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69. Y. Habu, T. Kakutani, J. Paszkowski, Curr. Opin. Genet. 79. C. Cogoni et al., EMBO J. 15, 3153 (1996). 89. P. SanMiguel et al., Science 274, 765 (1996). Dev. 11, 215 (2001). 80. G. Faugeron, Curr. Opin. Microbiol. 3, 144 (2000). 90. R. Mauricio, Nature Rev. Genet. 2, 370 (2001). 70. M. Wassenegger, Plant Mol. Biol. 43, 203 (2000). 81. L. Jackson-Grusby et al., Nature Genet. 27, 31 (2001). 91. P. Cubas, C. Vincent, E. Coen, Nature 401, 157 (1999). 71. M. A. Matzke, A. J. Matzke, J. M. Kooter, Science 293, 82. J. P. Vielle-Calzada, R. Baskar, U. Grossniklaus, Nature 92. R. Martienssen, Curr. Opin. Genet. Dev. 8, 240 1080 (2001). 404, 91 (2000). (1998). 72. J. Bender, Trends Biochem. Sci. 23, 252 (1998). Genes Dev. 11 73. E. U. Selker, Cell 97, 157 (1999). 83. P. S. Springer, D. R. Holding, A. Groover, C. Yordan, 93. Z. J. Chen, C. S. Pikaard, , 2124 (1997). 74. M. N. Raizada, M. I. Benito, V. Walbot, Plant J. 25,79 R. A. Martienssen, Development 127, 1815 (2000). 94. L. Comai et al., Plant Cell 12, 1551 (2000). (2001). 84. J. P. Vielle-Calzada et al., Genes Dev. 13, 2971 (1999). 95. H. S. Lee, Z. J. Chen, Proc. Natl. Acad. Sci. U.S.A. 98, 75. R. F. Ketting, T. H. Haverkamp, H. G. van Luenen, R. H. 85. R. Vinkenoog et al., Plant Cell 12, 2271 (2000). 6753 (2001). Plasterk, Cell 99, 133 (1999). 86. S. Adams, R. Vinkenoog, M. Spielman, H. G. Dickinson, 96. We thank E. Selker, E. Richards, V. Chandler, S. Kaep- 76. H. Tabara et al., Cell 99, 123 (1999). R. J. Scott, Development 127, 2493 (2000). pler, S. Jacobsen, and J. Bender for communicating 77. B. H. Ramsahoye et al., Proc. Natl. Acad. Sci. U.S.A. 87. M. Byrne, M. Timmermans, C. Kidner, R. Martienssen, results prior to publication, two anonymous referees 97, 5237 (2000). Curr. Opin. Plant Biol. 4, 38 (2001). for suggestions for improvement, and our colleagues 78. P. Svoboda, P. Stein, H. Hayashi, R. M. Schultz, De- 88. E. B. Cambareri, R. Aisner, J. Carbon, Mol. Cell. Biol. for many interesting discussions. R.M. and V.C. re- velopment 127, 4147 (2000). 18, 5465 (1998). ceive grant support from the NSF (DBI 1057338).

REVIEW Translating the Code Thomas Jenuwein1 and C. David Allis2

Chromatin, the physiological template of all eukaryotic genetic information, is transcriptionally inert are highly condensed subject to a diverse array of posttranslational modifications that largely in the interphase nucleus and remain cytolog- impinge on histone amino termini, thereby regulating access to the underly- ically visible as heterochromatic foci or as the ing DNA. Distinct histone amino-terminal modifications can generate syner- “Barr body,” which is the inactive X chromo- gistic or antagonistic interaction affinities for -associated proteins, some in female mammalian cells (2). The which in turn dictate dynamic transitions between transcriptionally active or distinct levels of chromatin organization are transcriptionally silent chromatin states. The combinatorial nature of histone dependent on the dynamic higher order struc- amino-terminal modifications thus reveals a “histone code” that considerably turing of , which represent the extends the information potential of the genetic code. We propose that this basic repeating unit of chromatin. In each epigenetic marking system represents a fundamental regulatory mechanism , roughly two superhelical turns that has an impact on most, if not all, chromatin-templated processes, with of DNA wrap around an octamer of core far-reaching consequences for cell fate decisions and both normal and patho- histone proteins formed by four histone part- logical development. ners: an H3-H4 tetramer and two H2A-H2B dimers (3). are small basic proteins Genomic DNA is the ultimate template of our nisms for how such a code is “read” and consisting of a globular domain and a more

