Perspective

PODs in the Nuclear Spot: Enigmas in the Magician's Pot

Science's STKE  21 Aug 2001:
Vol. 2001, Issue 96, pp. pe1
DOI: 10.1126/stke.2001.96.pe1

Abstract

The promyelocytic leukemia (PML) nuclear body, also known as the PML oncogenic domain (POD), is implicated in the pathophysiology of PML. These nuclear subcompartments are dynamic structures. The PML protein, which undergoes a fusion event in patients with promyelocytic leukemia, is normally found in PODs. The PML protein may be a major regulator of the constituents of PODs, controlling POD organization and function. Hatta and Fukamizu describe the functions of PML and discuss how the POD structure and organization may be regulated and affect apoptosis, gene expression, and cellular transformation.

Nuclear Subcompartments

This perspective focuses on the subnuclear structures called promyelocytic leukemia protein (PML)-containing nuclear bodies, also known as PML oncogenic domains (PODs), nuclear dots (ND10), or Kr bodies. The eukaryotic nucleus is organized into distinct domains. In interphase cells, chromatin of varying degrees of condensation occupies discrete spaces in the nucleus called chromatin territories (1). The interchromatin space, sometimes called the nuclear matrix, contains the nucleolus (the site of RNA synthesis) and many other morphologically distinct subnuclear structures and bodies (2, 3). Nuclear bodies and substructures serve as the sites of many critical metabolic activities, including transcription, DNA replication, pre-mRNA processing, ribonucleoprotein (RNP) assembly, protein modification, DNA condensation and fragmentation associated with apoptosis, sites for cell cycle control, and ribosome production. The precise roles for each type of nuclear body in these biological processes remain to be established; however, remarkable progress has recently been made in understanding the individual constituents of nuclear bodies and their potential importance for cellular functions (4-7).

The POD-Cancer Connection

PODs are 0.3- to 0.5-μm spherical structures in which the PML protein surrounds an electron-dense core associated with the nuclear matrix. They are dynamic structures that form between 10 to 20 distinct bodies distributed throughout the nucleus in most cell types (6, 7). The PML gene was originally identified because of its involvement in acute promyelocytic leukemia (APL). In APL patients, there is a reciprocal chromosomal translocation event t(15;17) of the PML gene, which results in fusion to the retinoic acid receptor α gene (RARα) (8, 9). In cells from APL patients, the PML-RARα fusion protein physically interacts with wild-type PML expressed from the nontranslocated locus, disrupts PODs in a dominant negative manner, and induces the delocalization of POD components, including wild-type PML. The integrity of PODs is lost in APL cells, in which the nuclear bodies are dispersed into a microspeckle pattern. This disorganization of the nuclear body structure is thought to be relevant to the pathogenesis of APL. Re-formation of the nuclear bodies occurs upon treatment with retinoic acid, which induces terminal differentiation, or arsenic trioxide (As2O3), which triggers apoptosis in APL cells (10). Based on the involvement of PML in APL, PML and PODs are proposed to have a role in control of cell growth, and PML may be a tumor suppressor.

Additional data support the hypothesis that PML and PODs are involved in tumorigenesis. For example, the number of PODs is regulated during progression of the cell cycle, and the highest number of PODs is found during G1 phase (11, 12). Furthermore, the potential role of PML in controlling cell division and suppression of oncogenic transformation of cells is supported by findings from mice with a targeted mutation of the PML gene (13). Fibroblasts derived from PML–/– mice lack PODs, exhibit an increased proportion of cells in S phase, and display a marked growth advantage compared to wild-type cells (13). PML–/– mice are viable, but more susceptible to tumorigenesis and bacterial infections. The PML–/– mice and the PML–/– fibroblasts are also less sensitive to lethal doses of γ irradiation or treatment with Fas antibody, both of which can induce apoptosis. This suggests that PML may be a proapoptotic protein (14). These observations imply that PML is a critical component of PODs involved in growth suppression and apoptosis, possibly because of its ability to recruit an apoptotic factor to PODs.

One model suggests that recruitment of the tumor suppressor p53 to PODs by PML could contribute to apoptosis and cell cycle regulation. The role of p53 in the induction of apoptosis and growth suppression is well established (15). Interactions between p53 and PML influence cell cycle arrest, apoptosis, and senescence induced by the guanosine triphosphatase Ras. Lack of PML dramatically impairs p53-dependent Ras-induced senescence, and the requirement for PML in this type of senescence is closely related to the formation of nuclear bodies and the recruitment of p53 to PODs (16, 17). POD-associated p53 was differentially modified by acetylation and phosphorylation compared to non-POD-associated p53, suggesting that p53 protein modifications may be important for suppression of Ras-dependent transformation. Adenosine 3′-5′ monophosphate (cAMP)-responsive element binding protein (CREB)-binding protein (CBP), a transcriptional coactivator, is a potential candidate for the acetyltransferase that acetylates p53, because CBP is recruited to PODs through interactions with PML (4, 17-20). How PML regulates phosphorylation of p53 remains to be resolved. PML is unlikely to be a kinase itself, but may be required to recruit protein kinases, which phosphorylate p53, to PODs.

PODs and Transcription

An emerging concept is that a functional relationship exists between nuclear compartments and gene expression (21). The nucleolus is the site of high transcriptional activity of rRNA genes. The Cajal body is frequently associated with specific histone and small nuclear RNA gene loci (3). PODs also represent active sites of transcriptional regulation, based on the inclusion of nascent RNA polymerase II transcripts, as well as CBP, in this nuclear body (22). PML may serve to recruit various transcriptional regulators to these structures and, depending on its interacting partners, activate or repress gene expression (Fig. 1).

