Pushing the Envelope

Molecular Cell 1156

tion, to propagate the parental chromatin structure. Accessibility may be a requirement for Dot1p modification, and SIR-protein binding in the condensed heterochromatin could prevent H3 K79 methylation. If methylation at H3 K79 prevents chromatin fiber condensation, the modification itself could in turn allow H3 K79 methylation by Dot1p of neighboring nucleosomes, establishing the epigenetic propagation of the euchromatic state (Figure, panel B). The phylogenetically conserved nature of Dot1p and its substrate suggests that related enzymes play an important role establishing the chromatin domains in higher eukaryotes and, indeed, Hui Ng et al. (2002) find methylated H3 K79 in calf thymus and human cells. The future will undoubtedly reveal a general importance of this expansion of the histone code to include the modification of cores as well as tails.

Selected Reading

Patrick D. Varga-Weisz and Jacob Z. Dalgaard Marie Curie Research Institute The Chart Oxted RH8 0TL, Surrey United Kingdom

Van Leeuwen, F., Gafken, P.R., and Gottschling, D.E. (2002). Cell 109, 745–756.

Pushing the Envelope: Chromatin Boundaries at the Nuclear Pore

silent information regulator proteins (SIRs), at the nuclear periphery (Maillet et al., 1996). If this serves as a specialized silencing compartment enriched in transcriptional repressors, turning genes off may be regulated by distance to the periphery or by looping that could move a gene into this compartment. There are data supporting both mechanisms. In the first case, silencing decreases as a function of distance from yeast telomeres (Gottschling et al., 1990). In the second case, defective silencing can be restored by repositioning a gene to the nuclear membrane (Andrulis et al., 1998). These results suggest two ways in which barriers might work as anti-silencers. A barrier might interfere with the propagation of heterochromatin by creating an obstacle or a nucleosomal gap or by serving as an entry site for remodeling activities (Bi and Broach, 2001; Donze and Kamakaka, 2002). In the second case, restricting the movement of a chromatin domain by as yet undescribed attachments or constraints could block repositioning to a silencing compartment. It is reasonable to imagine that barrier activity may be achieved by several different mechanisms (West et al., 2002). The two silent mating type loci in yeast, like telomeres, are specific heterochromatic domains that can mediate gene nonspecific silencing by virtue of flanking E and I silencers (Bi and Broach, 2001). The silencers are loading sites for Sir proteins that spread along chromatin to repress genes located in between. Silencing by E or I alone can be blocked by a single barrier inserted in between the silencer and the promoter. Barriers have been found to recruit specialized proteins, termed barrier activities (BA), such as BEAF-32 and CTCF, which are able to block the spreading of heterochromatin (West et al., 2002). The ability of barriers to establish a nonsilenced domain within heterochromatin was the

In a novel genetic screen, the nuclear-cytoplasmic transport system was found to reposition DNA to the nuclear pore and establish a barrier to the spread of heterochromatin. These data provide a mechanism for movement and attachment of DNA to a functional nuclear compartment. Heterochromatin is a repressive, self-propagating structure that, if unimpeded, can lead to the inappropriate silencing of nearby genes (Moazed, 2001). On the other hand, intact heterochromatin at telomeres and centromeres is necessary for proper chromosome segregation. Therefore, preserving the structural requirement of heterochromatin while inhibiting its ability to spread requires specialized elements. These are termed boundary elements or barriers and represent a collection of powerful, yet incompletely understood, determinants of chromatin structure and transcriptional regulation (West et al., 2002). It has been known for some time that heterochromatin is nonrandomly positioned in the nucleus. Bulk heterochromatin is localized to the nuclear periphery and is in close association with the nuclear membrane. In mammals, the constitutively condensed and transcriptionally inactive X chromosome, the Barr body, is stably located at the nuclear envelope. In yeast as well, telomeric heterochromatin and the silent mating type loci are found together in foci, along with the major cellular supply of

Grewal, S.I., and Elgin, S.C. (2002). Curr. Opin. Genet. Dev. 12, 178–187. Hui Ng, H., Feng, Q., Wang, H., Erdjument-Bromage, H., Tempst, P., Zhang, Y., and Struhl, K. (2002). Genes Dev. 16, 1518–1527. Jenuwein, T. (2001). Trends Cell Biol. 11, 266–273. Jenuwein, T., and Allis, C.D. (2001). Science 293, 1074–1080. Luger, K., Maeder, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T. (1997). Nature 389, 251–260. Nishioka, K., Rice, J.C., Sarma, K., Erdjument-Bromage, H., Werner, J., Wang, Y., Chuikov, S., Valenzuela, P., Tempst, P., Steward, R., et al. (2002). Mol. Cell 9, 1201–1213. Singer, M.S., Kahana, A., Wolf, A.J., Meisinger, L.L., Peterson, S.E., Goggin, C., Mahowald, M., and Gottschling, D.E. (1998). Genetics 150, 613–632. Strahl, B.D., and Allis, C.D. (2000). Nature 403, 41–45. Strahl, B.D., Grant, P.A., Briggs, S.D., Sun, Z.W., Bone, J.R., Caldwell, J.A., Mollah, S., Cook, R.G., Shabanowitz, J., Hunt, D.F., et al. (2002). Mol. Cell. Biol. 22, 1298–1306.

