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 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) 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 understanding 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 particular the role of noncoding RNA in DNA methylation and transcriptional silencing doi:10.1111/j.1742-4658.2010.07892.x Introduction Growing cells require continuous ribosome synthesis to ensure that subsequent generations are provided with the ribosomes necessary to support protein synthesis The more rapidly cells proliferate, the more rapidly ribosomes must be synthesized The synthesis of rRNA, the first event in ribosome synthesis, is a fundamental 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 transcription 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 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; TAFI, 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 I Grummt et al Regulation of rRNA synthesis encodes a long precursor RNA (45S pre-rRNA) that is processed and post-transcriptionally modified to generate 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) transcription machinery Given the repetitive nature of rRNA genes, two strategies for regulating rRNA synthesis are conceivable 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 transcription factors that affect the efficiency of transcription initiation and ⁄ or the rate of transcription from active rRNA genes, whereas long-term regulation during development and differentiation is achieved by epigenetic 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 unexpected 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 Altered rate of transcription initiation 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 pathways 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 modules 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 DNAdependent RNA polymerase III, form the RNA backbone of the ribosome Altered ratio of active versus silent genes Active copies Silent copies Fig 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 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS 4627 Regulation of rRNA synthesis I Grummt et al T1–10 T0 18S RNA 28S RNA TIF-IA TTF-I T0 UBF SL1 UCE CK2 Pol I Topo I SIRT7 NM1 Ac n CORE Fig 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 (T1–10) and upstream of the gene promoter (To) 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 tandem head-to-tail arrangement of rRNA genes that are separated by ‘nontranscribed spacers’ and the characteristic Christmas tree appearance of active transcription units (from Miller and Beatty [75]) Transcription initiation is a complex process that requires the assembly of a specific multiprotein complex 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 transcription 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 displacement of nonspecific DNA-binding proteins, such as histone H1, from rDNA [8] Depletion of UBF leads to stable and reversible repression of rDNA transcription by promoting histone H1-induced assembly of compact, transcriptionally inactive chromatin [9] Addi4628 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 differentiation [9] UBF acts synergistically with SL1, a complex containing the TATA-binding protein (TBP) and four Pol Ispecific 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 association of Pol I with the preinitiation complex involves interactions with UBF and PAF53 (53 kDa Pol I-associated 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 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS I Grummt et al transcription initiation complexes [16] Pol Ib is associated with numerous proteins, including the basal transcription factors, protein kinase CK2, nuclear actin, nuclear myosin (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, transcription 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 stochastic manner from freely diffusible subunits has been eclipsed by measurements of the movement of fluorescently tagged subunits of Pol I and basal transcription factors These studies revealed that the Pol I transcription machinery is highly dynamic, assembling in a stochastic fashion, sometimes individually and sometimes in subcomplexes [18] Quantitative single-cell imaging combined with computational modeling and biochemical 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 cellular metabolism, such as nutrient starvation, oxidative stress, inhibition of protein synthesis and cell confluence, 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-dependent kinase)1–cyclin B-dependent phosphorylation of TAFI110, a subunit of SL1 that nucleates PIC assembly, 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 formation [20,21] Thus, reversible phosphorylation of SL1 is used as a molecular switch to shut down rDNA transcription during mitosis Resetting of the Pol I transcription machinery at the end of mitosis is brought Regulation of rRNA synthesis Growth factors Nutrients Oncogenes Tumor suppressors Genotoxic stress Viral infection Metabolic stress Starvation Fig Extracellular signals impinge on transcription of rRNA genes The cartoon illustrates the signaling pathways that upregulate (green arrows) or downregulate (red arrows) nucleolar transcription, converging at Pol I transcription 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 transcription 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], underscoring 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 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS 4629 Regulation of rRNA synthesis I Grummt et al Cdk2–cyclin E P P P P TIF-IA ERK P UBF S6K Cdk4–cyclin D P G1 P S Cdk2–cyclin A UBF P P P M TIF-IA RSK G0 G2 UBF P SL1 Cdk1–cyclin B Fig 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-dependent pathways activate TIF-IA by phosphorylation of Ser44, Ser633 and Ser649 At entry into mitosis, Cdk1–cyclin B phosphorylates TAFI110, 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 phosphorylations are marked in green, and inhibiting ones in red Transcription is low in resting cells (G0), 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 is a major rate-limiting step for growth