Transcription of the major ribosomal RNAs by Pol I (RNA polymerase I) is a key determinant of ribosome biogenesis, driving cell growth and proliferation in eukaryotes. Hundreds of copies of rRNA genes are present in each cell, and there is evidence that the cellular control of Pol I transcription involves adjustments to the number of rRNA genes actively engaged in transcription, as well as to the rate of transcription from each active gene. Chromatin structure is inextricably linked to rRNA gene activity, and the present review highlights recent advances in this area.
A characteristic feature of the genes encoding the major rRNAs is their repetitive nature. Human cells have approx. 400 copies of a ribosomal DNA repeat that encode the rRNAs, distributed over the short arms of the five acrocentric chromosomes 13, 14, 15, 21 and 22. In yeast, 150–200 rDNA (ribosomal DNA) copies occur in tandem arrays on the right arm of chromosome XII. Intriguingly, only a proportion of these repeats are transcribed in actively growing cells of yeast or humans (∼50% of cells in interphase). The number of repeats engaged in active transcription can influence the overall level of rRNA synthesis. So, what determines whether a particular rDNA gene is transcriptionally active or silent? Active (transcriptionally competent or transcriptionally active) and inactive (silent) rDNA genes have distinctive chromatin states, therefore chromatin context might influence the transcriptional activity of the rDNA genes. Conversely, there is evidence that the transcriptional activity of the rDNA genes can influence the chromatin state of the rDNA. In the present review, we describe the known features of rDNA chromatin, the ability of the Pol (RNA polymerase) I transcription machinery to negotiate the rDNA chromatin template and the regulatory factors involved in, and alterations accompanying, the silencing or activation of rRNA genes.
Features of rDNA chromatin
Two distinct chromatin states: active and inactive
Regularly spaced nucleosomes are detectable at the rDNA repeats, by MNase (micrococcal nuclease) digestion analysis of bulk chromatin . However, the chromatin structures of individual rDNA repeats are not distinguishable by this method. Heterogeneity in the transcriptional state of rDNA repeats was revealed by electron microscopy analyses , with some repeats transcribed and others transcriptionally silent. The observed high density of loading of RNA polymerases on the actively transcribed rRNA genes might preclude a ‘normal’ nucleosomal arrangement.
Two distinct coexisting chromatin states correlating with transcriptional activity were defined by psoralen photo-cross-linking analysis of the rDNA repeats. rDNA chromatin that is relatively refractory to psoralen cross-linking, representing the inactive rDNA repeats, was organized in regular nucleosomal arrays similar to those observed for bulk chromatin . Other rDNA repeats (∼50% of the total) were found in a psoralen-accessible (‘open’) chromatin state lacking regular nucleosomal arrays  and were associated with nascent rRNA, indicating that they were actively transcribed . Active and inactive repeats are interspersed in yeast, arguing against control of rDNA gene activation via organization into specific ‘on’ and ‘off’ chromosomal domains and in favour of the independent regulation of each rDNA repeat [5,6], notwithstanding the global forces which operate to achieve nucleolar dominance in hybrids [7,8].
Recently, ChEC (chromatin endogenous cleavage) with MNase-fusion proteins, which allows for the precise localization of chromatin-associated factors on genomic DNA , was combined with psoralen photo-cross-linking analyses to demonstrate that, in contrast with the inactive rDNA repeats, the active repeats have few histones, hence few nucleosomes, associated with the transcribed regions . That at least some nucleosomes are associated with the actively transcribed rDNA is also suggested by the following. The rapid replication-independent exchange of histone H3/H4 tetramers at the transcribed regions of the rRNA gene loci in the slime mould Physarum polycephalum suggests a dynamic nucleosomal arrangement at the active rDNA . A dynamic chromatin structure of unphased nucleosomes has also been inferred from studies of the rDNA chromatin in a particular yeast mutant strain in which all rDNA repeats were active following a reduction in rDNA copy number , and histones H3 and H2B were present at the active rDNA repeats, albeit at a reduced level . Factors Spt4p and Spt5p, which positively affect Pol II transcription elongation through chromatin, influence Pol I transcription elongation in yeast , and chromatin-remodelling activities such as Chd1p, Isw1p and Isw2p have been found associated with the active rDNA repeats . In mammalian rDNA, nucleosomes are present at the rDNA promoter regions of both active and inactive repeats, and, significantly, the active and silent rDNA promoters are distinguishable by differential nucleosome positioning. At the active sites, the promoter-bound nucleosome covers nucleotides from −157 to −2, whereas, at silent loci, the nucleosome is 25 nt further downstream, at a position potentially incompatible with productive pre-initiation complex formation [14,15]. Inactive mammalian rDNA repeats are maintained in a silent state via association of NoRC (nucleolar-remodelling complex) with the rDNA promoter, as described below.
