Yahli Lorch

Twenty-Five Years of the Nucleosome, Fundamental Particle of the Eukaryote Chromosome

The chromatin field needs much more information about structure beyond the nucleosome. Even the trajectory of the DNA entering and exiting the nucleosome, immediately beyond the core particle, is unclear (reviewed by Prunell 1998xA topological approach to nucleosome structure and dynamics (the linking number paradox and other issues) . Prunell, A. Biophys. J. 1998; 74: 2531–2544Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesPrunell 1998). The pattern of coiling a chain of nucleosomes in a thicker fiber remains uncertain (reviewed by Ramakrishnan 1997xHistone H1 and chromatin higher-order structure. Ramakrishnan, V. Crit. Rev. Eukaryot. Gene Expr. 1997; 7: 215–230Crossref | PubMedSee all ReferencesRamakrishnan 1997). Whereas additional coiling in a succession of higher helices would be a most plausible mechanism of further condensation, alternative hypotheses have been advanced. Many of the difficulties of analyzing chromatin problems stem from the variability inherent in higher order chromatin structures. Existing methods of structure determination require averaging and thus are of limited use in the face of variability.Solution of the higher order structure problem is crucial for understanding chromatin function. Histone tail modifications and interactions with other proteins, important for regulation, seem likely to influence higher order structure more than core particle structure. There is, however, insufficient evidence that acetylation actually causes chromatin unfolding, and only a suggestion of the interplay between acetylation and the chromatin remodeling events that affect core particle structure (14xOrdered recruitment of transcription and chromatin-remodeling factors to a cell cycle– and developmentally regulated promoter. Cosma, M.P, Tanaka, T, and Nasmyth, K. Cell. 1999; 97: 299–311Abstract | Full Text | Full Text PDF | PubMed | Scopus (545)See all References, 81xThe nucleosome remodeling complex, Snf/Swi, is required for the maintenance of transcription in vivo and is partially redundant with the histone acetyltransferase, gcn5. Sudarsanam, P, Cao, Y, Wu, L, Laurent, B.C, and Winston, F. EMBO J. 1999; 18: 3101–3106Crossref | PubMed | Scopus (86)See all References). Histone phosphorylation, long correlated with chromosome condensation, has recently been linked to transcriptional activity (reviewed by Bjorklund et al. 1999xGlobal transcription regulators of eukaryotes. Bjorklund, S, Almouzni, G, Davidson, I, Nightingale, K.P, and Weiss, K. Cell. 1999; 96: 759–767Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesBjorklund et al. 1999). There is no information as to the structural or functional consequences of other modifications, such as ubiquitination and glycosylation. Histone H1 is thought to promote condensation, but an understanding of its structural role and the connection with gene activity is lacking. Elucidation of gene silencing by heterochromatin similarly depends on determination of its structure.Finally, functional analysis in cell-free systems must be extended beyond the nucleosome to the chromosomal context. Histone–DNA complexes assembled in vitro reveal effects of HAT and chromatin-remodeling complexes on transcription (56xRole of nucleosome remodeling factor NURF in transcriptional activation of chromatin. Mizuguchi, G, Tsukiyama, T, Wisniewski, J, and Wu, C. Mol. Cell. 1997; 1: 141–150Abstract | Full Text | Full Text PDF | PubMedSee all References, 49xRequirement of RSF and FACT for transcription of chromatin templates in vitro. LeRoy, G, Orphanides, G, Lane, W.S, and Reinberg, D. Science. 1998; 282: 1900–1904Crossref | PubMedSee all References, 88xTranscriptional activators direct histone acetyltransferase complexes to nucleosomes. Utley, R.T, Ikeda, K, Grant, P.A, Cote, J, Steger, D.J, Eberharter, A, John, S, and Workman, J.L. Nature. 1998; 394: 498–502Crossref | PubMed | Scopus (406)See all References), but in vivo, promoters are associated with additional, nonhistone proteins, which influence the locations of nucleosomes and undoubtedly the higher order configuration of chromatin as well. These associations extend, in the broadest sense, to such DNA elements as locus control regions, which regulate the structure and activity of entire chromosomal domains. Only when transcription, replication, recombination, and other DNA transactions have been reconstituted in vitro with naturally assembled chromatin templates will a full understanding of the nucleosome be achieved.*To whom correspondence should be addressed (e-mail: kornberg@ stanford.edu).

