating phosphoryla-
tions are marked in green, and inhibiting ones in red. Transcription is low in resting cells (G
0
), and resumption of full transcriptional activity
on re-entry into the cell cycle requires phosphorylation of TIF-IA by ERK ⁄ RSK and phosphorylation of UBF by ERK, Cdk4–cyclin D and S6K.
See text for details.
Regulation of rRNA synthesis I. Grummt et al.
4630 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS

stimulate growth and proliferation upregulate rRNA
synthesis [35–38]. A key player in growth-dependent
regulation of rDNA transcription is TIF-IA, the mam-
malian homolog of yeast RRN3 [39], an essential tran-
scription initiation factor that is associated with the
initiation-competent form of Pol I [40–42]. TIF-IA
interacts with Pol I, and with two Pol I-specific TAF
I
s,
thereby connecting Pol I with the preinitiation complex
[16,43]. The activity of TIF-IA is regulated by diverse
signals that affect cell growth and proliferation, thus
adapting Pol I transcription to different growth condi-
tions and environmental cues. TIF-IA is phosphory-
lated at multiple sites by various signaling cascades,
and changes in the phosphorylation pattern of TIF-IA
correlate with upegulation or downregulation of rRNA
synthesis in response to external signals (Fig. 5).
Specific phosphorylation of TIF-IA either facilitates or
impairs the interaction with Pol I and ⁄ or SL1, indicat-
ing that reversible phosphorylation of TIF-IA is an
effective way to rapidly and efficiently modulate rDNA
transcription in response to growth factors, nutrient
availability, or external stress. Conditions that support
growth and proliferation, such as nutrients and growth
factors, activate TIF-IA by mTOR-dependent and
ERK-dependent phosphorylation at Ser44, Ser633, and
Ser649. Conversely, stress-induced activation of c-Jun
N-terminal kinase (JNK)2 triggers phosphorylation of
TIF-IA at Thr200, and this phosphorylation impairs

the interaction of TIF-IA with both Pol I and SL1.
Thus, JNK2-dependent, mitogen-activated protein
kinase (MAPK)-dependent and mTOR-dependent
phosphorylation of TIF-IA affects the formation of
productive transcription complexes and adapts rRNA
synthesis to cell growth and proliferation. Moreover,
rDNA transcription and ribosome biogenesis are regu-
lated by the intracellular ATP levels [44]. The key
enzyme that translates changes in energy levels into
adaptive cellular responses is the AMP-activated pro-
tein kinase (AMPK). If energy levels are low and the
intracellular AMP ⁄ ATP ratio is elevated, AMPK
switches on energy-producing processes and switches
off energy-consuming pathways to restore cellular
ATP levels. Activation of AMPK triggers phosphory-
lation of TIF-IA at Ser635, which in turn inactivates
TIF-IA and inhibits rRNA synthesis [38]. This finding
reveals another level of regulation of Pol I transcrip-
tion, at which TIF-IA not only senses external signals
but also translates changes in intracellular energy
supply into upregulation or downregulation of rRNA
synthesis.
Oncogenes and tumor suppressors
affect rRNA synthesis
Consistent with rDNA transcription being tightly
linked to cell growth and proliferation, Pol I trans-
cription is regulated by a balanced interplay between
oncogene products and tumor suppressors (Fig. 6). In
healthy cells, Pol I transcription is restrained by tumor
suppressors, such as pRb, p53, ARF and PTEN (phos-

phatase and tensin homolog deleted on chromo-
some 10. Such restraints are compromised during cell
transformation and are accentuated by oncogene
products, such as c-Myc and nucleophosmin (NPM),
which stimulate Pol I transcription. Several oncogene
products have been demonstrated to directly regulate
rRNA biogenesis, whereas others affect signaling path-
ways that control Pol I transcription. It is therefore
plausible that cells might achieve a proliferative advan-
tage by elevating the level of specific oncogene prod-
ucts to increase the production of rRNA. For
example, the proto-oncogene product c-Myc was
shown to localize in nucleoli at sites of rRNA synthe-
sis, to interact with specific consensus elements at
rRNA genes, to associate with SL1, and to activate
rDNA transcription [45,46]. c-Myc appears to promote
cell growth, at least in part through facilitating recruit-
ment of the Pol I machinery to rDNA, thereby
enhancing production of components required for
ribosome biogenesis. Consistent with elevated levels of
specific oncogene products increasing the production
of rRNA, the nucleolar endoribonuclease NPM (also
Cell cycle
44
Cdk2
mTOR
199
635
649
RSK

