Nuclear tumor suppressors in space and time

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Nuclear tumor suppressors in space and time David A. Barbie1,*, Lindus A. Conlan2,* and Brian K. Kennedy 2 1 2

Department of Internal Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA Department of Biochemistry, University of Washington, Seattle, WA 98195, USA

Numerous studies have identified key binding partners and functional activities of nuclear tumor-suppressor proteins such as the retinoblastoma protein, p53 and BRCA1. Historically, less attention has been given to the subnuclear locations of these proteins. Here, we describe several recent studies that promote the view that regulated association with subcompartments of the nucleus is inherent to tumor-suppressor function.

Introduction Tumorigenesis is marked fundamentally by deregulation of the cell cycle and disruption of pathways that monitor genomic integrity [1,2]. The initiation of DNA replication at the G1–S-phase transition represents a key decision point in cell-cycle control because the cell commits to duplication on traversing this boundary. The retinoblastoma protein (pRB), which is a major substrate for the cyclin dependent kinases (CDKs) that regulate the G1–Sphase transition, is a crucial component of this process [3]. The activity of pRB is disrupted in most tumors, either by direct mutation or by unchecked activity of CDKs. Most human tumors also have either mutations or alterations in the p53 pathway that initiates responses to genotoxic insults and deregulated proliferation signals [4]. Loss of the key checkpoint protein p53 permits cells to survive anti-proliferative signals that are induced by DNA damage, hypoxia, telomere dysfunction and/or oncogene activation [5]. In a similar manner, loss of the BRCA1 tumor-suppressor protein leads to defective regulation of DNA double-strand-break repair (DSBR), which inactivates an important response to DNA damage and results in the accumulation of mutations that drive oncogenesis in familial breast cancer [6]. Many studies have identified binding partners for nuclear tumor suppressors such as pRB, p53 and BRCA1. There are, for example, greater than 100 binding partners reported for pRB [7]. However, until recently the localization in vivo of tumor-suppressor proteins has been less well investigated. It is increasingly apparent that large-scale processes such as DNA replication, gene transcription and RNA processing are coordinated through nuclear organization (Box 1). Recently, the relationship between the subnuclear distribution of Corresponding author: Kennedy, B.K. ([email protected]). * D.A.B. and L.A.C. contributed equally to this review. Available online 4 June 2005

tumor suppressors and their association with known binding partners (Table 1), nuclear structures and the nuclear matrix has come under intense study. Here, we summarize these studies and their importance to understanding tumor-suppressor function. Subnuclear localization of pRB Before entry into S phase hypophosphorylated pRB binds to the E2F family of transcription factors, which represses Box 1. Nuclear structure and nuclear bodies Rather than being distributed randomly throughout the nucleoplasm, chromosomes occupy distinct territories that are separated by interchromosomal domains. Genes that are expressed actively tend to face the surface of these domains and inactive genes are either buried in regions of heterochromatin or situated at the nuclear periphery [65]. How this localization is established and maintained in a cell-type-specific manner is understood poorly, but it appears that the nuclear matrix plays a key role in the organization of chromatin. The nuclear matrix represents the biochemically insoluble compartment of the nucleus, key components of which are the nuclear lamins. Lamins are intermediate filament proteins that form a latticework adjacent to the inner surface of the nuclear envelope, and distinct intranuclear structures [66]. The LMNA gene encodes all A-type lamins (primarily lamin A and lamin C), whereas two genes, LMNB1 and LMNB2, encode B-type lamins. Mutations in LMNA have been found in several human dystrophic and progeroid syndromes, which indicates that A-type lamins might have a role in maintaining healthy tissue [17]. Lamins might have a direct role in the organization of chromatin, and have been linked to processes such as DNA replication and transcriptional control [67]. Interspersed within chromatin are the nuclear matrix are several organized, structural elements. The most prominent of these is the nucleolus, which is the site of rRNA synthesis, processing and assembly into ribosomes. Several perinucleolar structures have been described recently, including the perinucleolar compartment, which is identified by localization of the polypyrimidine-tractbinding protein and is associated with cell transformation and RNA processing [68], Sam68 nuclear bodies, which are also involved in RNA metabolism, and perinucleolar foci, which contain cell-cycle and DNA-replication proteins and are affiliated with internal lamin A/C structures. The relationship between these structures has not been established. Other nuclear bodies, which have been identified by the immunolocalization of specific proteins, include speckles, gems, coiled bodies and PML-NBs. Speckles and gems appear to play a role RNA processing [65], whereas coiled bodies are affiliated with histone genomic loci and might help regulate their transcription [69]. PML bodies are revealed by immunofluorescent detection of PML, which was discovered by its fusion to the retinoic acid receptor in acute promyelocytic leukemia [65]. These structures are affiliated with the nuclear matrix, have been shown to play a role in antiviral defense, and interact with tumor-suppressor proteins in the nucleus (see text).

