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Regulation and epigenetic control of transcription at the nuclear periphery
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Regulation and epigenetic control of transcription at the nuclear periphery Sara Ahmed and Jason H. Brickner Department of Biochemistry, Molecular Biology and Cell Biology Northwestern University, Evanston, IL 60208, USA
The localization of DNA within the nucleus influences the regulation of gene transcription. Subnuclear environments at the nuclear periphery promote gene silencing and activation. Silenced regions of the genome, such as centromeres and telomeres, are statically tethered to the nuclear envelope. Recent work in yeast has revealed that certain genes can undergo dynamic recruitment to the periphery upon transcriptional activation. For such genes, localization to the periphery has been suggested to improve mRNA export and favor optimal transcription. In addition, maintenance of peripheral localization confers cellular memory of previous transcriptional activation, enabling cells to adapt rapidly to transcriptional cues. Localization at the nuclear periphery promotes transcriptional silencing and activation The eukaryotic nucleus has different compartments, such as the nucleolus, nuclear envelope and nuclear pores, each with distinct structures and functions. The nuclear pore provides a gateway to the nucleus, enabling exchange of proteins and mRNA with the cytoplasm, whereas the nucleolus serves as the site for ribosomal component assembly and synthesis [1,2]. Subnuclear compartments or domains have also been implicated in regulation of transcription. Although transcription is regulated mainly through DNA-binding proteins that affect RNA polymerase recruitment or local chromatin structure, there is also a functional relationship between gene expression and nuclear organization. Chromosomes are nonrandomly arranged within the nucleus in diverse eukaryotic organisms, suggesting that subnuclear domains might have specialized roles in chromatin organization and transcription. In many metazoan cells, chromosomes fold back onto themselves to create distinct subnuclear territories [3]. Transcriptionally active genes frequently localize at the edge of such territories, and it has been proposed that this localization enables better access to stable transcriptional ‘factories’ between territories [4]. The arrangement of these chromosome territories within the nucleus is uniform in cells within the same tissue and can be conserved between species [5]. One of the best-characterized subnuclear domains is the nuclear periphery. Localization of parts of the genome to the nuclear periphery has important effects on transcription. Constitutive heterochromatin localizes to the nuclear periphery in many cell types [6,7], as do other silenced loci, Corresponding author: Brickner, J.H. ([email protected]). Available online 12 June 2007. www.sciencedirect.com
such as the immunoglobulin genes in hematopoietic progenitors of B lymphocytes [8]. Localization of repressed parts of the genome to the nuclear periphery has been thoroughly explored in the budding yeast Saccharomyces cerevisiae, in which regions of transcriptionally silent chromatin, such as telomeres and the mating type loci, associate with the nuclear envelope [9]. Proximity to the nuclear periphery promotes efficient silencing of genes near telomeres [9]. Conversely, artificially tethering mating-type loci with crippled silencing elements to the nuclear envelope promotes silencing [10]. Recent work, however, indicates that transcriptional activation also takes place at the nuclear periphery in yeast. Several dynamically regulated genes are randomly distributed in the nucleoplasm when repressed but are recruited to the nuclear periphery when activated. These genes include many highly expressed genes such as INO1 (encoding an enzyme involved in phospholipid biosynthesis), HSP104 (a molecular chaperone), HXK1 (hexokinase), SUC2 (invertase), GAL1 (galactokinase), GAL2 (a hexose transporter), GAL10 (glucose epimerase) and mating pheromone-induced genes [11–17]. The recruitment of active genes to the nuclear periphery in yeast is reminiscent of the observation made over 20 years ago by Hutchison and Weintraub that in mouse fibroblasts DNase I-sensitive (and presumably transcriptionally active) regions of the genome were found localized along the nuclear rim [18]. This suggests that transcriptional activation at the nuclear periphery might not be a yeast-specific phenomenon. The mouse b-globin locus is transcriptionally active at the nuclear periphery before its relocalization toward the nuclear interior during erythroid maturation [19]. This indicates that localization at the nuclear periphery is not incompatible with transcription in mammalian cells. Also, the transcriptional upregulation of the X chromosome in males in Drosophila requires nuclear pore components Mtor
Glossary Mex: mRNA export Mlp: Myosin-like protein Mtor: Megator Nic: Nuclear pore interacting complex Nup: nucleoporin Sac: Suppressor of actin Thp: Tho/Hpr1 phenotype TPR: Translocated promoter region TREX: transcription and export complex, a complex involved in cotranscriptional mRNA processing and transport, composed of the THO complex (Hpr1, Tho2, Mft1 and Thp2) and the Yra1–Sub2 complex
0168-9525/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2007.05.009
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(also called TPR) and Nup153 (see Glossary) and the X chromosome localizes at the nuclear periphery [20]. In yeast, gene recruitment to the periphery changes the subnuclear distribution of genes and reduces their mobility to a constrained, two-dimensional movement near the nuclear envelope [13,21]. Genome-wide chromatin immunoprecipitation studies reveal that many transcriptionally active genes physically interact with components of the nuclear pore complex (NPC) and associated factors [14,15]. Intriguingly, the nuclear pore component Nup2 is necessary and sufficient (when tethered to DNA directly) to create boundary elements that block the spread of heterochromatin [22–24]. Nup2 also interacts with transcriptionally active loci [15,25], suggesting that the protein might help to shield these regions from neighboring silenced DNA (see below). The nuclear periphery could represent a subnuclear domain that is itself composed of distinct microenvironments with roles in either gene silencing or activation. This review focuses on the mechanism of gene recruitment to the nuclear periphery in yeast. In addition, we highlight the potential functional significance of localizing genes to the nuclear periphery, particularly in promoting rapid transcriptional activation of recruited genes and in establishing novel epigenetic states. The mechanism of gene recruitment to the nuclear periphery The nuclear pore complex One of the most fascinating features of gene recruitment is that cells dynamically control the localization of particular parts of the genome. The mechanism(s) by which this is achieved are not only interesting in themselves, but also could have more general implications for our understanding of how genomes are spatially organized. The nuclear pore complex (NPC) has been identified as the site to which these genes are recruited [15]. The NPC is a large assembly of 30 core nucleoporin proteins and numerous accessory proteins [26] that perforates the nuclear envelope and serves essential roles in nucleocytoplasmic trafficking [27]. As articulated by Blobel’s ‘gene-gating’ hypothesis, the processes of transcription and mRNA export might be coupled through interactions with the NPC [28]. According to this model, nuclear pore complexes might interact with expanded, transcriptionally active portions of the genome, promoting optimal export of the transcript through the pore to which it is gated [28]. The observation that many genes are recruited to the nuclear periphery upon activation and that they physically interact with the NPC has inspired a re-evaluation of this appealing hypothesis. The yeast nucleoporins Nup2 [15,25], Nup60, Nic96, Nup116 and the myosin-like proteins Mlp1 and Mlp2 [14,15] physically interact with active genes. The NPC has three distinct parts: the cytoplasmic filaments, the core channel and the nucleoplasmic basket [29,30]. The components of the nucleoplasmic basket interact with several proteins implicated in mRNA export – such as Mlp1, Mlp2, Sac3, Thp1 and Mex67 [31] – and transcription – such as the Spt–Ada–Gcn5 histone acetyltransferase (SAGA) complex [32] (Figure 1). Loss of many of these proteins blocks gene recruitment, suggesting a general www.sciencedirect.com