heredity. Yet despite the justifiable excitement translated into biological functions. flexible and charged NH2-terminus (histone over the human genome, many challenges re- Throughout this review, we have chosen “tail”) that protrudes from the nucleosome. It main in understanding the regulation and trans- epigenetic phenomena and underlying mecha- remains unclear how nucleosomal arrays con- duction of genetic information (1). It is unclear, nisms in two general categories: chromatin- taining linker histone (H1) then twist and fold for example, why the number of protein-coding based events leading to either gene activation or this chromatin fiber into increasingly more genes in humans, now estimated at ϳ35,000, gene silencing. In particular, we center our dis- compacted filaments leading to defined high- only doubles that of the fruit fly Drosophila cussion on examples where differences in “on- er order structures. melanogaster. Is DNA alone then responsible off ” transcriptional states are reflected by dif- Central to our current thinking is that for generating the full range of information that ferences in histone modifications that are either chromatin structure plays an important regu- ultimately results in a complex eukaryotic or- “euchromatic” (on) or “heterochromatic” (off ) latory role and that multiple signaling path- ganism, such as ourselves? (Fig. 1A). We also point out that, despite many ways converge on histones (4). Although We favor the view that , im- elegant genetic and biochemical insights into histone proteins themselves come in generic posed at the level of DNA-packaging proteins chromatin function and gene regulation in the or specialized forms (5), exquisite variation is (histones), is a critical feature of a genome- budding yeast Saccharomyces cerevisiae, some provided by covalent modifications (acetyla- wide mechanism of information storage and of the heterochromatic mechanisms (e.g., HP1- tion, , ) of the hi- retrieval that is only beginning to be under- based gene silencing) discussed here do not stone tail domains, which allow regulatable stood. We propose that a “histone code” ex- exist in an obvious form in this organism. Thus, contacts with the underlying DNA. The en- ists that may considerably extend the infor- we will need to pursue other model systems, zymes transducing these histone tail modifi- mation potential of the genetic (DNA) code. such as Schizosaccharomyces pombe, Caeno- cations are highly specific for particular ami- We review emerging evidence that histone rhabditis elegans, Drosophila, and mice, to no acid positions (6, 7), thereby extending proteins and their associated covalent modi- “crack” the histone code. the information content of the genome past fications contribute to a mechanism that can the genetic (DNA) code. This hypothesis pre- alter chromatin structure, thereby leading to Chromatin Template and Histone dicts that (i) distinct modifications of the inherited differences in transcriptional “on- Code off ” states or to the stable propagation of In the nuclei of all eukaryotic cells, genomic 1Research Institute of Molecular Pathology (IMP) at by defining a specialized high- DNA is highly folded, constrained, and com- the Vienna Biocenter, Dr. Bohrgasse 7, A-1030 Vi- er order structure at centromeres. Under the pacted by histone and nonhistone proteins in enna, Austria. E-mail: [email protected] 2Department of Biochemistry and Molecular Genetics, assumption that a histone code exists, at least a dynamic polymer called chromatin. For University of Virginia Health Science Center, Char- in some form, we discuss potential mecha- example, chromosomal regions that remain lottesville, VA 22908, USA. E-mail: [email protected]

1074 10 AUGUST 2001 VOL 293 SCIENCE www.sciencemag.org E PIGENETICS histone tails would induce interaction affini- the location of a gene within a distinct chro- order chromatin structure more closely re- ties for chromatin-associated proteins, and matin environment, the mat region, dictates sembling that of multicellular eukaryotes, in- (ii) modifications on the same or different the establishment of an active or a silent heritance of silenced chromatin domains has histone tails may be interdependent and gen- transcriptional state. In particular for S. been shown to be remarkably stable during erate various combinations on any one nu- pombe, which appears to contain a higher both mitosis and meiosis (16). cleosome. Here, we wish to extend this concept for overall structure by proposing that (iii) distinct qualities of higher order chromatin, such as euchromatic or heterochromatic do- mains (7), are largely dependent on the local concentration and combination of differentially modified nucleosomes (Fig. 1A). We envision that this “nucleosome code” then permits the assembly of different epigenetic states (7), leading to distinct “readouts” of the genetic information, such as gene activation versus gene silencing or, more globally, cell prolifer- ation versus cell differentiation. Any such mod- el must account for how these epigenetic states are established, maintained, and stably inherited through mitosis and meiosis. Although there is clear evidence for a “cellular memory” (8), sudden switches in cell fate do occur, leading to variegating phenotypes. If the histone code hy- pothesis is correct, at least in part, deciphering how that code is translated into biological re- sponse remains an important and nontrivial challenge. On the basis of current knowledge, other possibilities are likely to exist, including less stringent “charge patches” in histone tails (9). Clear evidence is beginning to link alter- ations in chromatin structure to cell cycle progression, DNA replication, DNA damage and its repair, recombination, and overall chromosome stability (10). It also seems like- ly that the fundamental nature of chromatin- based epigenetics will have an impact on X inactivation, imprinting, developmental re- programming of cell lineages, and the plas- ticity of stem cells. The implications for hu- man biology and disease, including cancer and aging, are far-reaching.

