Chromatin structure and transcription.

ANNUAL REVIEWS

Annu. Rev. Cell BioI. 1992. 8:563-87 Copyright © 1992 by Annual Reviews Inc. All rights reserved

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CHROMATIN STRUCTURE

Annu. Rev. Cell. Biol. 1992.8:563-587. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/04/13. For personal use only.

AND TRANSCRIPTION Roger D. Kornberg and Yahli Lorch Department of Cell Biology, Stanford University School of Medicine, Stanford,

California 94305 KEY WORDS: chromosome, nuc\eosome, histone, DNA

CONTENTS 563

INTRODUCTION

CHROMATIN STRUCTURE . . . . . . . . . . . . . Histone Octamer . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . Nucleosome Positioning In Vitro ...................... . ....... . ... . ... Nucleosome Positioning In Vivo ... . ... . ... . ............... . ..........

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TRANSCRIPTION OF CHROMATIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unfolding o/ Chromosomal Domains . .. . . . . . ... . . . . . . . .. . . . . . . .. . . . . . . . Activator Protein-Binding to Regulatory Sites in Chromatin ....... . .......... Initiation o/ Transcription in Chromatin: Biochemical Studies . ...... . . . . . . ... Initiation o/ Transcription in Chromatin: Genetic Studies .. .... . .. ........ Transcription Elongation in Chromatin '" ... . ..... . ...... . . . . . . . . . . . . ..

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CONCLUSIONS AND PERSPECTIVES ..................................

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INTRODUCTION The challenge of chromosome structure and transcription is to understand how DNA is coiled up in eukaryote chromosomes and uncoiled for gene activity. The coiling appears to be hierarchical, with several levels required to achieve the highest degree of condensation found in transcriptionally inactive regions. At the first level, the DNA is wrapped around a set of histones in the nucleosome. A chain of nucleosomes, or 10-nm fiber, is probably coiled in a 30 nm-fiber, which is further coiled or folded in a manner that remains obscure. A major goal of the studies reviewed here is to elucidate the process of uncoiling and determine the structure of transcriptionally active chromo somal material (chromatin). What is the extent of uncoiling for transcription? Are there special proteins or modifications of existing ones in transcribed 563

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regions? How does the control of uncoiling contribute to the overall process of gene regulation? Since the nucleosome is the fundamental unit of transcriptionally inactive chromatin, and most sequences are in the inactive state most of the time, the existence and structure of the nucleosome could be determined from studies of unfractionated chromosomal material. By contrast, structural studies of active chromatin are bedeviled by the scarcity, variety, and transient nature of transcribed regions. Isolation of active chromatin and specific detection of active regions with nucleic acid and antibody probes have provided clues, but have failed to give a full solution of the problem. Rather it appears that elUCidating the functional state(s) of chromatin will require functional studies, both reconstitution of transcription with chromatin templates in vitro, and genetic analysis of chromatin transcription in vivo. Reconstitution experi ments should help delineate the pathway of transcriptional activation and allow the preparation of chromatin in various states along the pathway for structural analysis. Genetic studies should identify some of the proteins and interactions involved. Here we review the current status of both biochemical and genetic analyses of transcriptionally active chromatin. We begin with a summary of new information on the structure of chromatin pertinent to transcriptional activa tion, and then take up various questions and topics in the approximate order that they arise along the pathway of activation. Due to space limitation, we deal only with transcriptional activation and not with the closely related processes of repression and silencing. There have been almost as many reviews as original papers published on some aspects of active chromatin in the past few years (Elgin 1990; Felsenfeld 1992; Grunstein 1990a,b; Herbomel 1990; Kornberg & Lorch 1991; Spencer & Groudine 1990; Svaren & Chalkley 1990; Thoma 1991; Wolffe 1990), so we do not feel compelled to cover the entire literature, but rather have compiled a representative list of references to document our perspective.