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requirement for the nucleoplasmic basket and associated factors in this phenomenon (Table 1). Although there is general agreement that the nuclear basket is important for gene recruitment, it is unclear if all basket proteins are essential. Although Nup2, a component of the nuclear basket [33], promotes boundary activity and physically interacts with the promoter of GAL1 under activating conditions [14,15,34], the role of the protein in recruitment of GAL1 and other genes remains controversial. We found that Nup2 is essential for recruitment of both GAL1 and INO1 to the nuclear periphery [35], whereas Cabal et al. reported that GAL1 recruitment takes place normally in a nup2-D mutant using a similar assay [13]. Likewise, they found that the gene is recruited in cells lacking Nup60 [13], which tethers Nup2 to the nuclear pore basket [36]. Similarly, the Stutz group found that the basket-associated protein Mlp1 is essential for recruitment of GAL10 and HSP104 [37] whereas Cabal et al. found it to be dispensable [13]. Therefore, although the nuclear pore basket is implicated in recruitment of several genes to the periphery, it is uncertain whether there are basket substructures that are sufficient for gene recruitment. Does transcription drive peripheral localization? When it was discovered that certain genes are recruited to the nuclear pore complex upon activation, it was unclear whether localization at the nuclear periphery is an active targeting process, coordinated with transcription and mRNA export, or simply an effect of coupling mRNA production with mRNA export through the NPC. Could a change in gene localization and physical association with the pore arise from association of nascent transcripts with the mRNA export machinery and NPC? Most genes that undergo recruitment are highly expressed [38,39]. Therefore, it is conceivable that the mRNA export machinery forms a bridge between the nascent message and the NPC, leading to peripheral localization of genes. Consistent with this possibility, the association of Mlp1 with pheromone-induced genes is sensitive to RNase treatment [14]. Similarly, recruitment of HXK1 and GAL1 to the nuclear periphery requires their 30 untranslated regions (UTRs), which are involved in efficient mRNA export and processing [11,16]. Finally, loss of Sac3 (part of the Sac3–Thp1–Cdc31 mRNA export complex [40]) or Mex67 (an mRNA export receptor) blocks recruitment of the GAL1–10 locus [13,37]. These results suggested that the nascent transcript and mRNA export machinery mediate gene recruitment. Alternatively, post-transcriptional mRNA might regulate maintenance of GAL1 at the nuclear envelope [11]. Heterologous mRNA expressed from a GAL1 promoter accumulates at foci near the nuclear periphery and this localization is modulated by the GAL1 30 -UTR [11]. The localization of these mRNA foci is distinct from the localization of the DNA, indicating that they represent post-transcriptional forms of the transcript. Furthermore, the foci persist at the nuclear periphery after transcriptional shutoff [11]. Thus, post-transcriptional events might also affect tethering of recruited genes to the NPC. In contrast to these results, other findings argue that gene recruitment is independent of transcription. Using a
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Figure 1. A model for gene recruitment at multiple stages of transcription. Gene recruitment has been shown to require factors involved in transcription and mRNA export (shown as blue proteins; see text and Table 1 for references). However, several groups have found that transcription is not required. The model proposes that there are different phases of recruitment that require different factors. (a) Initial recruitment occurs upstream of transcription and involves nuclear pore–promoter interactions, which are regulated by transcription factors. This can occur either after the association of nucleoplasmic transcription factors or through the interaction of the promoter with NPCassociated transcription factors, like the Nup84-bound Rap1–Gcr1–Gcr2 transcription machinery [44]. (b) Following recruitment, the NPC-associated SAGA histone acetyltransferase is recruited to the promoter, linking the DNA to the periphery through interactions involving the Sus1 and Sac3–Thp1 proteins. (c) Alternatively, the mRNA export receptor Mex67 can be recruited through an initial RNA-independent interaction with the TREX complex [63] and subsequently be transferred to the mRNA. Mex67 also directly interacts with components of the NPC. Steps proposed in (b) and (c) are not mutually exclusive and can be coordinated in the same pathway, as represented by the hatched arrow. Pol II, RNA polymerase II.
temperature-sensitive mutant in the large subunit of RNA polymerase II (rpb1–1), Schmid et al. showed that the nuclear pore protein Nup2 interacts with the promoter of GAL1–10 in the absence of RNA polymerase II function [25]. They also showed that Nup2 interaction was independent of the SAGA complex, suggesting that Nup2 interaction occurs before the preinitiation complex is formed [25]. Because Nup2 is rapidly exchanged between its nucleoplasmic and NPC-associated pools [33], the interaction of Nup2 with the promoter of GAL1 might not be a perfect indicator of gene relocalization to the nuclear
periphery. However, using a chromatin localization assay in the rpb1–1 mutant, we found that INO1 recruitment to the nuclear periphery occurs normally after global inactivation of RNA polymerase II [35]. Also, association of the mRNA export factor Mex67 with GAL10 is RNA-independent, indicating that Mex67 might function in gene recruitment by interacting with chromatin through adaptor proteins such as Hpr1 [37,41]. Furthermore, NPC components such as Nup2 interact preferentially with the 50 ends of active genes, rather than 30 ends, as would be expected for RNA-mediated tethering [25,28]. Finally,
Table 1. Summary of yeast genes recruited to the nuclear periphery Gene GAL1–10, GAL7
Genomic region implicated in relocalization to the nuclear periphery Promoter, 30 -UTR
GAL2 INO1 HXK1 HSP104 SUC2
Promoter – 30 -UTR – –
a
Proteins required for relocalization to the nuclear periphery Mlp1a, Mex67, Nup1, Nup2b, Nup60, Ada2, Sac3, Sus1, Gal4 Mlp1, Mex67 Nup2, H2A.Z c – Mlp1, Mex67 Nup84 complex d
Refs [13,25,35,37] [37] [35] [16] [37] [17]
Mlp1 was shown to be required for recruitment of GAL genes by Ref. [37] but found to be dispensable by Ref. [13]. Nup2 was shown to be required for recruitment of GAL genes by Ref. [35] but found to be dispensable by Ref. [13]. c H2A.Z is only required for retention of INO1 at the periphery after transcriptional repression. It is not essential for recruitment in the activated state of the gene [35]. d G. Santangelo, personal communication. b
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recruitment of GAL1 [11] and GAL2 [37] requires sequences in the promoter but is independent of the coding sequence. Therefore, although gene recruitment might function to couple transcription and mRNA export, the localization event itself is an active process that can be separated from transcription. It is worth pointing out that the gene-gating model does not demand that mRNA tethers genes to the NPC; proximity of genes to the nuclear pore might improve nuclear export regardless of the mechanism of localization. In this case, export and translation of mRNAs from genes with the same basal transcription rate would be improved by proximity to the pore. The recruited gene could therefore be tethered physically through sequences in the promoter and this tethering could still promote export of mRNA to the cytoplasm. The SAGA complex The SAGA (Spt–Ada–Gcn5 acetyltransferase) complex is a transcriptional coactivator that alters gene expression by acetylating histones in the promoters of target genes [42]. Gcn5 is the catalytic subunit of this complex [42]. The SAGA complex has been proposed to associate physically with the NPC through a bridging interaction involving the Sac3 and Sus1 proteins [32], and several studies have suggested a role for this complex in promoting gene recruitment to the nuclear periphery (Figure 1b; [13,43]). Cabal et al., using gene localization experiments, found that mutants lacking the SAGA components Sus1 and Ada2 were defective for GAL1 recruitment [13]. A recent study by Luthra et al. [43] described a link between the SAGA complex and the NPC-associated protein Mlp1. Chromatin immunoprecipitation experiments demonstrate that Mlp1 interacts with active genes at the nuclear periphery [14,15]. Furthermore, mutants lacking Mlp1 fail to recruit GAL1 to the nuclear periphery [37]. Luthra et al. found that Mlp1 interacts directly with SAGA. Similar to Nup2 [25], both Mlp1 and SAGA interact with the upstream activating sequence (UAS) elements in the GAL1–10 promoter [43]. The interaction of Mlp1 with the promoter requires the SAGA complex [43]. Intriguingly, the interaction of Nup2 with the GAL UAS is independent of SAGA [25]. Therefore, Nup2 and Mlp1 interact with the GAL locus by distinct mechanisms, perhaps representing distinct stages of recruitment (Figure 1). Nup2 interactions with the GAL UAS might represent an early event, preceding recruitment of the SAGA complex to the activated promoter. Because Nup2 dynamically exchanges between the nucleoplasm and the NPC [33], it is an attractive candidate for a shuttle protein that mediates the initial relocalization event. Mlp1 association might represent a second stage in the process, downstream of SAGA recruitment to the promoter and perhaps downstream of transcription [14] (Figure 1c). Functional significance Gene recruitment as a means of optimal expression Why are active genes recruited to the nuclear periphery? When INO1 gene recruitment was initially discovered, we showed that artificially tethering the gene to the nuclear envelope bypassed the requirement for a transcription www.sciencedirect.com
