chromatin and transcription in saccharomyces cerevisiae

Chromatin and transcription in Saccharomyces cerevisiae

Jose Perez-Mart|n *Department of Microbial Biotechnology, Centro Nacional de Biotecnolog|a, CSIC, Campus de Cantoblanco, Madrid 28049, Spain

Received 27 November 1998; accepted 4 April 1999

Abstract

A central problem in eukaryotic transcription is how proteins gain access to DNA packaged in nucleosomes. Research on theinterplay between chromatin and transcription has progressed with the use of yeast genetics as a useful tool to characterizefactors involved in this process. These factors have both positive and negative effects on the stability of nucleosomes, therebycontrolling the role of chromatin in transcription in vivo. The negative effectors include the structural components ofchromatin, the histones and non-histone chromatin associated proteins, as well as regulatory components like chromatinassembly factors and histone deacetylase complexes. The positive factors are involved in remodeling chromatin and severalmultiprotein complexes have been described: Swi/Snf, Srb/mediator and SAGA. The components of each of these complexes,as well as the functional relationships between them are covered by this review. ß 1999 Federation of European Micro-biological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords: Transcription; Chromatin; Swi/Snf complex; Histone acetylation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5042. Genetic links between transcription and chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5043. Chromatin and transcription: positive and negative regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5064. Structural negative elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506

4.1. Histones: the core elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5064.2. Histone tails . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5084.3. Linker histones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5084.4. Non-histone components of chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509

5. Regulatory negative elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5105.1. Accessory factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5105.2. Histone deacetylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511

6. Regulatory positive factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5126.1. The Swi/Snf complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5126.2. The Srb/mediator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514

0168-6445 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 1 6 8 - 6 4 4 5 ( 9 9 ) 0 0 0 1 8 - 2

* Tel. : +34 (91) 585 4704; Fax: +34 (91) 585 4506; E-mail: [email protected]

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6.3. The SAGA complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5157. Genetic interactions between di¡erent complexes: a matter of three . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5168. A model of interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518

1. Introduction

In eukaryotic cells, the DNA is packaged in ahighly condensed complex to ¢t in the nucleus. How-ever, all nuclear processes, including transcription,require that enzymes gain access to the DNA tem-plate despite the fact that it is complexed with his-tone and non-histone proteins to form chromatin. Aconsiderable amount of evidence has indicated some-thing obvious: the chromatin structure imposes adefault repressed state upon the genome. Recentstudies have focused on the mechanisms by whichthe nucleosome structure is destabilized in order tofacilitate the access of sequence-speci¢c transcriptionfactors and the general transcription machinery. It isnow clear that this process requires not only thetranscription factors, but also cooperation with his-tones and with cofactors that help to remodel ordisplace nucleosomes [1^3].

Chromatin is organized in a hierarchy of struc-tures (Fig. 1), from the basic repeat unit, i.e. thewinding of the DNA around the histone octamerto form nucleosomes, to the complex appearance ofmetaphase chromosomes [4]. Each level of chromatinorganization contributes to the dense packaging ofDNA, e¡ectively repressing gene expression. Singlepositioned nucleosomes and arrays of regularlyspaced nucleosomes can be reconstituted in cell-freesystems and, in these systems, the lower levels ofstructural organization and their functional implica-tions are amenable to detailed analysis [1]. However,although these studies represent true breakthroughsin the ¢eld, they do not necessarily re£ect the phys-iological situation. Some activators, such as Gal4,require highly specialized cellular machinery to dealwith the repressive e¡ects of chromatin in vivo [5] inspite of the fact that they can bind to their site invitro, even when it is complexed into nucleosomes[6]. These facts indicate that the complex folding ofthe nucleosomal ¢ber, collectively termed `higher or-der structures', plays a dramatic role in the transcrip-

tion-chromatin relationships. The caveat is that the`higher order structures' cannot be reconstituted incell-free systems and therefore our understanding ofthose structures and their implications in transcrip-tion is still di¡use.

Baker's yeast (Saccharomyces cerevisiae) is partic-ularly amenable to analyzing mechanisms in vivounder physiological conditions. Studies in yeasttake advantage of powerful genetic approaches thatare not available in other eukaryotic organisms. Theaim of this review is to summarize many old andrecent ¢ndings which have contributed to our appre-ciation of the active role played by the chromatin inregulated gene expression in S. cerevisiae. This re-view is organized into two di¡erent sections. The¢rst one summarizes the present knowledge wehave about the components of the multiprotein com-plexes whose primary function is to mediate changesin chromatin structure. The second section discussthe functional relationships between these complexesas well as their way of action. Finally a model isproposed about the relationships between chromatinand transcription.

2. Genetic links between transcription and chromatin

Our present view of the role of chromatin in tran-scriptional regulation in yeast comes from studiesaimed at the elucidation of the speci¢c regulationof particular systems. Di¡erent, independent ap-proaches have converged to the description of a ser-ies of genes related to the same process: transcrip-tion regulation facing the repressive constraints ofchromatin. The purpose of this section is not togive an exhaustive list of the di¡erent genes identi¢edbut to describe how di¡erent screens, aimed at an-swering system-speci¢c questions, yielded the samekind of genes and how these gene products are re-lated to chromatin architecture.

One of the earliest and most productive screens

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was the study of mutations able to suppress the tran-scriptional defects caused by Ty (a transposable ele-ment) insertion mutations at the promoter region ofthe LYS2 and HIS4 loci [7]. Cells carrying the lys2-128N or his4-912N mutant alleles are unable to growin synthetic media lacking lysine or histidine, respec-tively (Spt3 phenotype). The search for suppressorsof the Spt3 phenotype identi¢ed a group of genes(named SPT for suppressor of Ty) whose productsturned out to be involved both in chromatin struc-ture and in transcription regulation [8,9]. Amongthese genes were SPT11 and SPT12, encoding his-tones H2A and H2B [10], SPT2, which encodes anHMG1-like protein [11,12], and other genes whoseactivity could a¡ect chromatin and transcription(SPT4, SPT5 and SPT6) [13^15]. Also the gene en-coding the TBP protein (TATA binding protein,SPT15) [16,17] and a subset of genes encoding pro-teins that help TBP function at particular promoters(SPT3, SPT7, SPT8, SPT20) [18^22] were recoveredin this screen.

