- Email: [email protected]
Linking lipids to chromatin
Linking lipids to chromatin David R Jones and Nullin Divecha Dynamic regulation of chromatin structure is thought to be a prerequisite for nuclear functions that require accessibility to DNA such as replication, transcription and DNA repair. The phosphoinositide (PI) pathway is a second messenger signalling system regulated in response to a variety of extracellular (growth factors, differentiation signals) and intracellular (cell cycle progression, DNA damage) stimuli. The presence of a PI pathway in the nucleus together with the recent findings that specific nuclear proteins can interact with and are regulated by phosphoinositides suggest that changes in the nuclear phosphoinositide profile may have a direct role in modulating chromatin structure. Addresses Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands e-mail: [email protected]
Current Opinion in Genetics & Development 2004, 14:196–202 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by Stephen D Bell and Andy Bannister 0959-437X/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2004.02.003
Abbreviations ACF ATP-dependent chromatin-assembly factor ING2 inhibitor of growth protein 2 NHEJ non-homologous end joining NURF nucleosome remodelling factor PHD plant homedomain PI phosphoinositide
Introduction In the nucleus, the genome is packaged into a nucleoprotein structure called chromatin. The fundamental unit of chromatin, the nucleosome, is a repeating motif consisting of two turns of DNA wrapped around a histone octamer. Chromatin is a highly ordered repressive structure that limits nuclear processes requiring access to the DNA sequence including RNA transcription, DNA replication and DNA repair and it is widely held that disruption or remodelling of chromatin is a rate-determining step in these processes [1]. Remodelling of chromatin is the consequence of two distinct mechanisms. The first leads to covalent modification of nucleosomal histones [2], including phosphorylation, acetylation, methylation and ubiquitination, which can themselves effect chromatin structure or recruit proteins that do this. The second utilises the energy of ATP hydrolysis to alter chromatin Current Opinion in Genetics & Development 2004, 14:196–202
structure [1]. It should be noted that the two processes are by no means mutually exclusive as the first has been shown to lead to recruitment of factors, which carry out the second [3]. Phosphoinositides, which form a minor family of phospholipids, play a key role as intracellular second messengers [4]. The action of phospholipases, lipid kinases and lipid phosphatases in response to extracellular signals leads to remodelling of the phosphoinositide profile (Figure 1), which in turn regulates downstream targets to control diverse intracellular processes including vesicle trafficking [5], cell proliferation and survival [6], gene transcription and pathogen elimination [7]. Phosphoinositides are known to transduce signals essentially via two events: their modification and their interactions with specific proteins. Protein domains that interact with phosphoinositides include PH [8], ENTH [9], FYVE [10], PHOX [11] domains and lysine/arginine rich patches [12]. In addition to phosphoinositide signalling at the plasma membrane, biochemical studies have demonstrated the presence of the lipid kinases DGK, PtdIns-4K and PIPkinase in nuclear membranes [13–15] and within the nuclear matrix [16,17]. Localisation of phosphoinositides (particularly PtdIns[4,5]P2) using both antibodies [18,19] and the GST-tagged PH domain of PI-specific PLC-d1 [20] (a sensor for PtdIns[4,5]P2) indicate their presence in the nuclear membrane, interchromatin granules, heterochromatin, nucleolar-associated heterochromatin and sites of pre-mRNA processing [21]. Furthermore, exogenously added fluorescently-labelled PtdIns(4,5)P2 is found at the plasma membrane and as speckle staining within nuclei reminiscent of that described above [22]. In addition to phosphoinositides, phosphatidylcholine has also been shown to be present in the nuclear matrix [23]. The presence of both nuclear phosphoinositides and the enzymes responsible for their metabolism suggest that they may serve as a signalling system. Nuclear phosphoinositides are modulated in response to short-term growth factor signalling, cell-cycle progression and during differentiation. Perhaps central to the regulation of nuclear phosphoinositide signalling is PI–PLC. PI–PLC catalyses the hydrolysis of PtdIns(4,5)P2 to generate two very important second messengers: Ins(1,4,5)P3, which can be further phosphorylated to Ins(1,2,3,4,5,6)P6 (InsP6) and DAG. PtdIns–PLC regulation is also one mechanism by which the level of nuclear PtdIns(4,5)P2 and PtdIns(4)P may be controlled. Numerous isoforms of PI–PLC have been identified in the nucleus but the b1 isoform is the www.sciencedirect.com
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Figure 1
PtdIns(3,5)P2 PIKfyve PtdIns(3,4)P2 PtdIns(3)P Type II PIPkinase ? PI3K
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A map of phosphoinositide metabolism. The enzymes are indicated in blue.
best characterised. Upon short term stimulation with IGF-1, nuclear PI–PLCb1 is activated by phosphorylation by p42/p44 mitogen-activated protein kinase [24] and subsequently negatively regulated by the DAG-dependent bII isoform of protein kinase C [25]. Although upregulation of nuclear PI–PLCbI activity correlates with increased progression through the cell cycle, differentiation of murine erythroleukaemia (MEL) cells, induced by DMSO, causes downregulation of nuclear PI–PLCb1, an accumulation of nuclear polyphosphoinositides and a decrease in nuclear DAG [14,26,27]. Moreover, overexpression of PI–PLCb1 in the nucleus is sufficient to drive 3T3 cells into S-phase, whereas in MEL cells nuclear PI– PLCb1 expression can block differentiation [28]. In addition, activation of the enzymes that synthesise PtdInsP and PtdIns(4,5)P2 occurs as cells progress through G1 into S-phase [29]. Thus in response to a variety of signals activation/repression of PI-PLC, PIPkinase and phosphatase activities (Figure 1) will generate different patterns of PtdInsPs, PtdIns(4,5)P2 and inositol phosphates within the nucleus.
Chromatin remodelling and phosphoinositides Although phosphoinositides have been shown to be present in the nucleus and can change in response to external stimuli, there is a paucity of mechanistic data on how these changes may be transduced into functional regulation of nuclear processes. Previous data showed www.sciencedirect.com
that addition of phospholipids to purified nuclei could effect in vitro transcription and replication of DNA [30]. Indeed in vitro negatively charged lipids lead to chromatin decondensation, whereas positively charged lipids have the opposite effect [31]. PtdIns(4,5)P2 can bind to the C-terminal tail of histones H1 and H3 and in an in vitro assay counteracts histone H1-mediated repression of basal transcription [32]. A novel mechanism for the regulation of chromatin structure by phosphoinositides came with the unexpected discovery that the interaction of a chromatin-remodelling complex, BAF, can be regulated by the level of PtdIns(4,5)P2 [33]. Resting T cells have small, compact nuclei with dense heterochromatin, which upon antigenic activation increase in size with the appearance of euchromatin. These changes in chromatin are thought to be required for the subsequent activation of T-cell-specific genetic programmes. Upon T cell stimulation with anti-CD3 (an antibody against the T cell receptor) or with ionomycin/PMA, the BAF complex translocates from a nuclear soluble fraction to an insoluble fraction. This translocation can be mimicked by incubation of non-activated T cell nuclei with exogenously added PtdIns(4,5)P2 [33] Although the data are consistent with a role for PtdIns(4,5)P2 in regulating BAF complex localisation, there is no data to demonstrate that T cell activation leads to an increase in nuclear PtdIns(4,5)P2. Central to a chromatin-remodelling complex is an ATPase subunit related to the yeast SWI/SNF2 protein, Current Opinion in Genetics & Development 2004, 14:196–202
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which is a member of the DEAD/H superfamily of DNA and RNA helicases [34] and is the key to their various remodelling activities such as nucleosomal assembly, sliding and positioning. Among the other numerous subunits, most if not all mammalian chromatin remodelling complexes also contain actin or actin-related proteins, which modulate remodelling activity both in vitro and in vivo [35]. BRG-1, the ATPase subunit of the BAF complex, has two domains that can interact with actin, one of which contains a lysine-rich patch. This lysine-rich patch is required for BRG-1 function in vivo [36] and importantly can interact with PtdIns(4,5)P2 [37]. The authors suggest that the interaction of BRG-1 with actin is disrupted by PtdIns(4,5)P2 and consequently exposes a site on actin for interaction with components of the nuclear matrix. This is analogous to PtdIns(4,5)P2mediated uncapping of actin, which stimulates actin polymerisation [12]. Recent studies have demonstrated that upon transcriptional activation BRG-1 is recruited to the active promoter before other components of the BAF complex suggesting that a pre-associated BAF complex may not be recruited to active promoters [38]. Perhaps PtdIns(4,5)P2, present on histone H1/H3, or locally synthesised, may allosterically stimulate interaction of BRG-1 with other BAF complex components and/or lead to stabilisation of the BAF complex at the promoter by increasing its interaction with the nuclear matrix. How might localised synthesis of PtdIns(4,5)P2 be regulated? The retinoblastoma protein, which recruits the BAF complex to regulate gene expression, interacts with and activates type I PIPkinases [39], the enzymes responsible for the generation of nuclear PtdIns(4,5)P2. We suggest that transcriptional regulators and/or chromatin remodelling complexes may interact with and recruit type I PIPkinase to control localised synthesis of PtdIns(4,5)P2.