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Table 1. Factors that colocalize with pRB, p53 and BRCA1 Interacting factors ATM ATR BRG1 CBP CHK2 E2F HDAC1 HIPK Lamin A/C XIST RNA a

Function Phosphorylates p53 at Ser15, and BRCA1 at Ser1423 and Ser1524 Protein kinase that phosphorylates BRCA1 and p53 in response to DNA damage ATPase component of SWI/SNF-related chromatin-remodeling complex Transcriptional cofactor (histone acetyltransferase) Protein kinase that phosphorylates p53 at Ser20 and BRCA at Ser988 Transcriptional activation factor that interacts with pRB Transcriptional repressor that binds pRB and BRCA1 Phosphorylates p53 at PML-NBs Nuclear intermediate-filament proteins Heterochromatin silencing

Subnuclear sites of colocalization BRCA1 in IRIFsa, p53 at PML-NBs BRCA1 in IRIFs, p53 at PML-NBs BRCA1 at silenced X chromosome p53 at PML-NBs, pRB BRCA1 in IRIFs, p53 at PML-NBs pRB pRB, BRCA1 at heterochromatin p53 at PML-NBs pRB at perinucleolar locations Silenced X chromosome

Refs [70] [71] [61] [23,48] [51] [7] [3,61] [72] [17] [59]

Abbreviation: IRIFs, ionizing-radiation-induced foci.

the transcription of genes that are important for S-phase entry. Several mechanisms of repression have been proposed, including direct interference with the E2F transactivation domain and the recruitment of co-repressor complexes [3,8]. Phosphorylation of pRB by cyclin–CDKs dissociates these complexes, but pRB is maintained stably in the nucleus. Early immunofluorescence studies reported that pRB has a speckled nucleoplasmic appearance with small bright foci that are excluded from heterochromatic regions [9,10]. Biochemical studies demonstrate that hypophosphorylated (active) pRB is associated with the nuclear compartment, whereas hyperphosphorylated pRB can be eluted easily under hypotonic buffer conditions [10]. This association is perturbed in cells that express tumorassociated mutants of pRB [10,11], which indicates that association with the nuclear compartment is important for the function of pRB. After extraction from the nuclear matrix, pRB associates with nuclear matrix fibrogranular masses, the nucleolar remnant and the nuclear lamina in G1-arrested (but not S-phase) cells [12]. In primary mammalian cells, pRB localizes to perinucleolar foci that overlap with lamin A/C structures located at perinucleolar sites (Figure 1a) [13,14]. Lamin A/C is predominantly located at the nuclear periphery in differentiated cells, with a subpopulation located adjacent to the nucleolus. Association between pRB and A-type nuclear lamins (lamin A/C) has been reported previously, which indicates that these core constituents of the nuclear matrix might tether pRB directly [12]. More recent studies find that nuclear anchorage of pRB also requires LAP2a [15]. Lamin A/C might play a role in mediating the formation of the pRB foci because pRB is dispersed throughout the nucleus and fails to associate with E2F foci in their absence [16]. Furthermore, expression of a dominant-negative mutant of lamin A results in the redistribution of pRB into nuclear aggregates. Lamin A/C appears to play a role in cell differentiation, and its mutation results in several dystrophic and progeroid syndromes [17]. Tethering of pRB by lamin A/C might be required to maintain differentiation programs in these tissues. Perinucleolar foci of pRB are prominent during G1 phase and early S phase in normal diploid cells, and co-localize with E2F-1, E2F-4, histone deacetylases (HDACs) and early sites of DNA synthesis [13]. During S phase, pRB distributes to hundreds of small foci www.sciencedirect.com

