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Biochem. Biophys. Res. Commun. 128, 1079–1086 nonessential, substrate-specific role in protein receptors. EMBO J. 19, 187–198 16 Chau, V. et al. (1989) A multiubiquitin chain is confined turnover. Mol. Cell. Biol. 16, 6020–6028 35 Spence, J. et al. (1995) A ubiquitin mutant with to specific lysine in a targeted short-lived protein. 26 Fu, H. et al. (1998) Multiubiquitin chain binding and specific defects in DNA repair and multiubiquitination. Science 243, 1576–1583 protein degradation are mediated by distinct domains Mol. Cell. Biol. 15, 1265–1273 17 Finley, D. et al. (1994) Inhibition of proteolysis and cell within the 26S proteasome subunit Mcb1. J. Biol. 36 Arnason, T. and Ellison, M.J. (1994) Stress resistance cycle progression in a multiubiquitination-deficient Chem. 273, 1970–1981 in Saccharomyces cerevisiae is strongly correlated with yeast mutant. Mol. Cell Biol. 14, 5501–5509 27 Girod, P-A. et al. (1999) Multiubiquitin chain binding assembly of a novel type of multiubiquitin chain. Mol. 18 Thrower, J.S. et al. (2000) Recognition of the subunit MCB1 (RPN10) of the 26S proteasome is Cell. Biol. 14, 7876–7883 polyubiquitin proteolytic signal. EMBO J. 19, 94–102 essential for developmental progression in 37 Fisk, H.A. and Yaffe, M.P. (1999) A role for 19 Piotrowski, J. et al. (1997) Inhibition of the 26S Physcomitrella patens. Plant Cell 11, 1457–1471 ubiquitination in mitochondrial inheritance in proteasome by polyubiquitin chains synthesized to 28 Glickman, M.H. et al. (1998) A subcomplex of the Saccharomyces cerevisiae. J. Cell Biol. 145, have defined lengths. J. Biol. Chem. 272, proteasome regulatory particle required for ubiquitin- 1199–1208 23712–23721 conjugate degradation and related to the COP9- 38 Galan, J-M. and Haguenauer-Tsapis, R. (1997) Ubiquitin 20 Beal, R. et al. (1996) Surface hydrophobic residues of signalosome and eIF3. Cell 94, 615–623 Lys63 is involved in ubiquitination of a yeast plasma multiubiquitin chains essential for proteolytic targeting. 29 Hofmann, K. and Bucher, P. (1998) the PCI domain: a membrane protein. EMBO J. 16, 5847–5854 Proc. Natl. Acad. Sci. U. S. A. 93, 861–866 common theme in three multiprotein complexes. 39 Spence, J. et al. (2000) Cell cycle-regulated 21 Beal, R.E. et al. (1998) The hydrophobic effect Trends Biochem. Sci. 23, 204–205 modification of the ribosome by a variant multiubiquitin contributes to polyubiquitin chain recognition. 30 Ferrell, K. et al. (2000) Regulatory subunit interactions chain. Cell 102, 67–76 Biochemistry 37, 2925–2934 of the 26S proteasome, a complex problem. Trends 40 Koegl, M. et al. (1999) A novel ubiquitination factor, E4, 22 Cook, W.J. et al. (1994) Structure of tetraubiquitin Biochem. Sci. 25, 83–88 is involved in multiubiquitin chain assembly. Cell 96, shows how multiubiquitin chains can be formed. 31 Young, P. et al. (1998) Characterization of two 635–644 J. Mol. Biol. 236, 601–609 polyubiquitin binding sites in the 26S protease subunit 41 Hofmann, R.M. and Pickart, C.M. (1999) Noncanonical 23 Deveraux, Q. et al. (1994) A 26S protease subunit that 5a. J. Biol. Chem. 273, 5461–5467 MMS2-encoded ubiquitin conjugating enzyme functions binds ubiquitin conjugates. J. Biol. Chem. 269, 32 Wilkinson, K.D. and Hochstrasser, M. (1998) The in assembly of novel polyubiquitin chains for DNA 7059–7061 deubiquitinating enzymes. In Ubiquitin and the Biology repair. Cell 96, 645–653 24 van Nocker, S. et al. (1996) Arabidopsis MBP1 gene of the Cell (Peters, J-M., Harris, J.R. and Finley, D., 42 Haas, A.L. and Bright, P.M. (1985) The immunochemical encodes a conserved ubiquitin recognition component eds), pp. 99–126, Plenum Press detection and quantitation of intracellular ubiquitin- of the 26S proteasome. Proc. Natl. Acad. Sci. U. S. A. 33 Terrell, J. et al. (1998) A function for protein conjugates. J. Biol. Chem. 260, 12464–12473 93, 856–860 monoubiquitination in the internalization of a G protein- 43 Xie, Y. and Varshavsky, A. (2000) Physical association 25 van Nocker, S., et al. (1996) The multiubiquitin-chain- coupled receptor. Mol. Cell 1, 193–202 of ubiquitin ligases and the 26S proteasome. Proc. binding protein Mcb1 is a component of the 26S 34 Shih, S.C. et al. (2000) Monoubiquitin carries a novel Natl. Acad. Sci. U. S. A. 97, 2497–2502 proteasome in Saccharomyces cerevisiae and plays a internalization signal that is appended to activated