The problem with phosphoinositides and a solution in yeast Phosphoinositides have a hydrophilic head group coupled to two extremely hydrophobic fatty acyl chains. They are thus particularly suited to partitioning into membranes but are unlikely to move freely through the nucleus. Where they are generated is where they act! This poses a problem regarding PtdIns(4,5)P2 accessibility to chromatin-remodelling factors. Yeast have enzymes to generate PtdIns(4,5)P2 within the nucleus and have a nuclear PtdIns–PLC, which can generate Ins(1,4,5)P3. This Ins(1,4,5)P3 is rapidly phosphorylated to InsP6 via the action of two inositol polyphosphate kinases. Ipk2 phosphorylates Ins(1,4,5)P3 on the 6 and the 3 positions to generate inositol (1,3,4,5,6)pentakisphosphate (InsP5), which is then the substrate for Ipk1 to generate InsP6 [40]. Interestingly Ipk2 turns out to be a known transcription factor [41], ARG82, involved in responses to changes in nutrients and stress, sporulation and in mating. Steger et al. identified a requirement for ARG82 in the regulation Current Opinion in Genetics & Development 2004, 14:196–202
of PHO5 transcription in response to changes in the levels of phosphate [42]. Transcriptional activation of PHO5 requires the remodelling of one nucleosome, which is dependent on the SWI/SNF (yeast homologue of the BAF complex) and the INO80 chromatin-remodelling complexes. Yeast strains deficient in ARG82 (or importantly its inositol phosphate kinase activity) or PtdIns– PLC are unable to remodel this nucleosome, suggesting that the generation of highly phosphorylated inositol phosphates are important for chromatin remodelling at the PHO5 promoter. Using mutations in other inositol phosphate kinases, the authors conclude that the generation of InsP4/InsP5 is/are the important molecule(s), which likely act(s) through direct regulation of the remodelling complexes. In a complementary study, the activity of several different chromatin-remodelling complexes (Nurf [in Drosophila], ISW2 [in yeast], SWI/SNF and INO80) were found to be sensitive to inositol phosphates in vitro [43]. Whereas Nurf, ISW2 and INO80 activities were inhibited by high concentration of IP6, SWI/SNF activity was increased by the presence of InsP4/InsP5. This latter observation is in accord with data for an in vivo role of inositol phosphates on SWI/SNF remodelling activity. How do inositol phosphates regulate remodelling activities? InsP6 can bind to the NURF and ISW2 complexes and lead to a reduction of nucleosome-stimulated ATPase activity [43]. However, InsP4/InsP5-mediated activation of the SWI/SNF complex does not lead to enhanced ATPase activity. Moreover in vivo, both the INO80 and SWI/SNF complexes are not efficiently recruited to their promoters in the absence of higher phosphorylated inositol phosphates [42]. These data suggest that InsP4/InsP5 may regulate the interaction of SWI/SNF with chromatin. Although the above data are consistent with a role for inositol phosphates in the regulation of chromatin-remodelling complexes, it should be noted that activation of the SWI/SNF complex by InsP4/InsP5 was shown using a concentration of 500 mM, whereas estimates in yeast suggest that the level of InsP4/InsP5 is likely to be between 1–5 mM. Compartmentalisation of InsP4/InsP5 in the nucleus may represent a mechanism for increasing the local concentration. Alternatively, regulatory changes in interacting proteins or post-translational modification may increase the affinity of the complex for inositol phosphates. This change in affinity would alleviate a requirement for changes in the concentrations of InsP4/InsP5 to act as a signal to activate remodelling. It should also be noted that yeast lacking ARG82, or any of the genes involved in the synthesis of higher inositol phosphates, have profound defects in the function of the endocytic pathway, vacuole biogenesis and in mRNA transport [44]. These may lead to an impairment of signalling pathways involved in transducing changes in extracellular levels of phosphate to changes in gene expression rather than a direct effect on chromatin-remodelling complexes. www.sciencedirect.com
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Are inositol phosphates implicated in mammalian chromatin remodelling? A mammalian homologue of ARG82, IPMK, has been cloned and does show similar inositol phosphate kinase activity [45]. Interestingly IMPK appears to be localised within the nucleus suggesting a role in nuclear functions. To date, no data has demonstrated a role for inositol phosphates or IMPK in the regulation of mammalian chromatin remodelling complexes or in gene transcription. A role for inositol phosphates in DNA repair in mammalian cells has been suggested, as InsP6 is a potent co-activator of non-homologous end joining (NHEJ) when assessed in vitro [46]. Further studies have shown that InsP6 can interact with the Ku protein, although it is not clear if this interaction is required for the stimulation of end joining activity [47]. How InsP6 modulates Ku function is not clear. InsP6 does not appear to regulate Ku interaction with either DNA or with DNA-dependent protein kinase. Interestingly, a role for chromatin-remodelling complexes in nucleotide excision repair has been demonstrated [48,49].
Pointing the finger at PtdInsPs
The plant homedomain (PHD) finger is a Cys4–HisCys3 zinc finger [54,55] present in 66 different human proteins many of which are nuclear. Several PHD-encoding genes have been implicated in human diseases and many of the characterised mutations in these genes lead to a loss of function of the PHD domain [56–59]. Recently, the PHD finger of inhibitor of growth protein 2 (ING2) was found to interact with phosphomonoinositides and particularly with PtdIns(5)P [60]. ING2 is a potential tumour suppressor and can induce growth arrest and apoptosis in a p53-dependent manner. Gozani et al. [60] showed that ING2 function in vivo (induction of apoptosis and p53 acetylation) was compromised by point mutations that resulted in loss of PtdIns(5)P binding. Moreover, overexpression of a type II PIPkinase (Figure 1), assumed to phosphorylate and remove nuclear PtdIns(5)P, attenuated ING2 function. Proteins containing PHD fingers that modulate chromatin structure include p300 (histone acetylase), Mi2, ATP-dependent chromatin-assembly factor (ACF) and TIP5 (all chromatin-remodelling complex proteins), the ING family (interact with acetylase and deacetylase complexes), polycomb proteins (silenwww.sciencedirect.com
Do all PHD fingers interact with phosphoinositides? Gozani et al. [60] showed that other PHD-motif-containing proteins including ACF also bind to phosphoinositides in vitro. In addition, we have screened >40 different murine PHD motifs and found 10 that bind phosphoinositides. We also found that of two different PHD domains from the same protein, only one interacted with phosphoinositides, suggesting that it is likely that different PHD fingers have different functions. Furthermore we find that different PHD domains appear to be able to interact with different subsets of phosphomonoinositides. We expect that diverse and contextual modulation of PtdIns(3)P, PtdIns(4)P and PtdIns(5)P within the nucleus will differentially regulate PHD-containing protein function.
Figure 2
Signal input
PtdInsP2
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PtdInsPs are often thought of as just intermediates for the synthesis of PtdIns(4,5)P2 via their phosphorylation by either type I or type II PIPkinases (Figure 1) [50,51]. However, PtdIns(4)P and PtdIns(5)P mass accumulates as cells progress from G1 into S-phase [29] and in response to a variety of cellular stresses (UV, etoposide and oxidative damage; DR Jones, N Divecha, unpublished). In addition, nuclear PtdIns(3)P accumulates during G2/M phase in HL60 cells and in response to long-term treatment with all-trans-retinoic acid [52,53]. One mechanism for how changes in nuclear PtdInsPs may be transduced into a functional output has recently been elucidated.
cing genes) trithorax (gene activation) and the CpG binding protein (involved in regulating methylation of DNA). In the case of p300, the PHD finger has been shown to be essential for acetylation activity. Interestingly the ING family, of which ING1, 2 and 3 interact with phosphoinositides (DR Jones, N Divecha, unpublished), is intimately involved in the regulation of both histone and non-histone protein acetylation [61].