Su(var)s, , and It is now widely recognized that heritable, but Fig. 1. Models for euchromatic or hetero- reversible, changes in can chromatic histone tail modifications. (A) occur without alterations in DNA sequence. Schematic representation of euchromatin and heterochromatin as accessible or Pioneering studies on radiation-induced chro- condensed nucleosome fibers containing mosomal translocations (11) provided some acetylated (Ac), phosphorylated (P), and of the earliest findings that epigenetic “on- methylated (Me) histone NH2-termini. off ” transcriptional states are largely depen- (B) Generic model for antagonistic E(var) dent on the position of a gene within an and Su(var) gene function in adding eu- accessible (euchromatic) or an inaccessible chromatic (EU) or heterochromatic (HET ) modification marks onto a nucleosomal (heterochromatic) chromatin environment. template. In addition, Su(var)s also func- This phenomenon, known as position-effect tion in removing euchromatic signals and variegation (PEV), allowed the development E(var)s can destabilize the heterochro- of genetic screens in Drosophila (12) and S. matic state. (C) Examples of combinato- pombe (13, 14) that have identified ϳ30 to rial modifications in histone NH2-termini 40 loci involved in modifying PEV. Similar that are likely to represent “imprints” for active or inactive chromatin. Single-letter abbreviations for amino acid residues: A, Ala; E, Glu; G, Gly; H, His; K, Lys; L, Leu; M, Met; P, Pro; Q, Gln; R, Arg; to PEV, mating-type switching in budding S, Ser; and T, Thr. (D) Proposed synergistic (connected arrowheads) or antagonistic (blocked oval (15) and fission (16) yeast represents another line) modifications in and H4 NH2-termini. The arrow with the scissors indicates paradigm for a variegating mechanism where possible proteolytic cleavage of the H3 NH2-terminus.

www.sciencemag.org SCIENCE VOL 293 10 AUGUST 2001 1075 E PIGENETICS Among the modifier genes identified in the associated with a Pc-G complex containing subunit of the NuRD complex (44), which above model systems, one subclass suppresses HDAC activity (36), and trx or HRX have harbors two chromodomains and might con- variegation [the Su(var) group] and comprises been shown to interact with components of ceivably recognize dimethylated histone tails gene products such as histone deacetylases chromatin-remodeling machines (37). In gen- in a manner analogous to double bromodo- (HDACs), protein phosphatases (PPTases), and eral terms, Su(var) and Pc-G gene function mains. In this regard, we note that Lys9 and S-adenosylmethionine (SAM) synthetase (17), would be characterized by transducing the Lys27 in the H3 tail are embedded in similar as well as chromatin-associated components addition of heterochromatic marks and the sequence motifs, and both positions are “hot that are best characterized by the heterochroma- removal of euchromatic marks on the chro- spots” for methylation by the SET domain– tin protein HP1 [Su(var)2-5] (18). In addition to matin template. Conversely, the antagonizing containing HMTase G9a (45). the Su(var) group of genes, an antagonizing activity of E(var) and trx-G gene function Finally, a hallmark property of all HP1 class of PEV modifiers enhances variegation would involve the establishment of euchro- proteins is the combination of a chromodo- [E(var) group] (12) and counteracts the Su(var)- matic signals (e.g., increased nucleosome main with a chromoshadow domain that are induced silent chromatin state. Several E(var) mobility) and destabilize or degrade (see be- separated by a short but variable hinge re- gene products are components of adenosine low) heterochromatic “imprints” (Fig. 1B). gion. Because the chromoshadow domain of triphosphate (ATP)–dependent nucleosome-re- HP1 appears to self-dimerize in solution (46, modeling machines, such as the SWI/SNF and Translating the Histone Code 47), it is tempting to infer that full-length brahma complexes (19, 20), which increase The histone code hypothesis predicts that the HP1 may assemble intermolecular chromo- overall nucleosome mobility. modification marks on the histone tails domains, thereby generating a bifunctional Extending these parallels even further, should provide binding sites for effector pro- cross-linker that is likely to stabilize the more Su(var) and E(var) gene products contain teins. In agreement with this notion, the bro- rigid higher order structure of heterochroma- several conserved protein domains—the bro- modomain has been the first protein module tin (35, 48). mo-, chromo-, and SET domains—that are to be shown to selectively interact with a also shared with two other classes of antag- covalent mark (acetylated ) in the his- Combinations and Switches