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factor in its activation [12]. Subsequent work from our laboratory has shown that tethering results in more rapid activation of INO1 [35]. Likewise, LexA fusions to nucleoporins of the Nup84 subcomplex activate reporter genes, suggesting that localization at the NPC is sufficient to activate transcription [17,44]. In addition, transcription of the HXK1 gene is enhanced by tethering to the nuclear periphery [16]. Thus, localization at the nuclear periphery promotes enhanced transcription. Peripheral localization might promote transcription in several ways. Expression of genes could be enhanced through recruitment and proper assembly of the transcription initiation machinery. Recruitment of Rap1–Gcr1–Gcr2activated genes to the Nup84 subcomplex has been proposed to provide a platform for assembly of the transcription machinery [44]. Intriguingly, Rap1-binding sites are enriched in the promoters of genes that interact with the NPC by chromatin immunoprecipitation [15]. Alternatively, chromatin-remodeling events important for transcriptional activation might be promoted by localization at the nuclear periphery. This model is particularly intriguing in light of a requirement for the SAGA complex in gene recruitment [13]. SAGA is required for full transcriptional activation of GAL1 [45], INO1 [46] and SUC2 [47]. Thus, gene recruitment might directly or indirectly affect formation of the transcription preinitiation complex. Consistent with this possibility, upstream activating sequences control the interactions of certain genes with nuclear pore components [25]. These results suggest that gene recruitment is coordinated with the activation event and that localization at the periphery promotes transcription initiation, but is not absolutely required for transcription under steady-state conditions [13,37]. Despite coupling of transcription to peripheral localization, loss of Nup2, Mlp1 and SAGA components that are required for gene recruitment does not dramatically affect steady-state transcriptional activity of GAL1 [13,43]. Similarly, GAL10 expression is not disrupted in mlp1D cells [37]. Therefore, although gene recruitment might enhance the rate of transcriptional activation, it is not absolutely required for transcriptional activation. Gene localization as a source of transcriptional memory The INO1 and GAL1 genes are randomly localized when repressed and peripherally localized when activated. Analysis of the localization of INO1 and GAL1 upon shifting from activating to repressing conditions, however, revealed an unexpected result. Although both genes are transcriptionally repressed within minutes, they linger at the nuclear periphery for hours after repression [35]. Localization at the nuclear periphery is a heritable trait; after shifting from activating conditions, cells maintain INO1 and GAL1 at the nuclear periphery through multiple cell divisions [35]. Peripheral localization therefore represents a novel epigenetic state. This suggests a stable and active mechanism for retention of these genes at the nuclear periphery, even in the absence of ongoing transcription. The maintenance of genes at the nuclear periphery after repression suggests that repressed INO1 and GAL1 are present in two states: a long-term repressed form
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Figure 2. Chromatin-mediated transcriptional memory at the nuclear periphery. In their long-term repressed state, certain genes localize randomly within the nucleoplasm. Upon activation, relocalization to the nuclear periphery is mediated through interactions with the nuclear pore complex (NPC) and associated components. Following repression, incorporation of histone variant H2A.Z (red nucleosomes) marks these genes, leading to their retention at the nuclear periphery and promoting rapid reactivation. Adapted with permission from Ref. [35].
distributed randomly in the nucleoplasm and a recently repressed form localized at the nuclear periphery (Figure 2). This suggests that localization might enable cells to distinguish recently repressed genes from long-term repressed genes. This difference represents a form of cellular memory of recent transcription. This memory is adaptive; recently repressed GAL1 is activated much more rapidly than the long-term repressed form, even after many generations of repression [35]. Furthermore, reactivation of INO1 is delayed in a mutant lacking the Nup2 protein [35]. Considering that artificial tethering of INO1 causes more rapid activation, these results suggest that localization of genes at the periphery promotes more rapid activation or reactivation. This form of transcriptional memory requires the histone variant H2A.Z, which is frequently found in nucleosomes in the promoters of repressed genes and has been proposed to promote rapid activation [48–51]. Mutants lacking H2A.Z fail to retain INO1 and GAL1 at the nuclear periphery after repression and show a dramatic defect in reactivation of these genes [35]. Importantly, this effect is specific to the memory state. Mutants lacking H2A.Z recruit INO1 and GAL1 normally under activating conditions and have no defect in activation of long-term repressed forms of these genes [35]. Three important conclusions of this study are: (i) the repressed forms of certain genes assume two different distributions, enabling cells to ‘mark’ previously transcribed genes; (ii) transcriptional memory is an epigenetic phenomenon, being inherited for 3–4 generations for INO1 and >65 generations for GAL1; and (iii) memory is adaptive, enhancing the rate of reactivation of INO1 and GAL1. In the case of the GAL1 gene, reactivation rate is much more rapid following repression for 12 h than in long-term repressed cells. After 12 h, cells have undergone 6–7 doublings and thus only 1% of the cells have ever directly experienced growth in galactose. Therefore, this phenomenon is a type of adaptive www.sciencedirect.com
learning that can affect the responsiveness of a population of cells to changes in growth conditions for many generations. Recent work in Drosophila has shown that another histone variant, H3.3, can also serve as a marker of transcriptionally active regions of the genome [52]. This variant histone differs from histone H3 at four amino acids [52]. Unlike histone H3, histone H3.3 is incorporated into chromatin through a replication-independent mechanism, perhaps coupled to transcription [52]. Genome-wide chromatin immunoprecipitation has revealed two sites of enrichment of incorporation of H3.3: transcriptionally active genes and regulatory elements [53,54]. H3.3 is enriched in the dosagecompensated male X chromosome, which localizes to the nuclear periphery [20,53]. The incorporation of H3.3 into these regions seems to be the result of a greater turnover of these nucleosomes and the replacement of H3 with H3.3 [54]. Therefore, the incorporation of H3.3, like H2A.Z, could serve to mark active or poised parts of the genome. These marks could be faithfully replicated during DNA replication, enabling their epigenetic inheritance. Concluding remarks and future directions Recruitment of activated genes to the nuclear periphery in yeast is a new and exciting example of the relationship between subnuclear localization and transcriptional state. Although dynamic recruitment of activated genes to the nuclear periphery has not been described in other organisms, the conventional role of the periphery as a silencing environment needs revision. Examples from mice and Drosophila indicate that localization at the periphery is compatible with transcriptional activation [19,20,55]. Localization of genes at the nuclear pore might also have important medical relevance; several forms of acute myeloid leukemia result from the fusion of DNAbinding domains to nuclear pore proteins, presumably leading to relocalization of target genes to the nuclear periphery and altered gene expression [56,57]. A fundamental understanding of how subnuclear positioning affects normal regulation is essential to understanding such disease states. In their activated state, certain genes relocalize to the nuclear periphery by a rapid, active process that is independent of transcription. At the periphery, these genes interact with components of the nuclear pore and SAGA complex. Robust transcription occurs after these genes localize to the periphery [35], and artificially localizing genes to the periphery promotes stronger transcription [16,35]. Thus, recruitment might provide an optimal environment for expression and mRNA export. Localization is maintained long after transcriptional repression, promoting reactivation by a memory mechanism that requires the histone variant H2A.Z. Adaptive memory might enable genes to bypass rate-limiting steps in their activation, leading to more rapid reactivation when exposed to activating conditions once again. Recent studies have provided valuable insights into the mechanism and functional significance of gene recruitment. There are still many missing pieces to the puzzle. To date, nuclear pore, mRNA export and SAGA complex components have been shown to have a role in gene relocalization. However, because the histone variant H2A.Z is
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Box 1. Important future questions about adaptive transcriptional memory What controls H2A.Z incorporation in the recently repressed promoter? How does this incorporation promote faster transcriptional reactivation of genes that have been active in the recent past? What is the mechanism of H2A.Z-dependent targeting of recently repressed genes to the nuclear periphery? How widespread and conserved is the phenomenon of adaptive transcriptional memory? What determines the duration of memory? Do all loci at which H2A.Z is found localize to the nuclear periphery?
