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

Abbreviations AMPK, AMP-activated protein kinase; Cdk, cyclin-dependent kinase; CK2, casein kinase 2; CSB, Cockayne syndrome group B protein; DNMT, DNA methyltransferase; ERK, extracellu

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: i.grummt@dkfz.de

(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.

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 initiatranscrip-tion 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.

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-bindcontain-ing protein (TBP) and four Pol I-specific TBP-associated factors (TAFIs), which nucleates transcription complex assembly and confers promoter selectivity on Pol I [12,13] The TAFI 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-assoI-asso-ciated 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

18S RNA

Topo I SIRT7 CK2

AcƟn

Pol I

NM1 28S RNA

UBF

TIF-IA

T0

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]).

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-depen(cyclin-depen-dent phosphorylation of

TAFI110, a subunit of SL1 that nucleates PIC

assem-bly, causes shutdown of rDNA transcription during

mitosis Mitotic phosphorylation of TAFI110 at

Thr852 impairs the ability of SL1 to interact with

UBF, thereby abrogating transcription complex

forma-tion [20,21] Thus, reversible phosphorylaforma-tion of SL1 is

used as a molecular switch to shut down rDNA

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

G1-phase (Fig 4)

In early G1-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

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 downreguupregu-late (red arrows) nucleolar tran-scription, converging at Pol I transcription.

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 TAFI68 by the the histone acetyltransferase

PCAF augments SL1 activity and stimulates

transcrip-tion initiatranscrip-tion [30,31] PCAF-dependent acetylatranscrip-tion of

TAFI68 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 TAFI68, leading to impaired binding of

SL1 to the rDNA promoter and inhibition of

transcrip-tion initiatranscrip-tion 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

Cdk2–cyclin A ERK

Cdk1–cyclin B

P P P

TIF-IA

UBF

P P

UBF

P P

UBF

P P

P

TIF-IA

P P

SL1

P

S

G1

G2 M

Fig 4 Regulation of Pol I transcription during the cell cycle During progression through the G1-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 TAFI–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.

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 TAFIs,

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

635

649

RSK

JNK

200

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.

known as B23), was shown to increase the level of

TAFI48 and to stimulate proliferation of transformed

cells NPM shuttles between the nucleolus,

nucleo-plasm, and cytonucleo-plasm, 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 (p19ARF

in mouse and p14ARF 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 TAFI110, 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 transcripinhibi-tion, perturbainhibi-tion 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

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).

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 accumulatermina-tion 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–TAFI

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

Intact nucleolus

Perturbed nucleolus

MDM2

p53

rP rP rP

p53

rP MDM2

rP rP

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.

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 dimethylaacetyla-tion 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 (CH3) 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.

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 T0, the

promoter-proximal binding site of the transcription factor

TTF-I Truncated pRNA derivatives lacking the T0 sequence fail to trigger de novo methylation and rDNA silencing Strikingly, the upstream part of pRNA that

is complementary to T0 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 G

G A U

U U G

–126G –49

G

A U U U U

–1997

Spacer promoter pre rRNA promoter

T0

A C G

U U

G C A C

G C C U C U C

UCE CORE

U

U

A G G

C U U

G C G A U G

G C

(promoter-associated RNA)

150–250 nucleotides

‘pRNA’

C U U

G G

U

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  2 kb

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

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 T0 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.