Yeast Heterochromatin: Regulation of Its Assembly and Inheritance by Histones
1998; Cell Press; Volume: 93; Issue: 3 Linguagem: Inglês
10.1016/s0092-8674(00)81160-5
ISSN1097-4172
Autores Tópico(s)RNA Research and Splicing
ResumoRecent papers, reviewed here, suggest how yeast heterochromatin initiates and spreads and is assembled and inherited. Histones may play a crucial role in regulating each of these processes. Heterochromatin in more complex eukaryotes such as fruit flies and mammals was first identified cytologically. Unlike euchromatin, which contains most active genes, heterochromatin stains darkly and may be condensed even in interphase. Also, it is often at centromeres and telomeres where it has the ability to silence adjacent genes epigenetically (please see16Wakimoto B.T Cell. 1998; 93 (this issue): 321-324Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar [this issue of Cell). Yeast provides the possibility of genetic and molecular approaches for dissecting heterochromatin structure that are unavailable in more complex eukaryotes. However, yeast chromosomes are too small to allow heterochromatin to be seen by the same cytological procedures. Nevertheless, yeast has chromosomal regions with many of the features of heterochromatin. Like heterochromatin of fruit flies, these regions appear to be condensed since they prevent access to DNA-altering enzymes, are late replicating in S phase, and are associated in foci that appear to be at the nuclear periphery (6Gotta M Gasser S.M Experientia. 1996; 52: 1136-1147Crossref PubMed Scopus (53) Google Scholar). Moreover, as with fly heterochromatin, the yeast counterpart contains histone H4 that is uniquely hypoacetylated at lysines K5, K8, and K16 but not at K12 (references in11Lowell J.E Pillus L Cell. Mol. Life Sci., in press. 1998; Google Scholar). In Saccharomyces cerevisiae such regions are found at the telomeres and the silent (HM) mating loci. In fission yeast, Schizosaccharomyces pombe, which has much larger centromeres, heterochromatin is also centromeric (references in2Ekwall K Olsson T Turner B.M Cranston G Allshire R.C Cell. 1997; 91: 1021-1032Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Yeast heterochromatin can also repress adjacent genes in an epigenetic manner. For example, Gottschling and colleagues have shown that when a gene such as URA3 or ADE2 is integrated near the telomere it is repressed epigenetically. This telomere position effect, or TPE, is similar to position–effect variegation (PEV) at fly heterochromatin. Normally, TPE spreads into adjacent DNA only up to approximately 3 kb from the telomeric end. When the silencing information regulator SIR3 is overexpressed, TPE can extend to approximately 16 kb. These regions are referred to here as core and extended telomeric heterochromatin, respectively. By using genetic and molecular tools, it has been possible to identify molecular interactions that enable their assembly. The ends (∼300 bp) of yeast chromosomes consist of repeats of a simple sequence, C2–3A (CA)1–5/(TG)1–5TG2–3, commonly referred to as C1–3A. V. Zakian's laboratory has shown that this repeat may not be simply wrapped in nucleosomes. Instead it binds to the N terminus of RAP1, an 827 aa protein, on average every 18 bp to form the telosome, a nuclease-resistant structure. Adjacent to the telosome are a variable number of moderately repetitive Y′ (5.2 or 6.7 kb) and X (core consensus 560 bp) DNA sequence elements that are more clearly nucleosomal and whose function is unknown. Genetic, coimmunoprecipitation, and immunofluorescence data argue strongly that RAP1 participates in a large, macromolecular complex that includes SIR2, SIR3, SIR4, and the histones H3 and H4. RAP1 interacts genetically with the complex through its C terminus which is required for TPE. Pairwise direct binding and coimmunoprecipitation experiments have shown further that RAP1-SIR3-SIR4-SIR2 interact, in that order. In addition, both SIR3 and SIR4 interact directly with sequences containing the histone H3 (residues 4–20) and H4 (residues 16–29) N-terminal domains involved in telomeric and HM silencing. Moreover, SIR proteins form homotypic interactions (references in15Strahl-Bolsinger S Hecht A Luo K Grunstein M Genes Dev. 1997; 11: 83-93Crossref PubMed Scopus (582) Google Scholar, 11Lowell J.E Pillus L Cell. Mol. Life Sci., in press. 