required to maintain genes at the nuclear periphery, it is clear that nonperipheral proteins can also be involved. The mechanism of gene recruitment depends on both peripheral proteins that target genes to the NPC and cis-acting DNA elements in the vicinity of recruited genes. GAL1 reporter constructs exhibited similar relocalization behavior when placed at a nonendogenous locus [11]. These reporters contained only the GAL promoter and 30 -UTR encompassing a green fluorescent protein open reading frame, indicating that the GAL1 ORF and neighboring sequences are dispensable for recruitment to the nuclear periphery [11]. Studying the role of promoters and 30 -UTRs in recruitment of genes to the nuclear periphery will reveal whether their role is based on mRNA export and processing or in recruitment of proteins involved in gene localization. It is possible that microenvironments at the nuclear periphery are exploited by gene recruitment to the NPC. It remains unclear if recruitment occurs to any of the nuclear pores within the nuclear envelope, or to particular nuclear pores. In other words, can a gene be recruited to any nuclear pore or do constraints on mobility imposed by the position of a gene within the chromosome define which nuclear pores it can visit? Along these lines, are all NPCs equal or is there some specialization of function among the pores in a nucleus? Several results hint that not all NPCs are identical and that genes are recruited to a subset of pores. The myosin-like proteins Mlp1 and Mlp2, which have a role in gene recruitment [14,15, 37,43], localize in a crescent-shaped distribution at the nuclear periphery, colocalizing only with those nuclear pores on the side of the nucleus opposite the nucleolus [58,59]. Therefore, only a subset of NPCs are associated with Mlp1 and Mlp2. Intriguingly, the mating pheromone-induced gene FIG2 is recruited to the nuclear periphery in a polarized manner [14], localizing to a region close to the spindle pole body [60–62]. Therefore, activated genes might be recruited to distinct locations at the nuclear periphery, defined by the subset of pores to which the genes have access. The discovery that H2A.Z has a key role in maintaining peripheral localization of genes, and hence their faster reactivation after a short period of repression, raises several fascinating questions (Box 1). The answers to these questions will provide crucial insights into how the localization and history of a locus have long-lasting effects on its transcriptional regulation. www.sciencedirect.com
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Acknowledgements We thank Rick Gaber and Eric Weiss, in addition to members of the Brickner laboratory, for helpful comments on the manuscript. We also thank George Santangelo for communicating unpublished results.
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26 Rout, M.P. and Aitchison, J.D. (2001) The nuclear pore complex as a transport machine. J. Biol. Chem. 276, 16593–16596 27 Lim, R.Y. and Fahrenkrog, B. (2006) The nuclear pore complex up close. Curr. Opin. Cell Biol. 18, 342–347 28 Blobel, G. (1985) Gene gating: a hypothesis. Proc. Natl. Acad. Sci. U. S. A. 82, 8527–8529 29 Suntharalingam, M. and Wente, S.R. (2003) Peering through the pore: nuclear pore complex structure, assembly, and function. Dev. Cell 4, 775–789 30 Tran, E.J. and Wente, S.R. (2006) Dynamic nuclear pore complexes: life on the edge. Cell 125, 1041–1053 31 Fischer, T. et al. (2002) The mRNA export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores. EMBO J. 21, 5843–5852 32 Rodriguez-Navarro, S. et al. (2004) Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery. Cell 116, 75–86 33 Dilworth, D.J. et al. (2001) Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex. J. Cell Biol. 153, 1465– 1478 34 Schmid, M. et al. (2004) ChIC and ChEC; genomic mapping of chromatin proteins. Mol. Cell 16, 147–157 35 Brickner, D.G. et al. (2007) H2A.Z-mediated localization of genes at the nuclear periphery confers epigenetic memory of previous transcriptional state. PLoS Biol. 5, e81 36 Denning, D. et al. (2001) The nucleoporin Nup60p functions as a Gsp1pGTP-sensitive tether for Nup2p at the nuclear pore complex. J. Cell Biol. 154, 937–950 37 Dieppois, G. et al. (2006) Cotranscriptional recruitment to the mRNA export receptor Mex67p contributes to nuclear pore anchoring of activated genes. Mol. Cell. Biol. 26, 7858–7870 38 Hirsch, J.P. and Henry, S.A. (1986) Expression of the Saccharomyces cerevisiae inositol-1-phosphate synthase (INO1) gene is regulated by factors that affect phospholipid synthesis. Mol. Cell. Biol. 6, 3320–3328 39 St John, T.P. and Davis, R.W. (1981) The organization and transcription of the galactose gene cluster of Saccharomyces. J. Mol. Biol. 152, 285–315 40 Fischer, T. et al. (2004) Yeast centrin Cdc31 is linked to the nuclear mRNA export machinery. Nat. Cell Biol. 6, 840–848 41 Gwizdek, C. et al. (2006) Ubiquitin-associated domain of Mex67 synchronizes recruitment of the mRNA export machinery with transcription. Proc. Natl. Acad. Sci. U. S. A. 103, 16376–16381 42 Grant, P.A. et al. (1997) Yeast Gcn5 functions in two multisubunit complexes to acetylate nucleosomal histones: characterization of an Ada complex and the SAGA (Spt/Ada) complex. Genes Dev. 11, 1640– 1650 43 Luthra, R. et al. (2006) Actively transcribed GAL genes can be physically linked to the nuclear pore by the SAGA chromatin modifying complex. J. Biol. Chem. 282, 3042–3049 44 Menon, B.B. et al. (2005) Reverse recruitment: the Nup84 nuclear pore subcomplex mediates Rap1/Gcr1/Gcr2 transcriptional activation. Proc. Natl. Acad. Sci. U. S. A. 102, 5749–5754
45 Larschan, E. and Winston, F. (2005) The Saccharomyces cerevisiae Srb8-Srb11 complex functions with the SAGA complex during Gal4-activated transcription. Mol. Cell. Biol. 25, 114–123 46 Lo, W.S. et al. (2001) Snf1 – a histone kinase that works in concert with the histone acetyltransferase Gcn5 to regulate transcription. Science 293, 1142–1146 47 Roberts, S.M. and Winston, F. (1997) Essential functional interactions of SAGA, a Saccharomyces cerevisiae complex of Spt, Ada, and Gcn5 proteins, with the Snf/Swi and Srb/mediator complexes. Genetics 147, 451–465 48 Guillemette, B. et al. (2005) Variant histone H2A.Z is globally localized to the promoters of inactive yeast genes and regulates nucleosome positioning. PLoS Biol. 3, e384 49 Li, B. et al. (2005) Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling. Proc. Natl. Acad. Sci. U. S. A. 102, 18385–18390 50 Raisner, R.M. et al. (2005) Histone variant H2A.Z marks the 50 ends of both active and inactive genes in euchromatin. Cell 123, 233–248 51 Zhang, H. et al. (2005) Genome-wide dynamics of Htz1, a histone H2A variant that poises repressed/basal promoters for activation through histone loss. Cell 123, 219–231 52 Ahmad, K. and Henikoff, S. (2002) The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol. Cell 9, 1191–1200 53 Mito, Y. et al. (2005) Genome-scale profiling of histone H3.3 replacement patterns. Nat. Genet. 37, 1090–1097 54 Mito, Y. et al. (2007) Histone replacement marks the boundaries of cis-regulatory domains. Science 315, 1408–1411 55 Buscaino, A. et al. (2006) X-chromosome targeting and dosage compensation are mediated by distinct domains in MSL-3. EMBO Rep. 7, 531–538 56 Nakamura, T. et al. (1996) Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia. Nat. Genet. 