Zegerman, P., Canas, B., Pappin, D., and Kouzarides, T. (2002). J. Biol. Chem. 277, 11621–11624. Zhang, Y., and Reinberg, D. (2001). Genes Dev. 15, 2343–2360.

Previews 1157

The Boundary-Trap Assay The ADE2 and URA3 genes can adopt three different expression states when inserted into the silent mating type locus, HML. In the parental strain shown on the top line, both genes are repressed by heterochromatin spreading between E and I silencers (blue triangles) represented by the thick blue bar. This is described by the OFF/OFF state reflecting the expression of the ADE2 and URA3 genes, respectively. After the binding of a barrier activity (BA) to the Gal4 sites (UASg), shown on the middle line, heterochromatin spreading from E and I is disrupted to create an accessible minidomain between the UASg sites indicated by the thin beaded string. In these strains, ADE2 is derepressed while URA3 remains inactive due to silencing by I. As a result, the locus is in the ON/OFF state. On the other hand, when UASg is bound by a transcriptional activator (TA), silencing is lost at both ADE2 and URA3, producing the ON/ON state. The activity conferred by UASg in the presence of various Gal4 DNA binding domain (Gbd) fusions to the UASg is shown as yellow triangle when unbound (or bound by Gbd alone), red when bound by Gbd fused to a protein possessing barrier activity, and green when bound by Gbd fused to a protein possessing transcriptional activator function.

basis for a boundary-trap screen used by Ishii et al. (2002) in the June 14 issue of Cell to identify new components of barriers as well as pathways of barrier function. In this screen, Gal4 fusions having barrier activity can be distinguished from classical activators. The ADE2 and URA3 genes were inserted into the silent mating type locus (HML) with Gal4 sites placed both upstream and downstream of ADE2. URA3 is located 3⬘ to ADE2, positioning Gal4 sites within its promoter region (see Figure). In this configuration, both genes were in a repressed state (OFF/OFF). Gal4 fusions that could derepress ADE2 but not URA3 produce the ON/OFF state. Conversely, Gal4 fused to any domain capable of stimulating transcription would induce both ADE2 and URA3 (ON/ON state). Boundary activities in this screen are therefore defined as establishing a minidomain within preexisting heterochromatin and are transcriptionally neutral. Transforming the boundary trap strain with a yeast Gal4 fusion library and selecting for the ON/OFF phenotype yielded the unexpected finding that several members of the transportin family were able to establish

functional barriers within heterochromatin. The transportins included the importins Sxm1 and Srp1; exportins Cse1, Los1, and Mex67; and Gsp2, a yeast homolog of the small GTPase Ran. These proteins form complexes that move protein and RNA toward and away from the nuclear pore. Their identification in this screen raised the exciting possibility that these proteins could be part of a complex that moves DNA as well. How then can a transportin contribute to barrier activity? In a series of experiments, barrier activity was shown to require binding to the nuclear pore complex (NPC), and, when tethered to DNA, the transportin can reposition chromatin to the nuclear envelope. Strikingly, barrier activity of all Gal4-transportin fusions was dependent on Nup2. This was subsequently validated by real-time imaging and chromatin immunoprecipitation. In live cells, the binding of a lacI-Cse1 fusion led to the repositioning of a lac operator array to the nuclear rim by a Nup2-dependent process. Additionally, the ADE2 gene was isolated by immunoprecipitation of crosslinked chromatin using an anti-NPC antibody. Together, these observations confirm that transportin-mediated BA re-