1998; Google Scholar). These interactions suggest how heterochromatin initiates and spreads along the chromosome. While I will focus mainly on telomeric heterochromatin here, it should be mentioned that the rDNA locus in yeast can also repress genes transposed there, has a unique chromatin structure, and utilizes SIR2. For a review of this topic and its surprising association with cellular senescence, the reader is referred to Guarente, 1997. RAP1 is the only one of the proteins described that recognizes a specific DNA sequence. It is likely to specify where heterochromatin assembles by sequestering SIR proteins at the C1–3A repeats (14Moretti P Freeman K Coodly L Shore D Genes Dev. 1994; 8: 2257-2269Crossref PubMed Scopus (450) Google Scholar). Despite RAP1-SIR3 binding in vitro this may not occur independently of histone H4 in vivo. A substitution at acetylatable lysine 16 in H4 (K16Q), which prevents its interaction with SIR3 in cell extracts, prevents the coimmunoprecipitation of SIR3 with RAP1. Conversely, a deletion of the C terminus of RAP1 strongly decreases the coimmunoprecipitation of SIR3 with histones (9Hecht A Strahl-Bolsinger S Grunstein M Nature. 1996; 383: 92-96Crossref PubMed Scopus (437) Google Scholar). Finally, A. Lustig's laboratory found that a mutation of SIR3 (SIR3N205), which suppresses the effects of H4 mutations on silencing, requires the C terminus of RAP1 in this suppression (9Hecht A Strahl-Bolsinger S Grunstein M Nature. 1996; 383: 92-96Crossref PubMed Scopus (437) Google Scholar; references in15Strahl-Bolsinger S Hecht A Luo K Grunstein M Genes Dev. 1997; 11: 83-93Crossref PubMed Scopus (582) Google Scholar). These data argue for an interdependent RAP1-SIR3-histone containing complex that initiates or seeds heterochromatin. How much of the complex spreads into adjacent core telomeric heterochromatin? Antibodies to RAP1, SIR2, SIR3, and SIR4 were used to immunoprecipitate formaldehyde cross-linked chromatin that had been sonicated extensively. To detect these fragments PCR primers were used to amplify sequences at intervals throughout the chromosomal ends of Chr. VI-R, which contains no Y′ or X elements. It was found that SIR2, SIR3, and SIR4 all spread in a similar, histone-dependent manner up to 2.8 kb in core telomeric heterochromatin. Surprisingly, while RAP1 was expected only at the telosome due to its DNA binding properties, its antibody also immunoprecipitated every site up to 2.8 kb. To explain the unexpected localization of RAP1, 15Strahl-Bolsinger S Hecht A Luo K Grunstein M Genes Dev. 1997; 11: 83-93Crossref PubMed Scopus (582) Google Scholar have proposed that telosomal RAP1 sites fold back onto the subtelomeric region (Figure 1). This would allow SIR proteins to polymerize and spread along the face of the chromosome by interacting with the histone N termini while interacting with each other across the chromosome to further strengthen contacts within heterochromatin. This may also contribute to condensation at telomeric heterochromatin. Since SIR2 and SIR4 are also required for the extension of TPE by SIR3 overexpression, it may be that SIR3 is merely limiting for spreading of the entire repressive complex to 16 kb. However, this is unlikely to be the case. Strahl-Bolsinger and coworkers found that as SIR3 is overexpressed, some SIR4 and most SIR2 are lost from the core region. RAP1 binding appeared unchanged. Only SIR3 was seen to spread strongly (with some SIR4) up to approximately 15 kb (Chr. VI-R) or 17.5 kb (Chr. V-R). Since SIR3 spreading was dependent on RAP1, the SIR proteins, and the H4 N terminus, these data suggested that it was mainly SIR3 that spread from the initiation site by interacting with the histone N termini. It is perhaps for this reason that repression is weaker in the extended telomeric heterochromatin than in the core (references in11Lowell J.E Pillus L Cell. Mol. Life Sci., in press. 1998; Google Scholar). Recently, 12Luger K Mader A.W Richmond R.K Sargent D.F Richmond T.J Nature. 