throughout the nucleus, which indicates that progressive phosphorylation of pRB results in dissociation from these matrix-associated sites of function (Figure 1b). In response to intra-S phase DNA damage and activation of protein phosphatase 2A, hypophosphorylated pRB resumes a focal-staining pattern that is reminiscent of perinucleolar foci, inhibits origins of DNA replication and prevents endoreduplication [18]. Studies using GFP–pRB in live cells further support an affiliation between pRB and a matrix-associated nucleolar compartment. When overexpressed, GFP–pRB is apparent in the nucleolus and nucleoplasm, and the nucleolar fraction is immobile [19]. In this study, nucleolar association depends on the N-terminus of pRB. This region binds the p84 nuclear-matrix protein [20], which indicates that it might be important in mediating interactions with the nuclear matrix and the nucleolus. In addition, co-expression of cyclin E and SV40 large T antigen, which promote entry into S phase by inactivating pRB, disrupt nucleolar accumulation [19]. Consistent with these findings, localization of pRB to perinucleolar foci appears to depend on both cell-cycle position and cell-immortalization status because the foci are perturbed and distributed throughout the nucleus in several immortalized cell lines in G1 [13]. Accumulation of pRB in nucleoli is associated with the inhibition of UBF and repression of rDNA transcription in part through recruitment of histone deacetylases [21–23]. Thus, pRB is likely to coordinate the downregulation of rRNA synthesis with cell-cycle exit. In support of this hypothesis, re-introduction of exogenous GFP–pRB into SAOS-2 cells inhibits cell-cycle progression and increases the concentration of pRB in nucleolar regions [19]. It is important to distinguish the specific role of pRB in regulating the transcription of rDNA from its more general role in G1–S-phase transcription and, possibly, replication control at sites in and around the nucleolus. In addition to regulating transcription of S-phase genes and rDNA, it is suggested that pRB is involved directly in modulating the initiation of DNA replication. pRB inhibits DNA replication by interacting with components of the prereplication complex such as MCM7, ORC1 and ORC2 [24–27]. As mentioned above, the perinucleolar, lamin A/C-associated foci of pRB overlap with the first sites of DNA replication in S phase and colocalize with several replication proteins [13]. This finding differs from earlier studies where DNA replication was distributed throughout

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Figure 1. Subnuclear localization of pRB in primary and immortalized cells. (a) In primary human fibroblasts (upper panel) pRB (green) localizes to distinct perinucleolar foci in G1 and early S phase and distributes to hundreds of foci throughout the nucleus during mid–late S phase. By contrast, in actively cycling, immortalized 3T3 cells (lower panel), pRB foci are evenly distributed throughout most of the cell cycle. Images represent single slices through the nucleus. (b) Model of the regulation of pRB localization by phosphorylation (upper panel). As phosphorylation occurs during the G0–G1- and G1–S-phase transitions, pRB dissociates progressively from perinucleolar foci (clustered, more prominent dots) and distributes throughout the nucleus (smaller, more dispersed dots). It is hypothesized that early cyclins and CDKs associated with G0–G1 (cylin C, cylin D, CDK 3, CDK 4 and CDK 6) might promote early, site-specific phosphorylation events that trigger initial distribution of the foci, whereas cyclins and CDKs associated with late G1 and S phase (cyclin E, cyclin A and CDK 2) promote distinct phosphorylation events that lead to subsequent dispersal of the foci. In primary cells, this transition occurs in early S phase (upper panel). DNA damage during S phase results in dephosphorylation of pRB by protein phosphatase 2A (PP2A) and reformation of focal pRB structures. In immortalized 3T3 cells (lower panel), excess cyclin and CDK activity might cause early phosphorylation of pRB, so explaining the premature distribution of pRB foci during G1 and distributed replication sites at the onset of S phase. The effects of DNA damage during S phase on the localization of pRB have not been studied in this context, but it is possible that pRB remains hyperphosphorylated and distributed.