RSC, an Essential, Abundant Chromatin-Remodeling Complex

A novel 15-subunit complex with the capacity to remodel the structure of chromatin, termed RSC, has been isolated from S. cerevisiae on the basis of homology to the SWI/SNF complex. At least three RSC subunits are related to SWI/SNF polypeptides: Sth1p, Rsc6p, and Rsc8p are significantly similar to Swi2/Snf2p, Swp73p, and Swi3p, respectively, and were identified by mass spectrometric and sequence analysis of peptide fragments. Like SWI/SNF, RSC exhibits a DNA-dependent ATPase activity stimulated by both free and nucleosomal DNA and a capacity to perturb nucleosome structure. RSC is, however, at least 10-fold more abundant than SWI/SNF complex and is essential for mitotic growth. Contrary to a report for SWII/SNF complex, no association of RSC (nor of SWI/SNF complex) with RNA polymerase II holoenzyme was detected.

Nucleosomes inhibit the initiation of transcription but allow chain elongation with the displacement of histones

Promoters were assembled in nucleosomes or ligated to nucleosomes and transcribed with SP6 RNA polymerase or with mammalian RNA polymerase II and accessory factors. Neither polymerase would initiate transcription at a promoter in a nucleosome, but once engaged in transcription, both polymerases were capable of reading through a nucleosome. In the course of readthrough transcription, the histones were displaced from the DNA, as shown by the exposure of restriction sites and by a shift of the template to the position of naked DNA in a gel. It may be true, in general, that processive enzymes will traverse regions of DNA organized in nucleosomes and displace histones.

Histone octamer transfer by a chromatin-remodeling complex.

RSC, an abundant, essential chromatin-remodeling complex related to SWI/SNF complex, catalyzes the transfer of a histone octamer from a nucleosome core particle to naked DNA. The newly formed octamer-DNA complex is identical with a nucleosome in all respects. The reaction requires ATP and involves an activated RSC-nucleosome intermediate. The mechanism may entail formation of a duplex displacement loop on the nucleosome, facilitating the entry of exogeneous DNA and the release of the endogenous molecule.

Chromatin-modifying and -remodeling complexes.

Nucleosomes have long been known to inhibit DNA transactions on chromosomes and a remarkable abundance of multiprotein complexes that either enhance or relieve this inhibition have been described. Most is known about chromatin-remodeling complexes that perturb nucleosome structure.

Activated RSC-Nucleosome Complex and Persistently Altered Form of the Nucleosome

RSC, an abundant, essential chromatin-remodeling complex, related to SWI/SNF complex, binds nucleosomes and naked DNA with comparable affinities, as shown by gel shift analysis. The RSC-nucleosome complex is converted in the presence of ATP to a slower migrating form. This activated complex exhibits greatly increased susceptibility to endo- and exonucleases but retains a full complement of histones. Activation persists in the absence of ATP, and on removal of RSC, the nucleosome is released in an altered form, with a diminished electrophoretic mobility, greater sedimentation rate, and marked instability at elevated ionic strength. The reaction is reversible in the presence of RSC and ATP, with conversion of the altered form back to the nucleosome.

Structural basis of eukaryotic gene transcription

An RNA polymerase II promoter has been isolated in transcriptionally activated and repressed states. Topological and nuclease digestion analyses have revealed a dynamic equilibrium between nucleosome removal and reassembly upon transcriptional activation, and have further shown that nucleosomes are removed by eviction of histone octamers rather than by sliding. The promoter, once exposed, assembles with RNA polymerase II, general transcription factors, and Mediator in a ∼3 MDa transcription initiation complex. X‐ray crystallography has revealed the structure of RNA polymerase II, in the act of transcription, at atomic resolution. Extension of this analysis has shown how nucleotides undergo selection, polymerization, and eventual release from the transcribing complex. X‐ray and electron crystallography have led to a picture of the entire transcription initiation complex, elucidating the mechanisms of promoter recognition, DNA unwinding, abortive initiation, and promoter escape.