1997; 389: 251-260Crossref PubMed Scopus (6355) Google Scholar described an X-ray crystal structure of the nucleosome core particle that may help explain how H4-SIR3 interact. In this structure, the highly basic domain of H4 (residues 16–25) interacts with a negatively charged pocket of the H2A-H2B dimer in the adjacent nucleosome of the crystal lattice. Since this H4 sequence contains much of the silencing domain that interacts with SIR3, it is possible that in heterochromatin, SIR3 replaces the internucleosomal interaction. If so, the acetylation state of K16 may be important in regulating both interactions. HM heterochromatin is uniquely acetylated at histone H4 K12 only (1Braunstein M Sobel R.E Allis C.D Turner B.M Broach J Mol. Cell. Biol. 1996; 16: 4349-4356Crossref PubMed Scopus (321) Google Scholar). Is this hypoacetylated state important for silencing? K16 is part of the H4 silencing domain (residues 16–29) that recognizes SIR3 and SIR4. Changing this site to glutamine (K16Q) simulates the acetylated uncharged state and derepresses HML and telomeric repression of URA3 strongly. In contrast, the mutation to arginine (K16R) simulates the unacetylated residue and has a lesser effect on both HML and telomeric silencing. Moreover, K16Q disrupts interactions between H4 and SIR3 in heterochromatin (9Hecht A Strahl-Bolsinger S Grunstein M Nature. 1996; 383: 92-96Crossref PubMed Scopus (437) Google Scholar). Interestingly, Bryan Turner's laboratory has shown that monoacetylated H4 in yeast euchromatin is acetylated almost exclusively at K16. Therefore, hypoacetylation of K16 may be important to allow H4 to interact with SIR3 in heterochromatin and its acetylation may reduce SIR3 binding to histones of euchromatin (Figure 2). In addition, K16 acetylation in euchromatin may interfere with the binding of the H4 N terminus (residues 16–25) to the charged H2A-H2B pocket in nucleosomes adjacent to each other. If this occurs at promoters, it might facilitate access to transcription factors. The function of hypoacetylated K5 and K8 is less clear. While these sites do not appear to interact directly with SIR3, they may indirectly influence H4-SIR3 binding at residues 16–29 (8Hecht A Laroche T Strahl-Bolsinger S Gasser S Grunstein M Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (675) Google Scholar). Histone H4 K12 that is unmutagenized can suppress the defect in silencing caused by a combination of arginine substitutions at K5, K8, and K16 (1Braunstein M Sobel R.E Allis C.D Turner B.M Broach J Mol. Cell. Biol. 1996; 16: 4349-4356Crossref PubMed Scopus (321) Google Scholar). However, lesions at this site do not decrease telomeric silencing. An insight into this apparent discrepancy is provided by comparing in vivo and in vitro H4-SIR3 interactions. While H4 K16Q disrupts H4-SIR3 binding in chromatin when H4 may be acetylated at K12 (9Hecht A Strahl-Bolsinger S Grunstein M Nature. 1996; 383: 92-96Crossref PubMed Scopus (437) Google Scholar), H4 K16Q does not affect binding of the unacetylated H4 N terminus to SIR3 in vitro. It is only when K12 (or better yet K5 and K12) is mutated to glutamine that K16Q can disrupt binding in vitro (8Hecht A Laroche T Strahl-Bolsinger S Gasser S Grunstein M Cell. 1995; 80: 583-592Abstract Full Text PDF PubMed Scopus (675) Google Scholar). These in vitro experiments must be viewed cautiously since they do not utilize nucleosomal structures. Nevertheless, they suggest that acetylation at K12 in vivo may increase the specificity of H4-SIR3 interactions at residues 16–29 allowing K16Q to disrupt these contacts (Figure 2). The work of A. Miller and K. Nasmyth first demonstrated that yeast cells establish HM silencing coincident with S phase. It is at this time that DNA replication and nucleosome assembly occur. Some involvement for DNA replication is evident in that two proteins of the origin recognition complex (ORC2 and ORC5) also have a role in both telomeric and HM silencing and ORC1 shares extensive homology with SIR3 (for a review on the cell cycle and its relation to silencing, see5Fox C.A Rine J Curr. Opin. Cell. Biol. 1996; 8: 354-357Crossref PubMed Scopus (17) Google Scholar). How