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flects the stable association of barrier DNA at the NPC. One interesting exception is that the BA mediated by Gal4 fused to BEAF-32, a factor which binds to the Drosophila scs’ barrier/insulator, is functionally independent of Nup2 and does not require or lead to association with nuclear pores. This serves as an example of a second, NPC-independent, mechanism for achieving a transcriptionally neutral heterochromatin barrier (West et al., 2002). Among the surprising conclusions to be drawn from these findings is that despite several lines of evidence indicating that the nuclear periphery is heterochromatic, expression of ADE2 is facilitated by attachment to the envelope. Previously transcriptional repression at telomeres has been shown to require NPC interactions and is disrupted by the loss of two nucleoporins, Nup60 and Nup145 (Feuerbach et al., 2002). Photomicrographs show that envelope-associated heterochromatin is gapped by nuclear pore-associated channels of less condensed chromatin that might be shielded from repression. This provides a visual basis for considering how a pore-associated barrier may segregate neighboring chromatin into expressed (ADE2) and repressed (URA3) genes at the periphery. The nuclear envelope is looking like a traffic jam with possibly specialized NPC domains of repression and derepression residing in close proximity. It remains to be seen what cellular DNA sequences are associated with the NPC, if these can serve as barriers, and whether NPC targeting can establish a barrier in other eukaryotic cells. In considering that the organization of DNA within the interphase nucleus was unlikely to be random, Blobel

proposed the “gene-gate” hypothesis (Blobel, 1985). In this model, the primary determinant of the 3D organization of the interphase nucleus was the attachment of chromatin to nuclear pores. The latest findings of Laemmli and colleagues (Ishii et al., 2002) reflect on Blobel’s vision and portend a new level of interest in the NPC with regard to transcriptional control and DNA positioning for the future.

Histone Variants and Nucleosome Deposition Pathways

viding cells undergoing continued gene expression, strictly limiting histone deposition to S phase could destroy chromosome integrity. Thus, most organisms encode histone sequence variants that are expressed at low levels constitutively during the cell cycle. These variants serve as replacement histones for replicationindependent (RI) nucleosome assembly. An intriguing consequence of RI assembly is an obligatory change in the histone sequence composition of chromatin over time, and it has long been speculated that this might provide a mechanism for establishing different functional chromatin states (van Holde, 1989). One of the best studied of the replacement histones is H3.3, which differs from the major histone H3 protein at four amino acid residues, one in the N-terminal domain and three clustered within the histone fold domain (Figure, panel A; Thatcher et al., 1994). The relative importance of constitutive expression versus the variant amino acid sequence for RI assembly was first addressed by gene knockout experiments in Tetrahymena (Yu and Gorovsky, 1997). Tetrahymena contains two cell cycle regulated genes, HHT1 and HHT2, that encode the major histone H3 protein for RC assembly, and a third constitutively expressed gene, HHT3 (hv2), that encodes an H3.3-like variant histone for RI assembly.

In this issue of Molecular Cell, Ahmad and Henikoff show that the replication-independent pathway of chromatin assembly in vivo can discriminate between different histone variants on the basis of their primary amino acid sequences. These results have important implications for chromatin remodeling and epigenetic imprinting. During each division cycle, as cells replicate their DNA they must also synthesize an equal mass of histone proteins and assemble them together into nucleosomes. To meet this cyclical demand, bulk histone gene expression is tightly regulated during S phase (Osley, 1991). This replication coupled (RC) pathway involves a variety of components, including PCNA, histone acetylation, chromatin assembly factors such as CAF-1 and RCAF, Hir proteins, and other histone chaperones (Verreault, 2000; Mello and Almouzni, 2001). But for cells that encounter DNA damage outside of S phase, or for nondi-

Nobuhiko Shinkura and William C. Forrester Department of Pathology Harvard Medical School 200 Longwood Avenue Boston, Massachusetts 02115 Selected Reading Andrulis, E.D., Neiman, A.M., Zappula, D.C., and Sternglanz, R. (1998). Nature 394, 592–595. Bi, X., and Broach, J.R. (2001). Curr. Opin. Genet. Dev. 11, 199–204. Blobel, G. (1985). Proc. Natl. Acad. Sci. USA 82, 8527–8529. Donze, D., and Kamakaka, R.T. (2002). Bioessays 24, 344–349. Feuerbach, F., Galy, V., Trelles-Sticken, E., Fromont-Racine, M., Jacquier, A., Gilson, E., Olivo-Marin, J.C., Scherthan, H., and Nehrbass, U. (2002). Nat. Cell Biol. 4, 214–221. Gottschling, D.E., Aparicio, O.M., Billington, B.L., and Zakian, V.A. (1990). Cell 63, 751–762. Ishii, K., Arib, G., Lin, C., Van Houwe, G., and Laemmli, U.K. (2002). Cell 109, 551–562. Maillet, L., Boscheron, C., Gotta, M., Gilson, E., and Gasser, S.M. (1996). Genes Dev. 10, 1796–1811. Moazed, D. (2001). Mol. Cell 8, 489–498. West, A.G., Gaszner, M., and Felsenfeld, G. (2002). Genes Dev. 16, 271–288.