factor-dependent regulation of rDNA transcription [11] Acetylation is another post-translational modification that regulates the activity of UBF and SL1 Acetylation of TAFI68 by the the histone acetyltransferase PCAF augments SL1 activity and stimulates transcription initiation [30,31] PCAF-dependent acetylation of TAFI68 is counteracted by SIRT1, the founding member of a family of highly conserved NAD+-dependent histone deacetylases, termed sirtuins SIRT1 is conserved 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 transcription initiation In contrast, SIRT7, another member of the sirtuin family, exerts a positive effect on Pol I transcription SIRT7 localizes to nucleoli, is associated with active rDNA repeats, interacts with Pol I, and stimulates rDNA transcription by enhancing Pol I occupancy at rDNA [33] Knockdown of SIRT7 leads to cell cycle arrest and apoptosis, underscoring the pivotal 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 4630 rDNA transcription, SIRT1 repressing and SIRT7 activating rRNA genes, thereby linking Pol I transcription 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 nucleolar 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 histone 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, downregulate transcription of rDNA, whereas agents that FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS I Grummt et al Regulation of rRNA synthesis stimulate growth and proliferation upregulate rRNA synthesis [35–38] A key player in growth-dependent regulation of rDNA transcription is TIF-IA, the mammalian homolog of yeast RRN3 [39], an essential transcription 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 conditions and environmental cues TIF-IA is phosphorylated 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, indicating 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 Cell cycle Cdk2 Energy deprivation 635 44 AMPK 649 Nutrients mTOR 633 Stress JNK RSK 199 200 ERK2 Growth factors 172 CK2 170 Fig The transcription factor TIF-IA is targeted by multiple signaling 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 multiple sites by the indicated protein kinases (boxed) Specific phosphorylation 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 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 regulated by the intracellular ATP levels [44] The key enzyme that translates changes in energy levels into adaptive cellular responses is the AMP-activated protein 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 phosphorylation 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 transcription, 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 transcription 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 (phosphatase and tensin homolog deleted on chromosome 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 pathways that control Pol I transcription It is therefore plausible that cells might achieve a proliferative advantage by elevating the level of specific oncogene products to increase the production of rRNA For example, the proto-oncogene product c-Myc was shown to localize in nucleoli at sites of rRNA synthesis, 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 recruitment 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 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS 4631 Regulation of rRNA synthesis I Grummt et al Tumor suppressors Prevents TTF-I binding Inhibits UBF/SL1 interaction p53 GSK3β pRb ARF PTEN Induces UBF Inhibits SL1–UBF interaction degradation Disrupts SL1 TIF-IA TTF-I Pol I SL1 UBF Increases UBF expression Increases TAFI48 expression Stabilizes SL1–UBF Myc NPM Oncogenes known as B23), was shown to increase the level of TAFI48 and to stimulate proliferation of transformed cells NPM shuttles between the nucleolus, nucleoplasm, and cytoplasm, and its overexpression or mutation 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 4632 Fig Oncogene products and tumor suppressors control Pol I transcription Oncogene 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 macromolecular interactions required for transcription initiation complex assembly (marked by red lollypops) 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 Credependent homologous recombination leads to inhibition of Pol I transcription, perturbation of the nucleolar 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 cytoplasm After TIF-IA depletion, the p53–MDM2 complex 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 activity, reinforcing the idea that the nucleolus is a major cellular stress sensor that integrates and transmits signals for regulation of p53 activity (Fig 7) A key upstream controller of p53 is the tumor suppressor 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 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS I Grummt et al Regulation of rRNA synthesis TIF-IA+/+ TIF-IA–/– Intact nucleolus rP rP Fig 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 degradation 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 consequence, the amount and activity of p53 are enhanced, leading to cell cycle arrest and apoptosis Perturbed nucleolus rP MDM2 p53 p53 degradation rP MDM2 rP rP p53 stabilization p53 Cell growth 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 prerRNA 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 production of mature rRNA by inhibiting the processing of pre-rRNA Presumably, the primordial role of ARF is to slow ribosome production in response to hyperproliferative 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 termination 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 phosphatase activity, and is independent of the p53 status of the cell PTEN inhibits phosphoinositide 3-kinase signaling and triggers disruption of the TBP–TAFI complex SL1, thereby preventing the assembly of transcription initiation complexes [52] In Ras-trans- G1-arrest, apoptosis 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 constitutively active GSK3b mutant inhibits rDNA transcription by interaction with SL1 Thus, the interplay between PTEN and GSK3b represents a powerful mechanism the cell uses to ensure that ribosome biogenesis is coupled to growth control Chromatin modifications