12, 154–158 57 Lawrence, H.J. et al. (1999) Frequent co-expression of the HOXA9 and MEIS1 homeobox genes in human myeloid leukemias. Leukemia 13, 1993–1999 58 Galy, V. et al. (2004) Nuclear retention of unspliced mRNAs in yeast is mediated by perinuclear Mlp1. Cell 116, 63–73 59 Strambio-de-Castillia, C. et al. (1999) Proteins connecting the nuclear pore complex with the nuclear interior. J. Cell Biol. 144, 839–855 60 Baba, M. et al. (1989) Three-dimensional analysis of morphogenesis induced by mating pheromone alpha factor in Saccharomyces cerevisiae. J. Cell Sci. 94, 207–216 61 Yang, C.H. et al. (1989) Higher order structure is present in the yeast nucleus: autoantibody probes demonstrate that the nucleolus lies opposite the spindle pole body. Chromosoma 98, 123–128 62 Snyder, M. et al. (1991) Studies concerning the temporal and genetic control of cell polarity in Saccharomyces cerevisiae. J. Cell Biol. 114, 515–532 63 Strasser, K. et al. (2002) TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417, 304–308
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Regulation and epigenetic control of transcription at the nuclear periphery Sara Ahmed and Jason H. Brickner Department of Biochemistry, Molecular Biology and Cell Biology Northwestern University, Evanston, IL 60208, USA
The localization of DNA within the nucleus influences the regulation of gene transcription. Subnuclear environments at the nuclear periphery promote gene silencing and activation. Silenced regions of the genome, such as centromeres and telomeres, are statically tethered to the nuclear envelope. Recent work in yeast has revealed that certain genes can undergo dynamic recruitment to the periphery upon transcriptional activation. For such genes, localization to the periphery has been suggested to improve mRNA export and favor optimal transcription. In addition, maintenance of peripheral localization confers cellular memory of previous transcriptional activation, enabling cells to adapt rapidly to transcriptional cues. Localization at the nuclear periphery promotes transcriptional silencing and activation The eukaryotic nucleus has different compartments, such as the nucleolus, nuclear envelope and nuclear pores, each with distinct structures and functions. The nuclear pore provides a gateway to the nucleus, enabling exchange of proteins and mRNA with the cytoplasm, whereas the nucleolus serves as the site for ribosomal component assembly and synthesis [1,2]. Subnuclear compartments or domains have also been implicated in regulation of transcription. Although transcription is regulated mainly through DNA-binding proteins that affect RNA polymerase recruitment or local chromatin structure, there is also a functional relationship between gene expression and nuclear organization. Chromosomes are nonrandomly arranged within the nucleus in diverse eukaryotic organisms, suggesting that subnuclear domains might have specialized roles in chromatin organization and transcription. In many metazoan cells, chromosomes fold back onto themselves to create distinct subnuclear territories [3]. Transcriptionally active genes frequently localize at the edge of such territories, and it has been proposed that this localization enables better access to stable transcriptional ‘factories’ between territories [4]. The arrangement of these chromosome territories within the nucleus is uniform in cells within the same tissue and can be conserved between species [5]. One of the best-characterized subnuclear domains is the nuclear periphery. Localization of parts of the genome to the nuclear periphery has important effects on transcription. Constitutive heterochromatin localizes to the nuclear periphery in many cell types [6,7], as do other silenced loci, Corresponding author: Brickner, J.H. ([email protected]). Available online 12 June 2007. www.sciencedirect.com
such as the immunoglobulin genes in hematopoietic progenitors of B lymphocytes [8]. Localization of repressed parts of the genome to the nuclear periphery has been thoroughly explored in the budding yeast Saccharomyces cerevisiae, in which regions of transcriptionally silent chromatin, such as telomeres and the mating type loci, associate with the nuclear envelope [9]. Proximity to the nuclear periphery promotes efficient silencing of genes near telomeres [9]. Conversely, artificially tethering mating-type loci with crippled silencing elements to the nuclear envelope promotes silencing [10]. Recent work, however, indicates that transcriptional activation also takes place at the nuclear periphery in yeast. Several dynamically regulated genes are randomly distributed in the nucleoplasm when repressed but are recruited to the nuclear periphery when activated. These genes include many highly expressed genes such as INO1 (encoding an enzyme involved in phospholipid biosynthesis), HSP104 (a molecular chaperone), HXK1 (hexokinase), SUC2 (invertase), GAL1 (galactokinase), GAL2 (a hexose transporter), GAL10 (glucose epimerase) and mating pheromone-induced genes [11–17]. The recruitment of active genes to the nuclear periphery in yeast is reminiscent of the observation made over 20 years ago by Hutchison and Weintraub that in mouse fibroblasts DNase I-sensitive (and presumably transcriptionally active) regions of the genome were found localized along the nuclear rim [18]. This suggests that transcriptional activation at the nuclear periphery might not be a yeast-specific phenomenon. The mouse b-globin locus is transcriptionally active at the nuclear periphery before its relocalization toward the nuclear interior during erythroid maturation [19]. This indicates that localization at the nuclear periphery is not incompatible with transcription in mammalian cells. Also, the transcriptional upregulation of the X chromosome in males in Drosophila requires nuclear pore components Mtor
Glossary Mex: mRNA export Mlp: Myosin-like protein Mtor: Megator Nic: Nuclear pore interacting complex Nup: nucleoporin Sac: Suppressor of actin Thp: Tho/Hpr1 phenotype TPR: Translocated promoter region TREX: transcription and export complex, a complex involved in cotranscriptional mRNA processing and transport, composed of the THO complex (Hpr1, Tho2, Mft1 and Thp2) and the Yra1–Sub2 complex
0168-9525/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2007.05.009