the nucleus in early S phase [28], This discrepancy appears to be related to the sensitivity of nuclear structure to denaturating conditions. In addition, most previous studies have examined immortalized cells in which perinucleolar pRB and replication foci tend to be dispersed throughout the nucleus, even in early S phase [13]. Recent work indicates that the role of pRB in modulating the onset of DNA replication is more indirect and occurs www.sciencedirect.com

largely by transcriptional repression of genes that encode the replication machinery, PCNA in particular [29]. However, it should be noted that this has been observed in immortalized cell lines, which might be why pRB is not reported to associate directly with replication sites. Although there is a relationship between pRB and DNA replication, future experiments are needed to clarify its exact nature.

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Regulation of the localization of pRB Because the amount of pRB phosphorylation depends on cell type and cell-cycle position, it is important to note that different cell types and physiological conditions might result in variable dispersal of the perinucleolar foci. In immortalized cell lines such as U20S and 3T3 cells, in which loss of p16INK4A results in excessive activity of G1 cyclin–CDK, there are pRB foci distributed throughout the nucleus for much of the cell cycle (Figure 1a,b) [13]. By contrast, in primary cells that are arrested by confluence, serum starvation and senescence, perinucleolar pRB and DNA-replication foci persist throughout the S phase that precedes cell-cycle exit [14]. Under these conditions, accumulation of CDK inhibitors leads to hypophosphorylation of pRB, which might account for the persistence of perinucleolar foci of pRB. Alterations in the nuclear organization of DNA synthesis might provide a mechanism by which pRB coordinates epigenetic modification of chromatin with exit from the cell cycle. In cycling cells, pRB dissociates from replication foci when both distribute throughout the nucleus during S-phase progression. By contrast, during the S phase(s) that precede cell-cycle exit, pRB and HDACs remain affiliated with the perinucleolar foci throughout S phase, with redirection of chromatin through these central replication factories and the potential for histone modification during DNA synthesis [14]. Conversely, the premature distribution of the perinucleolar pRB and replication foci in immortalized cell lines might interfere with the normal regulation of chromatin. The restriction of pRB to the perinucleolar foci is most prominent during, but not limited to, cell-cycle exit and re-entry into G1 from G0 [13,30]. Recently, it has been shown that phosphorylation of pRB by cyclin C/cdk3 is responsible for the G0–G1-phase transition, which indicates that pRB can exist in distinct phosphorylated states in G0 and G1 [31]. Alternatively, the prominence of perinucleolar pRB foci during G0 might be related to the global changes in nuclear architecture that appear to accompany cell-cycle exit. One such example occurs during senescence, where heterochromatin is rearranged and is associated with stable silencing of E2F target genes, both of which depend on a functional p16/pRB pathway [32]. This phenomenon coincides with an increase in repressive histone methylation and recruitment of pRB to E2F-target promoters, and result in changes that are distinct from those in reversibly arrested cells. How this reorganization is regulated is yet to be established, but it might, in part, be related to restructuring of DNA replication before cell-cycle exit. Localization of p53 Commonly, p53 is regarded as the guardian of the genome because it is the main effector of the transcriptional response to genotoxic- and stress-induced checkpoints. The choice of cell fate such as cell-cycle arrest, senescence and apoptosis is dictated by the set of genes that p53 activates [33]. How p53 selects a cellular outcome remains an area of intense interest. It depends on several interdependent factors including (1) combinatorial posttranslational modifications of p53 that are conferred by www.sciencedirect.com