unique protein complexes have been purified whose apparent function is to What does ‘chromatin utilize the energy of ATP hydrolysis to re- move, displace, or destabilize nucleo- somes at specific chromosomal sites, in- remodeling’ mean? cluding promoters6–12,46. The scientist entering the chromatin field today has access to a variety of reagents that could scarcely be imagined ten years ago. Jeff D. Aalfs and Robert E. Kingston Not surprisingly, this rapid growth of information has complicated our under- The regulated alteration of chromatin structure, termed ‘chromatin remod- standing of the role of chromatin struc- eling’, can be accomplished by covalent modification of histones or by the ture in the regulation of nuclear events. action of ATP-dependent remodeling complexes. A variety of mechanisms The nucleosome was once thought of can be used to remodel chromatin; some act locally on a single nucleo- solely as a packaging unit for fitting DNA some and others act more broadly. It is critical to establish a direct con- into the nucleus. It is now clear that modi- nection between the remodeling events observed in vivo and the mecha- fication of nucleosome structure plays a nistic capabilities of remodeling complexes in vitro. critical role in the normal regulation of gene expression, and that nucleosomes interact with the transcription machin- THE CHROMATIN FIELD has undergone plexes that relocate nucleosomes, alter ery through a variety of mechanisms. a significant transition in the past the structure of nucleosomes and cova- The term ‘chromatin remodeling’ is decade. Previously, it had been gener- lently modify histones have been iso- widely used to describe changes in ally acknowledged that the incorpor- lated and characterized. It had long chromatin structure that occur during ation of eukaryotic DNA into protein been known that histones are acetylated regulatory processes; because it refers complexes, called nucleosomes, could at certain lysine residues, and that the to many events, it effectively describes affect gene regulation and that covalent acetylation state often correlates with none of them. Chromatin remodeling modification of the protein components gene expression and silencing1,2. Now, can generally be defined as any event of the nucleosome, or histones, was also both histone acetyltransferases and his- that alters the nuclease sensitivity of a likely to be important. Today, com- tone deacetylases have been identified region of chromatin. These events can and characterized, and their role in tran- occur independently or in concert with scriptional regulation can be studied di- other events, such as transcription. An J.D. Aalfs and R.E. Kingston are at the Dept rectly3–5. Ten years ago, it was not energy source, such as ATP, may or may of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA; and Dept known whether any activity beyond the not be required. Some events, but not of Genetics, Harvard Medical School, Boston, transcription machinery was needed to all, involve covalent modification of MA 02115, USA. remove the nucleosomes from promot- the histones. Given the variability of Email: [email protected] ers. Since then, more than half a dozen chromatin composition at different loci, 548 0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved. PII: SO968-0004(00)01689-3 TIBS 25 – NOVEMBER 2000 REVIEWS

(a) (b)

++

Transcription factor binds DNA Remodeler slides nucleosome

+ +

Transcription factor recruits remodeler; Transcription factor binds nucleosome- nucleosomes remodeled free DNA, acts as boundary to restrict nucleosome movement and allow other factors to bind

Additional factors bind remodeled nucleosomes

Key: Transcription Remodeled Remodeler Nucleosome factors nucleosome Ti BS

Figure 1 Hypothetical models for how remodeling complexes participate in gene regulation. In (a) a sequence-specific, DNA-binding factor binds to DNA and then recruits a remodeling complex by a direct protein–protein interaction. The remodeling complex then alters the structure of the nearby nucleosomes, increasing the accessibility of the nucleosomal DNA. Other factors can then bind. In (b) the remodeling complex, not targeted by the transcription factor, ‘slides’ the nucleosome away from the binding site to allow the transcription factor to bind. Once bound, the factor blocks further nucleosomal movement. remodeling might involve only the nu- the term has no mechanistic meaning; DNA, this factor then recruits a remodel- cleosomes, or it might involve the neu- a new and more precise vocabulary is ing complex. The remodeling complex tralization of repression complexes needed. then stably remodels the structure of such as the Drosophila Polycomb com- the surrounding nucleosomes, which al- plex or the yeast SIR (silent information Chromatin remodeling in vivo: chromatin lows other factors to bind to nearby repression) complex. Most researchers changes observed at promoters sites, preparing the gene to be tran- can agree on a set of observations, Remodeling of chromatin structure scribed. This ‘cascade’ of transcription in vivo and in vitro, which can be consid- has been observed in conjunction with factor binding, remodeling and addi- ered ‘chromatin remodeling’. What is transcriptional activation at several pro- tional binding events seems to occur at more difficult is to determine the exact moters. This remodeling involves the many promoters. In this example, a tran- physical changes underlying an in vivo loading of transcription factors onto scription factor targets a remodeling result, and to relate them to biochemi- their binding sites in the promoter, and activity to cause stable remodeling of cal activities observed in vitro. The term can involve changes in the positions several surrounding nucleosomes. ‘chromatin remodeling’ is defined of specific nucleosomes (sliding) or A different, hypothetical model is broadly, but the definition tells us very changes in the three-dimensional struc- shown in Fig. 1b. Here, a DNA-binding little about the mechanisms behind indi- ture of nucleosomes, or both. Binding of transcription factor and a remodeling vidual observations. transcription factors, sliding of nucleo- complex work together to initiate re- In this article, we examine a handful somes and conformational changes in modeling. The nucleosomes over this of observations loosely termed ‘chro- nucleosomes can all change the nu- promoter maintain their normal struc- matin remodeling’ events. First, we re- clease sensitivity of chromatin, and thus ture but are mobilized and slide away view the chromatin remodeling events each of these events can be construed as from the binding sites, enabling the tran- observed at different promoters in vivo, remodeling. There are several different scription factor to bind. The DNA-bind- comparing the requirements and most ways in which these events can combine ing factor, once bound to its site, ex- plausible mechanisms of each event. to remodel any given region of a pro- cludes nucleosomes and thus helps to We then compare two prominent fami- moter during transcriptional activation. define and stabilize a nucleosome-free lies of ATP-dependent remodeling com- In the hypothetical example of Fig. 1a, region. Other factors can then bind to plexes and discuss mechanistic simi- a transcription factor binds to nucleoso- the nucleosome-free region and enable larities and differences between these mal DNA independently; either the tran- transcription. This model differs from complexes. We contend that there are scription factor binds to a site between that shown in Fig. 1a because the re- so many distinct events that can be cat- nucleosomes, or it can bind to DNA modeler is required for the initial tran- egorized as chromatin remodeling that within a nucleosome. Once bound to scription factor binding, and because 549 REVIEWS TIBS 25 – NOVEMBER 2000