Nuclear phosphoinositide-binding proteins
Differentiation
Proliferation
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A variety of signal inputs lead to temporal remodelling of the inositide profile in the nuclear envelope and/or in the nuclear matrix (the size of the oval reflects the size of the inositide pool). The combinatorial changes in nuclear phosphoinositides are decoded through their interaction with specific phosphoinositide-binding proteins. This decoding leads to differential outputs, coordinating processes such as differentiation, proliferation or apoptosis. For example signal inputs that increase the level of nuclear PtdIns(5)P within the chromatin-enriched fraction lead to the translocation of ING2 that, in turn, may regulates p53 function and histone acetylation to coordinate an apoptotic response. Current Opinion in Genetics & Development 2004, 14:196–202
200 Chromosomes and expression mechanisms
Conclusions The identification of nuclear-specific phosphoinositide binding domains establishes potential novel functions for phosphoinositides within the nucleus. Signals that induce processes such as differentiation, cell proliferation and stress adaptation/apoptosis lead to a remodelling of nuclear inositide profiles (Figure 2). We suggest that the combinatorial spatial and temporal changes in nuclear PtdInsPs, PtdIns(4,5)P2 and inositol phosphates are decoded by specific phosphoinositide-binding proteins to elicit differential outputs. These may include changes in chromatin structure to regulate gene expression, replication or DNA repair. Furthermore, the presence of a dynamic pool of nuclear envelope or matrix phosphoinositides, which respond to external cues together with chromatin-associated proteins that can interact with phosphoinositides, would provide an ideal mechanism for controlling gene positioning shown to be important in regulating gene expression [62,63].
Acknowledgements
12. Yin HL, Janmey PA: Phosphoinositide regulation of the actin cytoskeleton. Annu Rev Physiol 2003, 65:761-789. 13. Smith CD, Wells WW: Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation. J Biol Chem 1983, 258:9368-9373. 14. Cocco L, Gilmour RS, Ognibene A, Letcher AJ, Manzoli FA, Irvine RF: Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation. Biochem J 1987, 248:765-770. 15. Capitani S, Bertagnolo V, Mazzoni M, Santi P, Previati M, Antonucci A, Manzoli FA: Lipid phosphorylation in isolated rat liver nuclei. Synthesis of polyphosphoinositides at subnuclear level. FEBS Lett 1989, 254:194-198. 16. Payrastre B, Nievers M, Boonstra J, Breton M, Verkleij AJ, Van Bergen en Henegouwen PM: A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix. J Biol Chem 1992, 267:5078-5084. 17. Vann LR, Wooding FB, Irvine RF, Divecha N: Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei. Biochem J 1997, 327:569-576. 18. Osborne SL, Thomas CL, Gschmeissner S, Schiavo G: Nuclear PtdIns(4,5)P2 assembles in a mitotically regulated particle involved in pre-mRNA splicing. J Cell Sci 2001, 114:2501-2511.
We apologise to those whose work was not cited for lack of space. We thank all the members of the Inositide Laboratory and Maria Carla Motta for helpful discussions. Work in the laboratory has been supported by grants from the Dutch Cancer Society and the European Commission.
19. Yokogawa T, Nagata S, Nishio Y, Tsutsumi T, Ihara S, Shirai R, Morita K, Umeda M, Shirai Y, Saitoh N, Fukui Y: Evidence that 3(-phosphorylated polyphosphoinositides are generated at the nuclear surface: use of immunostaining technique with monoclonal antibodies specific for PI 3,4-P(2). FEBS Lett 2000, 473:222-226.
References and recommended reading
20. Watt SA, Kular G, Fleming IN, Downes CP, Lucocq JM: Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1. Biochem J 2002, 363:657-666.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Peterson CL: Chromatin remodeling: nucleosomes bulging at the seams. Curr Biol 2002, 12:R245-R247.
2.
Turner BM: Cellular memory and the histone code. Cell 2002, 111:285-291.
3.
Agalioti T, Chen G, Thanos D: Deciphering the transcriptional histone acetylation code for a human gene. Cell 2002, 111:381-392.
21. Boronenkov IV, Loijens JC, Umeda M, Anderson RA: Phosphoinositide signaling pathways in nuclei are associated with nuclear speckles containing pre-mRNA processing factors. Mol Biol Cell 1998, 9:3547-3560. 22. Ozaki S, DeWald DB, Shope JC, Chen J, Prestwich GD: Intracellular delivery of phosphoinositides and inositol phosphates using polyamine carriers. Proc Natl Acad Sci USA 2000, 97:11286-11291. 23. Hunt AN, Clark GT, Attard GS, Postle AD: Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes. J Biol Chem 2001, 276:8492-8499.
4.
Payrastre B, Missy K, Giuriato S, Bodin S, Plantavid M, Gratacap M: Phosphoinositides: key players in cell signalling, in time and space. Cell Signal 2001, 13:377-387.
5.
De Camilli P, Emr SD, McPherson PS, Novick P: Phosphoinositides as regulators in membrane traffic. Science 1996, 271:1533-1539.
6.
Datta SR, Brunet A, Greenberg ME: Cellular survival: a play in three Akts. Genes Dev 1999, 13:2905-2927.
7.
Ellson CD, Andrews S, Stephens LR, Hawkins PT: The PX domain: a new phosphoinositide-binding module. J Cell Sci 2002, 115:1099-1105.
25. Xu A, Wang Y, Xu LY, Gilmour RS: Protein kinase C alpha -mediated negative feedback regulation is responsible for the termination of insulin-like growth factor I-induced activation of nuclear phospholipase C beta1 in Swiss 3T3 cells. J Biol Chem 2001, 276:14980-14986.
8.
Dowler S, Currie RA, Campbell DG, Deak M, Kular G, Downes CP, Alessi DR: Identification of pleckstrin-homology-domaincontaining proteins with novel phosphoinositide-binding specificities. Biochem J 2000, 351:19-31.
26. Divecha N, Letcher AJ, Banfic HH, Rhee SG, Irvine RF: Changes in the components of a nuclear inositide cycle during differentiation in murine erythroleukaemia cells. Biochem J 1995, 312:63-67.
9.
De Camilli P, Chen H, Hyman J, Panepucci E, Bateman A, Brunger AT: The ENTH domain. FEBS Lett 2002, 513:11-18.
27. Martelli AM, Cataldi A, Manzoli L, Billi AM, Rubbini S, Gilmour RS, Cocco L: Inositides in nuclei of Friend cells: changes of polyphosphoinositide and diacylglycerol levels accompany cell differentiation. Cell Signal 1995, 7:53-56.
10. Gaullier JM, Simonsen A, D’Arrigo A, Bremnes B, Stenmark H, Aasland R: FYVE fingers bind PtdIns(3)P. Nature 1998, 394:432-433. 11. Xu Y, Seet LF, Hanson B, Hong W: The Phox homology (PX) domain, a new player in phosphoinositide signalling. Biochem J 2001, 360:513-530. Current Opinion in Genetics & Development 2004, 14:196–202
24. Xu A, Suh PG, Marmy-Conus N, Pearson RB, Seok OY, Cocco L, Gilmour RS: Phosphorylation of nuclear phospholipase C beta1 by extracellular signal-regulated kinase mediates the mitogenic action of insulin-like growth factor I. Mol Cell Biol 2001, 21:2981-2990.