A second group of genes, the SWI/SNF genes,were obtained from genetic screens aimed at the in-dependent analysis of the transcriptional regulationof the HO gene (encoding an endonuclease required

for mating type switching; SWI stands for switching)[23] and the SUC2 gene (encoding an invertase, re-quired for growth on sucrose and ra¤nose; SNFstands for sucrose non-fermenting) [24]. Geneticanalyses of the genes required for the expression ofboth systems resulted in the isolation of a series ofgenes involved in gene activation (SWI1/ADR6,SWI2/SNF2, SWI3, SNF5, SNF6, SNF11, TFG3,SWP73) [25^30]. The link between the SWI/SNFgroup of genes and chromatin came from the studyof suppressors of defects in components of thisgroup. The SIN (for switch independent) [31,32]and SSN (for suppressors of snf mutations) [33]genes were identi¢ed as suppressors of swi and snfphenotypes. For instance, the sin2 mutation wasfound to lie in the HHT1 gene, which encodes his-tone H3 [34]. The SIN1 gene was found to be allelicto the SPT2 gene [11,12] and the SSN20 gene turnedout to be SPT6 [35]. A di¡erent gene called SSN4was found to be allelic to SIN4 [36] which was pre-viously obtained as a suppressor of swi mutations[31].

More recently, a group of genes involved in acet-ylation and deacetylation of histones has been rec-ognized. A genetic selection in yeast to identify co-

Fig. 1. Levels of chromatin packing. A model (top left) of nucleosome with associated proteins (black ball). In the middle a model of the`beads-on-a-string' form of chromatin. At right folding intermediates from `beads on a string' to the 30-nm £at ¢ber. Adapted from [4].

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factors which functionally interact with the acidicactivation domain of herpes simplex virus VP16 re-vealed the ADA genes (from alteration/de¢ciency inactivation) [37]. This group comprises, amongothers, the ADA2, ADA3 and GCN5 genes [37^39],which form part of a histone acetyltransferase com-plex, and are allelic to the genes SWI7, SWI8 andSWI9, respectively [40], all of them involved in thetranscriptional activation of the HO promoter. Anadditional gene, ADA5, turned out to be allelic toSPT20, which belongs to the SPT group of genes[21,22]. In a totally di¡erent screening, aimed atidentifying genes involved in the regulation of agene encoding a low-a¤nity K� transporter(TRK1), two di¡erent genes, RPD3 and RPD4(from reduced potassium dependence), were isolated[41]. These two genes turned out to be global tran-scriptional regulators [42,43]. Subsequent studiesshowed that the RPD3 gene encodes the catalyticsubunit of a histone deacetylase [44] while RPD4was found to be allelic to SIN3, one of the genesisolated as a suppressor of swi mutations [45].

3. Chromatin and transcription: positive and negativeregulators

Genetic studies have identi¢ed a large number offactors required for transcriptional regulation in re-lation to chromatin. In many cases these studies havesuggested that groups of factors might function by acommon mechanism. For some of these functionalgroups, subsequent biochemical analysis has demon-strated that their members function in large com-plexes; this is the case of the Swi/Snf complex[46,47], the Srb/mediator/holoenzyme complex[48,49] and the SAGA complex [50]. These com-plexes are required for transcription of a numberof genes and the genes encoding their componentswere obtained as positive global regulators.

In contrast, other genes were obtained as formalnegative regulators. Mutations in these genes oftensuppress mutations in the positive factors. Thesegenes encode products directly related to the chro-matin structure or to modi¢cation of the chromatin.

A simpli¢ed view of the relationships betweenchromatin and transcription results from the inter-play of the two kinds of e¡ectors: negative e¡ectors

do not allow transcription because they form andstabilize chromatin structure, while positive e¡ectorsallow transcription by remodeling the repressivechromatin structure. This simpli¢ed view is indeedmore complicated, because some negative compo-nents are part of complexes involved in the chroma-tin remodeling; however, for the sake of clarity wewill describe these complexes as positive and negativefactors.

4. Structural negative elements

4.1. Histones: the core elements

The nucleosome is composed of an octameric corewhich contains two subunits of each histone (H2A,H2B, H3 and H4). This core has 147 bp of DNAwrapped around it in 1.65 turns of a left-handedsuperhelix. The histone-histone and histone-DNA in-teractions are now understood in considerable struc-tural detail [51,52]. Each histone has a central coredomain (histone fold) and an amino-terminal taildomain that reaches outside the wrapped DNA.The histone fold is involved both in histone-histoneinteractions inside the nucleosomal core and in his-tone-DNA interactions [53,54]. One of the main ad-vantages of yeast, in addition to the ease of geneticmanipulation, is the presence of only two copies ofeach of the genes encoding the core histones perhaploid genome (by comparison, in Drosophila me-lanogaster, there are an estimated 110 copies of eachof the histone genes). Most studies of the histoneroles in transcription performed in yeast use twotypes of genetic manipulations: alterations of histoneexpression and mutations in the histone genes.

Strains with an altered histone gene dosage wereisolated from screens for suppressors of Ty insertions(SPT genes) [7^9] (see Section 2). Speci¢cally, theyeast histone locus HTA1-HTB1, which encodes his-tones H2A and H2B, was identi¢ed as composed ofthe SPT11 and SPT12 genes [10]. By variation of thein vivo gene doses of either H2A-H2B or H3-H4, itwas found that the suppression of the transcriptiondefects was due to an imbalance in the synthesis ofH2A-H2B relative to H3-H4 [10,55]. A more drasticchange in histone levels was achieved by placing theH4 histone gene under the control of the inducible

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promoter GAL1 in a yeast strain in which the twoendogenous H4 genes were deleted [56]. In this strainhistone H4 is depleted in the presence of glucose andthe chromatin structure is altered (`nucleosomeloss'). Such nucleosome loss led to activation of re-pressed genes like PHO5, CYC1 and GAL1 [57,58].These results indicated that the alteration of the sto-ichiometry of several chromatin components a¡ectedtranscription, through an alteration of the structureof chromatin.

More evidence supporting the role of nucleosomesas global repressors of transcription comes from thestudy of particular histone mutations called histonesin mutations [34]. These histone mutations were ob-tained as suppressors of defects in swi genes [31,32](see Section 2), although they are able to suppress anadditional number of phenotypic defects. Histone sinmutations allow growth in medium lacking lysine orhistidine of a strain carrying the mutant alleles lys2-

128N and his4-912N (Spt3 phenotype) [34,59]; theysuppress gcn5 defects [40,58], as well as transcrip-tional defects caused by partial deletions of the C-terminal domain of the largest subunit of RNA po-lymerase II (Srb3 phenotype) [60] and allow tran-scription of UAS-less promoters [61]. The histonesin mutations are partially dominant and cause singleamino acid substitutions con¢ned to the same regionon the surface of the histone octamer [34]. This ¢nd-ing led to the proposal that this region may de¢ne afunctional domain (the SIN domain), that behavesformally as a negative regulator of transcription.This SIN domain is located in one L-bridge motifwithin the H3-H4 heterodimers (Fig. 2A). Becauseof the juxtaposition of the two H3-H4 at the dyadaxis of the nucleosome, the sin mutations have thepotential to disrupt histone-DNA interactions in-volving the central turn of DNA at the dyad axis(Fig. 2B). Indeed, in vivo analysis of chromatin

Fig. 2. Nucleosome structure. A: Structural model for the interaction of the core histones with DNA in the nucleosome. The view is oneturn of DNA. For clarity only one molecule of H2A, H2B and H4 is shown. B: Scheme of the interactions between heterodimers ofH2A, H2B and H3-H4. The sites of primary interaction of the histone fold domains with DNA (the paired ends of helices and L-bridgemotifs) are indicated. Also indicated are the location of sin mutations as black stars and the location of the H2A `knuckle' as a whitestar. Adapted from [4].