CHROMATIN STRUCTURE Histone Octamer The recent X-ray structure determination of the histone octamer at 3.1 A resolution (Arents et al 1991)(Figure 1,2) is likely to have important consequences for understanding both the organization of DNA in the nucleosome and the fate of nucleosomes during transcription. The structure is generally consistent with previous results from electron microscope crystallography of the octamer at 22 A resolution and X-ray structure determination of the core particle at 7 A resolution. The shape and dimensions of the octamer and its organization as a left-handed superhelix of protein

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Figure 1

Structure of the histone oetamer at 3.1

A resolution. The path of the protein superhelix

is indicated by the line. The H2A-H2B dimers are darkly shaded at the beginning and end of the superhelix; the H3-H4 tetramer is lightly shaded in the middle (from Arents et al 19 91).

subunits for supercoiling DNA are confirmed. The superhelix has an inner diameter of about 65 A and length along the helix axis of about 60 A. In addition to these broad features of octamer structure, three new aspects are revealed that could not be discerned in previous structural analyses at lower resolution. First, there is a marked subdivision of the protein in dimers, arrayed in the order (H2B/H2A)-(H4/H3)-(H3/H4)-(H2A/H2B) along the superhelix. Within the dimers, the individual polypeptides interdigitate extensively, so regions attributable to the individual histones cannot be clearly defined. Second, the histones exhibit a common fold, with a long central helix flanked by shorter helices. There is no apparent relationship to other DNA-binding motifs. Third, the amino-terminal regions of the histones are not found in the map, so these regions may be disordered or flexibly linked to the rest of the structure. The size of the unstructured regions is comparable to that of the


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Figure 2

Stereo pai ax is. Amino-te rmin i are n id

domains removed from the core particle by proteolysis: the first ordered residue (amino terminus of cleaved protein) in the current model is 15 ( 12) for H2A, 36 (24) for H2B, 43 (27) for H3, and 26 (20) for H4. The agreement, approximate at this stage, is likely to improve as model-building extends the number of ordered residues and underscores the notion of the amino-terminal regions as distinct domains. In all cases, the first ordered residue lies at the periphery of the protein superhelix, and the locations of the amino-terminal regions will be of interest in regard to the involvement of these regions in gene activation and transcription (see below). The greatest significance of the high resolution structure of the octamer may lie in combination with the lower resolution structure of the core particle. Model-building could identify the precise path of the DNA around the octamer in the core particle. Details of the histone-DNA interaction would emerge, including amino acid side chains in contact with nucleotide bases or sugar-phosphate backbone. It may also be possible to deduce the detailed variation in helical periodicity of the DNA, an important aspect of previous interpretations of both structural and chemical analyses.

Nucleosome Positioning In Vitro Recent studies have advanced our understanding of both the structural basis for and the degree of nucIeosome positioning. The treatment here is confined to essential background and a discussion of results published during the past year, as excellent reviews of earlier work are available (van Holde 1989; Simpson 1991). It was previously shown that histone octamers bind prefer entially to certain DNA sequences in the formation of nucleoprotein com plexes in vitro. This effect could be attributed to the greater facility of certain