initiates transcription from the (a) (b) HSP70 gene, then pauses ap- Uninduced (non-heat-shock) conditions SWI/SNF proximately 40 bp downstream from the start site15,16. Upon heat shock, the inducible tran- TBP (1) Swi5p scription activator, heat-shock factor (HSF), binds to sites in GAGA Engaged, paused SAGA the DNase-hypersensitive re- factor polymerase (~ +40 nt) gion, stimulating re-initiation and elongation of the nascent Constitutive DNAse I (2) HSP70 transcript. Remodeled nucleosomes hypersensitive site The DNase-hypersensitive region is required for the nor- Induced (heat-shock) conditions mal expression of HSP70. It is (3) dependent on the presence of SBF GAGA several [GA]n repeats in the factor HSF TBP Remodeled, acetylated nucleosomes promoter, and on a sequence- specific transcription factor, GAGA factor, which binds con- HSF TBP Polymerase stitutively to these repeats17. A binds upon remains bound, elongates 500-kDa, four-peptide remodel- heat shock catalyzes (4) ing complex, the Drosophila re-initiation Polymerase NURF (nucleosome remodel-

Ti BS ing factor) complex, was puri- fied on the basis of its ability Figure 2 to act in concert with GAGA Models for the activation of the HSP70 and HO promoters. These models are not completely substan- factor to disrupt nucleosome tiated by experiment, but are consistent with what is currently known about each promoter. At HSP70 spacing over the HSP70 pro- (a), the promoter region is constitutively hypersensitive to DNase I and appears to be depleted of his- moter in vitro10. The core of the tones. Under non-heat-shock conditions, TBP binds at the TATA box, and RNA polymerase II holoenzyme initiates transcription, pausing 40 nt downstream. Under stressful conditions (e.g. heat shock) the NURF complex, and several heat-shock factor (HSF) binds to its sites in the nucleosome region and stimulates elongation by the other remodeling complexes paused polymerase. At HO (b), Swi5p binds DNA without the help of any known remodeling complex isolated from Drosophila, is an and then recruits SWI/SNF, which remodels several surrounding nucleosomes (1). SWI/SNF then ATP-hydrolysing protein called recruits SAGA, directly or indirectly (by providing better substrates for acetylation), which acetylates the imitation switch (ISWI) surrounding nucleosomes (2). Acetylation might be needed to ‘lock’ the nucleosomes into a more (Ref. 18). accessible form created by SWI/SNF. After SWI/SNF and SAGA have modified the DNA, SBF As will be discussed in de- (Swi4p–Swi6p) can bind to the remodeled DNA (3), where it presumably recruits the general transcrip- tion machinery (4). At HO, the remodeling appears to act over several nucleosomes (Ͼ1 kb of DNA). tail below, the ISWI-based re- Abbreviations and explanations: HSP, heat-shock protein; SAGA, Spt-Ada-Gcn5-acetyltransferase; modelers appear to act by SWI/SNF, nucleosome-remodeling complex named after mating-type switching (SWI), caused by defects ‘sliding’ nucleosomes over in the transcription of the HO endonuclease gene and sucrose non-fermenting (SNF), caused by defects DNA, leaving them otherwise in the transcription of the SUC2 invertase gene; TBP, TAT-binding protein. intact. This sliding activity ap- parently creates a nucleo- the binding of the transcription factor completely disparate mechanisms are some-free region over the HSP70 pro- plays a direct role in establishing a used. Two well-characterized examples moter, delimited by GAGA factor bound remodeled state by forming a barrier of remodeling illustrate these principles. at the [GA]n repeats, which allows TFIID to subsequent nucleosome movement. and RNA polymerase to bind and initi- The transcription factor acts locally, to Remodeling and activation of the HSP70 ate. Upon heat shock, this hypersensi- establish a boundary that blocks further promoter tive site appears to be essential for the nucleosome movement. In Drosophila, the promoters of sev- binding of HSF and subsequent tran- There are other theoretical schemes eral heat-shock response genes are re- scriptional activation. It is important to by which factor binding and remodeling modeled in vivo13; the properties of one point out that NURF has not yet been complexes can combine to remodel of these promoters, the 70-kDa heat- shown to interact directly with the chromatin at a promoter. The central shock protein (HSP70) promoter, are il- HSP70 promoter in vivo. point is that chromatin remodeling of a lustrated in Fig. 2a. The HSP70 promoter The HSP70 promoter illustrates the promoter will always involve multiple region is constitutively hypersensitive apparent use of nucleosome sliding as a steps, and might involve multiple tran- to DNase I digestion in vivo13, and ap- central mechanism of remodeling. scription factors and even multiple, dis- pears to be depleted of histones as de- Further work is needed to verify the tinct remodelers. Nucleosome movement termined by crosslinking analysis14. mechanism of establishment of the re- might be enhanced by remodelers or Furthermore, the general transcription modeled HSP70 promoter; however, at blocked by transcription factors, and nu- factor TATA-binding protein (TBP) is this point it appears that the chromatin cleosome conformation might be altered. constitutively bound to the TATA box, a remodeling observed on this promoter All of these potential changes will result key regulatory site just upstream of the is caused by ATP-dependent nucleo- in changes in nuclease sensitivity, and all transcription start site. Under non-heat- some sliding, stabilized by the constitu- will be seen as ‘remodeling’ even when shock conditions, RNA polymerase II tive binding of transcription factors. In 550 TIBS 25 – NOVEMBER 2000 REVIEWS contrast to the examples cited below, acetyltransferase activity of SAGA; an in- (homologous to the yeast SWI/SNF com- there are no data that any of these fac- active, but otherwise intact, SAGA will plex described above) acts in concert tors can bind to chromatin without the not allow SBF binding or transcription. with the glucocorticoid or estrogen re- help of the ATP-dependent remodeling These studies lead to a detailed, ceptors to remodel the nucleosomes complex. Further chromatin remodeling though still speculative, model for acti- over the promoter and activate tran- events are likely to play a role during vation of HO (Fig. 2b). Swi5p binds to its scription. Glucocorticoid receptor and subsequent activation by HSF, although sites in the HO promoter and then re- other nuclear receptors are able to bind these events are not well understood. cruits the remodeling complex SWI/SNF. independently to nucleosomal DNA, so SWI/SNF facilitates the binding and ac- remodeling at these promoters might in- The yeast HO promoter tivity of the SAGA in one (or both) of volve cascades conceptually similar to The yeast HO gene encodes an en- two ways: either by directly recruiting that described above for HO. In contrast donuclease involved in mating-type SAGA, or by remodeling the surrounding to HO, there is no requirement for acety- switching. A genetic screen for defects nucleosomes to make them better sub- lation at the MMTV promoter; in fact, in mating-type switching uncovered a strates for acetylation. Finally, after the deacetylation appears to be necessary number of genes, named the SWI (mat- action