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structure from yeast strains carrying sin mutations inthe histone H4 gene showed that there is an in-creased accessibility of nucleosomal DNA to Dammethyltransferase and micrococcal nuclease (MNase)[62]. However, in vitro reconstitution of nucleosomesusing puri¢ed histone variants carrying sin mutationsdoes not sustain this view [63]. Although all sin al-leles tested so far produce the same kind of e¡ects invivo, di¡erent sin mutations alter to various extentsthe in vitro stability of histone-DNA in the nucleo-some [63]. Alternative explanations of the e¡ects ob-served in histone sin mutations include defects in theinteraction with other proteins, which we will discussbelow.

Results from mutations in tyrosine residues of his-tone H4 that play a role in the interaction betweenthe H3-H4 tetramer core and the H2A-H2B dimersupport the importance of an intact nucleosome torepress transcription [64]. An exhaustive search forhistone H2A mutations which decrease SUC2 andGAL1 expression has identi¢ed a distinct region inthis histone [65]. This region, called `H2A knuckle',is located near the H2A amino tail and exposed inthe surface of the nucleosome, although not in aregion involved in DNA-histone interactions (Fig.2A). It has been proposed that this region of H2Amay be recognized by a histone binding factor thataids the dissociation of the H2A-H2B dimer fromthe nucleosome [66].

4.2. Histone tails

The amino-terminal regions of each histone, orhistone tails, constitute distinct functional proteindomains that extend from the nucleosomal core.They are positively charged, and are targets of rever-sible posttranslational modi¢cations which alter ei-ther their charge or their conformation and possibleinteractions with DNA or other proteins (Fig. 3).Some portions of the amino-terminal regions of thehistones can be deleted without loss of viability [67].A systematic set of deletions and substitutions inboth histone H3 and histone H4 revealed that his-tone tails function in both gene activation and re-pression. For instance, histone H3 tail deletions in-crease GAL1 mRNA levels, while similar deletions inthe histone H4 tail decrease them [68^70]. A moredetailed analysis has been performed with the H4

histone tail, where replacement of these lysine resi-dues with other amino acids causes transcriptionaldefects as high as or, in some cases, even higherthan deletion of these residues [68].

Although it was thought that these tails contrib-uted to DNA binding and stabilized the nucleosome[71], the histone tails are largely dispensable for ei-ther nucleosome assembly or stability in vitro [72].The present view of the role of histone tails is thatthey are involved in the formation of higher orderstructures, in which two nucleosome particles arestacked together by protein-protein interactions be-tween the basic residues in the amino-terminal tailsof H3 and H4 histones and the acidic region formedby H2A-H2B on the surface of the histone octamer[66].

4.3. Linker histones

An additional component of nucleosomes in high-er organisms is the histone H1, often referred to aslinker histone because it is associated with the linkerDNA between the nucleosomal cores. The histoneH1 facilitates the folding of the nucleosomes in theso-called 30-nm ¢ber and stabilizes the compactedstructure. There is considerable evidence that H1participates in transcriptional repression [1].

In S. cerevisiae, the presence of a version of his-tone H1 has been an unanswered question [73] but

Fig. 3. The histone tails. The N-terminal histone tails are shownas straight lines with lysine (K) and serine (S) residues indicated.Possible histone modi¢cations are indicated: acetylation (Ac),methylation (Me), phosphorylation (P) and ubiquitination (Ub).

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the sequencing of the yeast genome revealed thepresence of an open reading frame (YPL127c) withregions of signi¢cant sequence homology to the H1histones of higher eukaryotes [74^76]. The corre-sponding gene, which has been named HHO1, en-codes a protein with the biochemical features of aH1 histone [77]. However, deletion of the geneHHO1 produced no e¡ect in the cell [74^76]. Unlikemutants in core histones or other chromatin associ-ated proteins (see below), a mutant deleted in HHO1is unable to suppress transcriptional defects such asthe Spt3 or Sin3 phenotype. Since no di¡erences in aseries of DNA properties or in chromatin associatedphenomena [76] were found in the mutant, it appearsthat the histone-like H1 protein does not play animportant role in the regulation of gene transcrip-tion.

4.4. Non-histone components of chromatin

Chromatin structure can be modi¢ed by the selec-tive association of non-histone proteins that interactwith DNA histone complexes. Primary among theseare the high mobility group (HMG) of proteins [78].Cloning of several genes encoding these proteins hasrevealed three types of HMGs that can be distin-guished on the basis of their molecular masses,DNA binding characteristics and amino acid se-quence motifs : HMG-1/2, HMG-14/17, and HMG-I(Y) [79]. From these groups only members ofHMG-1/2 have been found in yeast. Members ofthe HMG-1/2 type are distinguished by the presenceof one or more copies of an 80-amino acid domaintermed the `HMG box' that is responsible for DNAbinding capacity [80]. Proteins containing the HMG

box comprise a superfamily that can be divide intotwo classes: transcription factors, able to recognizespeci¢c DNA sequences, and chromatin associatedproteins, which recognize DNA with little or no se-quence speci¢city [81].

In yeast, a number of HMG-like proteins with nosequence speci¢city have been reported (Fig. 4). Theproteins encoded by the NHP6A and NHP6B genesare closely related 10-kDa proteins that contain asingle HMG box [82]. The genes HMO1 andHMO2 encode related proteins which contain twocontiguous HMG boxes [83]. In both cases(Nhp6A/B and Hmo1/2) the biochemical character-ization of these proteins indicated that they can workas true HMG proteins, but there is no evidence asyet of any role in chromatin-mediated repression ofthe transcription [83,84]. However, studies with athird HMG-like protein, Sin1/Spt2, support a clearrole of HMG proteins in the chromatin-transcriptionrelationships [11,12].