JNK
200200
633
Growth
factors
ERK2
172
CK2
170
AMPK
Nutrients
Stress
Energy deprivation
Fig. 5. The transcription factor TIF-IA is targeted by multiple signal-
ing pathways. Growth-dependent control of rDNA transcription is
exerted by TIF-IA, a basal transcription factor that mediates the
interaction of Pol I with the PIC. TIF-IA is phosphorylated at multi-
ple sites by the indicated protein kinases (boxed). Specific phos-
phorylation enhances (green) or inhibits (red) the interaction with
Pol I and ⁄ or SL1. A two-dimensional tryptic phosphopeptide map of
in vivo labeled TIF-IA is shown. The encircled numbers indicate the
positions of the phosphorylated serines or threonines contained in
the respective tryptic peptides.
I. Grummt et al. Regulation of rRNA synthesis
FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS 4631
known as B23), was shown to increase the level of
TAF
I
48 and to stimulate proliferation of transformed
cells. NPM shuttles between the nucleolus, nucleo-

plasm, and cytoplasm, and its overexpression or muta-
tion has been associated with a broad range of human
cancers [47]. NPM is required for rRNA maturation,
and has been implicated in multiple cellular processes,
including genome stability, cell cycle progression,
response to genotoxic stress, DNA repair, maintenance
of chromatin structure, and regulation of the activity
and ⁄ or stability of the tumor suppressors p53 and
ARF.
The tumor suppressors pRb, p53 and ARF (p19
ARF
in mouse and p14
ARF
in human) are central players in
pathways that arrest cell cycle progression and induce
cell death in response to DNA damage and oncogenic
stress. These tumor suppressors restrain cell growth by
repressing Pol I transcription. pRb, the product of the
retinoblastoma susceptibility ( Rb) gene, accumulates in
nucleoli of differentiated or cell cycle-arrested cells
[48], and downregulates rRNA synthesis [49]. In
healthy cells, pRb restrains Pol I transcription by
interacting with UBF, leading to dissociation of UBF
from rDNA and to impaired transcription complex
formation [50,51]. The tumor suppressor p53, on the
other hand, represses Pol I transcription by association
with TBP and TAF
I
110, abrogating the formation of
PICs consisting of SL1 and UBF [52]. Under normal

conditions, p53 is a short-lived protein present at a
barely detectable level. On exposure to stress or after
inhibition of rDNA transcription, p53 levels increase,
triggering a cascade of events that finally lead to cell
cycle arrest or apoptosis. Actually, any agent that
inhibits ribosome biogenesis also disturbs the nucleolar
structure, and this, in turn, is translated into enhanced
p53 activity [53]. In support of nucleolar transcription
regulating p53, disruption of the TIF-IA gene by Cre-
dependent homologous recombination leads to inhibi-
tion of Pol I transcription, perturbation of the nucleo-
lar structure, and p53-dependent apoptosis [54].
Upregulation of p53 in response to TIF-IA deficiency
is caused by inhibition of MDM2 ⁄ HDM2, a specific
E3 ubiquitin ligase that controls p53 abundance by
proteasome-mediated degradation of p53 in the cyto-
plasm. After TIF-IA depletion, the p53–MDM2 com-
plex is disrupted and p53 levels are elevated. The
increase of p53 level in response to inhibition of rRNA
synthesis is caused by release of ribosomal proteins,
which bind to MDM2⁄ HDM2 and thereby inhibit its
E3 ligase activity, resulting in p53 being stabilized [55].
Thus, ongoing pre-rRNA synthesis is required for
nucleolar retention of proteins that control p53 activ-
ity, reinforcing the idea that the nucleolus is a major
cellular stress sensor that integrates and transmits sig-
nals for regulation of p53 activity (Fig. 7).
A key upstream controller of p53 is the tumor sup-
pressor ARF, which provides a first line of defense
against hyperproliferative signals that are provoked by

oncogenic stimuli. ARF is sequestered in the nucleoli
of unstressed cells. Nucleolar sequestration of ARF
depends on continuous transcription, and release of
ARF from the nucleolus is a plausible mechanism for
transmission of the stress signal. ARF activity is
induced upon nucleolar stress, which increases p53
Tumor suppressors
Disrupts SL1
Prevents
TTF-I binding
Inhibits
UBF/SL1
interaction
Increases TAF
I
48
expression
Increases UBF
expression
Stabilizes
SL1–UBF
Oncogenes
NPM
Myc
PTEN
p53
GSK3β
pRb
ARF
TTF-I