28. Faenza I, Matteucci A, Bavelloni A, Marmiroli S, Martelli AM, Gilmour RS, Suh PG, Manzoli L, Cocco L: Nuclear PLCbeta(1) acts as a negative regulator of p45/NF-E2 expression levels in Friend erythroleukemia cells. Biochim Biophys Acta 2002, 1589:305-310. www.sciencedirect.com
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29. Clarke JH, Letcher AJ, D’Santos CS, Halstead JR, Irvine RF, Divecha N: Inositol lipids are regulated during cell cycle progression in the nuclei of murine erythroleukaemia cells. Biochem J 2001, 357:905-910. 30. Capitani S, Cocco L, Maraldi NM, Papa S, Manzoli FA: Effect of phospholipids on transcription and ribonucleoprotein processing in isolated nuclei. Adv Enzyme Regul 1986, 25:425-438. 31. Kuvichkin VV: DNA-lipid interactions in vitro and in vivo. Bioelectrochemistry 2002, 58:3-12. 32. Yu H, Fukami K, Watanabe Y, Ozaki C, Takenawa T: Phosphatidylinositol 4,5-bisphosphate reverses the inhibition of RNA transcription caused by histone H1. Eur J Biochem 1998, 251:281-287. 33. Zhao K, Wang W, Rando OJ, Xue Y, Swiderek K, Kuo A, Crabtree GR: Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling. Cell 1998, 95:625-636. 34. Eisen JA, Sweder KS, Hanawalt PC: Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions. Nucleic Acids Res 1995, 23:2715-2723. 35. Shen X, Ranallo R, Choi E, Wu C: Involvement of actin-related proteins in ATP-dependent chromatin remodeling. Mol Cell 2003, 12:147-155. The authors use yeast genetics to demonstrate a role both in vivo and in vitro for the actin-related subunits in regulating chromatin-remodeling complexes. 36. Bourachot B, Yaniv M, Muchardt C: The activity of mammalian brm/SNF2alpha is dependent on a high-mobility-group protein I/Y-like DNA binding domain. Mol Cell Biol 1999, 19:3931-3939. 37. Rando OJ, Zhao K, Janmey P, Crabtree GR: Phosphatidylinositol dependent actin filament binding by the SWI/SNF-like BAF chromatin remodeling complex. Proc Natl Acad Sci USA 2002, 99:2824-2829. The paper demonstrates that the mammalian BAF complex binds to PtdIns(4,5)P2 in vitro and in vivo. PtdIns(4,5)P2 stimulates binding of BAF to actin pointed ends and branch points. The interaction between the BAF complex and actin is via the Brg1 protein which has two distinct actin-binding domains, only one of which is sensitive to PtdIns(4,5)P2. 38. Memedula S, Belmont AS: Sequential recruitment of HAT and SWI/SNF components to condensed chromatin by VP16. Curr Biol 2003, 13:241-246. This paper uses in vivo imaging techniques to demonstrate that transcriptional activation leads to the sequential recruitment of factors involved in regulating chromatin structure. The authors suggest that the recruitment to the promoter of the key ATPase of chromatin remodeling complexes may increase chromatin accessibility before the subsequent recruitment of other components of the complex. 39. Divecha N, Roefs M, Los A, Halstead J, Bannister A, D’Santos C: Type I PIPkinases interact with and are regulated by the Retinoblastoma susceptibility gene product - pRB. Curr Biol 2002, 12:582-587. The authors demonstrate that the retinoblastoma protein interacts both in vitro and in vivo with type I PIPkinase which is the enzyme responsible for almost all PtdIns(4,5)P2 synthesis in nuclei. This paper provides insight to how changes in the cell cycle may correlate with changes in nuclear PtdIns(4,5)P2. 40. York JD, Odom AR, Murphy R, Ives EB, Wente SR: A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 1999, 285:96-100. 41. Odom AR, Stahlberg A, Wente SR, York JD: A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science 2000, 287:2026-2029. 42. Steger DJ, Haswell ES, Miller AL, Wente SR, O’Shea EK: Regulation of chromatin remodeling by inositol polyphosphates. Science 2003, 299:114-116. The report shows that Arg82/Ipk2 is an inositol polyphosphate kinase responsible for the generation of InsP4/InsP5 in yeast. Mutants within the gene encoding the protein give rise to strains that have impaired remodelling of the PHO5 promoter and the ATP-dependent chromatinwww.sciencedirect.com
remodelling complexes SWI/SNF and INO80 are not efficiently recruited to phosphate-responsive promoters. 43. Shen X, Xiao H, Ranallo R, Wu WH, Wu C: Modulation of ATP-dependent chromatin-remodeling complexes by inositol polyphosphates. Science 2003, 299:112-114. The authors show that several chromatin-remodelling complexes can be modulated by inositol polyphosphates in vitro. InsP6 inhibits nucleosome mobilisation by NURF, ISW2 and INO80 complexes. By contrast, InsP4 and InsP5 can stimulate SWI/SNF activity. In vivo results show that InsP4, InsP5 and InsP6 are important for gene transcription in yeast. 44. Shears SB: Transcriptional regulation: a new dominion for inositol phosphate signaling? Bioessays 2000, 22:786-789. 45. Nalaskowski MM, Deschermeier C, Fanick W, Mayr GW: The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization. Biochem J 2002, 366:549-556. 46. Hanakahi LA, Bartlet-Jones M, Chappell C, Pappin D, West SC: Binding of inositol phosphate to DNA-PK and stimulation of double- strand break repair. Cell 2000, 102:721-729. 47. Ma Y, Lieber MR: Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs. J Biol Chem 2002, 277:10756-10759. 48. Gaillard H, Fitzgerald DJ, Smith CL, Peterson CL, Richmond TJ, Thoma F: Chromatin remodeling activities act on UV-damaged nucleosomes and modulate DNA damage accessibility to photolyase. J Biol Chem 2003, 278:17655-17663. 49. Frit P, Kwon K, Coin F, Auriol J, Dubaele S, Salles B, Egly JM: Transcriptional activators stimulate DNA repair. Mol Cell 2002, 10:1391-1401. 50. D’Santos CS, Clarke JH, Divecha N: Phospholipid signalling in the nucleus. Een DAG uit het leven van de inositide signalering in de nucleus. Biochim Biophys Acta 1998, 1436:201-232. 51. Doughman RL, Firestone AJ, Anderson RA: Phosphatidylinositol phosphate kinases put PI4,5P(2) in its place. J Membr Biol 2003, 194:77-89. 52. Visnjic D, Curic J, Crljen V, Batinic D, Volinia S, Banfic H: Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells. Biochim Biophys Acta 2003, 1631:61-71. 53. Visnjic D, Crljen V, Curic J, Batinic D, Volinia S, Banfic H: The activation of nuclear phosphoinositide 3-kinase C2beta in all-trans-retinoic acid-differentiated HL-60 cells. FEBS Lett 2002, 529:268-274. 54. Capili AD, Schultz DC, Rauscher FJ III, Borden KL: Solution structure of the PHD domain from the KAP-1 corepressor: structural determinants for PHD, RING and LIM zinc-binding domains. EMBO J 2001, 20:165-177. 55. Pascual J, Martinez-Yamout M, Dyson HJ, Wright PE: Structure of the PHD zinc finger from human Williams-Beuren syndrome transcription factor. J Mol Biol 2000, 304:723-729. 56. Heino M, Peterson P, Kudoh J, Shimizu N, Antonarakis SE, Scott HS, Krohn K: APECED mutations in the autoimmune regulator (AIRE) gene. Hum Mutat 2001, 18:205-211. 57. Vogel A, Strassburg CP, Obermayer-Straub P, Brabant G, Manns MP: The genetic background of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy and its autoimmune disease components. J Mol Med 2002, 80:201-211. 58. Gibbons RJ, Picketts DJ, Villard L, Higgs DR: Mutations in a putative global transcriptional regulator cause X-linked mental retardation with alpha-thalassemia (ATR-X syndrome). Cell 1995, 80:837-845. 59. Gibbons RJ, Bachoo S, Picketts DJ, Aftimos S, Asenbauer B, Bergoffen J, Berry SA, Dahl N, Fryer A, Keppler K et al.: Mutations in transcriptional regulator ATRX establish the functional significance of a PHD-like domain. Nat Genet 1997, 17:146-148. 60. Gozani O, Karuman P, Jones DR, Ivanov D, Cha J, Lugovskoy AA, Baird CL, Zhu H, Field SJ, Lessnick SL et al.: The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor. Cell 2003, 114:99-111. Current Opinion in Genetics & Development 2004, 14:196–202
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This paper describes phosphoinositide (predominantly PtdIns[5]P) binding to the PHD finger motif found within the putative tumour suppressor protein ING2. PtdIns(5)P binding in vivo is essential for ING2 function during p53-dependent apoptosis. This paper provides the first link between PtdInsPs and processes that regulate responses to DNA damage. 61. Feng X, Hara Y, Riabowol K: Different HATS of the ING1 gene family. Trends Cell Biol 2002, 12:532-538.
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62. Andrulis ED, Neiman AM, Zappulla DC, Sternglanz R: Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 1998, 394:592-595. 63. Feuerbach F, Galy V, Trelles-Sticken E, Fromont-Racine M, Jacquier A, Gilson E, Olivo-Marin JC, Scherthan H, Nehrbass U: Nuclear architecture and spatial positioning help establish transcriptional states of telomeres in yeast. Nat Cell Biol 2002, 4:214-221.