onizing chromatin regulators: the Polycomb tone NH2-terminal tail (23, 38, 39). In addi- The above examples provide support for mod- (Pc-G) and trithorax (trx-G) groups. The tion to the proteins discussed above, the bro- ification-induced recruitment of chromatin-as- Pc-G and trx-G genes are important for main- modomain is also present in many transcrip- sociated proteins to acetylated and methylated

taining the expression boundaries of the ho- tional regulators having intrinsic histone histone NH2-termini (Fig. 2A), and it is likely meotic selector genes and several other key acetyltransferase (HAT) activity (e.g., GCN5, that other modules exist that specifically recog-

developmental genes (21, 22), presumably by PCAF, TAFII250). Consistent with the sec- nize phosphorylation marks. Consistent with modulating the chromatin structure of their ond prediction of the histone code (that there the second prediction of the histone code hy-

target loci. The bromodomain (23) is found in be combinatorial readout), TAFII250, which pothesis, all four NH2-termini of the core his- SNF2, TAFII250, and mammalian trithorax itself harbors several histone-modifying ac- tones contain short “basic patches” that often (HRX/Mll); the chromodomain (24, 25)is tivities, contains two tandem copies of the comprise , phosphorylation, and shared between Polycomb and HP1; and the bromodomain. In this configuration it pref- methylation marks in close proximity on one SET domain (26) is found in Su(var)3-9, in erentially binds diacetylated histone pep- individual tail (4). All three of these modifica- the Pc-G member E(z), and in trithorax. tides presenting acetyl-lysine moieties that tions can be found both in active or silenced These modules have been widely used during are appropriately spaced (40). Use of the chromatin regions, which raises the question of evolution to generate a considerable function- Simple Modular Architectural Research how combinatorial specificity is used in defin- al diversity among proteins specialized in Tool (SMART; http://smart.embl-heidel- ing an imprint for euchromatin or heterochro- modulating chromatin structure. berg.de) indicates that there are ϳ75 bro- matin (Fig. 1, A and C). Histone acetylation (27, 28) and histone modomain-containing proteins in humans. Some evidence is emerging about a pos- phosphorylation (29) modification systems Several of these proteins, such as human sible combinatorial code. For example, the

have been characterized in detail. A further poly-bromodomain protein 1, exhibit many histone H3 NH2-terminus appears to exist in class of enzymatic activities that regulate the copies (six) of regularly spaced bromodo- two distinct modification states that are likely site-specific addition of methyl groups to hi- mains, which could conceivably bind to a to be regulated by a “switch” between Lys9 stones has recently been described. Original- specific combination of acetyl groups pre- methylation and Ser10 phosphorylation (Fig. ly identified as the PEV modifier Su(var)3-9 sented on one or several histone tails. 1D). Ser10 phosphorylation inhibits Lys9 in Drosophila, homologs from fission yeast Chromodomains, on the other hand, ap- methylation (30) but is synergistically cou- (Clr4) to human (SUV39H1) have been pear to be targeting modules for methylation pled with Lys9 and/or Lys14 acetylation dur- shown to encode histone methyltransferases marks. The chromodomain of HP1 is highly ing mitogenic and hormonal stimulation in (HMTases) that selectively methylate histone selective for methylated H3 at Lys9, and little mammalian cells (49–51). In this phos- H3 at Lys9 (30). The HMTase function in the if any binding is observed with H3 peptides phorylated-acetylated state, the modified H3 Su(var)3-9 family maps to the highly con- containing a methylated Lys4 position (32). tail marks transcriptional activation (Fig. 1C). served SET domain but also requires adjacent Thus, although chromodomains are highly H3 phosphorylation is also important for mi- Cys-rich regions. Notably, generation of the conserved, it seems likely that not all chro- totic chromosome condensation (52), where H3-Lys9 methyl epitope induces a hetero- modomains—nor their methyl targets—be- it may be linked to other secondary signal(s) chromatic affinity for HP1 proteins that rec- have similarly. In support, chromodomain such as the nucleosomal incorporation of the ognize this epigenetic signal through their swapping experiments have not uniformly pericentric H3 analog Cenp-A (53). Con- chromodomains (31, 32). These results pro- indicated functional conservation in silencing versely, aberrant Lys9 methylation antagoniz- vide a strong link among Su(var) function, assays (41, 42). Interestingly, Su(var)3-9 es Ser10 phosphorylation, leading to mitotic gene-silencing activity, and the assembly of HMTase family members also contain a chro- chromosome dysfunction (30, 54). Further, heterochromatin (31–35). modomain, whose integrity is critical for si- deacetylation of Lys14 in H3 (33) is required By contrast, an enzymatic HMTase func- lencing in vivo (33, 43). Several repressive to facilitate subsequent Lys9 methylation by tion has not yet been demonstrated for Pc-G chromatin-remodeling complexes comprise the Clr4 HMTase, again highlighting an or- and trx-G proteins. Instead, E(z) has been components such as the Mi-2/CHD ATPase dered interplay to establish distinct histone