Certain modifications of the DNA and associated histones also distinguish the active and silent rDNA genes. The promoters of actively transcribed mouse rRNA genes are hypomethylated [16,17], and the associated histones are highly acetylated, with the opposite being true of inactive gene promoters . Furthermore, the silent copies of the rDNA promoters have methylated histone H3K9 (histone H3 Lys9) and are associated with HP1 (heterochromatin protein 1). Active rDNA repeats have additional distinguishing features. Yeast Hmo1, an HMG (high mobility group)-box-containing protein, shown previously by chromatin immunoprecipitation to localize to the entire rDNA repeat [18,19], is specifically associated with the active rDNA chromatin. Hmo1 is not essential for establishment of the active chromatin state, however, and a role for Hmo1 has yet to be defined . The mammalian Pol I transcription factor UBF (upstream binding factor), which has multiple HMG boxes, activates promoterspecific Pol I transcription  through the stimulation of promoter escape by Pol I  and, perhaps, by increasing the local concentration of Pol I and transcription initiation factor SL1 (selectivity factor 1) at the rDNA [22,23]. At the promoter, UBF dimerizes and, in binding DNA, has the ability to induce formation of an ‘enhancesome’, in which ∼140 bp of DNA is organized in a 360° turn as a result of six in-phase bends generated by three of the six HMG boxes in each UBF monomer . The methylation of a single CpG dinucleotide at −133 in the mouse promoter is sufficient to prevent UBF from binding the nucleosomal rDNA promoter and to abolish rDNA chromatin transcription . The requirement for CpG methylation at specific positions appears to be echoed in the human system, where transcriptional silencing might be a consequence of the binding to these sites of MBD2 (methyl-CpG-binding protein 2), which could recruit HDAC (histone deacetylase) co-repressor complexes .
UBF and rDNA chromatin
UBF binding is not restricted to the promoter region and is found throughout the rDNA repeat [23,27], but, unlike yeast Hmo1, UBF is present at transcriptionally active and inactive loci . Large arrays of heterologous UBF-binding sequences integrated at ectopic sites in human chromosomes efficiently recruit UBF and form novel secondary constrictions, termed pseudo-NORs (nucleolar organizer regions) due to their morphological similarity to the decondensed rDNA repeat regions (NORs) on the acrocentric chromosomes . Furthermore, large-scale chromatin decondensation was induced when UBF was targeted, via a lac-repressor fusion protein, to a heterochromatic amplified chromosome region, which contains lac-operator repeats . These results suggest that UBF can dramatically influence chromatin structure , perhaps promoting steps to convert rDNA chromatin into a transcriptionally competent form. Interestingly, however, UBF does not appear to be required to maintain transcriptional competence, since adenoviral infection of cells, which leads to a sequestration of the majority of UBF away from the nucleolus, does not detectably affect rRNA synthesis by Pol I . Perhaps the chromatin alterations induced by UBF are associated with the events following DNA replication, involving the re-establishment of a nucleosomal structure on the newly replicated rDNA coding regions . UBF molecules bound to the transcribed regions of the rDNA impede elongation of transcription by Pol I [21,32], and this is modulated by the growth-factor regulated ERK (extracellular-signal-regulated kinase) phosphorylation of UBF [32,33]. Challenges for the future will be to understand how UBF remodels chromatin, the consequences for rDNA transcription and whether the role of UBF is related to the high affinity of UBF for nucleosomal DNA and/or to its ability to displace histone H1 in reconstituted chromatin .