Pol I
TIF-IA
SL1
UBF
Inhibits SL1–UBF
interaction
Induces UBF
degradation
Fig. 6. Oncogene products and tumor sup-
pressors control Pol I transcription. Onco-
gene products activate rRNA synthesis by
upregulating the level of transcription factors
and ⁄ or stabilizing protein–protein or protein–
DNA interactions (marked by green arrows),
whereas tumor suppressors inhibit rRNA
synthesis by interfering with macromolecu-
lar interactions required for transcription
initiation complex assembly (marked by
red lollypops).
Regulation of rRNA synthesis I. Grummt et al.
4632 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS
concentrations by binding to MDM2 ⁄ HDM2 and
inhibiting its ability to trigger p53 degradation. ARF
has been reported to downregulate Pol I transcription
through interaction with UBF and inhibition of pre-
rRNA processing, possibly by lowering the level
and ⁄ or activity of the endonuclease NPM, thereby
blocking a specific step in the maturation of rRNA
[56]. Thus, ARF not only triggers a p53 response that
represses Pol I transcription, but also blocks the pro-

duction of mature rRNA by inhibiting the processing
of pre-rRNA. Presumably, the primordial role of ARF
is to slow ribosome production in response to hyper-
proliferative stress provoked by oncogenic stimuli. Its
subsequent linkage to p53 may have then evolved to
improve its efficiency and provide a more adequate
checkpoint for coupling ribosome production with
p53-dependent inhibitors of cell cycle progression.
Moreover, a recent study demonstrated that ARF
inhibits the nucleolar import of transcription termina-
tion factor I (TTF-I), causing the accumulation of
TTF-I in the nucleoplasm [57].
The tumor suppressor PTEN is a phosphatase that
regulates cell growth by its ability to regulate Pol I
transcription. Overexpression of PTEN represses RNA
Pol I transcription, whereas decreased levels of PTEN
correlate with enhanced rRNA synthetic activity.
PTEN-mediated repression requires its lipid phospha-
tase activity, and is independent of the p53 status of
the cell. PTEN inhibits phosphoinositide 3-kinase sig-
naling and triggers disruption of the TBP–TAF
I
com-
plex SL1, thereby preventing the assembly of
transcription initiation complexes [52]. In Ras-trans-
formed cells, PTEN was found at the rDNA promoter
in a complex with another potential tumor suppressor,
glycogen synthase kinase (GSK)3b. Inhibition of
GSK3b upregulates rRNA synthesis, whereas a consti-
tutively active GSK3b mutant inhibits rDNA tran-

scription by interaction with SL1. Thus, the interplay
between PTEN and GSK3b represents a powerful
mechanism the cell uses to ensure that ribosome bio-
genesis is coupled to growth control.
Chromatin modifications and
epigenetic control of rDNA transcription
Transcription of rDNA is also modulated by epige-
netic mechanisms. Approximately half of the several
hundred copies of rRNA genes exhibit a heterochro-
matic chromatin structure and are transcriptionally
silent (Fig. 8). The fact that, even in proliferating cells
with a high demand for ribosome biogenesis, a signifi-
cant fraction of rRNA genes are epigenetically silent
provides a unique possibility to decipher the mecha-
nisms that establish a given epigenetic state of rDNA,
and to study the functional impact of balancing the
ratio of active and silent rDNA repeats on cell surveil-
lance and genomic stability. Specific epigenetic charac-
teristics distinguish active rDNA repeats from inactive
ones. Generally, transcriptionally active genes are char-
acterized by an ‘open’ euchromatic structure, whereas
silent ones exhibit a more compact heterochromatic
structure. Specific histone modifications are associated
with transcriptionally active and silent rDNA repeats,
acetylation of histone H4 and methylation of his-
TIF-IA
+/+
TIF-IA
–/–
Intact nucleolus