1076 10 AUGUST 2001 VOL 293 SCIENCE www.sciencemag.org E PIGENETICS tail modifications. Although the single H3- Indeed, methylation of Lys4 in H3 occurs may be recruited to their target loci in much Lys9 methyl epitope appears sufficient to re- in transcriptionally active macronuclei of Tet- the same way that Su(var)3-9–catalyzed H3- cruit HP1 to heterochromatic regions, acety- rahymena and appears to be a euchromatic Lys9 methylation triggers the recruitment of lation of Lys12 in H4 is another repressive imprint in a wide range of organisms (63). In HP1 to heterochromatin. mark (55) that may help to reinforce a silent addition, several arginine-directed HMTases, There are several intriguing candidates for chromatin state (Fig. 1C). such as the steroid receptor coactivators such positively acting methyl-docking part- The SUV39H1 HMTase also displays CARM1 and PRMT1, methylate selective ar- ners. The chromodomain-containing HAT, weak activity toward histone H1 (30), and ginine positions in H3 and H4 NH2-termini Esa1, is the only known essential HAT in S. this is likely to involve methylation of Lys26 and induce synergistic transcriptional activa- cerevisiae (27) and represents the catalytic (56). RNA interference (RNAi) for an H1 tion from transiently transfected reporter subunit of the NuA4 HAT complex, which variant was recently shown to phenocopy constructs (64, 65). In vivo evidence that has been linked to transcriptional activation silencing and proliferation defects in the C. histones are physiological targets of these and nucleosome remodeling in yeast and flies elegans germ line (57). These phenotypes are coactivators is beginning to emerge (66, 67). (68, 69). Because Esa1 displays robust in similar to those seen in mes-2 mutants. Mes-2 Assuming that euchromatic methylation vitro acetylation activity toward Lys5 in H4 is a homolog of the SET domain–containing marks exist (Fig. 1C), we predict that chro- (70, 71), it is possible that Arg3 methylation E(z) member of the Pc-G group (58). Su- modomain-containing, positive regulators in H4, catalyzed by the PRMT1 HMTase (66, (var)3-9 (59) and a few other Su(var) genes, such as E(Pc)(60), have also been shown to enhance Pc-G–dependent homeotic transfor- mations (60, 61). Is there a possible mecha- nistic link between Su(var) and Pc-G func- tion? Because the Polycomb protein contains a chromodomain, the dual methylation of Lys26 in H1 and of Lys9 in H3 could con- ceivably provide a combinatorial signal to recruit a Pc-G protein complex to develop- mentally regulated target loci (Fig. 2C). Collectively, these observations indicate that one histone modification can influence an- other in either a synergistic or an antagonistic way (Fig. 1D), providing a mechanism to gen- erate and stabilize specific imprints. During development, stem cell divisions are often char- acterized by one daughter cell that continues to proliferate while the other daughter cell starts to differentiate. Could the proposed “Lys9/Ser10” switch or the discussed synergisms provide an early clue about a more general mecha- nism for how these cell fates are chosen and maintained? Do other histone tails or entire nucleosomes contain similar switches, and to what extent has this theme been used in other nonhistone proteins?