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Current Opinion in Genetics & Development 2004, 14:196–202 This review comes from a themed issue on Chromosomes and expression mechanisms Edited by Stephen D Bell and Andy Bannister 0959-437X/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.gde.2004.02.003
Abbreviations ACF ATP-dependent chromatin-assembly factor ING2 inhibitor of growth protein 2 NHEJ non-homologous end joining NURF nucleosome remodelling factor PHD plant homedomain PI phosphoinositide
Introduction In the nucleus, the genome is packaged into a nucleoprotein structure called chromatin. The fundamental unit of chromatin, the nucleosome, is a repeating motif consisting of two turns of DNA wrapped around a histone octamer. Chromatin is a highly ordered repressive structure that limits nuclear processes requiring access to the DNA sequence including RNA transcription, DNA replication and DNA repair and it is widely held that disruption or remodelling of chromatin is a rate-determining step in these processes [1]. Remodelling of chromatin is the consequence of two distinct mechanisms. The first leads to covalent modification of nucleosomal histones [2], including phosphorylation, acetylation, methylation and ubiquitination, which can themselves effect chromatin structure or recruit proteins that do this. The second utilises the energy of ATP hydrolysis to alter chromatin Current Opinion in Genetics & Development 2004, 14:196–202
structure [1]. It should be noted that the two processes are by no means mutually exclusive as the first has been shown to lead to recruitment of factors, which carry out the second [3]. Phosphoinositides, which form a minor family of phospholipids, play a key role as intracellular second messengers [4]. The action of phospholipases, lipid kinases and lipid phosphatases in response to extracellular signals leads to remodelling of the phosphoinositide profile (Figure 1), which in turn regulates downstream targets to control diverse intracellular processes including vesicle trafficking [5], cell proliferation and survival [6], gene transcription and pathogen elimination [7]. Phosphoinositides are known to transduce signals essentially via two events: their modification and their interactions with specific proteins. Protein domains that interact with phosphoinositides include PH [8], ENTH [9], FYVE [10], PHOX [11] domains and lysine/arginine rich patches [12]. In addition to phosphoinositide signalling at the plasma membrane, biochemical studies have demonstrated the presence of the lipid kinases DGK, PtdIns-4K and PIPkinase in nuclear membranes [13–15] and within the nuclear matrix [16,17]. Localisation of phosphoinositides (particularly PtdIns[4,5]P2) using both antibodies [18,19] and the GST-tagged PH domain of PI-specific PLC-d1 [20] (a sensor for PtdIns[4,5]P2) indicate their presence in the nuclear membrane, interchromatin granules, heterochromatin, nucleolar-associated heterochromatin and sites of pre-mRNA processing [21]. Furthermore, exogenously added fluorescently-labelled PtdIns(4,5)P2 is found at the plasma membrane and as speckle staining within nuclei reminiscent of that described above [22]. In addition to phosphoinositides, phosphatidylcholine has also been shown to be present in the nuclear matrix [23]. The presence of both nuclear phosphoinositides and the enzymes responsible for their metabolism suggest that they may serve as a signalling system. Nuclear phosphoinositides are modulated in response to short-term growth factor signalling, cell-cycle progression and during differentiation. Perhaps central to the regulation of nuclear phosphoinositide signalling is PI–PLC. PI–PLC catalyses the hydrolysis of PtdIns(4,5)P2 to generate two very important second messengers: Ins(1,4,5)P3, which can be further phosphorylated to Ins(1,2,3,4,5,6)P6 (InsP6) and DAG. PtdIns–PLC regulation is also one mechanism by which the level of nuclear PtdIns(4,5)P2 and PtdIns(4)P may be controlled. Numerous isoforms of PI–PLC have been identified in the nucleus but the b1 isoform is the www.sciencedirect.com
Linking lipids to chromatin Jones and Divecha 197
Figure 1
PtdIns(3,5)P2 PIKfyve PtdIns(3,4)P2 PtdIns(3)P Type II PIPkinase ? PI3K
Type I PIPkinase
SHIP
PtdIns(3,4,5)P3
PTEN PtdIns
PtdIns-4K
Type I PIPkinase PtdIns(4)P PtdIns(4,5)P2
PI3K
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PIKfyve? Type II PIPkinase
PtdIns(5)P PtdIns synthase
DGK PtdOH
DAG
Ins(1,4,5)P3 Ins(P)x kinases higher inositol phosphates Current Opinion in Genetics & Development
A map of phosphoinositide metabolism. The enzymes are indicated in blue.
best characterised. Upon short term stimulation with IGF-1, nuclear PI–PLCb1 is activated by phosphorylation by p42/p44 mitogen-activated protein kinase [24] and subsequently negatively regulated by the DAG-dependent bII isoform of protein kinase C [25]. Although upregulation of nuclear PI–PLCbI activity correlates with increased progression through the cell cycle, differentiation of murine erythroleukaemia (MEL) cells, induced by DMSO, causes downregulation of nuclear PI–PLCb1, an accumulation of nuclear polyphosphoinositides and a decrease in nuclear DAG [14,26,27]. Moreover, overexpression of PI–PLCb1 in the nucleus is sufficient to drive 3T3 cells into S-phase, whereas in MEL cells nuclear PI– PLCb1 expression can block differentiation [28]. In addition, activation of the enzymes that synthesise PtdInsP and PtdIns(4,5)P2 occurs as cells progress through G1 into S-phase [29]. Thus in response to a variety of signals activation/repression of PI-PLC, PIPkinase and phosphatase activities (Figure 1) will generate different patterns of PtdInsPs, PtdIns(4,5)P2 and inositol phosphates within the nucleus.
Chromatin remodelling and phosphoinositides Although phosphoinositides have been shown to be present in the nucleus and can change in response to external stimuli, there is a paucity of mechanistic data on how these changes may be transduced into functional regulation of nuclear processes. Previous data showed www.sciencedirect.com
that addition of phospholipids to purified nuclei could effect in vitro transcription and replication of DNA [30]. Indeed in vitro negatively charged lipids lead to chromatin decondensation, whereas positively charged lipids have the opposite effect [31]. PtdIns(4,5)P2 can bind to the C-terminal tail of histones H1 and H3 and in an in vitro assay counteracts histone H1-mediated repression of basal transcription [32]. A novel mechanism for the regulation of chromatin structure by phosphoinositides came with the unexpected discovery that the interaction of a chromatin-remodelling complex, BAF, can be regulated by the level of PtdIns(4,5)P2 [33]. Resting T cells have small, compact nuclei with dense heterochromatin, which upon antigenic activation increase in size with the appearance of euchromatin. These changes in chromatin are thought to be required for the subsequent activation of T-cell-specific genetic programmes. Upon T cell stimulation with anti-CD3 (an antibody against the T cell receptor) or with ionomycin/PMA, the BAF complex translocates from a nuclear soluble fraction to an insoluble fraction. This translocation can be mimicked by incubation of non-activated T cell nuclei with exogenously added PtdIns(4,5)P2 [33] Although the data are consistent with a role for PtdIns(4,5)P2 in regulating BAF complex localisation, there is no data to demonstrate that T cell activation leads to an increase in nuclear PtdIns(4,5)P2. Central to a chromatin-remodelling complex is an ATPase subunit related to the yeast SWI/SNF2 protein, Current Opinion in Genetics & Development 2004, 14:196–202
198 Chromosomes and expression mechanisms
which is a member of the DEAD/H superfamily of DNA and RNA helicases [34] and is the key to their various remodelling activities such as nucleosomal assembly, sliding and positioning. Among the other numerous subunits, most if not all mammalian chromatin remodelling complexes also contain actin or actin-related proteins, which modulate remodelling activity both in vitro and in vivo [35]. BRG-1, the ATPase subunit of the BAF complex, has two domains that can interact with actin, one of which contains a lysine-rich patch. This lysine-rich patch is required for BRG-1 function in vivo [36] and importantly can interact with PtdIns(4,5)P2 [37]. The authors suggest that the interaction of BRG-1 with actin is disrupted by PtdIns(4,5)P2 and consequently exposes a site on actin for interaction with components of the nuclear matrix. This is analogous to PtdIns(4,5)P2mediated uncapping of actin, which stimulates actin polymerisation [12]. Recent studies have demonstrated that upon transcriptional activation BRG-1 is recruited to the active promoter before other components of the BAF complex suggesting that a pre-associated BAF complex may not be recruited to active promoters [38]. Perhaps PtdIns(4,5)P2, present on histone H1/H3, or locally synthesised, may allosterically stimulate interaction of BRG-1 with other BAF complex components and/or lead to stabilisation of the BAF complex at the promoter by increasing its interaction with the nuclear matrix. How might localised synthesis of PtdIns(4,5)P2 be regulated? The retinoblastoma protein, which recruits the BAF complex to regulate gene expression, interacts with and activates type I PIPkinases [39], the enzymes responsible for the generation of nuclear PtdIns(4,5)P2. We suggest that transcriptional regulators and/or chromatin remodelling complexes may interact with and recruit type I PIPkinase to control localised synthesis of PtdIns(4,5)P2.