The SIN1/SPT2 gene was obtained in two di¡er-ent genetic screens [8,31,32]. Sin1 protein is highlycharged and shows two regions of similarity to themammalian HMG1 protein (Fig. 4). The sequencecharacteristics of Sin1 protein, its nuclear localiza-tion, its abundance, and its ability to bind DNA ina non-speci¢c way suggest that this protein may be achromatin component [12]. In fact, using hydroxy-apatite fractionation of a nuclear extract, Sin1 elutesat the same salt conditions as the H3 and H4 his-tones (J. Perez-Mart|n, unpublished observations).Additional links between Sin1 and histones camefrom genetic studies. Both sin1 and histone sinmutations show the same range of phenotypic ef-fects. They are able to suppress Spt3, Sin3, Srb3

Fig. 4. HMG1/2-like proteins from yeast. Schematic alignment of yeast Hmg1/2-like proteins. The `HMG boxes' are represented as whiteboxes. In addition, basic regions (black boxes) and acidic regions (gray boxes) are indicated.

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and Gcn53 phenotypes [11,12,34,40,59,60]. Further-more, the double mutant with sin1 and histone sinmutations displays the same e¡ects as do the singlemutations, reinforcing the view that Sin1 and thehistones H3 and H4 work together in the same ge-netic pathway [85]. A particularly revealing datum isthe ability to suppress the sin histone mutations byhigh gene dosage of SIN1 [85]. This suppression isgene-speci¢c (SIN1 overdose is unable to suppressother spt or sin mutations) but not allele-speci¢c(SIN1 suppress all the histone sin mutations de-scribed). A molecular explanation for this suppres-sion derives from the observation that the Sin1 pro-tein is present at signi¢cantly reduced levels in astrain carrying sin histone alleles. Since SIN1mRNA levels are unchanged in the histone mutants,the reduction in Sin1 levels must occur posttranscrip-tionally. An attractive possibility is that Sin1 that isnot complexed in chromatin is degraded. It has beenreported that the N-terminal domain of Sin1 inter-acts with Cdc23 [86] and a protein with homologiesto the AAA family of proteasome components [87].It is possible that these factors a¡ect the stability ofSin1. This result suggests that the ability of sin his-tone mutations to suppress swi defects could bemediated by the e¡ects on the levels of Sin1 andnot by the ability to produce a less stable nucleo-some, about which explanation some controversy re-mains [62,63]. The proposed role of Sin1 is to worklike a staple in the nucleosome, interacting with bothDNA and histones and stabilizing the nucleosome.

Interestingly, both genetic and biochemical datapoint to an interaction between the Sin1 proteinand the Swi/Snf complex [85]. The defects observedwhen a portion of Sin1 (the carboxy-terminal half) isoverexpressed, as well as the range of the mutationswhich suppress such defects, indicate that the over-expression of the Sin1 C-terminal half interferes withthe Swi/Snf complex. Furthermore, the ability ofhigh levels of SWI1 to correct these defects supportsthis view. Finally, results of co-puri¢cation experi-ments indicate that the C-terminal half of Sin1 isphysically associated with at least three componentsof the Swi/Snf complex. The Sin1 protein interacts ina regulated way with both histones and componentsof the Swi/Snf complex, which suggests that Sin1mediates the e¡ects of the Swi/Snf complex on chro-matin.

5. Regulatory negative elements

5.1. Accessory factors

In addition to mutations in histone and HMGgenes, other genes have been shown to be targetsof mutations that overcome transcriptional defects.The SPT4, SPT5 and SPT6 genes were obtained assuppressors of Ty insertions [13^15], although theyare also able to suppress swi and snf mutations(SPT6 is allelic to SSN20, one of the genes obtainedafter a search for suppressors of snf defects [33]).Mutations in these three genes confer similar pleio-tropic phenotypes. Genetic analyses indicated thatthese three genes cooperate in the same process[88]. The combination of non-lethal mutations inany two of these three genes causes lethality in hap-loids, and some recessive mutations in di¡erentmembers of this set fail to complement each other.Finally mutations in all three genes alter transcrip-tion in similar ways. However, SPT4 is distinct inother ways. For example, the SPT4 gene is non-es-sential, while disruption of either SPT5 or SPT6results in lethality. Furthermore, an altered genedose of either SPT5 or SPT6 suppresses the sametranscriptional defects as single mutations do, butnot an altered dose of SPT4.

The biochemical results are confusing. Results ofco-immunoprecipitation experiments indicate thatthe Spt5 and Spt6 proteins interact physically [88].However, more recent data indicated that Spt4 andSpt5 form a tight complex that, at most, associatesonly weakly with Spt6 [89]. Given the genetic evi-dence that Spt4, Spt5 and Spt6 work together, anSpt4-Spt5 complex may interact in a dynamic, butbiochemically unstable fashion in vivo.

Several data show that Spt6 interacts physicallyand functionally with histones [90]. Lethality of aspt6 mutation is suppressed by overexpression ofH3 or H3-H4, whereas overexpression of H4 aloneor of H2A-H2B has no signi¢cant e¡ect. SPT6 en-codes a large acidic protein which has been shown tobind directly to H3-H4. The region of histone H3involved in both complementation of in vivo defectsand binding in vitro is the histone fold. Addition ofSpt6 protein to a mixture of histones and DNA re-sults in the assembly of nucleosomes [90]. Interest-ingly, Spt6 contains three highly acidic aspartate-,

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glutamic-rich regions which were also present in thehistone assembly factors N1/N2 from Xenopus laevis[91]. All these data led to the proposal that Spt6,probably in concert with Spt4 and Spt5, could beinvolved in chromatin assembly or could serve as adonor or acceptor of histones in genomic regionsundergoing extensive chromatin reorganization,such as those that occur at highly regulated genes.

The Spt5 protein interacts functionally and physi-cally with the RNA polymerase II (Pol II) [89]. Thisgenetic interaction is allele-speci¢c between speci¢cmutations in SPT5 and the RPB1 gene, encodingthe largest subunit of RNA Pol II. Co-puri¢cationexperiments indicate that Rpb1 could be recoveredassociated with Spt5, albeit substoichiometrically.This interaction has been proposed to be importantfor transcriptional elongation in vivo [89].

5.2. Histone deacetylases

The amino-terminal tails of histones are the tar-gets for speci¢c modi¢cations like acetylation, meth-ylation, phosphorylation and ADP-ribosylation [92](Fig. 3). Increased levels of histone acetylation at agene or chromosomal region are associated withtranscriptional activity whereas underacetylation ofhistones is observed in non-transcriptionally activeregions ([93], reviewed in [94]). Thus, it has beenthought that core histone deacetylation leads to tran-scriptional repression. This hypothesis gains supportwith the characterization of histone deacetylases asnegative transcriptional regulators.

Two distinct yeast histone deacetylase complexesfrom S. cerevisiae have been puri¢ed to homogeneityby standard biochemical methods [44,95]. These twocomplexes, HDA and HDB (with molecular massesof approximately 350 and 600 kDa, respectively)show di¡erent sensitivity to trichostatin A (a speci¢cinhibitor of histone deacetylases). In vitro, the HDAactivity deacetylates all four core histones, althoughit has a preference for histone H3, and is stronglyinhibited by trichostatin A. HDB is considerably lesssensitive to trichostatin A. Genes encoding compo-nents of both complexes have been cloned. One ofthe subunits of HDB turned out to be the polypep-tide encoded by the RPD3 gene [44], already knownas a general regulatory factor for which no biochem-ical function for the protein was postulated

[32,41,43,96,97]. A subunit from HDA was encodedby a previously unidenti¢ed gene (designated HDA1gene) which shares signi¢cant sequence similaritywith Rpd3 [44].

