A Mark in the Core

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neuronal cells, GPCR stimulation leads to activation of the calcium- and cell adhesion-dependent FAK family kinase, Pyk2. Tyrosine autophosphorylated Pyk2 then binds the c-Src SH2 domain, triggering the phosphorylation of adaptor proteins and Ras-dependent ERK activation (Dikic et al., 1996). Another mechanism involves the release of preformed ligands for the epidermal growth factor (EGF) from the cell surface in response to G protein-dependent activation of matrix metalloproteinases (Prenzel et al., 1999). The local generation of EGF-like hormones, such as heparin binding EGF, results in autocrine or paracrine activation of EGF receptors and Rasdependent signaling. This pathway too involves Src, although it is less clear whether Src activity is involved upstream or downstream of the EGF receptor. Finally, the c-Src catalytic domain has been found to bind to ␤ arrestins, proteins that complex with agonist-occupied GPCRs and mediate the process of homologous receptor desensitization (Luttrell et al., 1999). B-arrestindependent Src activation has been implicated in the control of ERK activation, receptor endocytosis, and neutrophil degranulation in some systems. Each new example of the regulation of tyrosine kinase activity by heterotrimeric G proteins further blurs the once sharp line of distinction drawn between signals emanating from classical tyrosine kinase growth factor receptors and heptahelical GPCRs. With Csk now shown to be under G protein control, heptahelical receptors are poised to finely tune the balance between Srcdependent cell proliferation and cytoskeletal disassembly and Csk-dependent Src inhibition and cytoskeletal organization.

Louis M. Luttrell The Department of Medicine Duke University Medical Center Durham, North Carolina 27710 The Geriatrics Research, Education and Clinical Center Durham Veterans Affairs Medical Center Durham, North Carolina 27705

A Mark in the Core: Silence No More!

labs, centers on the conserved tails of the histones, the proteins that wrap the DNA to form the basic subunits of chromatin called nucleosomes. The hypothesis states that posttranslational modifications of histone tails play a central role in establishing chromatin domains. The histone tails protrude from the nucleosome core body and thus are readily available for interaction with other proteins. The tails of each of the histones H1-H4 offer residues that can be subject to various modifications, including acetylation, phosphorylation, methylation, ubiquitination, and poly-ADP-ribosylation. Combinations of these specific modifications are thought to produce a nucleosomal “barcode,” which is then read out by other factors, e.g., ATP-dependent chromatin remodeling factors. These histone modifications can, under certain circumstances, be heritable, and thus represent a possible mechanism of epigenetic inheritance. Enzymes that methylate histones have only recently been identified (reviewed in Zhang and Reinberg, 2001). Other proteins recognize the specific methylation status of histones and mediate the formation of specific chromatin structures: heterochromatin protein 1 (and its fission yeast homolog Swi6) binds specifically to histone H3 methylated at lysine 9 and forms condensed chromatin (reviewed in Jenuwein, 2001; Grewal and Elgin, 2002),

The histone modification repertoire has recently been expanded. Dot1p is a new type of methyltransferase that methylates lysine 79 in the histone H3 core only in its nucleosomal context and has a possible role in marking open chromatin regions. Elucidating the mechanisms by which DNA is packed into specialized chromatin domains is of profound importance for the understanding of gene regulation. Unlike in higher eukaryotes, most of the chromatin in budding yeast is quite “open” or accessible to incoming factors, and only a few areas (less than 10% of the genome) are packaged into transcriptionally silenced heterochromatin-like structures. These structures include the chromatin close to telomeres, the silent mating-type loci, HML and HMR, that contain information for mating-type switching, and the rDNA locus. The histonecode hypothesis (Strahl and Allis, 2000; Jenuwein and Allis, 2001), currently being verified by the work of many

Selected Reading Arthur, W.T., Petch, L.A., and Burridge, K. (2000). Curr. Biol. 10, 719–722. Chang, J.H., Gill, S., Settleman, J., and Parsons, S.J. (1995). J. Cell Biol. 130, 355–386. Dikic, I., Tokiwa, G., Lev, S., Courtneidge, S.A., and Schlessinger, J. (1996). Nature 383, 547–550. Lowry, W.E., Huang, J., Ma, Y.-C., Ali, S., Wang, D., Williams, D.M., Okada, M., Cole, P.A., and Huang, X.-Y. (2002). Dev. Cell 2, 733–744. Luttrell, L.M., Ferguson, S.S., Daaka, Y., Miller, W.E., Maudsley, S., Della Rocca, G.J., Lin, F., Kawakatsu, H., Owada, K., Luttrell, D.K., et al. (1999). Science 283, 655–661. Ma, Y.C., Huang, J., Ali, S., Lowry, W., and Huang, X.Y. (2000). Cell 102, 635–646. Nada, S., Okada, M., MacAuley, A., Cooper, J.A., and Nakagawa, H. (1991). Nature 351, 69–72. Prenzel, N., Zwick, E., Daub, H., Leserer, M., Abraham, R., Wallasch, C., and Ullrich, A. (1999). Nature 402, 884–888. Ridley, A.J., and Hall, A. (1994). EMBO J. 13, 2600–2610. Thomas, S.M., Soriano, P., and Imamoto, A. (1995). Nature 376, 267–271. van Biesen, T., Hawes, B.E., Luttrell, D.K., Krueger, K.M., Touhara, K., Porfiri, E., Sakaue, M., Luttrell, L.M., and Lefkowitz, R.J. (1995). Nature 376, 781–784.