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(also called TPR) and Nup153 (see Glossary) and the X chromosome localizes at the nuclear periphery [20]. In yeast, gene recruitment to the periphery changes the subnuclear distribution of genes and reduces their mobility to a constrained, two-dimensional movement near the nuclear envelope [13,21]. Genome-wide chromatin immunoprecipitation studies reveal that many transcriptionally active genes physically interact with components of the nuclear pore complex (NPC) and associated factors [14,15]. Intriguingly, the nuclear pore component Nup2 is necessary and sufficient (when tethered to DNA directly) to create boundary elements that block the spread of heterochromatin [22–24]. Nup2 also interacts with transcriptionally active loci [15,25], suggesting that the protein might help to shield these regions from neighboring silenced DNA (see below). The nuclear periphery could represent a subnuclear domain that is itself composed of distinct microenvironments with roles in either gene silencing or activation. This review focuses on the mechanism of gene recruitment to the nuclear periphery in yeast. In addition, we highlight the potential functional significance of localizing genes to the nuclear periphery, particularly in promoting rapid transcriptional activation of recruited genes and in establishing novel epigenetic states. The mechanism of gene recruitment to the nuclear periphery The nuclear pore complex One of the most fascinating features of gene recruitment is that cells dynamically control the localization of particular parts of the genome. The mechanism(s) by which this is achieved are not only interesting in themselves, but also could have more general implications for our understanding of how genomes are spatially organized. The nuclear pore complex (NPC) has been identified as the site to which these genes are recruited [15]. The NPC is a large assembly of 30 core nucleoporin proteins and numerous accessory proteins [26] that perforates the nuclear envelope and serves essential roles in nucleocytoplasmic trafficking [27]. As articulated by Blobel’s ‘gene-gating’ hypothesis, the processes of transcription and mRNA export might be coupled through interactions with the NPC [28]. According to this model, nuclear pore complexes might interact with expanded, transcriptionally active portions of the genome, promoting optimal export of the transcript through the pore to which it is gated [28]. The observation that many genes are recruited to the nuclear periphery upon activation and that they physically interact with the NPC has inspired a re-evaluation of this appealing hypothesis. The yeast nucleoporins Nup2 [15,25], Nup60, Nic96, Nup116 and the myosin-like proteins Mlp1 and Mlp2 [14,15] physically interact with active genes. The NPC has three distinct parts: the cytoplasmic filaments, the core channel and the nucleoplasmic basket [29,30]. The components of the nucleoplasmic basket interact with several proteins implicated in mRNA export – such as Mlp1, Mlp2, Sac3, Thp1 and Mex67 [31] – and transcription – such as the Spt–Ada–Gcn5 histone acetyltransferase (SAGA) complex [32] (Figure 1). Loss of many of these proteins blocks gene recruitment, suggesting a general www.sciencedirect.com
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requirement for the nucleoplasmic basket and associated factors in this phenomenon (Table 1). Although there is general agreement that the nuclear basket is important for gene recruitment, it is unclear if all basket proteins are essential. Although Nup2, a component of the nuclear basket [33], promotes boundary activity and physically interacts with the promoter of GAL1 under activating conditions [14,15,34], the role of the protein in recruitment of GAL1 and other genes remains controversial. We found that Nup2 is essential for recruitment of both GAL1 and INO1 to the nuclear periphery [35], whereas Cabal et al. reported that GAL1 recruitment takes place normally in a nup2-D mutant using a similar assay [13]. Likewise, they found that the gene is recruited in cells lacking Nup60 [13], which tethers Nup2 to the nuclear pore basket [36]. Similarly, the Stutz group found that the basket-associated protein Mlp1 is essential for recruitment of GAL10 and HSP104 [37] whereas Cabal et al. found it to be dispensable [13]. Therefore, although the nuclear pore basket is implicated in recruitment of several genes to the periphery, it is uncertain whether there are basket substructures that are sufficient for gene recruitment. Does transcription drive peripheral localization? When it was discovered that certain genes are recruited to the nuclear pore complex upon activation, it was unclear whether localization at the nuclear periphery is an active targeting process, coordinated with transcription and mRNA export, or simply an effect of coupling mRNA production with mRNA export through the NPC. Could a change in gene localization and physical association with the pore arise from association of nascent transcripts with the mRNA export machinery and NPC? Most genes that undergo recruitment are highly expressed [38,39]. Therefore, it is conceivable that the mRNA export machinery forms a bridge between the nascent message and the NPC, leading to peripheral localization of genes. Consistent with this possibility, the association of Mlp1 with pheromone-induced genes is sensitive to RNase treatment [14]. Similarly, recruitment of HXK1 and GAL1 to the nuclear periphery requires their 30 untranslated regions (UTRs), which are involved in efficient mRNA export and processing [11,16]. Finally, loss of Sac3 (part of the Sac3–Thp1–Cdc31 mRNA export complex [40]) or Mex67 (an mRNA export receptor) blocks recruitment of the GAL1–10 locus [13,37]. These results suggested that the nascent transcript and mRNA export machinery mediate gene recruitment. Alternatively, post-transcriptional mRNA might regulate maintenance of GAL1 at the nuclear envelope [11]. Heterologous mRNA expressed from a GAL1 promoter accumulates at foci near the nuclear periphery and this localization is modulated by the GAL1 30 -UTR [11]. The localization of these mRNA foci is distinct from the localization of the DNA, indicating that they represent post-transcriptional forms of the transcript. Furthermore, the foci persist at the nuclear periphery after transcriptional shutoff [11]. Thus, post-transcriptional events might also affect tethering of recruited genes to the NPC. In contrast to these results, other findings argue that gene recruitment is independent of transcription. Using a
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Figure 1. A model for gene recruitment at multiple stages of transcription. Gene recruitment has been shown to require factors involved in transcription and mRNA export (shown as blue proteins; see text and Table 1 for references). However, several groups have found that transcription is not required. The model proposes that there are different phases of recruitment that require different factors. (a) Initial recruitment occurs upstream of transcription and involves nuclear pore–promoter interactions, which are regulated by transcription factors. This can occur either after the association of nucleoplasmic transcription factors or through the interaction of the promoter with NPCassociated transcription factors, like the Nup84-bound Rap1–Gcr1–Gcr2 transcription machinery [44]. (b) Following recruitment, the NPC-associated SAGA histone acetyltransferase is recruited to the promoter, linking the DNA to the periphery through interactions involving the Sus1 and Sac3–Thp1 proteins. (c) Alternatively, the mRNA export receptor Mex67 can be recruited through an initial RNA-independent interaction with the TREX complex [63] and subsequently be transferred to the mRNA. Mex67 also directly interacts with components of the NPC. Steps proposed in (b) and (c) are not mutually exclusive and can be coordinated in the same pathway, as represented by the hatched arrow. Pol II, RNA polymerase II.