Histones are the physiological substrates for Rpd3and Hda1 histone deacetylases. In agreement withthe enzymatic speci¢cities of these deacetylases, yeaststrains lacking either Rpd3 and Hda1 show increasedacetylation at lysines 5 and 12 of histone H4 [98].Furthermore, mutant derivatives of Rpd3 that abol-ish histone deacetylase activity in vitro but do nota¡ect the ability of the polypeptide to form highmass complexes are defective for transcriptional re-pression in vivo [99].

In addition to the RPD3 and HDA1 genes, a data-base search showed that at least four di¡erent genesare related in sequence to RPD3 and HDA1 [44],although the function of these other genes has notbeen characterized.

In the same screens which produced RPD3, anoth-er gene was obtained, RPD1, which turned out to beallelic to SIN3, one of the swi/snf suppressors [31,42].Genetic [100] and biochemical [101] criteria indicatedthat Rpd3 and Sin3 interact and that they are com-ponents of the large HDB complex with deacetylaseactivity [44,102]. The arti¢cial recruitment of Sin3 toa promoter by using a LexA-Sin3 fusion repressestranscription from promoters containing a LexAbinding site [103]. This repression requires the pres-ence of a wild-type RPD3 allele in the cell. Thisresult suggests that the deacetylation complex hasto be targeted somehow. Additional support forthis view comes from the study of the repressionmediated by the protein Ume6 [101]. This DNAbinding protein represses several promoters in amanner dependent on Sin3 and Rpd3. Furthermore,Ume6 interacts with Sin3 protein via a short domainthat by itself is able to mediate Sin3-, Rpd3-depend-ent transcriptional repression. Additional workshowed that transcriptional repression by Ume6 in-volves deacetylation of lysine 5 of histone H4 byRpd3 [98].

The prevailing idea is that the Rpd3-Sin3 deacetyl-ation complex may be targeted to speci¢c promotersvia interaction with di¡erent DNA binding proteins.Sin3 is well suited for such a task. Sin3 is a 175-kDaprotein that contains four paired amphipathic helixmotifs [104]. Each motif consists of two amphipathic

FEMSRE 658 28-6-99


helices separated by a short amino acid linker. Sim-ilar structural motifs have been identi¢ed in tetratrico-peptide repeats (TPR) proteins, one of them,Tup1, is part of a repression complex which is tar-geted to di¡erent promoters by interaction with spe-ci¢c DNA binding proteins via the TPR motifs [105].

It has been assumed, yet not proven, that theHda1-containing complex would work in a similarway, although no targets have been identi¢ed so far.

6. Regulatory positive factors

6.1. The Swi/Snf complex

Swi/Snf is a protein complex that activates expres-sion of several genes in yeast. The description of theSwi/Snf complex was the result of a convergence ofgenetic and biochemical studies.

The SWI genes were identi¢ed as being importantfor transcription of the HO gene that encodes anendonuclease required for mating type switching[23]. On the other hand, SNF genes were identi¢edto be required for transcription of the SUC2 genethat encodes invertase, the enzyme required by S.cerevisiae to catabolize sucrose or ra¤nose [24].

The ¢rst indication of the function of SWI andSNF genes outside the transcription of HO andSUC2 was that mutants defective in some of thesegenes exhibit slow growth and other phenotypes likeinositol auxotrophy or inability to use galactose as acarbon source [25]. It was found that SWI2 wasidentical to SNF2, and hence, this gene is referredto as SWI2/SNF2 [25]. All these genes do not encodesequence-speci¢c DNA binding proteins but are re-quired to achieve the proper amount of transcriptionfrom a number of promoters [25]. Further experi-ments revealed a functional interdependence amongsome of the Swi and Snf proteins (speci¢cally Swi1,Swi2/Snf2, Swi3, Snf5 and Snf6) which indicated thatthey may act together as a complex [25,106]. Bio-chemical studies of Swi/Snf proteins led to the puri-¢cation of a 2-MDa protein complex (about half thesize of a ribosome), which is commonly known asthe Swi/Snf complex [46,47]. This complex is com-posed of the Swi1, Swi2/Snf2, Swi3, Snf5 and Snf6proteins and ¢ve additional polypeptides, some ofwhich have been cloned: Swp82, Swp73 (SWP73

[30]), Swp61, Swp59 and Swp29 (TFG3/TAF30/ANC1 [29]).

The connection between the function of the Swi/Snf complex and chromatin was established throughgenetic studies. In wild-type cells, the chromatinstructure surrounding the SUC2 promoter changesin response to the induction of transcription of thisgene. Mutations in either SWI2/SNF2 or SNF5 re-sult in a decrease in transcription and in a chromatinstructure more resistant to digestion by micrococcalnuclease, even in induced conditions [107]. This re-sult was interpreted as an indication of a failure ofthe mutant cells to antagonize nucleosomal organi-zation at the promoter region. Furthermore, thetranscriptional defects in strains lacking these SNFgenes are suppressed by a deletion of HTA1 orHTB1 genes encoding histones H2A and H2B(hta1-htb1) and the chromatin structure in these mu-tants is more accessible to micrococcal nuclease (re-sembling the wild-type structure in induced condi-tions) [108]. A second piece of evidence was thefact that the sin1 and sin2 suppressors of swi muta-tions encode mutant proteins implicated in chroma-tin structure. SIN2 is identical to HHT1, one of thetwo genes of S. cerevisiae that encode histone H3[34], and SIN1 is identical to SPT2, which encodesa protein similar to HMG1 and is a component ofchromatin in yeast [11,12] (see Sections 4.1 and 4.4).

The biochemical characterization of the puri¢edyeast Swi/Snf complex provides direct evidence thatthe Swi/Snf complex might function by disruptingnucleosome structure. Binding of Gal4 derivativesto a reconstituted mononucleosome carrying a singleGal4 binding site is substantially facilitated by puri-¢ed Swi/Snf complex in a reaction that requires ATPhydrolysis and is independent of the presence or ab-sence of activation domains in the Gal4 derivatives[109]. Puri¢ed Swi/Snf complex is also able to disruptan array of preassembled nucleosomes reconstitutedwith puri¢ed histones in an ATP-dependent manner[110]. However, a demonstration of Swi/Snf-depend-ent transcriptional activation of a preassembledchromatin template in vitro has not yet been re-ported.