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Proposed Roles of Dot1p Function in Regulating Chromatin Structure (A) Schematic outline of two opposing pathways for establishing euchromatin and heterochromatic domains through histone H3 K79. Black line, DNA; green clover, nucleosome; wiggly green lines, histone tails; blue triangles, acetyl groups; red circle, methylated K79 in histone H3; blue ellipse, SIR protein complex. (B) Mechanism for epigenetic inheritance of the euchromatic states involving Dot1p-mediated methylation of H3.

and methylation of histone H3 lysine 4 prevents the transcriptional repressor NuRD from interacting with H3 (Zegerman et al., 2002). Exciting papers by Van Leeuwen et al. (2002), in a recent issue of Cell, and by Hui Ng et al. (2002), establish a new histone methylation site at the histone H3 core (lysine 79) rather than at the tail. This lysine residue is located on the accessible surface on the outside (the “top” and “bottom”) of the nucleosome core and does not contact DNA or other histones in the crystal structure (Luger et al., 1997). Van Leeuwen et al. and Hui Ng et al. demonstrate that S. cerevisiae DOT1 encodes a new type of histone methyltransferase (without the characteristic SET domain) that methylates histone H3 exclusively at K79. Both groups also demonstrate that H3 K79 is essential for establishment of silenced regions as point mutations abolish silencing and reduce Sir2p and Sir3p association with silenced regions. Sir2p and Sir3p belong to the so-called silencing information regulator (SIR) proteins and are involved in setting up condensed chromatin structures in budding yeast. Dot1p was originally identified as causing derepression of silenced telomeric reporter genes when overexpressed (Singer et al., 1998). A deletion of DOT1 also leads to derepression of telomeric marker genes. However, Dot1p function is not limited to heterochromatin. Indeed, Van Leeuwen et al. establish that in wild-type strains an astonishing 90% of the histone H3s are to a varying degree (mono-, di-, or tri-) methylated at position K79 in a Dot1p-dependent manner, and overexpression of Dot1p leads to close to 100% trimethylation of this residue. In support of this observation, Hui Ng et al. (2002) detect global H3 K79 methylation, including in a telomeric region, by chromatin immunoprecipitation using an antiserum specific against methylated H3 K79. However, it still remains unknown whether there is a differ-

ence in the amount of mono-, di-, and tri-methylated H3 K79 between open and silenced chromatin. The global presence of Dot1p-methylated H3 leads Van Leeuwen et al. to argue that since only a reduction is observed in Sir2p and Sir3p localization at telomeric and matingtype donor loci, both when Dot1p is deleted or overexpressed, but almost abolished when H3 K79 is mutated, Dot1p-mediated methylation might indeed act to define euchromatic regions. They suggest that the observed derepression of silenced regions is an indirect effect due to a distortion of an equilibrium, which both groups agree may encompass dislocalization of Sir proteins. Two competitive processes centering on H3 K79 to establish this equilibrium might exist (Figure, panel A). The first process depends on methylation of H3 K79 by Dot1p and may play a role in positively marking euchromatin. H3 K79 methylation could directly affect the chromatin fiber structure by preventing interactions between nucleosomes or may promote specific interactions of other proteins. The second process positively marks heterochromatin and acts through proteins that interact with unmethylated H3 K79. Possible candidates are Sir2p and Sir3p. Thus, these two competing processes could stabilize the presence and boundaries of both types of chromatin structures. Both groups suggest that Dot1p might also have a role in epigenetic inheritance. Notably, unlike all the other identified HMTs with the exception of the recently identified yeast and PR-Set7 (Strahl et al., 2002; Nishioka et al., 2002), Dot1p does not methylate isolated histones, but only H3 in the nucleosome context. This specificity of Dot1p may allow for newly assembled and parentally inherited nucleosomes to be distinguished after DNA replication. Absence of K79 methylation in the newly synthesized chromatin could mark newly formed nucleosomes for other modifications, such as acetyla-

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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

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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.