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sequences for bcnding as required by the superhelical path in the nucleosome. Such "bendability", or anisotropic tlexibility, can arise from the interspersion of A/T- and G/C-rich sequences with the periodicity of the double helix. DNA isolated from natural nucleosome core particles exhibits this feature, with a tendency of A/T-rich (G/C-rich) di- and tri-nucleotides to occur every 10.02 residues from base pairs 5 to 55 (10 to 50) and base pairs 85 to 135 (90 to 130) along the core length of 146 base pairs. If this sequence periodicity retlects the structural periodicity of the DNA, then a (local) helical repeat of 10.02 base pairs per tum may be inferred for the 55 base pairs at either end of the core particle, appreciably less than the 10. 5 base pairs per turn found for DNA in solution. I t further follows that A/T-rich regions occur where the minor groove of the double helix faces inward towards the superhe1ix axis, while G/C-rich regions are to be found where the minor groove faces outward. An exception to this pattern is in the central three turns of DNA in the core particle, where a deviation from a smooth superhelical path occurs, and a helical periodicity of approximately 10. 7 base pairs per tum is inferred. Early evidence for variation of structural periodicity within core particle DNA came from DNase I digestion results, indicative of 10.c�10. 2 base pairs per turn near the ends and 10. 5 base pairs per tum in the middle (PruneII et al 1979). Finer discrimination has now been achieved with hydroxyl radical, which cleaves core particle DNA every 10.05 base pairs, except in the central three turns, where cutting occurs every 10. 7 base pairs (Hayes et al 1990). Although the coincidence with the sequence periodicity is striking, direct evidence of the structural periodicity from X-ray analysis at high resolution is still needed. A key question for the significance of nucIeosome positioning concerns the magnitude of the effect. What is the degree of sequence preference in histone octamer-binding to DNA? How large is the range from the strongest to the weakest interactions? The answer would be given by the relative affinities of the octamer for various DNAs under physiologic solution conditions, but the affinities cannot be measured because of a lack of exchange of octamers between DNAs under these conditions. It has been argued that relative affinities at elevated ionic strength can be obtained from competitive reconstitution experiments in which a limited amount of histones is mixed with a radiolabeled DNA fragment and bulk unlabeled DNA in 1 M NaCl, a salt concentration at which octamers exchange rapidly. Following dilution to 0. 1 M NaCl to terminate exchange, the extent of nucIeosome formation on the labeled DNA is determined. To the extent that dilution merely freezes an existing equilibrium, the ratio of nucleosoine formation on two fragments corresponds to the relative affinities of the octamer for these fragments, which may alternatively be expressed as an energy difference between them (Shrader & Crothers 1989). Results obtained with repetitive DNAs containing A/T- and G/C-regions


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(artificial nucleosome positioning sequences) attest to the validity of the competitive reconstitution approach (Shrader & Crothers 1990). A sequence periodicity of 10. 1 base pairs was clearly optimal for nucleosome formation. Moreover, insertion of a central region between two 40-base pair arms of a periodic sequence enhanced nucleosome formation, with a higher efficiency for a 2 l-base pair insert than for 20- or 22-base pair inserts. A structural periodicity of 10. 5 base pairs per tum in the central region would seem to be indicated, although the authors prefer another interpretation. Relative affinities from the competitive reconstitution approach therefore accord well with findings from nucleosome positioning analysis (sequences of core DNAs described above) and from digestion experiments. Competitive reconstitution reveals about a 100-fold preference for nucle osome formation on a strong natural positioning sequence, containing a gene for 5S ribosomal RNA, over bulk DNA. The highest affinity observed, with artificial repetitive arms flanking a 21-base pair central region, was another l O-fold greater. This relatively modest degree of specificity in histone octamer-DNA interaction seems consistent with the requirement for packag ing a wide variety of DNA sequences in nucleosomes. It raises important questions for the functional significance of nucleosome positioning discussed below.

Nucleosome Positioning In Vivo Many examples have been reported of both random and nonrandom locations of nucleosomes in a wide range of organisms. In some cases, small DNA fragmcnts have bcen shown to form nucleosomes in vitro at the same positions as found in vivo. Recently, however, repetitive sequences with among the highest affinities for the histone octamer measured in vitro were incorporated in a yeast plasmid and found not to form uniquely positioned nucleosomes in vivo (Tanaka et al 1992). It remains to be seen whether insertion of an appropriate ccntral region betwcen arms of repetitive sequences will lead to a greater degree of positioning. Two mechanisms have been shown to account for nonrandom l ocations of nucleosomes in cases that have been analyzed in detail. First, nonhistone proteins that bind with very high specificity and affinity to particular DNA sequences introduce a strong constraint: histones may be excluded from the nonhistone binding sites and, in some instances, histones are also absent from regions flanking these sites (called nuclease-sensitive regions or DNase I hypersensitive sites). An unavoidable consequence of nuc1eosome exclusion is statistical positioning, seen in the average over an ensemble of genomes, but not in any member of the ensemble. The second mechanism of positioning is sequence-preferential histone-DNA interaction along the lines discussed a bove, which comes into play at some distance from a nonhistone protein-