The way in which the Swi/Snf complex facilitatesthe accessibility to nucleosomal DNA is not known.It is clear that the activity of the Swi/Snf complexrequires continuous ATP hydrolysis. The SWI2 gene

FEMSRE 658 28-6-99


encodes a protein that contains motifs similar tothose found in DNA-stimulated helicases. In fact,the Swi2 protein has a DNA-dependent ATPase ac-tivity [111]. Mutagenesis of the conserved NTP bind-ing motif in Swi2/Snf2 results in a signi¢cant reduc-tion of the ATPase activity and facilitatedtranscription factor binding to mononucleosomes,as mediated by the Swi/Snf complex [112]. Severalmodels have been proposed. A model (Fig. 5A)[113] suggests that these factors may function asATP-driven motors that translocate along DNAand destabilize DNA-protein interactions. In thismodel, a DNA translocation protein uses the energyderived from hydrolysis of ATP to transverse a nu-cleosome in a wave-like manner that results in onlypartial disruption of the nucleosome at any particu-lar point. The transcription factors use this transi-tory disruption to reach its DNA targets. This modelis similar to the `spooling' mechanism that has beensuggested for the procession of polymerases throughnucleosomes [114]. However, this model of Swi/Snf

action does not explain the recent observation thatthe action of this complex on nucleosomes results ina stable remodeled form of nucleosome [115,116]. Analternative model proposes that the Swi/Snf complexinteracts with nucleosomal DNA and uses the energyof ATP hydrolysis to alter DNA-histone interaction(Fig. 5B). In its original conception it was supposedthat the action of the Swi/Snf complex promoted theloss of one or both H2A-H2B dimers from the nu-cleosome core [117], but recent data about in vitroSwi/Snf-altered nucleosomes indicate that there is noloss of histones [118].

An interesting issue is the question of how the Swi/Snf complex is targeted to the correct chromosomalposition. Since this complex is not abundant (ap-proximately 100 copies per cell) the possibility thatit is a general chromatin component is ruled out.There are several possible mechanisms but none ofthem has been clearly substantiated. One possibilityis that the Swi/Snf complex associates with activatorproteins then targets it to speci¢c genes. However,

Fig. 5. Possible models of Swi/Snf action. A: The ATP-driven motor model. The Swi/Snf complex moves across the nucleosome-associ-ated DNA using the energy of ATP hydrolysis. The movement of the complex across the nucleosome would result in a change of the po-sition of the DNA relative to the histone octamer. In this conformation the activator molecule binds to the DNA. B: The nucleosomemodi¢cation model. The Swi/Snf action involves the interaction with the nucleosome resulting in a modi¢ed nucleosome to which the acti-vators are able to bind. This process uses the energy of ATP hydrolysis. Adapted from [115,119].

FEMSRE 658 28-6-99


recent results suggest that it is rather the chromoso-mal context of the binding site of the activator thatdetermines the Swi/Snf dependence of transcription[118]. Another possibility would be that the Swi/Snfcomplex is recruited to promoters along with thetranscriptional machinery [119]. However, a strongor stable association with the RNA polymerase holo-enzyme has been questioned [120]. Still another alter-native would be to assume that one of the Swi/Snfsubunits has sequence-speci¢c DNA binding a¤nitythat provides promoter speci¢city [121]. An interest-ing clue is the interaction of the Swi/Snf complexwith the SAGA complex, which is recruited by spe-ci¢c activators (see Section 6.3).

A second large complex able to remodel chromatincalled RSC (from remodeling the structure of chro-matin) has been isolated in yeast [120]. This complex,composed of 15 subunits, is 10-fold more abundantthan the Swi/Snf complex and its properties are strik-ingly similar to those of the Swi/Snf complex; it hasa DNA-dependent ATPase activity and the capacityto alter nucleosome structure. The protein sequencesof four RSC subunits, Sth1/Nps1, Sfh1, Rsc6 andRsc8 are known; they show similarity to that ofthe Swi/Snf subunits, Swi2/Snf2, Snf5, Swp73, andSwi3, respectively [120,122,123] but unlike Swi/Snf,RSC is essential for mitotic growth. The defects incomponents of RSC cannot be suppressed by muta-tions in chromatin components and no associationbetween RSC and the RNA polymerase has beenobserved. A temperature-sensitive allele of theSFH1 gene arrests the cell in the G2/M phase atthe non-permissive temperature [122] and a mutationin NSP1/STH1 alters the chromatin structure of cen-tromeres [124]. All these data suggest that, althoughthe genetic targets of RSC remain unidenti¢ed, thiscomplex may be involved in the chromatin remodel-ing activity required for progression of the cell cyclemore than in transcriptional regulation.

6.2. The Srb/mediator

The SIN4/SSN4 gene was obtained as a suppres-sor of swi and snf requirements [31,36]. This gene isrequired both for repression and for full activationof several genes [125]. Sin4 is associated in vivo withRgr1 and mutations in SIN4 or RGR1 exhibit similarphenotypes [126]. In addition to suppressing swi/snf

defects, mutations in these genes also suppress tran-scriptional defects due to N insertion in promoters(Spt3 phenotype), and allow transcription of UAS-less promoters. These phenotypes are similar to thosefound in mutations or depletion of histones, whichsuggests some relation with chromatin. The similar-ities between RGR1 and SIN4 extend to a third gene,GAL11. The GAL11 gene was found to be allelic toSPT13 and mutations in this gene suppress not onlythe Spt3 phenotype but also bypass the requirementfor UAS [127]. In view of these genetic relationshipsit is not surprising that the proteins encoded by thesegenes form a complex with the addition of an un-characterized 50-kDa polypeptide [128]. This com-plex is actually a subcomplex of the mediator andRNA polymerase II holoenzyme [128].

The 12-subunit core of RNA Pol II associated in acomplex with a number of accessory factors will bereferred to here as holoenzyme. The characterizationof this complex was the result of the convergence ofgenetic and biochemical approaches. One feature ofthe RNA Pol II largest subunit is the presence of along C-terminal tail composed of 26 nearly identicalrepeats of a heptapeptide sequence [129]. While par-tial (up to 10 copies) truncation of the C-terminaltail results in weakened responses to some activators,more severe truncations (leaving just two or threerepeats) are lethal. In a search for extragenic sup-pressors of the more severe truncation of the C-ter-minal tail of Pol II, the SRB genes (from suppressorof RNA polymerase B) were obtained [130]. Bio-chemical analysis showed that the proteins encodedby SRB genes were associated in a complex requiredfor the response of Pol II to activators such as Gal4-VP16 and Gcn4 in a partially puri¢ed in vitro tran-scription reactions (and hence the name of mediatorto this complex) [131^133].