Turning the Histone Code Upside Down Although HP1 and H3-Lys9 methylation are mainly associated with heterochromatic re- gions, HP1 also interacts with a variety of transcriptional coactivators involved in gene regulation in euchromatin (17, 25). Likewise, whereas histone hypoacetylation correlates most often with transcriptionally silent chro- matin domains, acetylation of Lys12 in H4 has been reported to be a hallmark property of heterochromatin in organisms ranging Fig. 2. Translating the “histone code.” (A) Described protein modules of histone-modifying from yeast to flies (7, 55). Also counterintui- enzymes that have been shown to interact with site-specific methylation (chromodomain) or tive are the findings that mutations in the acetylation (bromodomain) marks in histone NH2-termini. A protein module that would selectively HDAC Rpd3 are enhancers rather than sup- recognize phosphorylated positions is currently not known. Abbreviations: HMT, histone methyl- pressors of PEV (62). These observations transferase; HAT, histone acetyltransferase; HDM, histone demethylase; PPTase, protein phospha- suggest that not all histone methylation marks tase; HDAC, . (B) Proposed histone tail interactions for a “reversed” histone correspond with gene silencing, and that code, showing a chromodomain-containing HAT (e.g., Esa1) and part of a nucleosome-remodeling complex that may comprise a bromodomain-containing, inactive HMTase (dashed lettering), such some histone acetylation events may repress as the trx-G protein HRX. (C) Possible functional interactions between Su(var) and Pc-G proteins rather than stimulate the readout of the genet- or between histone- and DNA-methylating enzymes that could be induced or stabilized by ic information. site-selective combinations of methylation marks.

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67), might play a role in recruiting Esa1 to HDMases do not exist, histone lysine meth- histone turnover (78). Further, the TAFII250- active chromatin regions (Fig. 2B). Another ylation would be a nearly perfect long-term mediated monoubiquitination of H1 has been chromodomain-containing HAT, Mof, has epigenetic mark for maintaining chromatin shown to correlate with transcriptional stim- been shown to display strong selectivity for states. In contrast to DNA methylation— ulation (79). Whether ubiquitination may be acetylation of Lys16 in H4, a hallmark mod- where the methylated imprint can be removed linked to the proteolytic removal of more ification correlated with the doubling of tran- by nucleotide excision followed by repair— stable methylation marks in histone tails—or scriptional up-regulation observed on the DNA replication and semiconservative nu- whether, in certain cases, it could even rep- male X chromosome in Drosophila (7). The cleosome distribution appears as the sole resent a synergistic signal for their addition— chromodomain of Mof has been suggested to means to “dilute” histone lysine methylation is not known, but remains an intriguing pos- bind RNA (72), raising the possibility that below a critical threshold level. sibility (Fig. 3). A putative -specific association with RNA—or even with meth- Another potential mechanism for remov- protease is encoded by an E(var) gene in ylated RNA—may contribute to the recruit- ing methylation marks from histone tails is Drosophila (80), and the DNA repair and

ment of Mof-containing complexes, which proteolytic processing. Histone NH2-termini histone-ubiquitinating rph6 protein has been also include another chromodomain compo- are exposed and labile to proteolysis (56), implicated in post-replication remodeling of nent, Msl3. Because Lys20 in H4 is a well- and portions of certain histone tails are the chromatin structure at the silent mating- documented methylation site (56), it is con- known to be clipped at precise stages in the type loci in fission yeast (81). Similarly, SIR- ceivable that this methylation mark may be cell cycle (74) or at specific stages of devel- dependent gene silencing in S. cerevisiae also involved in stabilizing the fly dosage com- opment (75). For example, in Tetrahymena, appears to be coregulated by a de-ubiquitinat- pensation complex, thereby facilitating Mof- the first six amino acids are removed from the ing enzyme (82). 16 dependent acetylation of adjacent Lys . NH2-terminus of H3 in transcriptionally si- The extent to which male versus female According to these views, appropriate lent micronuclei, but not in transcriptionally genomes are marked differentially by histone methylation mark(s) would dictate the re- active macronuclei. H3 is ubiquitinated at methylation is not known, but it seems likely cruitment of different chromodomain-con- specific stages of mouse spermatogenesis that imprinting mechanisms may well use taining complexes, which in turn contribute (76), and H3 is also degraded at a low level epigenetic marks outside of DNA methyl- to gene activation or gene silencing. It re- in many organisms in what is most often ation. Nearly complete removal of histones mains an intriguing, but undocumented, pos- assumed to be uncontrolled proteolysis oc- from the genome is known to occur during sibility that distinct histone methylation curring during isolation. Ubiquitin-based pro- vertebrate spermatogenesis and other special- marks may also interfere with the association tein processing, as opposed to degradation, ized developmental situations (83). Bulk dis- of repressive chromatin complexes, in much can occur (77). Conserved in the placement of histones during spermatogene- the same way that nearby modifications may COOH-terminal tails of histones H2A and sis would provide a means to “erase” poten- influence bromodomain recognition and H2B are also subjected to monoubiquitina- tial male marks in the germ line, allowing the binding (39). Finally, the molecular func- tion in a pathway that seems not to be tied to reprogramming of developmental imprints. tion(s) of the bromodomain-containing HRX and SNF2 proteins are characterized by tran- scriptional stimulation and nucleosome re- modeling. HRX also contains a SET domain that appears to be catalytically inactive (30) but has been shown to interact with a SWI/ SNF subunit (37), suggesting that some re- modeling complexes could transiently incor- porate a “mute” HMTase (Fig. 2B). Thus, intrinsically impaired HMTase function in HRX could preclude methylation-dependent binding of proteins, thereby rein- forcing an activated chromatin state. It there- fore seems plausible that the activities of several E(var) and trx-G proteins may be facilitated by the recruitment to - ally positive histone tail modifications and by subsequently antagonizing the establishment of negative marks.