Transcription through chromatin
The rate of rRNA synthesis, and the linked processing events , could depend upon the efficiency by which Pol I transcribes through nucleosome-containing regions of the active rDNA genes. Biochemical analyses have established that Pol III and SP6 RNA polymerase have the intrinsic ability to transcribe through a nucleosome, and this involves the translocation of the nucleosome to a position upstream of its original site during polymerase passage, without the disruption of the majority of octamer–DNA contacts [36,37]. Nucleosomes impede Pol II transcription, and chromatin transcription is facilitated by histone chaperones, such as the FACT (facilitates chromatin transcription) complex, which induce transient release of an H2A–H2B dimer and so destabilize the nucleosome to facilitate Pol II passage [38–40], consistent with the observed rapid exchange of these dimers in cells . Chromatin transcription by mammalian Pol I also appears to require histone chaperone activities provided, for example, by nucleolin [42,43], nucleophosmin (B23) [44,45] and/or FACT (J.L. Birch and J.C.B.M. Zomerdijk, unpublished work). Histone chaperones are important in the reassembly of nuclesomes in the wake of passing polymerases, and so could prevent spurious transcription initiation from otherwise exposed cryptic start sites in the transcribed region [46,47].
Alterations in rDNA chromatin: silencing and activation
Epigenetic inheritance of chromatin modifications in mammalian cells maintains the active and silent chromatin states of the rDNA repeats in the daughter cells. In mammalian cells, the inheritance of specific cytosine methylations is important . Chromatin modification in yeast, however, does not involve methylation of the rDNA, since yeast have no DNA methylase activities. In yeast, the active or silent state of the rDNA repeats are also re-established following DNA replication [31,49], but it is unclear how yeast cells select an rDNA repeat for activation or repression; it appears to be stochastic, although the ratio of active to inactive genes does not change drastically. Chromatin structures in yeast and mammalian systems are dynamically altered by the (combined) actions of ATP-dependent chromatin-remodelling activities and activities that covalently modify histones . The open chromatin structure of yeast rDNA requires transcribing Pol I complexes , suggesting that Pol I transcription might remodel chromatin.
Growth-factor-induced and nutrient-regulated signalling pathways have been shown to regulate several chromatin-remodelling complexes in mammalian and yeast cells [51,52] and, consequently, could affect rRNA gene promoter accessibility . For example, stationary-phase yeast cells repress rRNA transcription in part through reducing the number of active rDNA repeats. Furthermore, the targeting of the Rpd3–Sin3 HDAC co-repressor complex to the rDNA can lead to deacetylation of histone H4 and inactivation of individual rRNA genes, and this is controlled by the TOR (target of rapamycin) signalling pathway [53,54].
TTF-I (transcription termination factor I), which can bind to the mammalian rDNA promoter proximal terminator element (T0) in addition to the terminators downstream of the rRNA genes, is key in the recruitment of chromatin-remodelling complexes that either co-activate  or co-repress transcription from the mammalian rDNA chromatin [16,56].
Silencing of rDNA chromatin
In silencing the rDNA promoter, TTF-I recruits NoRC, a complex of TIP5 (TTF-I-interaction protein 5) and SNF2h, which induces sliding in an ATP- and histone H4 tail-dependent manner of the promoter-bound nucleosome, and this contributes to the repression of Pol I transcription [15,57,58]. The binding of the bromodomain of TIP5 to acetylated Lys16 of histone H4 is a prerequisite to NoRC function . NoRC interacting with TTF-I near the promoter recruits SNF2h, involved in nucleosome sliding, HDAC1 and DNMTs (DNA methyltransferases) 1 and 3 through the bromodomain and PHD (plant homeodomain) finger of TIP5 . Although the consequent deacetylation of H4 is not sufficient for silencing, this deacetylation is a prerequisite to the CpG methylation of the rDNA promoter, which contributes to the establishment and maintenance of a silent rDNA chromatin state [16,60–63]. Additionally, the establishment and maintenance of a specific heterochromatic configuration at the rDNA promoter, as reflected in histone H3K9 and H4K20 methylation and HP1 recruitment, requires rDNA intergenic spacer transcripts to interact with TIP5 of NoRC . In nucleolar dominance (the NORs of one parental species are dominant over the other in interspecies hybrids), the mechanisms of rDNA silencing also involve the co-ordinated and interdependent action of chromatin remodellers, histone and DNA modifiers [65–68].