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binding site or boundary of a nuclease-sensitive region. In addition to these mechanisms, both clearly documented and well understood, other factors that may influence the distribution of nucleosomes include any tendency towards a regular spacing of nucleosomes along a chromatin fiber and effects of higher coiling or folding of chromatin fibers (Thoma & Zatchej 1988). The central issue in the context of nucleosome positioning in vivo is whether the degree of positioning can be great enough to have functional consequences. It has long been imagined that the presence or absence of a nucleosome on a particular sequence might determine its accessibility to enzymes and other molecules and thus that the location of nucleosomes might play a regulatory role in replication, transcription, and so forth. For such involvement of nucleosomes in the activity of a chromosomal locus, the location of nuc1eosomes at this locus would have to be the same in all cells of a given type. The preference of specific gene regulatory proteins for their cognate binding sequences over nonspecific DNA, typically six orders of magnitude or more, provides a benchmark, The WOO-fold greater affinity of the histone octamer for the strongest nucleosome positioning sequences over bulk DNA, described above, falls far short of this standard. It may be argued, however, that the actual degree of positioning in the chromosomal context can be greater. A series of appropriately spaced positioning sequences could define the locations of nucleosomes far better than a single sequence: nucleosomes at each position would form boundaries, constraining the locations of their neighbors. The main difficulty in ad dressing this issue is the lack of a method of assessing the degree of positioning to the precision required. Nuclease digestion and chemical cleavage techniques commonly used to reveal locations of nucleosomes are capable of discrimination to a 1 6 2 part in 10 _10 , far less than the part in 10 or more needed. One must therefore take the opposite approach, of deliberately disrupting nucleosome positioning and determining the consequences for function, to establish whcther position ing plays an important role. Studies along these lines, though not directly to the point, are described below, and more are needed.

TRANSCRIPTION OF CHROMATIN The broad outlines of a general pathway of gene activation and transcription have emerged from work done over many decades. Logic would d ictate and classical ultrastructural analyses of nuclei and chromosomes clearly indicate that decondensation of chromatin accompanies transcription. As the structure of chromosomes is evidently hierarchical, based on a series of levels of coiling, decondensation must also occur in stages. At least three stages may be distinguished: unfolding of large chromosomal domains, typically 25-100,000 base pairs in extent; remodeling of the chromatin structure of promoters and


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regulatory regions; and disruption of nucleosome structure in transcribed regions. The entire pathway is presumably triggered by specific factor-binding to regulatory sites, and the way in which factors gain access to sites in condensed chromatin is a major enigma and object of current investigation. The accessibility problem is a recurring theme in gene activation and may itself be broken down into stages. Early acting factors appear to create conditions conducive to binding of later acting ones, through displacement of chromo somal proteins and exposure of the DNA. Most is known about this process as it occurs in the vicinity of a promoter, and both genetic studies to reveal the components involved in vivo and biochemical work to reconstitute the process in vitro are underway.

Unfolding of Chromosomal Domains Although the decondensation of chromatin undergoing transcription is plainly apparent, as in light and electron micrographs of polytene and lampbrush chromosomes, the molecular basis of the decondensation is obscure. Neither the higher order structure of chromatin whose unfolding is at issue nor the proteins and DNA sequences that govern the process have been determined. Several recent findings, however, suggest approaches to the problem and outlines of the solution. Molecular genetic experiments have identified two types of DNA sequences that may be involved in the unfolding of chromosomal domains for transcrip tion. A sequence 50-60,000 base pairs upstream of the human l3-globin gene, termed a locus control region (LCR), is required for high levels of regulated expression in transgenic mice (Grosveld et al 1987). Only in the presence of an LCR is expression independent of the site of integration in the mouse genome and therefore proportional to the number of integrated copies. The LCR also causes changes in chromatin structure distributed over the approx imately 80,000 base pairs of the globin locus (Forrester et al 1987). It is possible that the LCR initiates a pathway of decondensation leading to a structure of chromatin conducive to transcription. In the absence of an LCR, expression of a transgene would depend on whether the site of integration happens to lie within a chromosomal region already transformed to the transcriptionally active state. Distinctions between the LCR and transcriptional enhancers are summarized elsewhere (Felsenfeld 1992). The second type of DNA sequence whose function may relate to the establishment of transcriptionally active chromosomal domains appears to define the boundaries of the domains. Sequences termed A-elements bracket a 24,000 base pair region of altered structure (see Enhanced Susceptibility to Nuclease Digestion, below) containing the transcriptionally active chicken lysozyme gene (Fritton et al 1988). On placing a reporter gene between such