and epigenetic control of rDNA transcription Transcription of rDNA is also modulated by epigenetic mechanisms Approximately half of the several hundred copies of rRNA genes exhibit a heterochromatic chromatin structure and are transcriptionally silent (Fig 8) The fact that, even in proliferating cells with a high demand for ribosome biogenesis, a significant fraction of rRNA genes are epigenetically silent provides a unique possibility to decipher the mechanisms 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 surveillance and genomic stability Specific epigenetic characteristics distinguish active rDNA repeats from inactive ones Generally, transcriptionally active genes are characterized 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- FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS 4633 Regulation of rRNA synthesis I Grummt et al Active copies H4ac H3ac TIP5 UBF H3K4me2 TIP5 Silent copies SNF2h H3K9me CH3 H4K20me CH3 NoRC HP1 Chromatin remodeling Heterochromatin formation DNA methylation CH3 Fig 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, acetylation 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 (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 deconvolution 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 tone H3 Lys correlating with transcriptional activity, whereas histone H4 hypoacetylation and methylation of H3K9, histone H3 Lys27 and histone H4 Lys20 correlate 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 chromatin domain that is less accessible to the cellular recombination 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-Iinteracting protein [60]) NoRC interacts with DNA methyltransferase(s), histone deacetylase(s), and histone methyltransferase(s), thereby recruiting the enzymes required for heterochromatin formation and rDNA silencing In the mouse, NoRC-dependent 4634 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, heterochromatization and transcriptional silencing of rRNA genes [62,63] In addition, NoRC shifts the promoterbound 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 coordinating 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 demonstrates the profound and complex role that noncoding FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS I Grummt et al Regulation of rRNA synthesis G U A U A G U –126 G U –49 G U G C U U C C A U U G UU U UU G C G C C G C UC C C A U C U C U U C C CA CAC GU A U G C G C U G A U G G U G A U C C U U C U U C U C A A GGU G Fig Model depicting the origin of pRNA that is associated with NoRC Intergenic transcripts (dotted line) are synthesized from a Pol I promoter located  kb upstream of the pre-RNA promoter The primary intergenic transcripts are degraded by the exosome, except for 150–250 nucleotide transcripts that match the rDNA promoter (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 heterochromatin formation Spacer promoter pre-rRNA promoter T0 CORE UCE –1997 RNAs play in regulating gene expression [65,66] Noncoding RNAs are integral components of chromatin, 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 regulation probably represents an intimate and balanced interplay of both RNA and chromatin fields [67,68] In support of this notion, NoRC function requires binding of TIP5, the large subunit of NoRC, to 150– 250 nucleotide RNA, termed pRNA, because it is complementary in sequence to the rDNA promoter [63] pRNA originates from a Pol I promoter located within the intergenic spacer  kb upstream of the 45S prerRNA coding region (Fig 9) Intergenic transcripts are of low abundance and usually 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 displacement 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 nucleolar 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- 150–250 nucleotides ‘pRNA’ (promoter-associated RNA) TIP5 SNF2h U U proximal binding site of the transcription factor TTFI 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 methyltransferase (DNMT)3b to the promoter of specific rDNA repeats (Fig 10) HDAC HMT CH3 T0 UCE TIP5 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 T0 pRNA base pairs with T0, 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 FEBS Journal 277 (2010) 4626–4639 ª 2010 The Author Journal compilation ª 2010 FEBS 4635 Regulation of rRNA synthesis I Grummt et al Histone lysine methylation is dynamically regulated In contrast to NoRC-dependent heterochromatin formation and rDNA silencing, little is known about the mechanisms that counteract heterochromatin formation 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 transcriptional 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 methylation 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 posttranslational 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 elongation 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 (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 (H3K9me1/2) and activates rDNA transcription, transcriptional activation requiring both the JmjC domain and the PHD finger Strikingly, 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 readout, a finding that links dynamic histone methylation to rDNA transcription 4636 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 demethylation Knockdown of KDM2B leads to a significant increase in pre-rRNA transcription, cell size, and proliferation, suggesting that KDM2B is a repressor of Pol I transcription By contrast, PHF8 activates Pol I transcription, thereby promoting cell growth and proliferation 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 transition from one transcriptional state to another Conclusions Significant advances have been made in our understanding 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 during 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 perturbations 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 systems 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 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in the regulation of. .. elongation, whereas hypophosphorylated UBF inhibits elongation, demonstrating that transcription elongation FEBS Journal 277 (2010) 462 6–4 639 ª 2010 The Author Journal compilation ª 2010 FEBS 4629 Regulation