may nucleosome assembly occur at heterochromatin? The human chromatin assembly factor (CAF-1) contains three subunits (p150, p60, and p48) implicated in assembling acetylated histones H3 and H4 preferentially onto replicating DNA. H4 associated with human CAF-1 has been shown to be acetylated at K5, K8, or K12 or a combination of these sites. Yeast contains chromatin assembly complex proteins CAC1 (p90), CAC2 (p60), and CAC3 (p50) that are similar in sequence to the respective human factors (references in10Kaufman P.D Kobayashi R Stillman B Genes Dev. 1997; 11: 345-357Crossref PubMed Scopus (318) Google Scholar). Surprisingly, while the yeast CAF1 complex can assemble histones in vitro, it is not essential for viability and for nucleosome assembly in yeast cells. However, CAF1 absence may affect chromatin more subtly since it does result in increased UV sensitivity (10Kaufman P.D Kobayashi R Stillman B Genes Dev. 1997; 11: 345-357Crossref PubMed Scopus (318) Google Scholar), and increasing evidence points to CAF1 involvement in assembling histones at telomeric and HM heterochromatin. For example, cac1, cac2, and cac3 mutations result in decreased telomeric silencing (3Enomoto S McCune-Zierath P.D Gerami-Nejad M Sanders M.A Berman J Genes Dev. 1997; 11: 358-370Crossref PubMed Scopus (136) Google Scholar, 10Kaufman P.D Kobayashi R Stillman B Genes Dev. 1997; 11: 345-357Crossref PubMed Scopus (318) Google Scholar, 13Monson E.K de Bruin D Zakian V.A Proc. Natl. Acad. Sci. USA. 1997; 94: 13081-13086Crossref PubMed Scopus (113) Google Scholar, 4Enomoto S Berman J Genes Dev. 1998; 12: 219-232Crossref PubMed Scopus (167) Google Scholar) and a small decrease in HML repression as measured by a sensitive α-factor response assay (4Enomoto S Berman J Genes Dev. 1998; 12: 219-232Crossref PubMed Scopus (167) Google Scholar). cac1 mutants also mislocalize RAP1 from telomeric foci (3Enomoto S McCune-Zierath P.D Gerami-Nejad M Sanders M.A Berman J Genes Dev. 1997; 11: 358-370Crossref PubMed Scopus (136) Google Scholar). In addition cac mutants that are MATa mating type respond in an unusual manner to α-factor arrest by forming multiple, elongated “shmoo-like” projections. Interestingly, lesions that destroy certain acetylatable lysines at the H3 and H4 N termini also form these unusual shmoo-like clusters. Together, these data argue that the yeast CAF1 complex is required for the assembly of histones at heterochromatin, which will eventually have the heterochromatin-specific acetylation pattern. In the absence of CAF1, redundant factors may assemble improperly acetylated histones. Perhaps, RAP1 is mislocalized from the RAP1-SIR-histone complex in a cac1 mutant strain when H4-SIR interactions are weakened by a faulty H4 acetylation pattern. To investigate this possibility, 13Monson E.K de Bruin D Zakian V.A Proc. Natl. Acad. Sci. USA. 1997; 94: 13081-13086Crossref PubMed Scopus (113) Google Scholar used an antibody to tetra-acetylated H4 to immunoprecipitate chromatin fragments. They showed that H4 at telomeres is still hypoacetylated in a cac1 mutant. However, individual acetylation site differences would not be easily detected unless antibodies to individual acetylated lysines were used in these experiments. Thus, CAF1 may still be involved in assembling appropriately acetylated histones at heterochromatin and elsewhere. How CAF1 and the enzymes of acetylation and deacetylation cooperate in this respect is entirely unclear. However, it is important to note that the Sternglanz and Gottschling laboratories (references in3Enomoto S McCune-Zierath P.D Gerami-Nejad M Sanders M.A Berman J Genes Dev. 1997; 11: 358-370Crossref PubMed Scopus (136) Google Scholar) have shown that HAT1, a cytoplasmic acetyltransferase, acetylates H4 specifically at K12. Whether CAF1 assembles this uniquely acetylated H4 at heterochromatin remains to be determined. 