Irresistible force meets immovable object: Transcription and the nucleosome

Roger D. Kornberg and Yahli Larch Department of Cell Biology Stanford University School of Medicine Stanford, California 94305 A remarkable finding from a great many experiments done over the past 15 years is that the precise location of a regulatory DNA element in the vicinity of a promoter makes little difference to its effect on transcription. This general result is surprising in light of the organization of eukaryotic DNA in linear arrays of nucleosomes, thought to be further condensed through level upon level of higher coiling. It is as if the chromosomal proteins responsible for this elabo- rate structure were invisible, revealing the underlying DNA without impediment to the transcription apparatus. Many have thought, some even stated, that the master molecule has no clothes; the structure of chromatin is irrelevant to transcriptional regulation. Recent evidence reviewed here suggests the opposite: many regulatory sequences may be concerned with remodeling the structure of chromatin for transcription, Various observations hint at a machinery for chromatin assembly and disassembly, which may be mobilized by regulatory elements to enable the gene acti- vation process” The very power of this machinery may until now have obscured its existence. If the structure of chromatin is based on a hierarchy of levels of coiling, then decondensation for transcription presumablyvoccurs through sequential uncoiling. Genes may become accessible for transcription in a stepwise pro- cess We focus here on the final stages of the process, in which activator proteins and the transcription machinery confront the nucleosome. Most is known about this end- game of the gene adtivation process, and the underlying principles are beginning to emerge. While these principles may :be general, the present discussion is limited to tran- scription by,HNA polymerase II; the accessibility problem in transcription by RNA polymerase Ill and that in DNA replication are treated elsewhere (Wolffe and Brown, 1 Q&; Wolff<, 1990; Simpson, 1990). ?irinscf~pkbnal Activator Protein Binding to Ekha’nhdb in Chromatin Activator proteins bound to enhancer DNA elements stim- ulatethe initiation of transcription. Whatever the mecha- nismrof thiseffect, the activators must gain access to their re~oQnitic+iequences, and access is limited by packag- ing of the see Piria et al., 1990; Archer et al., 1991) and the yeast PH02/ PH04 proteins (Fascher et al., lQQO), may occur in ex- posed locations, either outward-facing on the surface of nucleosome or in a linker region between nucleosomes. It seems doubtful, however, that recognition sequences are generally exposed in this way-as mentioned above, they may be placed anywhere in the vicinity of a promoter and still function effectively, and in some locations they must be inward-facing on the surface of a nucleosome. Another possibility, suggested by recent studies of GAL4 protein binding (Taylor et al., 1991), is that some acti’vator proteins may interact with their recognition sequencles regardless of location in a nucleosome, albeit with much lower affinity than in free solution. It remains to be seen wlhether activa- tor proteins commonly bind to sites that are? at least par- tiallyobscured, and whethersuch binding can have biolog- ical consequences. Once an activator protein has breached the accessibility barrier and bound to a site in chromatin, it may bring about the displacement of histones, clearing way for addi- tional activator or other protein binding. Displacement of histones is evidenced by the formation of nucleosome-free regions in chromosomes known as DNAase I hypersensi- tive sites (reviewed in Elgin, 1988, and Gross and Garrard, 1988). Typically one to several hundred base pairs in ex- tent, these regions are apparently naked aside from fac- tors bound at specific sites. Almost all enhancers arefound in such regions tissues or at times development when their functions are required. Glucocorticoid receptor and PH02/PH04 proteins appear to induce the formatton of nucleosome-free regions, enlarging their initial sites of en- try into chromatin. Other proteins, such as yeast GRF2 (Chasman et al., 1990), may be specialized for removing obstacles to the binding of other activators that in turn enhance transcription. GRFP both creates nucleosome- free ‘regions and accentuates the effect of neighboring activators 1 OO-fold or more. Formation of Transcription Initiation Complexes in CAromafin Following activator protein binding, the next landmark in the transcription process is the assembly of general initia- tion factors in a complex at the promoter. The problem of #ac

Structure of a RSC–nucleosome complex and insights into chromatin remodeling

essibility arises anew at this stage process. Sevetal studies have shown that assembly of nucleo- someson a promoter prevents initiation of transcription in vitro (Wasylyk and Chambon, 1979; Knezetic and Luse, 1986; Larch et al., 1987; Workman and Roeder, 1987). Evidence that ~nucleosomes are; inhibitory inI vivo comes from lthe construction of yeast strains in which histone synth’esis can, be arrested. The iresulting loss of nucleo- somes ‘is accompanied by transcription of many pre- viously inactive genes (Han and Grunstein, 1988): In all likelihood, histones must be fully displaced from a

Interplay between chromatin structure and transcription.

ATP-dependent chromatin-remodeling complexes, such as RSC, can reposition, evict or restructure nucleosomes. A structure of a RSC–nucleosome complex with a nucleosome determined by cryo-EM shows the nucleosome bound in a central RSC cavity. Extensive interaction of RSC with histones and DNA seems to destabilize the nucleosome and lead to an overall ATP-independent rearrangement of its structure. Nucleosomal DNA appears disordered and largely free to bulge out into solution as required for remodeling, but the structure of the RSC–nucleosome complex indicates that RSC is unlikely to displace the octamer from the nucleosome to which it is bound. Consideration of the RSC–nucleosome structure and published biochemical information suggests that ATP-dependent DNA translocation by RSC may result in the eviction of histone octamers from adjacent nucleosomes.