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upstream, stress-specific, signal transducers such as CHK2, (2) the assembly of the transcriptional-activation complexes at target promoters, and (3) the regulated association of p53 with its nuclear sites of activity, which include promoter elements and PML-NBs. The access of p53 to nuclear sites is regulated primarily by cytoplasmic–nuclear transport. In normal cells, p53 is maintained actively at low levels in the cytoplasm through proteasome-dependent degradation. In response to checkpoint signals, it is shuttled rapidly into the nucleus, where it remains until surveillance tasks have been completed (Figure 2) [34]. Nucleocytoplasmic shuttling of p53 depends on the presence of nuclear-export- and nuclear-import motifs (NES and NLS, respectively). Post-translational modifications of p53 including ubiquitination, phosphorylation and acetylation [35,36] can block access to the NES and NLS directly, alter p53 conformation and affect oligomerization status. The E3 ubiquitin ligase murine double minute 2 (MDM2) is a key negative regulator of the nuclear localization of p53. Ubiquitination by MDM2 leads to export of p53 from the nucleus and its subsequent degradation [35]. In tumors in which MDM2 is amplified, p53 is sequestered constitutively in the cytoplasm. By contrast, DNA damage in primary cells leads to the phosphorylation of the N-terminus of p53 by kinases including CHK2, ATM and ATR. When phosphorylated by these kinases, p53 might be prevented from binding to MDM2. Phosphorylation of p53 at Ser392 can also promote tetramerization, which leads to nuclear retention and increased activity. Access of MDM2 to p53 in the nucleus is controlled tightly by p14ARF. p14ARF, which shares a genomic locus with p16INK4a, encodes a tumor suppressor that functions, in part at least, to upregulate the activity of p53 (Figure 2) [37]. It has been proposed that p14ARF inhibits the association of MDM2 with p53 by one or more mechanisms. In some circumstances p14ARF can sequester MDM2 in the nucleolus, which physically separates MDM2 and its primary target [38,39]. However, under other conditions it is reported that p14ARF (1) does not require nucleolar residence to inactivate MDM2 [40,41], (2) can form a ternary complex with MDM2 and p53 in the nucleus that blocks the nuclear export of both proteins [42], and (3) can inhibit the activity of MDM2 ubiquitin ligase [43,44]. Indeed, one study concludes that retention of p14ARF in the nucleolus inhibits association with MDM2 in the nucleoplasm [45]. p53 and PML-NBs At the subnuclear level, p53 associates with promyelocytic leukemia-nuclear bodies (PML-NBs) [46]. The promyelocytic leukemia protein (PML) itself is a tumor suppressor because chromosomal rearrangement of the gene encoding PML is a causal factor of acute promyelocytic leukemia that is accompanied by failure to form PML-NBs. These bodies are implicated in a myriad functions, including gene transcription and the induction of apoptosis and senescence, and, in addition, are targets for responses to viral and chemical insults (Box 1) [46]. PML-NBs might perform a ‘depot’ role that coordinates interactions between proteins of the p53 pathway.