2Ekwall K Olsson T Turner B.M Cranston G Allshire R.C Cell. 1997; 91: 1021-1032Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar have recently reported a surprising finding that also implicates histone acetylation in the propagation of chromosomal structures during cell division. They found that treating S. pombe for five cell doublings with the histone deacetylase inhibitor trichostatin A (TSA) causes centromeric heterochromatin to become hyperacetylated. Instead of the heterochromatin-specific pattern (here too, H4 acetylation mainly at K12), sites K5, K8, and K16 were more strongly acetylated. This was correlated with derepression of reporter genes at centromeric heterochromatin and defects in chromosomal segregation. Moreover, derepression of the marker genes was tightly linked to the centromeric region (cen1) itself, arguing that derepression is not due to the effect of TSA on a chromosomal gene in euchromatin whose derepression could act in trans on cen1. Surprisingly, the hyperacetylated state and the defects in repression and segregation were maintained even 80–100 generations after TSA was removed (2Ekwall K Olsson T Turner B.M Cranston G Allshire R.C Cell. 1997; 91: 1021-1032Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Perhaps, the hyperacetylated state prevents heterochromatin formation, thereby increasing access to histone acetyltransferases. This may ensure a decondensed state that may be acetylated and propagated as such in subsequent generations. Therefore, as in S. cerevisiae telomeric heterochromatin, the acetylation state of H4 may serve as a platform for the assembly of S. pombe centromeric factors—in particular, those required for repression and segregation. Moreover, the acetylation state resulting from TSA treatment may provide a chromosomal imprint that serves as a template for its inheritance in subsequent generations. This has profound implications for the inheritance of the normal hypoacetylated state of heterochromatin (telomeric or centromeric). It suggests that the H4 acetylation state is a template for its propagation during cell division (Figure 3). This may occur if the heterochromatin-specific state is recognized by histone deacetylases (or deacetylases and acetyltransferases) that duplicate this state on newly assembled histones during heterochromatin replication. Alternatively, some other aspect of the heterochromatin-specific structural state, perhaps the assembled SIR proteins, may be recognized. It is also possible that templating of the acetylation state from old to new nucleosomes involves the CAF1 complex. This could occur if the complex recognizes some aspect of mature heterochromatin and can then transfer appropriately acetylated H4 to new nucleosomes. In either case, once assembled, the SIR complex may prevent access to enzymes that promote further cycles of acetylation and deacetylation, thus protecting the heterochromatin-specific acetylation pattern. The inheritance of a particular acetylated state may also explain epigenetic inheritance. If the balance shifts to hypoacetylation in some cells, this would allow subsequent inheritance of the repressed state of heterochromatin; shifting the balance to hyperacetylation would propagate the active state. In conclusion, recent observations have pointed to new and surprising functions for histones and histone acetylation in regulating heterochromatin structure and its inheritance. Histone acetylation has long been thought to affect histone–DNA interactions. While this may still be true, there are cases where it may regulate protein–protein interactions that determine heterochromatin initiation and which allow heterochromatin to spread. In addition, acetylation may differentially regulate histone–histone internucleosomal interactions in heterochromatin and euchromatin. This has important physiological considerations. Heterochromatin is implicated in numerous functions, including the protection of chromosomal ends, genetic repression, chromosomal segregation, telomere length regulation, and even cellular senescence (11Lowell J.E Pillus L Cell. Mol. Life Sci., in press. 1998; Google Scholar). Since certain of the factors involved in yeast heterochromatin (e.g., RAP1, SIR2, histones) are strongly conserved or have homologs in higher cells, including humans, the study of yeast heterochromatin should be applicable to understanding its many important functions in all eukaryotes.
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