The problem with phosphoinositides and a solution in yeast Phosphoinositides have a hydrophilic head group coupled to two extremely hydrophobic fatty acyl chains. They are thus particularly suited to partitioning into membranes but are unlikely to move freely through the nucleus. Where they are generated is where they act! This poses a problem regarding PtdIns(4,5)P2 accessibility to chromatin-remodelling factors. Yeast have enzymes to generate PtdIns(4,5)P2 within the nucleus and have a nuclear PtdIns–PLC, which can generate Ins(1,4,5)P3. This Ins(1,4,5)P3 is rapidly phosphorylated to InsP6 via the action of two inositol polyphosphate kinases. Ipk2 phosphorylates Ins(1,4,5)P3 on the 6 and the 3 positions to generate inositol (1,3,4,5,6)pentakisphosphate (InsP5), which is then the substrate for Ipk1 to generate InsP6 [40]. Interestingly Ipk2 turns out to be a known transcription factor [41], ARG82, involved in responses to changes in nutrients and stress, sporulation and in mating. Steger et al. identified a requirement for ARG82 in the regulation Current Opinion in Genetics & Development 2004, 14:196–202
of PHO5 transcription in response to changes in the levels of phosphate [42]. Transcriptional activation of PHO5 requires the remodelling of one nucleosome, which is dependent on the SWI/SNF (yeast homologue of the BAF complex) and the INO80 chromatin-remodelling complexes. Yeast strains deficient in ARG82 (or importantly its inositol phosphate kinase activity) or PtdIns– PLC are unable to remodel this nucleosome, suggesting that the generation of highly phosphorylated inositol phosphates are important for chromatin remodelling at the PHO5 promoter. Using mutations in other inositol phosphate kinases, the authors conclude that the generation of InsP4/InsP5 is/are the important molecule(s), which likely act(s) through direct regulation of the remodelling complexes. In a complementary study, the activity of several different chromatin-remodelling complexes (Nurf [in Drosophila], ISW2 [in yeast], SWI/SNF and INO80) were found to be sensitive to inositol phosphates in vitro [43]. Whereas Nurf, ISW2 and INO80 activities were inhibited by high concentration of IP6, SWI/SNF activity was increased by the presence of InsP4/InsP5. This latter observation is in accord with data for an in vivo role of inositol phosphates on SWI/SNF remodelling activity. How do inositol phosphates regulate remodelling activities? InsP6 can bind to the NURF and ISW2 complexes and lead to a reduction of nucleosome-stimulated ATPase activity [43]. However, InsP4/InsP5-mediated activation of the SWI/SNF complex does not lead to enhanced ATPase activity. Moreover in vivo, both the INO80 and SWI/SNF complexes are not efficiently recruited to their promoters in the absence of higher phosphorylated inositol phosphates [42]. These data suggest that InsP4/InsP5 may regulate the interaction of SWI/SNF with chromatin. Although the above data are consistent with a role for inositol phosphates in the regulation of chromatin-remodelling complexes, it should be noted that activation of the SWI/SNF complex by InsP4/InsP5 was shown using a concentration of 500 mM, whereas estimates in yeast suggest that the level of InsP4/InsP5 is likely to be between 1–5 mM. Compartmentalisation of InsP4/InsP5 in the nucleus may represent a mechanism for increasing the local concentration. Alternatively, regulatory changes in interacting proteins or post-translational modification may increase the affinity of the complex for inositol phosphates. This change in affinity would alleviate a requirement for changes in the concentrations of InsP4/InsP5 to act as a signal to activate remodelling. It should also be noted that yeast lacking ARG82, or any of the genes involved in the synthesis of higher inositol phosphates, have profound defects in the function of the endocytic pathway, vacuole biogenesis and in mRNA transport [44]. These may lead to an impairment of signalling pathways involved in transducing changes in extracellular levels of phosphate to changes in gene expression rather than a direct effect on chromatin-remodelling complexes. www.sciencedirect.com
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Are inositol phosphates implicated in mammalian chromatin remodelling? A mammalian homologue of ARG82, IPMK, has been cloned and does show similar inositol phosphate kinase activity [45]. Interestingly IMPK appears to be localised within the nucleus suggesting a role in nuclear functions. To date, no data has demonstrated a role for inositol phosphates or IMPK in the regulation of mammalian chromatin remodelling complexes or in gene transcription. A role for inositol phosphates in DNA repair in mammalian cells has been suggested, as InsP6 is a potent co-activator of non-homologous end joining (NHEJ) when assessed in vitro [46]. Further studies have shown that InsP6 can interact with the Ku protein, although it is not clear if this interaction is required for the stimulation of end joining activity [47]. How InsP6 modulates Ku function is not clear. InsP6 does not appear to regulate Ku interaction with either DNA or with DNA-dependent protein kinase. Interestingly, a role for chromatin-remodelling complexes in nucleotide excision repair has been demonstrated [48,49].
Pointing the finger at PtdInsPs
The plant homedomain (PHD) finger is a Cys4–HisCys3 zinc finger [54,55] present in 66 different human proteins many of which are nuclear. Several PHD-encoding genes have been implicated in human diseases and many of the characterised mutations in these genes lead to a loss of function of the PHD domain [56–59]. Recently, the PHD finger of inhibitor of growth protein 2 (ING2) was found to interact with phosphomonoinositides and particularly with PtdIns(5)P [60]. ING2 is a potential tumour suppressor and can induce growth arrest and apoptosis in a p53-dependent manner. Gozani et al. [60] showed that ING2 function in vivo (induction of apoptosis and p53 acetylation) was compromised by point mutations that resulted in loss of PtdIns(5)P binding. Moreover, overexpression of a type II PIPkinase (Figure 1), assumed to phosphorylate and remove nuclear PtdIns(5)P, attenuated ING2 function. Proteins containing PHD fingers that modulate chromatin structure include p300 (histone acetylase), Mi2, ATP-dependent chromatin-assembly factor (ACF) and TIP5 (all chromatin-remodelling complex proteins), the ING family (interact with acetylase and deacetylase complexes), polycomb proteins (silenwww.sciencedirect.com
Do all PHD fingers interact with phosphoinositides? Gozani et al. [60] showed that other PHD-motif-containing proteins including ACF also bind to phosphoinositides in vitro. In addition, we have screened >40 different murine PHD motifs and found 10 that bind phosphoinositides. We also found that of two different PHD domains from the same protein, only one interacted with phosphoinositides, suggesting that it is likely that different PHD fingers have different functions. Furthermore we find that different PHD domains appear to be able to interact with different subsets of phosphomonoinositides. We expect that diverse and contextual modulation of PtdIns(3)P, PtdIns(4)P and PtdIns(5)P within the nucleus will differentially regulate PHD-containing protein function.
Figure 2
Signal input
PtdInsP2
PtdInsP2
PtdInsP2
PtdInsP
PtdInsP
PtdInsP
InsPx
InsPx
InsPx
Nuclear compartment
PtdInsPs are often thought of as just intermediates for the synthesis of PtdIns(4,5)P2 via their phosphorylation by either type I or type II PIPkinases (Figure 1) [50,51]. However, PtdIns(4)P and PtdIns(5)P mass accumulates as cells progress from G1 into S-phase [29] and in response to a variety of cellular stresses (UV, etoposide and oxidative damage; DR Jones, N Divecha, unpublished). In addition, nuclear PtdIns(3)P accumulates during G2/M phase in HL60 cells and in response to long-term treatment with all-trans-retinoic acid [52,53]. One mechanism for how changes in nuclear PtdInsPs may be transduced into a functional output has recently been elucidated.
cing genes) trithorax (gene activation) and the CpG binding protein (involved in regulating methylation of DNA). In the case of p300, the PHD finger has been shown to be essential for acetylation activity. Interestingly the ING family, of which ING1, 2 and 3 interact with phosphoinositides (DR Jones, N Divecha, unpublished), is intimately involved in the regulation of both histone and non-histone protein acetylation [61].