Transient Versus “Stable” Epigenetic Imprints Given that histone methylation is linked with both euchromatic and heterochromatic states, how stable is this histone modification? On the basis of thermodynamic principles alone, methyl groups, in particular methyl-lysine, have a considerably lower turnover than do acetyl or phosphoryl groups. The latter two modifications can be removed from histone Fig. 3. A proteolytic model to remove “stable” methylation marks from histone H3. Abbreviations: Ub, ubiquitin-conjugating activity; Ub protease, ubiquitin-directed proteolytic activity. Depending tails by the activity of HDACs or phospha- on the chromatin environment and/or the nature of the ubiquitin signal, a methylated H3 tases (29, 73), whereas histone demethylases NH2-terminus may be removed by proteolytic processing (left; see also Fig. 1D), or the entire H3 (HDMases) have yet to be characterized. If molecule may be degraded (right).

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87. C. M. Disteche, Trends Genet. 11, 17 (1995). 93. A. S. J. Klar, Trends Genet. 14, 299 (1998). and the Vienna Economy Promotion Fund ( T.J.), 88. P. Jeppesen, B. M. Turner, Cell 74, 281 (1993). 94. We thank G. Reuter and members of our laborato- and by NIH grant GM53512 and an NIH MERIT 89. D. R. Roth, S. Y. Roth, Cell 103, 699 (2000). ries for allowing us to cite unpublished observa- award (C.D.A.). 90. R. I. Gregory et al., Mol. Cell. Biol, 21, 5426 (2001). tions. We are particularly grateful to S. Rea for his This article is dedicated to the memory of Alan 91. G. Cavalli, R. Paro, Science 286, 955 (1999). assistance in preparing the figures. Supported by Wolffe, an inspirational leader to all of us who 92. A. H. F. M. Peters, D. O’Carroll, T. Jenuwein, the IMP through Boehringer Ingelheim and by have pondered the mysteries of chromatin and unpublished data. grants from the Austrian Research Promotion Fund gene regulation.

VIEWPOINT RNA: Guiding Gene Silencing Marjori Matzke,1* Antonius J. M. Matzke,1 Jan M. Kooter2