of the SWI/SNF and SAGA com- for full activation24. ing-type switching) genes, required for plexes, SBF (Swi4p–Swi6p) is able to At the yeast PHO5 gene, four pos- the normal transcriptional regulation of bind the promoter, where it might then itioned nucleosomes are either removed the HO gene19. Recently, an elegant se- recruit the general transcription ma- or destabilized in response to phos- ries of experiments from the Nasmyth chinery and activate the gene itself. phate starvation; the gene is concomi- and Peterson groups have provided an The HO promoter is remodeled by a tantly activated25. This remodeling does idea of the sequence of remodeling cascade of interactions. This cascade is not require histone acetylation or any events involved in the activation of HO triggered by the binding of Swi5p; to date, known remodeling complex, and might transcription20,21. there is no evidence that Swi5p requires require only components of the tran- The HO promoter is bound by at least any remodeling activity to bind, although scription machinery26. PHO5 might rep- two sequence-specific activating fac- several other putative remodelers remain resent an extreme example, where chro- tors. Swi5p and SBF (Swi4p–Swi6p cell- to be tested in this system. Swi5p recruits matin remodeling does not require cycle box factor, a heteromeric complex SWI/SNF and SAGA; judging by the size dedicated chromatin modifying com- of the proteins Swi4p and Swi6p) bind to of the promoter and the spacing of the plexes, but occurs as a result of acti- distinct sites upstream of the HO open elements, these complexes remodel vated transcription. These and other reading frame, and both are required for approximately 1 kb of chromatin. Sub- studies highlight the diversity of mecha- the normal expression of the HO gene. sequent binding by activators and the nistic paths that can lead to chromatin Also, a multiprotein nucleosome- general machinery requires this remod- remodeling. remodeling complex, SWI/SNF (for an eled stretch of chromatin. explanation of the term SNF, see below), The histone acetyltransferase activity Chromatin remodeling in vitro: SWI/SNF was identified by its critical role in HO of SAGA is required for the normal and ISWI-based protein complexes transcription22; SWI/SNF, a 2-MDa, 12- expression of HO, and ChIP analysis The previous section introduced two peptide complex, contains several detects acetylated histones over the classes of remodeling complexes, the genes identified in the original SWI promoter following activation, implying SWI/SNF complexes (yeast SWI/SNF and screen. Furthermore, another multipro- that nucleosomes are not removed dur- its yeast, human and fly homologs), and tein complex called SAGA (Spt-Ada- ing remodeling. The precise positions the ISWI-based complexes (Drosophila Gcn5-acetyltransferase) has been and structures of the nucleosomes over NURF and other complexes purified shown to be required for normal HO ac- the HO promoter following activation from Drosophila, yeast and humans). We tivation23. SAGA contains the histone are not known. SWI/SNF is able to create now examine some of the recent bio- acetyltransferase encoded by GCN5; the stably remodeled nucleosomal struc- chemical studies of these complexes, acetylation of certain lysine residues of tures in vitro (see below), so a simple examining the mechanisms by which the histones is highly correlated with hypothesis is that SWI/SNF and SAGA they remodel nucleosomes. Finally, we transcriptional activity of many genes. work together to create stably remod- will speculate on the relationship be- Chromatin immunoprecipation assays eled nucleosome structures that are tween the activities observed in vitro, (ChIPs) were used to detect the binding required for subsequent steps. Thus, and the effects on promoters observed of Swi5p, SWI/SNF, SBF (Swi4p–Swi6p), remodeling on HO might involve the cre- in vivo. and SAGA to the HO upstream regula- ation of altered nucleosome structures tory sequences in a variety of genetic over a wide region, in contrast to remod- The SWI/SNF complexes backgrounds, and to examine the acety- eling on Drosophila HSP70 where nucleo- A set of complexes have been purified lation status of the promoter (Fig. 2b). somes appear to have been removed containing members of the yeast SWI Swi5p binds the HO promoter indepen- over a shorter region. and SNF gene families (SNF is named dently, binding transiently before any after sucrose non-fermenting, caused by other protein. SWI/SNF is the next factor Other promoters: a lack of generality defects in the transcription of the SUC2 recruited to the promoter; its binding is There are several other promoters invertase gene)27. This group of com- dependent on the presence of Swi5p. with well-characterized chromatin plexes includes the SWI/SNF complexes, SAGA binding and histone acetylation structure. At the mouse mammary purified from yeast8, humans6,7, and are dependent on both Swi5p and tumor virus (MMTV) promoter, six pre- Drosophila46, and the RSC (remodels the SWI/SNF activity. Finally, SBF binding is cisely positioned nucleosomes play a structure of chromatin) and RSCA com- dependent on Swi5p, SWI/SNF and SAGA. key role in the regulation of transcrip- plexes from yeast9 (Fig. 3a). These com- Importantly, SBF is dependent on the tion; the human SWI/SNF complex plexes all contain an ATP-hydrolysing 551 REVIEWS TIBS 25 – NOVEMBER 2000 subunit homologous to the yeast SWI2/SNF2 gene. Each complex con- tains between eight and 16 distinct BAP60 ISWI BRM peptides; there are four subunits PP1 which are conserved between all of BAP47 BAP47 NURF38 NURF55 the known complexes. The core BAP155 BAP155 ATPase peptide from the human NURF (Drosophila) BAP55 BAP111 SWI/SNF complex, the BRG1 protein SNR1 (BRG: Brahma-related gene; Brahma is a Drosophila homolog of yeast ISWI SWI2/SNF2), can be purified as a ho- dSWI/SNF (Drosophila) mogenous peptide that has many of ACF1 the same activities of the SWI/SNF complex28. BAF60a,b,c p250 CHRAC (Drosophila) The ISWI-based complexes The NURF complex is one of three ISWI-containing, chromatin remodel- BRG1/hBRM ISWI BAF155 ing activities purified from Drosophila; ACF1 the other two complexes, CHRAC BAF170 BAF170 (chromatin accessibility complex)12 BAF53 INI1 BAF57 and ACF (ATP-utilizing, chromatin as- ACF (Drosophila) sembly and remodeling factor)11, puri- hSWI/SNF (human) fied independently, might be similar or the same. Two yeast complexes, ISWI1 SNF2h and ISWI2, were purified based on the SNF11 huACF1 presence of yeast homologs of ISWI SWI1 SWP73 (WSTF) (Ref. 29). Also, a human complex, RSF 30 SWP82 (remodeling and spacing factor) , as SWI2/SNF2 huACF (human) well as the human CHRAC complex31, have been purified and shown to con- tain the human ISWI homolog SNF2H SWI3 SWI3 (SNF2 homolog). The ISWI-based com- plexes are much smaller than the ARP7 SNF5 ARP9 P325 SWI/SNF complexes, containing be- SNF6 SWP29 tween two and six peptides (Fig. 3b). hSNF2H ISWI and its homologs hydrolyse ATP ySWI/SNF (human) and are distantly related to the SWI2/SNF2 family of ATPases. ISWI RSF (human) has been purified to homogeneity and has remodeling activity32,33; similar to RSC2 RSC1 yISWI1 results seen with BRG1, its specific RSC6 activity is much lower than that of P74 RSC7 P110 ISWI-containing complexes. STH1 RSC3 P105