The phenomenon of transcriptional silencing of Pol II-transcribed genes by rDNA chromatin (commonly referred to as rDNA silencing) is not to be confused with silent rDNA chromatin (i.e. not active for Pol I transcription). SIR (silent information regulator) 2, which is associated with yeast rDNA chromatin, is an NAD-dependent histone deacetylase  that represses recombination between the tandemly repeated ribosomal RNA genes  and is involved in Pol II transcriptional silencing by influencing rDNA chromatin accessibility [71–75]. A mammalian Sir2 homologue, SIRT7 (sirtuin 7), is associated with active rRNA genes, and with Pol I and histones, and functions as a positive regulator of Pol I transcription .
Activation of rDNA chromatin
Little is known of the selective TTF-I mediated recruitment of remodelling activities that activate rDNA genes, other than that it is independent of histone H4 tails . However, chromatin-remodelling complexes specific for active rDNA repeats have been identified and include WICH, a chromatin-remodelling complex containing WSTF (William's syndrome transcription factor) and SNF2h , and CSB (Cockayne syndrome group B) protein, a member of the SWI/SNF family of ATP-dependent chromatin-remodelling activities. CSB is required for rDNA transcription and is part of a protein complex that contains Pol I, TFIIH (transcription factor IIH), and basal Pol I transcription initiation factors . CSB is found at active rDNA associated with histone methyltransferase G9a, which methylates histone H3 on Lys9 in the pre-rRNA coding region and this appears to be important for transcription elongation through chromatin  (note that this modification at the promoter is associated with inactive rDNA).
Collectively, the data suggest that rDNA has a complex and dynamic chromatin structure and that actively transcribed rDNA chromatin is distinct both from that of silent rDNA genes and from that of other (Pol II-) transcribed loci. Future genetic analysis in combination with biochemical analysis of isolated active rDNA chromatin will provide additional clues to the constituents and structure of active rDNA repeats, define proteins required in the establishment and/or maintenance of active rDNA chromatin, and identify factors that facilitate chromatin transcription elongation by Pol I.
Ribosomal RNA synthesis by Pol I is crucially dependent upon nutrient availability and drives ribosome biogenesis, a process important for cell growth and proliferation [80–82]. Deregulation of Pol I transcription perturbs normal cell growth, leading to cell death or uncontrolled cell growth and proliferation, potentially resulting in developmental defects or cancer in mammals [83–86]. It is therefore important to further our understanding of the regulation of rDNA chromatin organization and its influence on rRNA gene transcription by Pol I.
Transcription: A Biochemical Society Focused Meeting held at the University of Manchester, U.K., 26–28 March 2008 as part of the Gene Expression and Analysis Linked Focused Meetings. Organized and Edited by Stefan Roberts (Manchester, U.K.) and Robert White (Beatson Institute, Glasgow, U.K.).
Cockayne syndrome group B
facilitates chromatin transcription
(etc.), histone H3 Lys9 (etc.)
high mobility group
hetreochromatin protein 1
nucleolar organizer region
transcription termination factor I
TTF-I-interaction protein 5
upstream binding factor
We thank Dr Jackie Russell for her interest, critical reading of the manuscript and helpful suggestions. We apologize to our colleagues whose work could not be cited owing to space limitations. We thank the Wellcome Trust for support of the research in the Zomerdijk laboratory, and the Biotechnology and Biological Sciences Research Council for a Ph.D. studentship of J.L.B.