In diverse organisms, small RNAs derived from cleavage of double-strand- ent ribonuclease complex, RISC (RNA-induced ed RNA can trigger epigenetic gene silencing in the cytoplasm and at the silencing complex), which cleaves the homolo- genome level. Small RNAs can guide posttranscriptional degradation of gous single-stranded mRNAs. RISC from Dro- complementary messenger RNAs and, in plants, transcriptional gene si- sophila extracts cofractionates with siRNAs lencing by methylation of homologous DNA sequences. RNA silencing is a that guide sequence-specific mRNA cleavage potent means to counteract foreign sequences and could play an impor- (12). RISC cuts the mRNA approximately in tant role in plant and animal development. the middle of the region paired with antisense siRNA (14) (Fig. 1), after which the mRNA is RNA silencing is a new field of research that sult from the same highly conserved mecha- further degraded. Although most protein com- has coalesced during the last decade from inde- nism, indicating an ancient origin (5–10). The ponents of RISC have not yet been identified, pendent studies on various organisms. Scien- basic process involves a dsRNA that is pro- they might include an endonuclease, an exonu- tists who study plants and fungi have known cessed into shorter units that guide recogni- clease, a helicase, and a homology-searching since the late 1980s that interactions between tion and targeted cleavage of homologous activity (6, 10). A candidate for a 3Ј,5Ј-exonu- homologous DNA and/or RNA sequences can mRNA. dsRNAs that trigger PTGS/RNAi clease is C. elegans MUT7, an RNase D–like silence genes and induce DNA methylation (1). can be made in the nucleus or cytoplasm in a protein recovered in a screen for RNAi mutants The discovery of RNA interference (RNAi) in number of ways, including transcription (10). Another component of RISC is a protein Caenorhabditis elegans in 1998 (2) focused through inverted DNA repeats, simultaneous of the PAZ/Piwi family (17), which could in- attention on double-stranded RNA (dsRNA) as synthesis of sense and antisense RNAs, viral teract with Dicer through their common PAZ an elicitor of gene silencing, and indeed, many replication, and the activity of cellular or viral domains (18) to incorporate the siRNA into gene-silencing effects in plants are now known RNA–dependent RNA polymerases (RdRP) RISC (17). Genes encoding members of the to be mediated by dsRNA (3). RNAi is usually on single-stranded RNA templates (Fig. 1). In PAZ/Piwi family (Arabidopsis: AGO1; N. described as a posttranscriptional gene-silenc- C. elegans, dsRNAs can be injected or intro- crassa: QDE2; C. elegans: RDE1), which are ing phenomenon in which dsRNA triggers deg- duced simply by soaking the worms in a homologous to the translation factor eIF2C, radation of homologous mRNA in the cyto- solution containing dsRNA or feeding them have been shown to be required for PTGS/ plasm (4). However, the potential for nuclear bacteria expressing sense and antisense RNA RNAi in several mutant screens (3, 5, 8, 10). dsRNA to enter a pathway leading to epigenetic (10). A putative RdRP was the first cellular pro- modifications of homologous DNA sequences Genetic and biochemical approaches are be- tein shown to be required for PTGS/RNAi in and silencing at the transcriptional level should ing used to dissect the mechanism of PTGS/ genetic screens (N. crassa: QDE1; C. elegans: not be discounted. Although the nuclear aspects RNAi. Putative RdRPs, putative helicases, and Ego1; Arabidopsis: SGS2/SDE1) (3, 5, 8, 10), of RNA silencing have been studied primarily members of the PAZ/Piwi family are some of but its exact role is unclear and the predicted in plants, there are hints that similar RNA- the common proteins identified in genetic enzyme activity remains to be established. This directed DNA or chromatin modifications screens in N. crassa, C. elegans, and Arabidop- protein might be dispensible when large might occur in other organisms as well. Here sis (3, 5, 8, 10). Although these proteins provide amounts of dsRNA are produced from trans- we adopt a broad definition of RNA silencing clues about dsRNA synthesis and processing, genes or when viral RdRPs are present (5). that encompasses effects in the cytoplasm and the most detailed insight into the two-step RNA RdRP might be needed only when dsRNA is the nucleus, and consider their possible devel- degradation process has come from biochemi- synthesized to initiate silencing—for example, opmental roles and evolutionary origins. cal experiments with cytoplasmic extracts from from “aberrant” sense RNAs that are prema- Drosophila (11–15) (Fig. 1). The first step in- turely terminated or processed improperly (19). RNA Guiding Homologous RNA volves a dsRNA endonuclease [ribonuclease III RISC-cleaved mRNAs may also be used as Degradation (RNase III)–like] activity that processes dsRNA templates and converted into dsRNA, increas- Although they may differ in detail, RNAi in into sense and antisense RNAs 21 to 25 nucle- ing the level of siRNAs and enhancing PTGS/ animals and the related phenomena of post- otides (nt) long. These small interfering RNAs RNAi (Fig. 1). transcriptional gene silencing (PTGS) in (siRNAs), which were first described in a plant Putative helicases are another class of en- plants and quelling in Neurospora crassa re- system (16), are generated in Drosophila by an zyme found repeatedly in mutant screens (N. RNase III–type protein termed Dicer. Orthologs crassa: QDE3; C. elegans: SMG-2; Chlamy- of Dicer, which contains a helicase, dsRNA domas: MUT6; Arabidopsis: SDE3) (3, 5, 8, 1 Institute of Molecular Biology, Austrian Academy of binding domains, and a PAZ domain, have 10). Those recovered so far are not highly Sciences, A-5020 Salzburg, Austria. 2Department of Developmental Genetics, Vrije Universiteit, Amster- been identified in Arabidopsis, C. elegans, related and have not yet been characterized dam, Netherlands. mammals, and Schizosaccharomyces pombe biochemically. A DNA helicase (QDE3) and *To whom correspondence should be addressed. E- (15). In the second step, the antisense siRNAs members of two RNA helicase superfamilies mail: [email protected] produced by Dicer serve as guides for a differ- (MUT6 and SMG2/SDE3, respectively) have

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