Sliding, a common mechanism yISWI1 (yeast) The NURF, CHRAC and yeast RSC8 RSC8 SWI/SNF complexes have all been shown to catalyse the cis-displace- yISWI2 ARP7 SFH1 ARP9 ment, or sliding, of a nucleosome p140 along a stretch of DNA (Fig. 4a). NURF RSC4 can move a nucleosome from the mid- dle of a 359-bp DNA fragment to two RSC (yeast) yISWI2 (yeast) distinct positions near each end of the Ti BS 34 fragment . Homogenous ISWI peptide Figure 3 can catalyse the same movement but The SWI/SNF and ISWI-based families of remodeling complexes. The core ATPase subunit of with 100-fold lower specific activity. In the SWI/SNF complexes is shown in green, whereas that of the ISWI complexes is blue. Violet a separate system, CHRAC can move a subunits are conserved among each of the SWI/SNF complexes; ACF1 is conserved among nucleosome from either end of a 248- CHRAC, ACF and huACF (light blue). Gray subunits do not appear to be conserved among bp fragment (completely unrelated to known complexes. ACF, ATP-utilizing, chromatin assembly and remodeling factor; CHRAC, chro- matin accessibility complex; ISWI, an ATP-hydrolysing protein called imitation switch; NURF, nu- the 359-bp fragment) to the middle of cleosome remodeling factor; RSC, remodels the structure of chromatin; RSF, remodeling and 33 the fragment . In this system, ISWI spacing factor; SWI/SNF, nucleosome-remodeling complex named after mating-type switching catalyses the opposite movement, (SWI), caused by defects in the transcription of the HO endonuclease gene and sucrose non- transferring the nucleosome from the fermenting (SNF), caused by defects in the transcription of the SUC2 invertase gene. 552 TIBS 25 – NOVEMBER 2000 REVIEWS