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Figure 2. Distribution of p53 in normal conditions (green arrows) and after either DNA damage or senescence signals (red arrows). At the subnuclear level, p53 colocalizes to PML-NBs where it is acetylated and/or phosphorylated by partner proteins. Modified, oligomerized forms of p53 have been localized to the promoters of genes that are involved in cell-cycle arrest and apoptosis. Nuclear export and degradation of p53 in the cytoplasm is controlled through ubiquitination, which is mediated by Mdm2 and blocked by phosphorylation of p53 on Ser15 by Chk2. Although the Mdm2 activity is limited by p14ARF, where this occurs in the nucleus is controversial, with evidence for (i) nucleoplasmic p14ARF causing Mdm2 ubiquitination and relieving nucleoplasmic p53, (ii) p14ARF escorting Mdm2 to the nucleolus or (iii) PML-NB-directed sequestration of Mdm2 to the nucleolus where p14ARF is known to reside. It is unknown whether p53 is in the tetrameric or monomeric form at PML-NBs (dashed lines). Dotted lines indicate that PML-NBs might be sites of transcription of p53-target genes.

Localization of p53 at PML-NBs enhances its acetylation by the cyclic-AMP-response-element-binding protein (CBP) complex, which is required to initiate either apoptosis in response to ionizing radiation or senescence in response to Ras [47,48]. Two DNA-damage-responsive p53 kinases, CHK2 and ATR, are linked to PML-NBs. Normally, ATR is present in nuclear bodies, but it relocalizes to sites of damage by ionizing radiation [49]. CHK2-mediated phosphorylation of p53 is linked to its ability to interact with PML, but this remains controversial [50,51]. How p53 accumulates at PML-NBs is unknown; it will be interesting to determine how this occurs in response to damage signals and whether modifications of PML-NBs confer specific p53-mediated transcriptional responses. Interestingly, pRB localizes to PML-NBs in immortalized www.sciencedirect.com

IB4 and U937 cells [52], which might be important in cell senescence because pRB foci in senescent primary cells overlap with PML-NBs [53]. Temporal and spatial regulation of the recruitment of p53 to specific target promoters might contribute to cellfate decisions in response to stress. Immediately after damage Espinosa and colleagues [54] have demonstrated the colocalization of transcription of the gene that encodes p21 with PML-NB-independent foci that contain p53 that is phosphorylated at Ser15 (p53-Ser15-P). Later, transcription of p21 is diminished, coincident with the redistribution of p53-Ser15-P throughout the nucleus and the transcription of ‘late-response’ p53-target genes. Such changes in the subnuclear distribution of modified p53 relative to different target promoters might coordinate temporal patterns of transcription.

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and the related kinase ATM following damage, provide further evidence that BRCA1 functions in a DNA-damage response at these sites [56]. Nonetheless, the identity of BRCA1 foci in untreated cells during S phase has remained obscure until recently. BRCA1 has been found to assist silencing of the inactive X chromosome by XIST RNA, a cis-acting transcriptional product of the X-chromosome-inactivation center that coats the inactive X chromosome [59]. In female somatic cells, including primary fibroblasts and telomeraseimmortalized mammary epithelial cells, one of the prominent BRCA1 nuclear foci colocalizes with the inactive X chromosome and RNA that encodes XIST. Moreover, loss of BRCA1 results in disruption of the association between XIST and the inactive chromosome, dispersal of focal staining of H3mK9 (a marker of heterochromatic regions) [60] and loss of asynchronous X-chromosome replication [59]. The authors suggest that other BRCA1 nuclear foci in S phase might target heterochromatic elements, and that BRCA1 might regulate the structure and function of heterochromatin, perhaps by assisting in the surveillance and repair of DNA damage at these sites. In support of this

BRCA1 nuclear foci Whereas pRB and p53 pathways are targeted for inactivation in nearly all somatic tumors, the BRCA1 tumorsuppressor gene was identified by virtue of its mutation in hereditary breast and ovarian cancer [55]. BRCA1 localizes to distinct intranuclear foci that appear in S phase, are larger and more widespread than perinucleolar pRB foci, and are disrupted in subsets of breastcancer and ovarian-cancer cells (Figure 3) [56]. Rad51 resides in these same nuclear foci during S phase in MCF7 cells, which indicates that BRCA1 is involved in the response to DNA damage and in DSBR [57]. In addition, both proteins overlap in meiosis during chromosome condensation along developing synaptonemal complexes that are undergoing homologous recombination, which further supports a role for BRCA1 in DSBR. Following DNA damage during S phase, BRCA1 foci redistribute and colocalize with Rad51 and the BRCA-interacting protein BARD1 at sites of DNA replication, which indicates that this complex might be involved in the repair of replicationassociated DNA damage [58]. Co-localization of BRCA1 with ATR, and phosphorylation of BRCA1 by both ATR