Perturbed nucleolus
p53 degradation p53 stabilization
Cell growth
G
1
-arrest, apoptosis
MDM2
p53
rP
rP
rP
p53
rP
MDM2
rPrP
Fig. 7. Ablation of TIF-IA leads to cell cycle
arrest and apoptosis. In TIF-IA-containing
cells, the nucleolus is transcriptionally
active, and p53 is maintained at low levels
through ubiquitination by MDM2 and degra-
dation by proteasomes. In TIF-IA-deficient
cells, the nucleolar structure is perturbed
and ribosomal proteins (rP) are released into
the nucleoplasm, where they associate with
MDM2 to inhibit its activity. As a conse-
quence, the amount and activity of p53 are
enhanced, leading to cell cycle arrest and
apoptosis.
I. Grummt et al. Regulation of rRNA synthesis
FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS 4633

tone H3 Lys 4 correlating with transcriptional activity,
whereas histone H4 hypoacetylation and methylation
of H3K9, histone H3 Lys27 and histone H4 Lys20 cor-
relate with transcriptional silencing [58,59]. Regarding
the functional relevance of heterochromatin formation
and rDNA silencing, hypomethylation of rRNA genes
decreases genomic stability, suggesting that silencing
entails the assembly of a generally repressive chroma-
tin domain that is less accessible to the cellular recom-
bination machinery.
NoRC – a chromatin remodeling
complex that mediates transcriptional
silencing
Switching between the active and silent state of rRNA
genes is mediated by a chromatin remodeling
complex, termed NoRC, a member of ATP-dependent
chromatin remodeling machines comprising the
ATPase SNF2h and a large subunit, TIP5 (TTF-I-
interacting protein 5 [60]). NoRC interacts with DNA
methyltransferase(s), histone deacetylase(s), and his-
tone methyltransferase(s), thereby recruiting the
enzymes required for heterochromatin formation and
rDNA silencing. In the mouse, NoRC-dependent
transcriptional silencing involves methylation of a
critical CpG residue in the upstream control element
(UCE) of the rDNA promoter. Methylation prevents
binding of the Pol I-specific transcription factor UBF
to nucleolar chromatin, and impairs the formation of
transcription initation complexes [61]. Thus, targeting
NoRC to rDNA leads to rewriting of the histone code,

changes in DNA methylation, and, ultimately, hetero-
chromatization and transcriptional silencing of rRNA
genes [62,63]. In addition, NoRC shifts the promoter-
bound nucleosome downstream of the transcription
start site into a translational position that is unfavorable
for transcription complex formation [64]. Thus, NoRC
serves at least two functions: first, as a remodeling
complex that alters the position of the nucleosome at
the rDNA promoter; and second, as a scaffold coordi-
nating the activities of macromolecular complexes that
modify histones, methylate DNA, and establish a
‘closed’ heterochromatic state.
A noncoding RNA is required for NoRC
function
Evidence from several experimental systems demon-
strates the profound and complex role that noncoding
Active copies
TIP5
UBF
Silent copies
NoRC
Chromatin remodeling
Heterochromatin formation
DNA methylation
CH
3
CH
3
CH
3

TIP5
SNF2h
H4ac H3ac
H3K4me2
H3K9me
H4K20me HP1
Fig. 8. NoRC triggers the establishment of the silent, heterochromatic state of rRNA genes. Potentially active rRNA genes exhibit an ‘open’
chromatin structure, are associated with Pol I and nascent pre-rRNA (green lines), and are characterized by DNA hypomethylation, acetyla-
tion of histone H4 (H4ac), and dimethylation of histone H3 Lys4 (H3K4me2). Epigenetically silenced rRNA genes are demarcated by
histone H4 hypoacetylation, methylation of H3K9 (H3K9me) and histone H4 Lys20 (H4K20me), association with heterochromatin protein 1
(HP1) and CpG methylation (CH
3
). Methylation prevents UBF binding and impairs transcription complex formation. The silent state of rRNA
genes is mediated by the NoRC, a complex comprising SNF2h and TIP5, which interacts with pRNA and histone-modifying enzymes. A de-
convolution micrograph of interphase nuclei in U2OS cells, showing the nucleolar localization of TIP5 (red) and UBF (green) combined with
4¢,6-diamidino-2-phenylindole-stained chromatin, is shown at the right.
Regulation of rRNA synthesis I. Grummt et al.
4634 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS
RNAs play in regulating gene expression [65,66].
Noncoding RNAs are integral components of chroma-
tin, acting as key regulators of gene expression and
genome stability. Although the mechanistic details of
how RNA and chromatin are connected remain
unclear, there is increasing evidence that epigenetic reg-
ulation probably represents an intimate and balanced
interplay of both RNA and chromatin fields [67,68]. In
support of this notion, NoRC function requires bind-
ing of TIP5, the large subunit of NoRC, to 150–
250 nucleotide RNA, termed pRNA, because it is com-
plementary in sequence to the rDNA promoter [63].