Fig. 1.

Schematic illustration of nuclear bodies and their representative components. A number of proteins are recruited to nuclear bodies, such as PODs, the Cajal body, the nucleolus, and HDAC-enriched foci. In addition to the nuclear factors discussed in the text, retinoblastoma protein (Rb) (38), and β-catenin (39) also accumulate in PODs. The heterogeneity of constituents in PODs may influence their number, size, and shape, and thus affect local nuclear functions. A, acetylation; P, phosphorylation; S, SUMOylation; SMRT, silencing-mediator for retinoid/thyroid hormone receptors; MTA2, metastasis-associated gene protein; MBD3, methyl-CpG-binding domain-containing protein.

Several transcriptional regulators can be found in PODs. Their localization in this structure may have a positive or negative regulatory effect depending on the transcriptional regulator. PML promotes CBP localization to PODs and enhances the transcriptional activity of nuclear receptors, such as glucocorticoid, retinoic acid, and retinoid-X-receptors (4). The Sp100 family of proteins includes Sp100, Sp140, and the recently cloned Sp110 protein (23). These proteins were originally identified in patients with primary biliary cirrhosis. Sp110, like PML, functions as a nuclear hormone coactivator (23), and Sp100 behaves as a transcriptional repressor (24). The ability of Sp110 to activate nuclear hormone receptors is possibly mediated by an LXXLL nuclear receptor-binding motif present in Sp110. This motif is absent in both Sp100 and Sp140. However, Sp140 may recruit Sp110 and Sp100 to PODs (23). Certain transcriptional regulators are inactive when associated with the POD. For example, PML inhibited Daxx (a transcriptional repressor)-mediated suppression of transcription by sequestering Daxx at PODs (25).

PML may also act directly as a transcriptional repressor. When fused to the Gal4 DNA binding domain, PML acts as a transcriptional suppressor of the Gal4-directed promoter (26). PML also suppresses the promoter activity of epidermal growth factor receptor (EGFR) by inhibiting its Sp1-dependent activity (27). The repressive action of PML (28) may relate to its functional and physical interaction with histone deacetylases (HDACs), a family of chromatin remodeling factors that achieve transcriptional silencing of the target promoter by deacetylation of histones (29). However, HDACs failed to overlap with PODs and localized to a unique nuclear domain (30). On the other hand, when NB4 cells (a human acute promyelocytic cell line) were treated with retinoic acids, PML colocalized with c-Ski or N-CoR, which are components of HDAC complex that are required for transcriptional repression (31). Therefore, the colocalization of PML and co-repressor factors in the nuclear bodies might be dependent upon cell types or cellular physiological conditions.

POD Dynamics

PODs are dynamic structures in composition, number, and size. Alterations in the protein constituents of PODs correlate with changes in the modification of PML by the small ubiquitin-like modifier (SUMO-1) protein (32). SUMO-1-conjugated PML is exclusively localized to PODs. The physiological importance of SUMOylation of PML is illustrated by the fact that SUMO-1-modified PML recruits both Daxx and p53 to PODs, which enhances Fas-dependent cell death (33) and stimulates the proapoptotic activity of p53 (34).

Conjugation of ubiquitin to proteins is a well-established modification for tagging proteins for degradation by the 26S proteasome (35). PODs may represent an intermediate reservoir for ubiquitinated proteins targeted for degradation (36). Treatment of cells with a proteasome inhibitor resulted in a dramatic redistribution and movement of POD-associated proteins, such as PML, Sp100, and SUMO-1, to the nucleolus (37). In contrast, several non-POD-associated proteins, such as p21, p37, and cyclin D3, did not change their distribution. The observed movement of POD-associated proteins to the nucleolus may reflect a normal trafficking of these proteins that is not readily detected until proteasome-dependent protein degradation is blocked.

It is becoming apparent that PODs are dynamic nuclear structures whose constituents can assemble to form PODs, or disassemble and disperse or redistribute the components to other nuclear structures. Stochastic associations of factors to a nuclear compartment are usually interpreted as defining a stable, persistent underlying nuclear architecture. However, it appears that particular proteins are likely to be recruited to PODs dynamically in response to a variety of physiological alterations, such as the cell cycle, disease state, viral infection, or external stimuli, through signaling cascades mediated by membrane-bound and nuclear receptors. The dynamic nature of PODs suggests that they may serve as a flexible protein-based scaffold to control protein interactions. The components of PODs can be stable or dynamic: PML and Sp100 are consistently found in PODs, but CBP is recruited to a subset of PODs only under certain conditions, such as exposure of cells to α-interferon (20). The heterogeneity of the constituents found in PODs may change the number, size, and shape of PODs and affect the local nuclear functions occurring at PODs. How members of the heterogeneous POD population communicate and how the PODs interact with other types of nuclear bodies to lead to integrated and organized nuclear activity remain to be determined.

Disruption of PML function and loss of the integrity of the POD structure are implicated in the oncogenic transformation that accompanies APL. Knowledge of the nuclear network and the components and functions of PODs may facilitate the development of potent and specific modular compounds that can be used therapeutically for inducing the reformation of PODs in these transformed cells. Investigations aimed at increasing our understanding of the underlying molecular pathophysiology of cellular transformation as it relates to alterations in nuclear structure and function may provide additional venues for improving the treatment or prevention of disease.

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