Nuclear phosphoinositide-binding proteins
Differentiation
Proliferation
Apoptosis
Current Opinion in Genetics & Development
A variety of signal inputs lead to temporal remodelling of the inositide profile in the nuclear envelope and/or in the nuclear matrix (the size of the oval reflects the size of the inositide pool). The combinatorial changes in nuclear phosphoinositides are decoded through their interaction with specific phosphoinositide-binding proteins. This decoding leads to differential outputs, coordinating processes such as differentiation, proliferation or apoptosis. For example signal inputs that increase the level of nuclear PtdIns(5)P within the chromatin-enriched fraction lead to the translocation of ING2 that, in turn, may regulates p53 function and histone acetylation to coordinate an apoptotic response. Current Opinion in Genetics & Development 2004, 14:196–202
200 Chromosomes and expression mechanisms
Conclusions The identification of nuclear-specific phosphoinositide binding domains establishes potential novel functions for phosphoinositides within the nucleus. Signals that induce processes such as differentiation, cell proliferation and stress adaptation/apoptosis lead to a remodelling of nuclear inositide profiles (Figure 2). We suggest that the combinatorial spatial and temporal changes in nuclear PtdInsPs, PtdIns(4,5)P2 and inositol phosphates are decoded by specific phosphoinositide-binding proteins to elicit differential outputs. These may include changes in chromatin structure to regulate gene expression, replication or DNA repair. Furthermore, the presence of a dynamic pool of nuclear envelope or matrix phosphoinositides, which respond to external cues together with chromatin-associated proteins that can interact with phosphoinositides, would provide an ideal mechanism for controlling gene positioning shown to be important in regulating gene expression [62,63].
Acknowledgements
12. Yin HL, Janmey PA: Phosphoinositide regulation of the actin cytoskeleton. Annu Rev Physiol 2003, 65:761-789. 13. Smith CD, Wells WW: Phosphorylation of rat liver nuclear envelopes. II. Characterization of in vitro lipid phosphorylation. J Biol Chem 1983, 258:9368-9373. 14. Cocco L, Gilmour RS, Ognibene A, Letcher AJ, Manzoli FA, Irvine RF: Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation. Biochem J 1987, 248:765-770. 15. Capitani S, Bertagnolo V, Mazzoni M, Santi P, Previati M, Antonucci A, Manzoli FA: Lipid phosphorylation in isolated rat liver nuclei. Synthesis of polyphosphoinositides at subnuclear level. FEBS Lett 1989, 254:194-198. 16. Payrastre B, Nievers M, Boonstra J, Breton M, Verkleij AJ, Van Bergen en Henegouwen PM: A differential location of phosphoinositide kinases, diacylglycerol kinase, and phospholipase C in the nuclear matrix. J Biol Chem 1992, 267:5078-5084. 17. Vann LR, Wooding FB, Irvine RF, Divecha N: Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei. Biochem J 1997, 327:569-576. 18. Osborne SL, Thomas CL, Gschmeissner S, Schiavo G: Nuclear PtdIns(4,5)P2 assembles in a mitotically regulated particle involved in pre-mRNA splicing. J Cell Sci 2001, 114:2501-2511.
We apologise to those whose work was not cited for lack of space. We thank all the members of the Inositide Laboratory and Maria Carla Motta for helpful discussions. Work in the laboratory has been supported by grants from the Dutch Cancer Society and the European Commission.
19. Yokogawa T, Nagata S, Nishio Y, Tsutsumi T, Ihara S, Shirai R, Morita K, Umeda M, Shirai Y, Saitoh N, Fukui Y: Evidence that 3(-phosphorylated polyphosphoinositides are generated at the nuclear surface: use of immunostaining technique with monoclonal antibodies specific for PI 3,4-P(2). FEBS Lett 2000, 473:222-226.
References and recommended reading
20. Watt SA, Kular G, Fleming IN, Downes CP, Lucocq JM: Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1. Biochem J 2002, 363:657-666.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Peterson CL: Chromatin remodeling: nucleosomes bulging at the seams. Curr Biol 2002, 12:R245-R247.
2.
Turner BM: Cellular memory and the histone code. Cell 2002, 111:285-291.
3.
Agalioti T, Chen G, Thanos D: Deciphering the transcriptional histone acetylation code for a human gene. Cell 2002, 111:381-392.
21. Boronenkov IV, Loijens JC, Umeda M, Anderson RA: Phosphoinositide signaling pathways in nuclei are associated with nuclear speckles containing pre-mRNA processing factors. Mol Biol Cell 1998, 9:3547-3560. 22. Ozaki S, DeWald DB, Shope JC, Chen J, Prestwich GD: Intracellular delivery of phosphoinositides and inositol phosphates using polyamine carriers. Proc Natl Acad Sci USA 2000, 97:11286-11291. 23. Hunt AN, Clark GT, Attard GS, Postle AD: Highly saturated endonuclear phosphatidylcholine is synthesized in situ and colocated with CDP-choline pathway enzymes. J Biol Chem 2001, 276:8492-8499.
4.
Payrastre B, Missy K, Giuriato S, Bodin S, Plantavid M, Gratacap M: Phosphoinositides: key players in cell signalling, in time and space. Cell Signal 2001, 13:377-387.
5.
De Camilli P, Emr SD, McPherson PS, Novick P: Phosphoinositides as regulators in membrane traffic. Science 1996, 271:1533-1539.
6.
Datta SR, Brunet A, Greenberg ME: Cellular survival: a play in three Akts. Genes Dev 1999, 13:2905-2927.
7.
Ellson CD, Andrews S, Stephens LR, Hawkins PT: The PX domain: a new phosphoinositide-binding module. J Cell Sci 2002, 115:1099-1105.
25. Xu A, Wang Y, Xu LY, Gilmour RS: Protein kinase C alpha -mediated negative feedback regulation is responsible for the termination of insulin-like growth factor I-induced activation of nuclear phospholipase C beta1 in Swiss 3T3 cells. J Biol Chem 2001, 276:14980-14986.
8.
Dowler S, Currie RA, Campbell DG, Deak M, Kular G, Downes CP, Alessi DR: Identification of pleckstrin-homology-domaincontaining proteins with novel phosphoinositide-binding specificities. Biochem J 2000, 351:19-31.
26. Divecha N, Letcher AJ, Banfic HH, Rhee SG, Irvine RF: Changes in the components of a nuclear inositide cycle during differentiation in murine erythroleukaemia cells. Biochem J 1995, 312:63-67.
9.
De Camilli P, Chen H, Hyman J, Panepucci E, Bateman A, Brunger AT: The ENTH domain. FEBS Lett 2002, 513:11-18.
27. Martelli AM, Cataldi A, Manzoli L, Billi AM, Rubbini S, Gilmour RS, Cocco L: Inositides in nuclei of Friend cells: changes of polyphosphoinositide and diacylglycerol levels accompany cell differentiation. Cell Signal 1995, 7:53-56.
10. Gaullier JM, Simonsen A, D’Arrigo A, Bremnes B, Stenmark H, Aasland R: FYVE fingers bind PtdIns(3)P. Nature 1998, 394:432-433. 11. Xu Y, Seet LF, Hanson B, Hong W: The Phox homology (PX) domain, a new player in phosphoinositide signalling. Biochem J 2001, 360:513-530. Current Opinion in Genetics & Development 2004, 14:196–202
24. Xu A, Suh PG, Marmy-Conus N, Pearson RB, Seok OY, Cocco L, Gilmour RS: Phosphorylation of nuclear phospholipase C beta1 by extracellular signal-regulated kinase mediates the mitogenic action of insulin-like growth factor I. Mol Cell Biol 2001, 21:2981-2990.