pRNA originates from a Pol I promoter located within
the intergenic spacer  2 kb upstream of the 45S pre-
rRNA coding region (Fig. 9). Intergenic transcripts are
of low abundance and usually do not accumulate
in vivo, because they are rapidly degraded, unless they
are shielded from degradation by binding to NoRC.
Antisense-mediated depletion of pRNA leads to dis-
placement of NoRC from nucleoli, hypomethylation of
rDNA, and activation of Pol I transcription. pRNA
folds into a stem–loop structure, and this specific
structure is conserved in several mammals. Mutations
that prevent formation of the stem–loop structure
impair binding of pRNA to TIP5 and abolish nucleo-
lar targeting of NoRC [69].
Analysis of the silencing capacity of wild-type or
mutant forms of pRNA revealed that the specific
stem–loop structure of pRNA is indispensable for
NoRC function [69]. Although pRNA sequences that
fold into the specific stem–loop structure are required
for NoRC binding and recruitment to rDNA, this part
of pRNA is not sufficient for NoRC-directed DNA
methylation and transcriptional silencing. Current
results show that pRNA sequences upstream of the
stem–loop structure interact with T
0
, the promoter-
proximal binding site of the transcription factor TTF-
I. Truncated pRNA derivatives lacking the T
0
sequence fail to trigger de novo methylation and rDNA

silencing. Strikingly, the upstream part of pRNA that
is complementary to T
0
is itself able to direct DNA
methylation and transcriptional silencing. We postulate
that this region of pRNA may form a specific RNA–
DNA structure, such as Watson–Crick base pairing or
Hoogsteen or reversed Hoogsteen base pairing, that
serves as an anchor module guiding DNA methyltrans-
ferase (DNMT)3b to the promoter of specific rDNA
repeats (Fig. 10).
G
U
U
A
G
G
A
U
U
U
-
U
U
U
U
U
G
U
–126

–49
G
G
G
G
U
A
C
U
C
U
C
U
U
U
–1997
Spacer promoter pre rRNA promoter
T
0
A
C
G
U
U
G
G
U
C
C
A

C
C
C
U
C
A
G
C
C
U
U
C
C
U
C
C
C
UCE
CORE
U
U
A
G
A
C
G
C
U
G
C

U
U
G
C
G
A
U
G
G
C
C
U
(promoter-associated RNA)
150–250 nucleotides
‘pRNA’
C
U
U
U
G
G
C
A
A
U
U
C
TIP5
SNF2h
Fig. 9. Model depicting the origin of pRNA

that is associated with NoRC. Intergenic
transcripts (dotted line) are synthesized
from a Pol I promoter located  2kb
upstream of the pre-RNA promoter. The pri-
mary intergenic transcripts are degraded by
the exosome, except for 150–250 nucleo-
tide transcripts that match the rDNA pro-
moter (pRNA), which are stabilized by
binding to NoRC. pRNA folds into a specific
stem–loop structure (shown at the right),
and this secondary structure is recognized
by TIP5. The association of pRNA with the
TAM domain of TIP5 is required for NoRC
binding to rDNA and NoRC-dependent het-
erochromatin formation.
HDAC
HMT
T
0
CH
3
TIP5
UCE
CORE
DNMT
Fig. 10. Model illustrating the role of NoRC and pRNA in rDNA
methylation and silencing. NoRC is recuited to the rDNA promoter
by interaction with TTF-I bound to its target site T
0
. pRNA base

pairs with T
0
, leading to displacement of TTF-I and recruitment of
DNMT3b, which mediates methylation of the rDNA promoter.
Methylation of CpG-133 impairs transcription complex assembly.
Triplex formation allows the neighboring hairpin structure of pRNA
to bring NoRC close to the rDNA promoter and to consolidate rDNA
repression by recruiting histone-modifying enzymes. HDAC, histone
deacetylase; HMT, histone methyltransferase.
I. Grummt et al. Regulation of rRNA synthesis
FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS 4635
Histone lysine methylation is
dynamically regulated
In contrast to NoRC-dependent heterochromatin for-
mation and rDNA silencing, little is known about the
mechanisms that counteract heterochromatin forma-
tion and promote the establishment and maintenance
of the euchromatic state of active rDNA repeats. We
have shown that Cockayne syndrome group B protein
(CSB), a DNA-dependent ATPase that plays a role in
both transcription-coupled DNA repair and transcrip-
tional regulation, activates rDNA transcription [70].
CSB localizes in the nucleolus at sites of active rDNA
transcription, and is part of a protein complex that
contains Pol I, TFIIH and basal Pol I transcription
initiation factors [71]. Importantly, CSB works
together with the H3K9 histone methyltransferase
G9a, which promotes dimethylation of H3K9 in the
pre-rRNA coding region. Given the well-established
role of di- and trimethylation of histone H3 at Lys9