more accessible to regulatory factor (a) (b) Dinucleosome formation binding in vivo than a standard nucleo- some. All current data are consistent with the idea that the formation of stably remodeled structures by SWI/SNF and octamer transfer by SWI/SNF might Stable remodeled species: use a common reaction intermediate altered nuclease accessibility (see Fig. 4b). Thus, these two remodel- ing reactions, neither of which can be Octamer transfer performed by ISWI-based remodeling complexes, might proceed by a mecha- nism that is not shared between the ISWI and SWI/SNF families. Based on the in vitro activities of the two classes of remodeler, one might ex- Holliday (fourway) pect SWI/SNF to be involved in the acti- junction blocks sliding vation of promoters where the nucleo- Ti BS somes are not removed or repositioned, Figure 4 such as the HO promoter. Conversely, Possible mechanisms for nucleosome remodeling. (a) Nucleosome sliding appears to in- one might expect an ISWI-based com- volve altered histone–DNA contacts (translational movement of the histone octamer), but plex to be involved in the activation of not altered histone–histone contacts (three-dimensional nucleosome structure). The block- promoters where nucleosomes are ei- ing of sliding by a fourway junction seems to rule out the dismantling and reassembly of the ther absent or re-positioned to make a nucleosome. (b) The stably remodeled species created by SWI/SNF might function as a re- promoter more accessible, such as the modeled, more accessible template for factor binding. The same intermediate (SWI/SNF, HSP70 promoter. At present, however, green, violet and gray; DNA, blue and yellow; histone core, light green) might be used to cre- ate the stably remodeled dimer and to promote the transfer of a histone octamer to nonad- there are not enough data to assess jacent DNA (trans-displacement). The binding of a nucleosome and a region of bare DNA this hypothesis. More experiments are could create an intermediate product, which would be converted back into a nucleosome needed to determine the exact fates of and a region of bare DNA; octamer transfer would be the result of the octamer re-assem- nucleosomes over specific promoters. bling onto the new (acceptor) piece of DNA instead of its original (donor) site. Other observed differences between the SWI/SNF and ISWI-based remodelers middle of the fragment to either end of somes. SWI/SNF can significantly support the notion that the remodelers the fragment. The two complexes have change the topology of a plasmid nucle- operate through different mechanisms. not been tested side by side in either osomal array7,37. The nucleosomes do system. not appear to be removed from the plas- Differences between the SWI/SNF The yeast SWI/SNF complex can slide a mid to produce these topological shifts; complexes and the ISWI-based complexes: nucleosome from the end of a 2000-bp rather, they appear to be restructured. interactions with DNA and nucleosomes DNA fragment to several internal pos- SWI/SNF has also been shown to create The two families of complexes appear itions35. SWI/SNF from both yeast and hu- a stably remodeled nucleosomal species, to recognize their substrate, the nucleo- mans has also been shown to catalyse in which two mononucleosomes are some, differently. Both complexes hy- the trans-displacement of a nucleosome, joined together into a single remodeled drolyse ATP, and this hydrolysis activity moving it to a second piece of DNA (‘oc- structure38,39. The DNA path around the is significantly stimulated in the pres- tamer transfer’; Fig. 4b)36. To differenti- histones is dramatically altered in this ence of nucleosomes. However, al- ate between the possibilities of cis- and structure as determined by nuclease ac- though NURF, CHRAC and ISWIp are trans-displacement, the authors showed cessibility, so this structure is unlikely stimulated only by nucleosomes10,32, that the SWI/SNF sliding reaction can be to result from sliding of DNA on the his- SWI/SNF is stimulated by both nucleo- blocked by the presence of a Holliday tone octamer. This stable structure somes and bare DNA (Ref. 8); in fact, junction (a fourway junction or ‘cruci- formed from mononucleosomes might SWI/SNF appears to bind bare DNA form’ DNA structure) upstream of the reflect the same altered nucleosomal with a higher affinity than nucleosomes. nucleosome35. In this sliding assay, the conformation that causes changes in This strong affinity for DNA might authors were able to see the trans-dis- the topology of arrays; there is no infor- help explain the profound structural placement reaction, but only at higher mation at present concerning this possi- changes caused by SWI/SNF. By strongly molar ratios of SWI/SNF to substrate, bility. These observations – changes in binding the DNA within a nucleosome, suggesting that the sliding reaction is topology and the stable remodeled nu- SWI/SNF might displace the histones to more efficient. Although it is clear that cleosome – could be caused by a stable create a more accessible DNA path. both classes of remodelers can promote peeling of DNA away from the nucleo- Alternatively, the ISWI complexes might sliding, there is evidence that not all re- some, conformational changes that interact primarily with the histones to modeling activities can be explained by significantly alter the histone-DNA path create a more mobile nucleosome. sliding alone. on the nucleosome, or a combination of these effects. Interactions with the histone tails Beyond sliding The stably remodeled structure cre- Each of the core histones within a nu- SWI/SNF complexes cause changes in ated by SWI/SNF is more accessible to cleosome is composed of a globular cen- chromatin structure that cannot be the restriction enzymes and to DNA binding tral domain, flanked by N- and C-termi- result of a simple sliding of nucleo- by GAL4, and is therefore likely to be nal tails40. The tails play important roles 553 REVIEWS TIBS 25 – NOVEMBER 2000 in gene regulation. In particular, histone found over the Drosophila promoters, poorly defined. To define these mecha- acetylation, correlated with transcrip- there are very few examples where nu- nisms, it will be necessary to develop a tional activity, is directed exclusively at cleosomes are clearly absent. Increased vocabulary that can distinguish be- lysine residues in the N-terminal tails of nuclease sensitivity, seen at several pro- tween the stages of promoter remodel- the histone proteins. The SWI/SNF com- moters, can be consistent with either ing and the types of remodeling. This plexes do not require an interaction the absence of nucleosomes, rearrange- vocabulary might be similar to that of with the histone tails; SWI/SNF can alter ment of nucleosome position, or the cre- the transcription field, which uses terms topology, alter DNase I accessibility of ation of a more accessible conformation such as ‘open complex formation’ and mononucleosomes and create the dinu- of the nucleosome. Experiments that ‘promoter clearance’ to define discrete cleosome species just as well on nucleo- can differentiate between the absence of mechanistic