What is the actual role of the mediator in chro-matin remodeling? This question has no answer sofar. It is clear that the Sin4-containing subcomplex isnot responsible for any functional attributes of me-diator in transcription with pure general factors[134]. On the other hand, RNA Pol II holoenzymerecruitment is su¤cient to remodel chromatin at thePHO5 promoter [135]. Although the Gal11 protein isnot required for this chromatin remodeling activity,it is not known if other components of the Sin4subcomplex are required. An interesting possibility

FEMSRE 658 28-6-99


is that this subcomplex could be involved in the com-petition between the transcription machinery andchromatin templates.

6.3. The SAGA complex

Histone acetylation has a role in chromatin assem-bly and transcription. Acetylation in vivo occursonly at speci¢c lysines in the amino-terminal tail ofhistones. Although these tails are not needed tomaintain the structural integrity of the nucleosome,they have roles in higher order chromatin structureand in interactions with non-histone chromosomalproteins (see above). Acetylation of the histone tailsmay introduce allosteric changes in nucleosome con-formation and inhibit the higher order folding ofnucleosome arrays that are repressive to transcrip-tion [136].

Two major histone acetyltransferase (HAT) activ-ities have been described in eukaryotic cells. The ¢rstis a cytoplasmic enzyme, called HAT-B, involved inthe deposition-related acetylation of H4 onto repli-cated DNA. The second kind of acetyltransferaseactivity, HAT-A, has been associated with the nu-cleus and it was thought to be responsible for tran-scription-associated acetylation.

The gene encoding HAT-B was cloned from S.cerevisiae [137,138]. This enzyme is composed oftwo polypeptides: Hat1, the catalytic subunit, andHat2, which is also a component of the chromatinassembly factor, CAF1. No role has been describedfor the HAT-B complex in transcriptional regula-tion.

The HAT-A activity is encoded by the GCN5 gene[139]. The GCN5 gene was initially identi¢ed in agenetic screen designed to isolate mutants unable togrow under conditions of amino acid limitation[39,140]. A second screen looking for mutants thatreversed the toxicity (squelching) caused by overpro-duction of the strong activator, Gal4-VP16, alsogave this gene. This last screen also led to the iso-lation of two other genes, ADA2 and ADA3 [37,38].

These three gene products are required for thefunction of several activators. Genetic and biochem-ical studies revealed that Gcn5, Ada2 and Ada3 forma complex, called the ADA complex [141,142]. Puri-¢ed Gcn5 protein shows histone acetyltransferase ac-tivity and it is able to acetylate free histones at spe-

ci¢c lysine positions (K14 in H3; K8, K16 in H4)[143]. Interestingly, these positions do not overlapsites used by cytoplasmic HAT-B for histone depo-sition and nucleosome assembly (K5, K12 in H4).However, puri¢ed Gcn5 protein is unable to acety-late in vitro histones already assembled in nucleo-somes, suggesting the possibility that other proteinsare required to direct Gcn5 in the acetylation ofnucleosomes. A biochemical search for native com-plexes able to acetylate in vitro nucleosomes yieldedthe isolation of two high molecular mass complexes(0.8 and 1.8 MDa) [50]. Both complexes containGcn5, Ada2 and Ada3. The larger of these two com-plexes turned out to contain Spt proteins (Spt20,Spt3, Spt8 and Spt7) and it is called SAGA (Spt/Ada/Gcn5 acetyltransferase). The relationship be-tween the 0.8-MDa Ada-Gcn5 complex and the1.8-MDa SAGA complex is not yet clear. Both com-plexes contain Gcn5 as the catalytic HAT subunit aswell as Ada2 and Ada3. One possibility is that the0.8-MDa complex lacks the Spt gene products, andactually it is a subcomplex of the larger SAGA com-plex. An alternative possibility is that, although bothcontain Gcn5 and Ada proteins, each complex mightrepresent quite distinct nucleosomal HAT activitieswith unique functions in the cell.

Recent work strongly suggests that nucleosomesare physiologically relevant substrates for these com-plexes and that histone acetyltransferase activity iscritical for transcriptional regulation. In one of thesestudies, a detailed mutational analysis of Gcn5 wasperformed in the context of the SAGA complex andon nucleosomal substrates [144]. The conclusion wasthat critical residues for histone acetylation by Gcn5are also required for transcriptional function in vivo.In the other study, additional evidence for physio-logical relevance was obtained by analyzing directlythe acetylation state of chromatin in yeast cells [145].Overexpression of Gcn5 leads to increased acetyla-tion of core histones. More interestingly, Gcn5 in-creases histone acetylation at promoter regions in amanner that is correlated with Gcn5-dependent tran-scriptional activation and histone acetylase activityin vitro.

The way in which the histone acetylase complexselectively a¡ects gene expression is an open ques-tion. It has been suggested that Gcn5 might be se-lectively recruited to promoters. Gcn5 has been

FEMSRE 658 28-6-99


shown to interact directly or indirectly through Ada2with a number of transactivators, for example VP16[146], Gcn4 [147] and Adr1 [148]. In addition to theability of acidic activators to physically interact withpuri¢ed native SAGA complex, it has been shownthat a Gal4-VP16 fusion targets acetylation andtranscriptional enhancement by SAGA [149].

An additional link between histone acetylation,activators and the basal transcriptional machineryis the recent characterization of several TBP-relatedproteins as components of the SAGA complex. The¢rst group of these components comprises all mem-bers of the TBP-related set of Spt proteins (Spt3,Spt7, Spt8 and Spt20), with the exception of TBPitself [150]. The second group is composed of severalTAFII (TATA binding protein-associated factors)[151]. This association of multiple transcriptionalregulatory proteins may confer upon SAGA the abil-ity to respond to a range of stimuli and to interactwith numerous activators, with the potential to reg-ulate a broad range of promoters.

7. Genetic interactions between di¡erent complexes:a matter of three

The presence of several complexes, acting to op-pose the contacts between nucleosomal histones andDNA, raises the question of whether all of them arenecessary for transcription of a single promoter or ifthey are redundant in function. In principle all thesecomplexes could act in a same pathway or throughdi¡erent pathways to activate gene expression. Theanalysis of the genetic interactions displayed betweencomponents of di¡erent complexes provides an ex-tremely powerful tool to garner new informationabout these systems.