(H3K9me3) in heterochromatin formation and rDNA
silencing [62], the finding that G9a-dependent methyla-
tion of H3K9 in the transcribed region is required for
activation of Pol I transcription indicates that the
function of these chromatin markers is more complex
than previously thought. Furthermore, it suggests that
histone markers may serve distinct functions in
transcription, depending on the context of other post-
translational histone modifications. CSB and G9a may
promote Pol I transcription elongation by depositing a
specific histone modification pattern that is recognized
by other chromatin-modifying activities or by elonga-
tion factors that are required for transcription through
chromatin.
As many of the covalent modifications that take
place on the histone tails are enzymatically reversible,
we searched for enzymes that may promote rDNA
transcription by removing repressive histone markers.
PHD finger protein 8 (PHF8), a ubiquitously
expressed member of the JmjC family of histone
demethylases that carries a PHD finger in addition to
the JmjC domain, localizes within nucleoli and is
associated with hypomethylated rRNA genes [72,73].
PHF8 demethylates histone H3 that is mono- or
dimethylated at Lys 9 (H3K9me1/2) and activates
rDNA transcription, transcriptional activation requir-
ing both the JmjC domain and the PHD finger. Strik-
ingly, demethylation of H3K9me1 and H3K9me2 is
enhanced by adjacent histone H3 trimethylated at
Lys3 (H3K4me3). Thus, the combination of specific

histone modifications determines the functional read-
out, a finding that links dynamic histone methylation
to rDNA transcription.
A previous study has shown that the histone
demethylase KDM2B (alias JHDM1B ⁄ FBXL10) is
also a nucleolar protein that influences cell growth and
proliferation [74]. Like PHF8, KDM2B is associated
with unmethylated rDNA. However, KDM2B and
PHF8 target different histone modifications and serve
opposite functions. Whereas KDM2B demethylates
H3K4me3, PHF8 targets H3K9me1 and H3K9me2
and requires adjacent H3K4me3 for efficient demethy-
lation. Knockdown of KDM2B leads to a significant
increase in pre-rRNA transcription, cell size, and pro-
liferation, suggesting that KDM2B is a repressor of
Pol I transcription. By contrast, PHF8 activates Pol I
transcription, thereby promoting cell growth and pro-
liferation. Thus, the histone demethylases KDM2B
and PHF8 counteract specific histone modifications
that oppose the epigenetic state of rRNA genes,
removing methyl groups from lysines during the transi-
tion from one transcriptional state to another.
Conclusions
Significant advances have been made in our under-
standing of the Pol I transcription machinery and the
sophisticated mechanisms that cells use to adapt rRNA
synthesis to the number of ribosomes required to
promote cell growth and proliferation. Research dur-
ing the last two decades has shown that regulation of
rDNA transcription is manifested by multiple pathways

that either act synergistically or operate in parallel.
The combination of these regulatory pathways appears
to be dependent on both the individual cell types and
their physiological state. Whether or not rDNA
transcription link the growth capacity of cells to cell
cycle progression or growth arrest in response to DNA
damage or stress remains to be investigated. It will be
interesting and challenging to determine whether per-
turbations of these regulatory systems are necessary or
sufficient to allow passage along multistep pathways to
carcinogenesis. A strong indication that this may be
the case is the finding that a growing number of tumor
suppressors and oncogene products target these sys-
tems directly and control their output. The fact that
elevated rRNA synthesis accelerates the proliferation
of transformed cells [66] implies that deregulation of
rDNA transcription may have a profound impact on
cancer biology. Understanding the intimate link
between deregulated rRNA synthesis and tumorigene-
sis will be instrumental for the development of strate-
gies leading to the molecular characterization of
neoplastic diseases, and will drive the design and devel-
opment of novel drugs to combat cancer through
targeted downregulation of Pol I transcription.
Regulation of rRNA synthesis I. Grummt et al.
4636 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS
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