steps in transcription. This somes where the tails have been pro- nucleosomes and the presence of re- vocabulary is needed to establish clear teolytically removed37,41. On the other modeled nucleosomes will be needed lines of thought regarding the sequence hand, the histone tails are required for to distinguish between the current hy- and nature of remodeling events during both ATPase and remodeling activities potheses for remodeling. gene regulation, and should accompany of the ISWI-based complexes42. It is not Which remodeling complexes are es- the design of experiments that examine yet known whether the sliding activity sential for the activation of a particular the surprisingly complex processes by of the ISWI-based complexes requires in- promoter? Some remodeling activities which individual genes are expressed. tact histone tails. have been genetically connected to Although the in vitro experiments some promoters, such as the SWI/SNF References with both the SWI/SNF complexes and complex at the HO promoter; SWI/SNF is 1 Allegra, P. et al. (1987) Affinity chromatographic purification of nucleosomes containing transcriptionally the ISWI-based complexes have offered known to be essential for the expression active DNA sequences. J. Mol. Biol. 196, 379–388 43 a few clues about what the complexes of many genes . What is not known, 2 Walker, J. et al. (1990) Affinity chromatography of can and might be doing, there is still however, is whether SWI/SNF is suffi- mammalian and yeast nucleosomes. Two modes of binding of transcriptionally active mammalian very little information about the exact cient for the expression of these genes, nucleosomes to organomercurial-agarose columns, and mechanisms by which they act. There is or if other remodeling complexes are contrasting behavior of the active nucleosomes of yeast. J. Biol. Chem. 265, 5736–5746 strong evidence that the two classes of needed. In particular, there are very few 3 Brownell, J.E. and Allis, C.D. (1995) An activity gel complexes differ in their recognition of examples of specific remodeler require- assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei. the nucleosome substrate, and in their ments for mammalian genes; perhaps Proc. Natl. Acad. Sci. U. S. A. 92, 6364–6368 mechanisms for displacing nucleo- the best current example is the require- 4 Taunton, J. et al. (1996) A mammalian histone somes from DNA. Side-by-side com- ment for BRG1 (and, presumably, the deacetylase related to the yeast transcriptional regulator Rpd3p. Science 272, 408–411 parisons of the various complexes, com- BRG1-nucleated human SWI/SNF com- 5 Xue, Y. et al. (1998) NURD, a novel complex with both bined with rigorous enzymological plex) for activation of the MMTV pro- ATP-dependent chromatin-remodeling and histone 44 deacetylase activities. Mol. Cell 2, 851–861 analysis of their activities, should lead moter . SWI/SNF also appears to be 6 Imbalzano, A.N. et al. (1994) Facilitated binding of to a more complete model of their activ- necessary for activation of the human TATA-binding protein to nucleosomal DNA. Nature 370, 45 481–485 ities both in vitro and in vivo. HSP70 gene . However, it is not known 7 Kwon, H. et al. (1994) Nucleosome disruption and whether other remodeling activities are enhancement of activator binding by a human SWI/SNF Conclusion complex. Nature 370, 477–481 involved in the regulation of either pro- 8 Cote, J. et al. (1994) Stimulation of GAL4 derivative Recent experiments, both in vivo and moter. binding to nucleosomal DNA by the yeast SWI/SNF in vitro, are supplying more and more Finally, although an in-depth discus- complex. Science 265, 53–60 9 Cairns, B.R. et al. (1996) RSC, an essential, abundant information regarding the role of chro- sion would go beyond the scope of this chromatin-remodeling complex. Cell 87, 1249–1260 matin remodeling in gene regulation. article, it is not clear how nucleosome 10 Tsukiyama, T. and Wu, C. (1995) Purification and properties of an ATP-dependent nucleosome The primary difficulty in understanding remodeling complexes relate to other remodeling factor. Cell 83, 1011–1020 this problem is the integration of mech- chromatin-related activities such as 11 Ito, T. et al. (1997) ACF, an ISWI-containing and ATP- utilizing chromatin assembly and remodeling factor. anistic data obtained in vitro with histone acetyltransferases and histone Cell 90, 145–155 molecular and physiological data ob- deacetylases. Although histone acety- 12 Varga-Weisz, P.D. et al. (1997) Chromatin-remodelling tained in vivo. We are learning more lation is required for normal transcrip- factor CHRAC contains the ATPases ISWI and topoisomerase II. [published erratum appeared in Nature about what remodeling complexes are tion of many genes and has been linked (1997) 389, 1003]. Nature 388, 598–602 capable of doing, but do not know which to ATP-dependent remodeling geneti- 13 Wu, C. (1980) The 5Ј ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I. biochemical activities are physiologi- cally, it is not known how acetylation Nature 286, 854–860 cally relevant. For example, the stably relates to remodeling at a mechanistic 14 Nacheva, G.A. et al. (1989) Change in the pattern of histone binding to DNA upon transcriptional activation. remodeled nucleosome species created level. Deacetylation complexes fre- Cell 58, 27–36 by SWI/SNF has not been detected quently contain remodeling proteins. 15 Gilmour, D.S. and Lis, J.T. (1986) RNA polymerase II in vivo. Conversely, molecular genetic Although remodeling appears to in- interacts with the promoter region of the noninduced HSP70 gene in Drosophila melanogaster cells. Mol. experiments such as the ones described crease the rate of deacetylation moder- Cell. Biol. 6, 3984–3989 here (the HSP70 and HO promoters) tell ately, it is not clear whether there are 16 Rougvie, A.E. and Lis, J.T. (1988) The RNA polymerase II molecule at the 5Ј end of the uninduced us what factors are involved in the acti- other mechanistic links. hsp70 gene of Drosophila melanogaster is vation of promoters, but do not tell us The definition of the term ‘chromatin transcriptionally engaged. Cell 54, 795–804 17 Wu, C. (1984) Activating protein factor binds in vitro to what those factors are actually doing. remodeling’ is not merely a question of upstream control sequences in heat shock gene There are several key questions that will semantics. The remodeling field’s great- chromatin. Nature 311, 81–84 further our understanding of chromatin est weakness at present is a failure to 18 Tsukiyama, T. et al. (1995) ISWI, a member of the SWI2/SNF2 ATPase family, encodes the 140 kDa remodeling and gene activation. make meaningful connections between subunit of the nucleosome remodeling factor. Cell 83, What actually happens to nucleo- in vivo and in vitro data, partially be- 1021–1026 19 Stern, M. et al. (1984) Five SWI genes are required for somes when a promoter is activated? cause many of the mechanisms ob- expression of the HO gene in yeast. J. Mol. Biol. 178, Except for the nucleosome-free regions served both in vivo and in vitro are 853–868 554 TIBS 25 – NOVEMBER 2000 REVIEWS