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Figure 3. BRCA1 foci coincide with altered chromatin structures. Within the undamaged nucleus, BRCA1 associates with microscopically visible structures called foci that contain chromatin and vary in their protein constituents. Under normal conditions, foci overlap with the inactive X chromosome in female somatic cells to assist in maintaining its silenced state, meiotic synaptonemal junctions in spermatocytes to assist in resolution of chromatin structures, chromatin modifiers in S-phase foci, possibly to assist in replication that is associated with heterochromatin, and ALT-associated PML-NBs, possibly for telomere processing. After DNA damage during S phase, BRCA1 localizes with components of the BRCA1-associated surveillance complex (BASC) at stalled replication forks during late S phase; these might be heterochromatic, which might assist in signaling and the resolution of this abnormal chromatin structure. Colocalization of BRCA1 with several chromatin-associated foci has been used to identify the functions of BRCA1. Dynamic association of BRCA1 with protein complexes that are associated with irregular chromatin structures in both undamaged and damaged nuclei indicate that appropriate spatial partitioning is an important aspect of its role as tumor suppressor. www.sciencedirect.com

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idea, BRCA1 interacts with several members of the histone deacetylase complex, such as HDAC1, HDAC2, RbAp46 and RbAp48, and with chromatin-remodeling factors such as BRG1 [61,62]. Further evidence that BRCA1 functions in heterochromatin maintenance in late S phase comes from studies that examine telomere replication in late S phase. Human cells that are undergoing alternative lengthening of telomeres (ALT) accumulate BRCA1 with the MRN complex (MRE11, Rad 50 and NBS1), telomere-binding proteins and replication factors at ALT-associated PML-NBs, which occur primarily in late S–G2 phase [63]. Moreover, it has been reported recently that, in yeast, an analogous MRE11-containing complex is recruited to telomeres during DNA replication in late S phase, which indicates that the normal terminus of a chromosome is recognized by DSBR during replication [64]. These results highlight the intriguing possibility that BRCA1 might play a role in regulating telomere replication. Concluding remarks Recent studies have begun to elucidate the importance of nuclear architecture in coordinating cell differentiation and tumorigenesis. In this review we have focused on three of the most studied nuclear tumor suppressors, pRB, p53 and BRCA1. Each is linked, in one or more ways, to either nuclear structures or proteins that are involved in nuclear organization. Although these links are not understood in detail, they provide a framework to understand the myriad protein–protein interactions and activities that are attributed to these three tumor suppressors. There are several outstanding questions regarding the links between nuclear organization and tumor-suppressor function. What are the activities of pRB at perinucleolar foci, and how does the organization of these structures by lamin A/C affect cell differentiation? How is rRNA synthesis linked to the control of cell proliferation? What role(s) does the nucleolus play as a coordination point for tumor suppressors such as pRB and p14ARF? Does p53-mediated transactivation occur directly at PML-NBs? What is the role of BRCA1 in the formation and maintenance of heterochromatin? Cell-biological approaches directed at pRB, p53, BRCA1 and other nuclear tumor suppressors have already provided a new framework to think about the functions of these crucial regulatory proteins, and continued investigation will provide the answers to many of these outstanding, important questions. Acknowledgements We apologize to authors whose work is not cited here because of space constraints. We thank Brian Kudlow for comments on the manuscript. Research in the authors’ laboratory is supported grant R01AG024287 (BKK). DAB has been supported by a Howard Hughes Medical Research Training Fellowship. BKK is a Searle Scholar.

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