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elements, its expression in stably transfected cells becomes proportional to the number of integrated copies (Stief et al 1989), which demonstrates independence of the site of integration. Sequences flanking the hsp70 genes at the 87A7 locus of Drosophila melanogaster. termed scs elements, have also been shown to confer position-independence of expression (Kellum & Schedl 1992). A Drosophila protein associated with boundaries of transcrip tionally active regions has recently been identified (Champlin et al 1991). Histone HI may interact preferentially with boundary elements as well (Izaurralde et al 1989).

Activator Protein-Binding to Regulatory Sites in Chromatin Activator protein-binding to DNA sequences in chromatin is paradoxical. Many studies have shown that the sequence requirements for activator function in vivo are closely correlated with those for binding to naked DNA. It is as if activator-binding sites in chromatin were invariably exposed in naked regions. And yet, activator-binding sites can be placed at virtually any location in the vicinity of a promoter with comparable effects on transcription in vivo. In some locations, a site will lie within a nucleosome, either inward- or outward-facing on the surface of the histone octamer, while in other locations the site will lie in a linker region between nucleosomes. Location within a nucleosome can impede access, as is most clearly shown by inhibition of cleavage by restriction enzymes and nonspecific endonucleases. How, then, do activators invariably find and interact with sites in chromatin? Early evidence bearing on the activator-accessibility problem came from studies of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter (Perlmann & Wrange 1988; Pina et al 1990; Archer et al 1991) and the yeast PH05 promoter (Almer et al 1986). The results of these studies were similar in outline, although different in detaiL Transcription of both promoters is inducible, and prior to induction, nucleosomes were shown to occupy nonrandom locations, placing two of four activator-binding sites in an outward-facing orientation on the surface of a nucIeosome for the MMTV LTR promoter, and placing one of four activator-binding sites in a linker region between nucIeosomes in the case of the PH05 promoter. Following induction of transcription, nucleosomes were disrupted or displaced, as shown by increased susceptibility to cleavage by enzymes and chemical reagents. The region of altered structure was limited but contained additional activator-binding sites, with the loss of a single nucleosome from the MMTV L TR promoter exposing two more sites for thc activator and one site for another transcription factor, and the loss of four nucleosomes from the PH05 promoter exposing three more sites for the first activator and one for a second activator. The conclusions from these studies were twofold: nucleosome positioning can solve the accessibility problem for initial activator-binding,