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changing mutational loads during the life of the patient, and in different mu- tational loads in different cells and tis- Mitochondrial genetics sues (mitotic segregation); (vi) because different cell types have different mini- and disease mal oxidative energy requirements (thresholds), the level of heteroplasmy and the dynamics of mitotic segregation play a critical role in determining the Eric A. Schon clinical presentation and outcome.

Mitochondrial respiratory chain diseases are a highly diverse group of dis- Pathogenic mutations associated with orders whose main unifying characteristic is the impairment of mitochon- generalized defects in OXPHOS function drial function. As befits an organelle containing gene products encoded by Mutations impairing the function of both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), these diseases two or more respiratory chain com- can be caused by inherited errors in either genome, but a surprising num- plexes are currently associated only ber are sporadic, and a few are even caused by environmental factors. with mutations in mtDNA, and all such mutations affect mitochondrial protein synthesis globally, either indirectly, via HUMAN MTDNA IS a 16.6-kb circular Mitochondria follow the rules of pop- deletions that remove large segments of DNA1 that contains only 37 genes ulation genetics. Six aspects of their be- the mitochondrial genome, or directly, (Fig. 1). Twenty-two genes specify trans- havior are critical for understanding the via mutations in specific tRNA and rRNA fer RNAs and two specify ribosomal etiology and pathogenesis of mitochon- genes. Interestingly, the diseases associ- RNAs; only 13 genes encode polypep- drial disorders: (i) they are maternally ated with the former are quite different tides, all of which are components of the inherited; (ii) cells typically contain from those associated with the latter. respiratory chain–oxidative phosphory- hundreds of organelles and thousands of Large-scale mtDNA rearrangements. lation (OXPHOS) system. The respira- mitochondrial genomes; (iii) mutations The most prominent disorders associ- tory complexes also contain approxi- can arise in a mtDNA population, re- ated with large-scale (kilobase-sized) mately 70 nuclear-encoded structural sulting in the coexistence of two or partial deletions of mtDNA are subunits that are synthesized in the more mtDNA genotypes within a single Kearns–Sayre syndrome (KSS), a fatal cytosol and are imported into the or- cell, organ or individual (heteroplasmy); multisystemic disorder, progressive ex- ganelle, where they are co-assembled (iv) if the mutation is pathogenic, the ternal ophthalmoplegia (PEO), a myo- with the mtDNA-encoded subunits into proportion of mutated molecules in a pathy characterized by paralysis of the the respective holoenzymes (Fig. 2). heteroplasmic population (mutational extraocular muscles and Pearson’s mar- load) affects the severity of the bio- row or pancreas syndrome (PS). In all E.A. Schon is at the Depts of Neurology and chemical defect, but not necessarily in three disorders, which are sporadic (i.e. of Genetics and Development, Columbia a linear fashion; (v) mtDNA replication mothers and siblings are unaffected), University, 630 West 168th St, New York, NY and inheritance in lineages of somatic patients harbor a single species of deleted 10032, USA. Email: [email protected] cells is stochastic, often resulting in mtDNA that co-exists with wild-type 0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved. PII: S0968-0004(00)01688-1 555