Genetic analyses indicate that the ADA/GCN5products function in concert with the Swi/Snf com-plex. ADA/GCN5 products are required for expres-sion of several SWI/SNF-dependent genes such asHO, SUC2, Ty and INO1 [40,59]. The combinedGcn5 and Swi/Snf activities are important forADH2 [25,148] and both complexes are requiredfor the function of the Gcn4 activator [37,39] andthe mammalian glucocorticoid receptor, when ex-pressed in yeast [152,153]. Additional data suggesta functional interaction between the SAGA and

Swi/Snf complexes. Although ada/gcn5 and swi singlemutants are viable, the ada2 swi1, ada3 swi1, andgcn5 swi1 double mutants are inviable [40,154].This lethality is rescued by mutations in the SIN1gene [40]. Additional synthetic lethality has been de-scribed between mutations in SPT20 or SPT7 (bothcomponents of the SAGA complex) and mutationsin SWI/SNF genes (particularly SWI1 and SWI2)[154]. However, these genetic interactions do not nec-essarily mean that both complexes are physically as-sociated. There are some contradictory results onthis issue. Mutation in the ADA3 gene has a dramat-ic e¡ect on the stability or assembly of the Swi/Snfcomplex [40]. The reason for this is not known; onepossibility is that the SAGA complex is associatedwith the Swi/Snf complex and this interaction is re-quired for Swi/Snf stability. In disagreement withthis interaction, the two complexes elute at di¡erentpeaks in chromatographic fractionation procedures[40].

Several patterns of genetic interactions have beendescribed between the Srb/mediator and the Swi/Snfcomplexes. Defects in components of both complexesare suppressed by similar mutations in chromatincomponents: histones (sin mutations) [34] and asso-ciated factors (sin1 mutations) [61]. Several swi/snfmutations are partially suppressed by a deletion ofthe SIN4 gene [31,100], but are synthetically lethalwith a deletion of the GAL11 gene [154]. These genes(SIN4 and GAL11) are members of a subcomplex ofmediator, and each has shown both positive andnegative roles in transcriptional regulation [134].Formally, these genetic data argue that some Swi/Snf and Srb/mediator activities function in parallelpathways, as suggested by their similar mutant phe-notypes and synthetic lethality. In other cases theymay be antagonistic, as suggested by partial suppres-sion. These types of genetic interaction ¢t with thefunctional heterogeneity among mediator compo-nents and they suggest a functional relationship be-tween the two complexes in vivo. This functionalrelationship is supported by biochemical data indi-cating a physical interaction between Swi/Snf and theholoenzyme [119], although the strength or stabilityof this association has been questioned [120].

Finally, the third corner in the triad, the interac-tions between SAGA and the Srb/mediator, is sup-ported by results from studies of other genetic inter-

FEMSRE 658 28-6-99


actions. The deletion of the SPT20 or the SPT7 geneshows synthetic lethality with a deletion of eitherSIN4 or GAL11 [154]. Interestingly, the double mu-tant gcn5 sin4 or gcn5 gal11 is viable, suggesting thatthe interactions between SAGA and Srb/mediatorare unrelated to histone acetylation [154]. Substantialevidence indicates an association between SAGA andTBP. First, SAGA contains two groups of proteinsthat interact with TBP: Spt20, Spt3 and Ada2 onone side [150,154] and TAFs on the other [151]. Sec-ond, certain missense mutations in SPT15 whichencodes TBP cause phenotypes similar to thosecaused by null mutations in these SAGA functions[155]. However, TBP has not been observed in thepuri¢ed SAGA complex [151,154].

8. A model of interaction

The genetic interactions described above suggest athree-step model to account for the di¡erent chro-matin remodeling activities present in the cell (Fig.6). Since the SAGA complex appears to bind toacidic activation domains [146^149], this model sug-gests that this complex is involved in the targeting ofother interacting complexes, such as the Swi/Snfcomplex, to speci¢c activator proteins. In addition,the histone acetyltransferase activity of the SAGAcomplex may disrupt the higher order structures byimpairing the ability of acetylated histone tails tosustain such structures, allowing the Swi/Snf com-plex to gain access to the nucleosome template.The interaction of the Swi/Snf complex with eitherthe histones or non-histone proteins, such as Sin1,produces the complete spectrum of changes in chro-matin structure that have been observed in vivo. Thishierarchy of gene function is well supported by sup-pression analyses between several components ofthese complexes. In the HO promoter, loss of Swi5(the major activator protein for the HO gene) can bepartially suppressed by sin1, sin2 (histone H3), sin3and rpd3 mutations [12,31,32,34,100]. Loss of Gcn5(one of the components of the histone acetyltransfer-ase complex required for HO transcription) can besuppressed by the same mutations [48,50]. However,while defects in the Swi/Snf complex can be sup-pressed by sin1 (which is thought to be a target ofthe Swi/Snf complex [85]) and sin2 mutations, they

cannot be suppressed e¤ciently by sin3 or rpd3 mu-tations. These results suggest that histone acetylationat the HO promoter functions upstream of the Swi/Snf complex. Consistent with this view there is astrong synergy (synthetic enhancement) betweenrpd3 mutations (which a¡ect the acetylation of his-tone tails) and sin1 and sin2 mutations (which cir-cumvent the need for the Swi/Snf complex) [59].

Results with speci¢c histone mutations in theknuckle suggested an additional step in overcomingchromatin-mediated repression that is downstreamof the step mediated by the Swi/Snf proteins, butprobably occurs before the transcription machinery

Fig. 6. Model of chromatin remodeling in a yeast promoter. Theshaded ovals represent nucleosomes starting from a highly com-pacted conformation (top) due to the interaction between histonetails (not shown) and core from neighboring nucleosomes. Theaction of histone acetyltransferases (like the GCN5-containingSAGA complex) acetylates the tails (shown by stars) and disruptsthe interactions responsible for the compacted conformation.This more relaxed chromatin is the target for the Swi/Snf com-plex which modi¢es single nucleosomes (open oval). In addition,accessory factors like Sin1 protein (small shaded ovals) could beremoved. Finally the RNA Pol II complex, containing the Srb/mediator complex, displaces the nucleosome allowing the tran-scription to start.

FEMSRE 658 28-6-99


is totally assembled in the promoter. An interestingpossibility is that this step is performed by the chro-matin remodeling activity associated with the Srb/mediator. Mutations in the SIN4 gene partially sup-press transcriptional defects caused by mutations inthe SWI1 and SWI2 genes [96]. Studies designed toaddress if genetic interactions occur between holoen-zyme components and histone H2A-H2B mutationsare required to support this view.

An important aspect to the understanding of thismodel is that not every promoter will require all thedi¡erent activities present in the several complexes inorder to start transcription. The requirement for anyof these activities will re£ect the chromosomal con-text where the promoter lies. Another importantpoint is that a single complex can participate inmore than one step even when these are not con-nected in the temporal sequence. For example, theSAGA complex is proposed to work in the ¢rst placeby opening the chromatin structure via acetylation ofhistone tails, but may be involved in later steps at thesame time, because it is composed of proteins able tointeract with the transcriptional machinery, like theSpt and the TAFII proteins.

Acknowledgements

The author thanks Fernando Rojo (CNB, Madrid)and Carlos Gancedo (IIB, Madrid) for critically re-viewing the manuscript. Work reported from the au-thor's laboratory was supported by CAM Grant07B-0030.

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chromatin and transcription in saccharomyces cerevisiae

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