temperature-sensitive mutant in the large subunit of RNA polymerase II (rpb1–1), Schmid et al. showed that the nuclear pore protein Nup2 interacts with the promoter of GAL1–10 in the absence of RNA polymerase II function [25]. They also showed that Nup2 interaction was independent of the SAGA complex, suggesting that Nup2 interaction occurs before the preinitiation complex is formed [25]. Because Nup2 is rapidly exchanged between its nucleoplasmic and NPC-associated pools [33], the interaction of Nup2 with the promoter of GAL1 might not be a perfect indicator of gene relocalization to the nuclear
periphery. However, using a chromatin localization assay in the rpb1–1 mutant, we found that INO1 recruitment to the nuclear periphery occurs normally after global inactivation of RNA polymerase II [35]. Also, association of the mRNA export factor Mex67 with GAL10 is RNA-independent, indicating that Mex67 might function in gene recruitment by interacting with chromatin through adaptor proteins such as Hpr1 [37,41]. Furthermore, NPC components such as Nup2 interact preferentially with the 50 ends of active genes, rather than 30 ends, as would be expected for RNA-mediated tethering [25,28]. Finally,
Table 1. Summary of yeast genes recruited to the nuclear periphery Gene GAL1–10, GAL7
Genomic region implicated in relocalization to the nuclear periphery Promoter, 30 -UTR
GAL2 INO1 HXK1 HSP104 SUC2
Promoter – 30 -UTR – –
a
Proteins required for relocalization to the nuclear periphery Mlp1a, Mex67, Nup1, Nup2b, Nup60, Ada2, Sac3, Sus1, Gal4 Mlp1, Mex67 Nup2, H2A.Z c – Mlp1, Mex67 Nup84 complex d
Refs [13,25,35,37] [37] [35] [16] [37] [17]
Mlp1 was shown to be required for recruitment of GAL genes by Ref. [37] but found to be dispensable by Ref. [13]. Nup2 was shown to be required for recruitment of GAL genes by Ref. [35] but found to be dispensable by Ref. [13]. c H2A.Z is only required for retention of INO1 at the periphery after transcriptional repression. It is not essential for recruitment in the activated state of the gene [35]. d G. Santangelo, personal communication. b
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recruitment of GAL1 [11] and GAL2 [37] requires sequences in the promoter but is independent of the coding sequence. Therefore, although gene recruitment might function to couple transcription and mRNA export, the localization event itself is an active process that can be separated from transcription. It is worth pointing out that the gene-gating model does not demand that mRNA tethers genes to the NPC; proximity of genes to the nuclear pore might improve nuclear export regardless of the mechanism of localization. In this case, export and translation of mRNAs from genes with the same basal transcription rate would be improved by proximity to the pore. The recruited gene could therefore be tethered physically through sequences in the promoter and this tethering could still promote export of mRNA to the cytoplasm. The SAGA complex The SAGA (Spt–Ada–Gcn5 acetyltransferase) complex is a transcriptional coactivator that alters gene expression by acetylating histones in the promoters of target genes [42]. Gcn5 is the catalytic subunit of this complex [42]. The SAGA complex has been proposed to associate physically with the NPC through a bridging interaction involving the Sac3 and Sus1 proteins [32], and several studies have suggested a role for this complex in promoting gene recruitment to the nuclear periphery (Figure 1b; [13,43]). Cabal et al., using gene localization experiments, found that mutants lacking the SAGA components Sus1 and Ada2 were defective for GAL1 recruitment [13]. A recent study by Luthra et al. [43] described a link between the SAGA complex and the NPC-associated protein Mlp1. Chromatin immunoprecipitation experiments demonstrate that Mlp1 interacts with active genes at the nuclear periphery [14,15]. Furthermore, mutants lacking Mlp1 fail to recruit GAL1 to the nuclear periphery [37]. Luthra et al. found that Mlp1 interacts directly with SAGA. Similar to Nup2 [25], both Mlp1 and SAGA interact with the upstream activating sequence (UAS) elements in the GAL1–10 promoter [43]. The interaction of Mlp1 with the promoter requires the SAGA complex [43]. Intriguingly, the interaction of Nup2 with the GAL UAS is independent of SAGA [25]. Therefore, Nup2 and Mlp1 interact with the GAL locus by distinct mechanisms, perhaps representing distinct stages of recruitment (Figure 1). Nup2 interactions with the GAL UAS might represent an early event, preceding recruitment of the SAGA complex to the activated promoter. Because Nup2 dynamically exchanges between the nucleoplasm and the NPC [33], it is an attractive candidate for a shuttle protein that mediates the initial relocalization event. Mlp1 association might represent a second stage in the process, downstream of SAGA recruitment to the promoter and perhaps downstream of transcription [14] (Figure 1c). Functional significance Gene recruitment as a means of optimal expression Why are active genes recruited to the nuclear periphery? When INO1 gene recruitment was initially discovered, we showed that artificially tethering the gene to the nuclear envelope bypassed the requirement for a transcription www.sciencedirect.com
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factor in its activation [12]. Subsequent work from our laboratory has shown that tethering results in more rapid activation of INO1 [35]. Likewise, LexA fusions to nucleoporins of the Nup84 subcomplex activate reporter genes, suggesting that localization at the NPC is sufficient to activate transcription [17,44]. In addition, transcription of the HXK1 gene is enhanced by tethering to the nuclear periphery [16]. Thus, localization at the nuclear periphery promotes enhanced transcription. Peripheral localization might promote transcription in several ways. Expression of genes could be enhanced through recruitment and proper assembly of the transcription initiation machinery. Recruitment of Rap1–Gcr1–Gcr2activated genes to the Nup84 subcomplex has been proposed to provide a platform for assembly of the transcription machinery [44]. Intriguingly, Rap1-binding sites are enriched in the promoters of genes that interact with the NPC by chromatin immunoprecipitation [15]. Alternatively, chromatin-remodeling events important for transcriptional activation might be promoted by localization at the nuclear periphery. This model is particularly intriguing in light of a requirement for the SAGA complex in gene recruitment [13]. SAGA is required for full transcriptional activation of GAL1 [45], INO1 [46] and SUC2 [47]. Thus, gene recruitment might directly or indirectly affect formation of the transcription preinitiation complex. Consistent with this possibility, upstream activating sequences control the interactions of certain genes with nuclear pore components [25]. These results suggest that gene recruitment is coordinated with the activation event and that localization at the periphery promotes transcription initiation, but is not absolutely required for transcription under steady-state conditions [13,37]. Despite coupling of transcription to peripheral localization, loss of Nup2, Mlp1 and SAGA components that are required for gene recruitment does not dramatically affect steady-state transcriptional activity of GAL1 [13,43]. Similarly, GAL10 expression is not disrupted in mlp1D cells [37]. Therefore, although gene recruitment might enhance the rate of transcriptional activation, it is not absolutely required for transcriptional activation. Gene localization as a source of transcriptional memory The INO1 and GAL1 genes are randomly localized when repressed and peripherally localized when activated. Analysis of the localization of INO1 and GAL1 upon shifting from activating to repressing conditions, however, revealed an unexpected result. Although both genes are transcriptionally repressed within minutes, they linger at the nuclear periphery for hours after repression [35]. Localization at the nuclear periphery is a heritable trait; after shifting from activating conditions, cells maintain INO1 and GAL1 at the nuclear periphery through multiple cell divisions [35]. Peripheral localization therefore represents a novel epigenetic state. This suggests a stable and active mechanism for retention of these genes at the nuclear periphery, even in the absence of ongoing transcription. The maintenance of genes at the nuclear periphery after repression suggests that repressed INO1 and GAL1 are present in two states: a long-term repressed form
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Figure 2. Chromatin-mediated transcriptional memory at the nuclear periphery. In their long-term repressed state, certain genes localize randomly within the nucleoplasm. Upon activation, relocalization to the nuclear periphery is mediated through interactions with the nuclear pore complex (NPC) and associated components. Following repression, incorporation of histone variant H2A.Z (red nucleosomes) marks these genes, leading to their retention at the nuclear periphery and promoting rapid reactivation. Adapted with permission from Ref. [35].