28. Faenza I, Matteucci A, Bavelloni A, Marmiroli S, Martelli AM, Gilmour RS, Suh PG, Manzoli L, Cocco L: Nuclear PLCbeta(1) acts as a negative regulator of p45/NF-E2 expression levels in Friend erythroleukemia cells. Biochim Biophys Acta 2002, 1589:305-310. www.sciencedirect.com
Linking lipids to chromatin Jones and Divecha 201
29. Clarke JH, Letcher AJ, D’Santos CS, Halstead JR, Irvine RF, Divecha N: Inositol lipids are regulated during cell cycle progression in the nuclei of murine erythroleukaemia cells. Biochem J 2001, 357:905-910. 30. Capitani S, Cocco L, Maraldi NM, Papa S, Manzoli FA: Effect of phospholipids on transcription and ribonucleoprotein processing in isolated nuclei. Adv Enzyme Regul 1986, 25:425-438. 31. Kuvichkin VV: DNA-lipid interactions in vitro and in vivo. Bioelectrochemistry 2002, 58:3-12. 32. Yu H, Fukami K, Watanabe Y, Ozaki C, Takenawa T: Phosphatidylinositol 4,5-bisphosphate reverses the inhibition of RNA transcription caused by histone H1. Eur J Biochem 1998, 251:281-287. 33. Zhao K, Wang W, Rando OJ, Xue Y, Swiderek K, Kuo A, Crabtree GR: Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling. Cell 1998, 95:625-636. 34. Eisen JA, Sweder KS, Hanawalt PC: Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions. Nucleic Acids Res 1995, 23:2715-2723. 35. Shen X, Ranallo R, Choi E, Wu C: Involvement of actin-related proteins in ATP-dependent chromatin remodeling. Mol Cell 2003, 12:147-155. The authors use yeast genetics to demonstrate a role both in vivo and in vitro for the actin-related subunits in regulating chromatin-remodeling complexes. 36. Bourachot B, Yaniv M, Muchardt C: The activity of mammalian brm/SNF2alpha is dependent on a high-mobility-group protein I/Y-like DNA binding domain. Mol Cell Biol 1999, 19:3931-3939. 37. Rando OJ, Zhao K, Janmey P, Crabtree GR: Phosphatidylinositol dependent actin filament binding by the SWI/SNF-like BAF chromatin remodeling complex. Proc Natl Acad Sci USA 2002, 99:2824-2829. The paper demonstrates that the mammalian BAF complex binds to PtdIns(4,5)P2 in vitro and in vivo. PtdIns(4,5)P2 stimulates binding of BAF to actin pointed ends and branch points. The interaction between the BAF complex and actin is via the Brg1 protein which has two distinct actin-binding domains, only one of which is sensitive to PtdIns(4,5)P2. 38. Memedula S, Belmont AS: Sequential recruitment of HAT and SWI/SNF components to condensed chromatin by VP16. Curr Biol 2003, 13:241-246. This paper uses in vivo imaging techniques to demonstrate that transcriptional activation leads to the sequential recruitment of factors involved in regulating chromatin structure. The authors suggest that the recruitment to the promoter of the key ATPase of chromatin remodeling complexes may increase chromatin accessibility before the subsequent recruitment of other components of the complex. 39. Divecha N, Roefs M, Los A, Halstead J, Bannister A, D’Santos C: Type I PIPkinases interact with and are regulated by the Retinoblastoma susceptibility gene product - pRB. Curr Biol 2002, 12:582-587. The authors demonstrate that the retinoblastoma protein interacts both in vitro and in vivo with type I PIPkinase which is the enzyme responsible for almost all PtdIns(4,5)P2 synthesis in nuclei. This paper provides insight to how changes in the cell cycle may correlate with changes in nuclear PtdIns(4,5)P2. 40. York JD, Odom AR, Murphy R, Ives EB, Wente SR: A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 1999, 285:96-100. 41. Odom AR, Stahlberg A, Wente SR, York JD: A role for nuclear inositol 1,4,5-trisphosphate kinase in transcriptional control. Science 2000, 287:2026-2029. 42. Steger DJ, Haswell ES, Miller AL, Wente SR, O’Shea EK: Regulation of chromatin remodeling by inositol polyphosphates. Science 2003, 299:114-116. The report shows that Arg82/Ipk2 is an inositol polyphosphate kinase responsible for the generation of InsP4/InsP5 in yeast. Mutants within the gene encoding the protein give rise to strains that have impaired remodelling of the PHO5 promoter and the ATP-dependent chromatinwww.sciencedirect.com
remodelling complexes SWI/SNF and INO80 are not efficiently recruited to phosphate-responsive promoters. 43. Shen X, Xiao H, Ranallo R, Wu WH, Wu C: Modulation of ATP-dependent chromatin-remodeling complexes by inositol polyphosphates. Science 2003, 299:112-114. The authors show that several chromatin-remodelling complexes can be modulated by inositol polyphosphates in vitro. InsP6 inhibits nucleosome mobilisation by NURF, ISW2 and INO80 complexes. By contrast, InsP4 and InsP5 can stimulate SWI/SNF activity. In vivo results show that InsP4, InsP5 and InsP6 are important for gene transcription in yeast. 44. Shears SB: Transcriptional regulation: a new dominion for inositol phosphate signaling? Bioessays 2000, 22:786-789. 45. Nalaskowski MM, Deschermeier C, Fanick W, Mayr GW: The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization. Biochem J 2002, 366:549-556. 46. Hanakahi LA, Bartlet-Jones M, Chappell C, Pappin D, West SC: Binding of inositol phosphate to DNA-PK and stimulation of double- strand break repair. Cell 2000, 102:721-729. 47. Ma Y, Lieber MR: Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs. J Biol Chem 2002, 277:10756-10759. 48. Gaillard H, Fitzgerald DJ, Smith CL, Peterson CL, Richmond TJ, Thoma F: Chromatin remodeling activities act on UV-damaged nucleosomes and modulate DNA damage accessibility to photolyase. J Biol Chem 2003, 278:17655-17663. 49. Frit P, Kwon K, Coin F, Auriol J, Dubaele S, Salles B, Egly JM: Transcriptional activators stimulate DNA repair. Mol Cell 2002, 10:1391-1401. 50. D’Santos CS, Clarke JH, Divecha N: Phospholipid signalling in the nucleus. Een DAG uit het leven van de inositide signalering in de nucleus. Biochim Biophys Acta 1998, 1436:201-232. 51. Doughman RL, Firestone AJ, Anderson RA: Phosphatidylinositol phosphate kinases put PI4,5P(2) in its place. J Membr Biol 2003, 194:77-89. 52. Visnjic D, Curic J, Crljen V, Batinic D, Volinia S, Banfic H: Nuclear phosphoinositide 3-kinase C2beta activation during G2/M phase of the cell cycle in HL-60 cells. Biochim Biophys Acta 2003, 1631:61-71. 53. Visnjic D, Crljen V, Curic J, Batinic D, Volinia S, Banfic H: The activation of nuclear phosphoinositide 3-kinase C2beta in all-trans-retinoic acid-differentiated HL-60 cells. FEBS Lett 2002, 529:268-274. 54. Capili AD, Schultz DC, Rauscher FJ III, Borden KL: Solution structure of the PHD domain from the KAP-1 corepressor: structural determinants for PHD, RING and LIM zinc-binding domains. EMBO J 2001, 20:165-177. 55. Pascual J, Martinez-Yamout M, Dyson HJ, Wright PE: Structure of the PHD zinc finger from human Williams-Beuren syndrome transcription factor. J Mol Biol 2000, 304:723-729. 56. Heino M, Peterson P, Kudoh J, Shimizu N, Antonarakis SE, Scott HS, Krohn K: APECED mutations in the autoimmune regulator (AIRE) gene. Hum Mutat 2001, 18:205-211. 57. Vogel A, Strassburg CP, Obermayer-Straub P, Brabant G, Manns MP: The genetic background of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy and its autoimmune disease components. J Mol Med 2002, 80:201-211. 58. Gibbons RJ, Picketts DJ, Villard L, Higgs DR: Mutations in a putative global transcriptional regulator cause X-linked mental retardation with alpha-thalassemia (ATR-X syndrome). Cell 1995, 80:837-845. 59. Gibbons RJ, Bachoo S, Picketts DJ, Aftimos S, Asenbauer B, Bergoffen J, Berry SA, Dahl N, Fryer A, Keppler K et al.: Mutations in transcriptional regulator ATRX establish the functional significance of a PHD-like domain. Nat Genet 1997, 17:146-148. 60. Gozani O, Karuman P, Jones DR, Ivanov D, Cha J, Lugovskoy AA, Baird CL, Zhu H, Field SJ, Lessnick SL et al.: The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor. Cell 2003, 114:99-111. Current Opinion in Genetics & Development 2004, 14:196–202
202 Chromosomes and expression mechanisms
This paper describes phosphoinositide (predominantly PtdIns[5]P) binding to the PHD finger motif found within the putative tumour suppressor protein ING2. PtdIns(5)P binding in vivo is essential for ING2 function during p53-dependent apoptosis. This paper provides the first link between PtdInsPs and processes that regulate responses to DNA damage. 61. Feng X, Hara Y, Riabowol K: Different HATS of the ING1 gene family. Trends Cell Biol 2002, 12:532-538.
Current Opinion in Genetics & Development 2004, 14:196–202
62. Andrulis ED, Neiman AM, Zappulla DC, Sternglanz R: Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 1998, 394:592-595. 63. Feuerbach F, Galy V, Trelles-Sticken E, Fromont-Racine M, Jacquier A, Gilson E, Olivo-Marin JC, Scherthan H, Nehrbass U: Nuclear architecture and spatial positioning help establish transcriptional states of telomeres in yeast. Nat Cell Biol 2002, 4:214-221.
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