distributed randomly in the nucleoplasm and a recently repressed form localized at the nuclear periphery (Figure 2). This suggests that localization might enable cells to distinguish recently repressed genes from long-term repressed genes. This difference represents a form of cellular memory of recent transcription. This memory is adaptive; recently repressed GAL1 is activated much more rapidly than the long-term repressed form, even after many generations of repression [35]. Furthermore, reactivation of INO1 is delayed in a mutant lacking the Nup2 protein [35]. Considering that artificial tethering of INO1 causes more rapid activation, these results suggest that localization of genes at the periphery promotes more rapid activation or reactivation. This form of transcriptional memory requires the histone variant H2A.Z, which is frequently found in nucleosomes in the promoters of repressed genes and has been proposed to promote rapid activation [48–51]. Mutants lacking H2A.Z fail to retain INO1 and GAL1 at the nuclear periphery after repression and show a dramatic defect in reactivation of these genes [35]. Importantly, this effect is specific to the memory state. Mutants lacking H2A.Z recruit INO1 and GAL1 normally under activating conditions and have no defect in activation of long-term repressed forms of these genes [35]. Three important conclusions of this study are: (i) the repressed forms of certain genes assume two different distributions, enabling cells to ‘mark’ previously transcribed genes; (ii) transcriptional memory is an epigenetic phenomenon, being inherited for 3–4 generations for INO1 and >65 generations for GAL1; and (iii) memory is adaptive, enhancing the rate of reactivation of INO1 and GAL1. In the case of the GAL1 gene, reactivation rate is much more rapid following repression for 12 h than in long-term repressed cells. After 12 h, cells have undergone 6–7 doublings and thus only 1% of the cells have ever directly experienced growth in galactose. Therefore, this phenomenon is a type of adaptive www.sciencedirect.com
learning that can affect the responsiveness of a population of cells to changes in growth conditions for many generations. Recent work in Drosophila has shown that another histone variant, H3.3, can also serve as a marker of transcriptionally active regions of the genome [52]. This variant histone differs from histone H3 at four amino acids [52]. Unlike histone H3, histone H3.3 is incorporated into chromatin through a replication-independent mechanism, perhaps coupled to transcription [52]. Genome-wide chromatin immunoprecipitation has revealed two sites of enrichment of incorporation of H3.3: transcriptionally active genes and regulatory elements [53,54]. H3.3 is enriched in the dosagecompensated male X chromosome, which localizes to the nuclear periphery [20,53]. The incorporation of H3.3 into these regions seems to be the result of a greater turnover of these nucleosomes and the replacement of H3 with H3.3 [54]. Therefore, the incorporation of H3.3, like H2A.Z, could serve to mark active or poised parts of the genome. These marks could be faithfully replicated during DNA replication, enabling their epigenetic inheritance. Concluding remarks and future directions Recruitment of activated genes to the nuclear periphery in yeast is a new and exciting example of the relationship between subnuclear localization and transcriptional state. Although dynamic recruitment of activated genes to the nuclear periphery has not been described in other organisms, the conventional role of the periphery as a silencing environment needs revision. Examples from mice and Drosophila indicate that localization at the periphery is compatible with transcriptional activation [19,20,55]. Localization of genes at the nuclear pore might also have important medical relevance; several forms of acute myeloid leukemia result from the fusion of DNAbinding domains to nuclear pore proteins, presumably leading to relocalization of target genes to the nuclear periphery and altered gene expression [56,57]. A fundamental understanding of how subnuclear positioning affects normal regulation is essential to understanding such disease states. In their activated state, certain genes relocalize to the nuclear periphery by a rapid, active process that is independent of transcription. At the periphery, these genes interact with components of the nuclear pore and SAGA complex. Robust transcription occurs after these genes localize to the periphery [35], and artificially localizing genes to the periphery promotes stronger transcription [16,35]. Thus, recruitment might provide an optimal environment for expression and mRNA export. Localization is maintained long after transcriptional repression, promoting reactivation by a memory mechanism that requires the histone variant H2A.Z. Adaptive memory might enable genes to bypass rate-limiting steps in their activation, leading to more rapid reactivation when exposed to activating conditions once again. Recent studies have provided valuable insights into the mechanism and functional significance of gene recruitment. There are still many missing pieces to the puzzle. To date, nuclear pore, mRNA export and SAGA complex components have been shown to have a role in gene relocalization. However, because the histone variant H2A.Z is
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Box 1. Important future questions about adaptive transcriptional memory What controls H2A.Z incorporation in the recently repressed promoter? How does this incorporation promote faster transcriptional reactivation of genes that have been active in the recent past? What is the mechanism of H2A.Z-dependent targeting of recently repressed genes to the nuclear periphery? How widespread and conserved is the phenomenon of adaptive transcriptional memory? What determines the duration of memory? Do all loci at which H2A.Z is found localize to the nuclear periphery?
required to maintain genes at the nuclear periphery, it is clear that nonperipheral proteins can also be involved. The mechanism of gene recruitment depends on both peripheral proteins that target genes to the NPC and cis-acting DNA elements in the vicinity of recruited genes. GAL1 reporter constructs exhibited similar relocalization behavior when placed at a nonendogenous locus [11]. These reporters contained only the GAL promoter and 30 -UTR encompassing a green fluorescent protein open reading frame, indicating that the GAL1 ORF and neighboring sequences are dispensable for recruitment to the nuclear periphery [11]. Studying the role of promoters and 30 -UTRs in recruitment of genes to the nuclear periphery will reveal whether their role is based on mRNA export and processing or in recruitment of proteins involved in gene localization. It is possible that microenvironments at the nuclear periphery are exploited by gene recruitment to the NPC. It remains unclear if recruitment occurs to any of the nuclear pores within the nuclear envelope, or to particular nuclear pores. In other words, can a gene be recruited to any nuclear pore or do constraints on mobility imposed by the position of a gene within the chromosome define which nuclear pores it can visit? Along these lines, are all NPCs equal or is there some specialization of function among the pores in a nucleus? Several results hint that not all NPCs are identical and that genes are recruited to a subset of pores. The myosin-like proteins Mlp1 and Mlp2, which have a role in gene recruitment [14,15, 37,43], localize in a crescent-shaped distribution at the nuclear periphery, colocalizing only with those nuclear pores on the side of the nucleus opposite the nucleolus [58,59]. Therefore, only a subset of NPCs are associated with Mlp1 and Mlp2. Intriguingly, the mating pheromone-induced gene FIG2 is recruited to the nuclear periphery in a polarized manner [14], localizing to a region close to the spindle pole body [60–62]. Therefore, activated genes might be recruited to distinct locations at the nuclear periphery, defined by the subset of pores to which the genes have access. The discovery that H2A.Z has a key role in maintaining peripheral localization of genes, and hence their faster reactivation after a short period of repression, raises several fascinating questions (Box 1). The answers to these questions will provide crucial insights into how the localization and history of a locus have long-lasting effects on its transcriptional regulation. www.sciencedirect.com
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Acknowledgements We thank Rick Gaber and Eric Weiss, in addition to members of the Brickner laboratory, for helpful comments on the manuscript. We also thank George Santangelo for communicating unpublished results.
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