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and subsequent nucleosome loss may allow further activator- and tranf>cription factor-binding, leading to transcription. The role of nucleosome positioning in the gene activation process deserves close scrutiny, both in regard to the examples studied and to the general solution of the activator-accessibility problem. Since the MMTV LTR and PH05 promoters each contain four activator-binding sites, it is not surprising that one or more sites is outward-facing or located in a linker in a particular arrangement of nucleosomes. Key questions are whether this exposure of sites is essential for transcription, and whether the arrangement of nucleosomes is sufficiently well determined that the sites will be exposed in every cell where transcription is required. Both questions might be addressed by systematic variation of promoter structure to expose or cover up activator-binding sites in nucleosomes. Such analyses would best be performed with a promoter containing a single activator-binding site, which could be moved a few base pairs at a time, with the nucleosomal location and effect on transcription determined in each case. If transcriptional activity is modulated by nucleoso mal location, as the proposed role of nucleosome positioning posits, then the question arises of whether the result is general. Studies with many other promoters, in which activator-binding sites were placed at arbitrary locations and invariably enhanced transcription, suggest otherwise. At least three further possibilities may be considered for solution of the activator-accessibility problem. First, nucleosomes might rapidly exchange on and off or slide along DNA, so all sites are available for activator-binding. While such movement does not occur in vitro, there may be factors that catalyze the process in vivo. Second, some activators may be capable of finding their sites regardless of location in a nucleosome. Evidence for such facilitated binding has been presented (Workman et al 1991), but details of the process remain to be established. Finally, there may be additional factors that help activators gain access to sites in nucleosomes. Both biochemical studies (Chasman et al 1990) and genetic analysis (see below) have revealed proteins that might play such a role. Many regulatory DNA elements contain multiple activator-binding sites, and these elements often appear in nuclease-sensitive regions, devoid of nucleosomes, under conditions of activity. It would seem, as suggested for the MMTV LTR and PH05 promoters, that binding of the first activator sets off a process that clears the region for binding of other activators. In the case of the PH05 promoter, a study has been performed to assess the importance of nucleosome loss for activation of transcription (Straka & Horz 1991). DNA associated with one of the nuc1eosomes lost d uring activation was replaced either by a satellite sequence, known to form a positioned nuc1eosome and thus to bind the histone oetamer with very high affinity, or by a sequence of bacterial origin. The satellite sequence ex hibited no nucleosome loss under


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inducing conditions, and transcription was abolished. The bacterial sequence, on the other hand, allowed some escape from repression and displayed nucleosome loss with a high level of transcription upon induction. While it may be argued that the satellite sequence was inhibitory for some reason other than its high affinity for the histone octamer, these experiments provide a useful starting point and might be ex tended by replacement of the satellite sequence with a series of DNAs of varying affinity for the oetamer (see above).

Initiation of Transcription in Chromatin: Biochemical Studies Whatever the steric requirements for activator-binding, they are dwarfed by those for assembly of a transcription initiation complex. Therefore, if nucleosomes prevent activator-binding, they would certainly be expected to impede transcription. Early studies with nuc1eosomes assembled at random and with nonspecific initiation by various polymerases bore out this expec tation. The analysiS has been refined over the years, with the use of promoters assembled in nucleosomes and the full set of RNA polymerase II initiation factors, and the result has come into sharper focus: nucleosomes block the initiation of transcription. As activators trigger the process of gene activation and transcription, they are ultimately responsible for relieving inhibition caused by nucleosomes. There have been several reports that activators can relieve inhibition by nuc1eosomes in vitro. The templates were promoters on plasmids, assembled in nucleosomes, with or without histone HI . The results depended to some extent on the activator, promoter, and experimental protocol used: in some cases, both activator and the general initiation factor TFIID were required during chromatin assembly for subsequent transcription (Workman et a1 1988, 1990); in other instances, either activator (Workman et al 1991; Laybourn & Kadonaga 1991) or TFIID (Workman & Roeder 1987; Becker ct al 1991) alone during assembly would suffice. A further variation employed only Hl, without core nuc1eosomes, to inhibit transcription, and again relief of inhibition by activators (added before HI to the template) was observed (Croston et al 1991). The only significant discrepancy among these studies is in regard to the activator Ga14-VPI6, variously reported as able to relieve inhibition by nucleosomes (Workman et al 1991) or not able to do so (Laybourn & Kadonaga 1991). The chief concern about activation of transcription with chromatin tem plates in vitro is whether the process corresponds with that occurring in vivo. In most cases, the use of crude assembly and transcription systems and the limited characterization of the templates with regard to locations of nucle osomes and presence of other proteins allow alternative interpretations. Indeed, the disparate observations with regard to relief of inhibition by Ga14-VP16


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mentioned above were obtained with a template assembled in a Xenopus oocyte supernatant and used directly (Workman et al 1991), or a template assembled with pure histones in the presence of polyglutamate and cleaved with a restriction enzyme to remove residual free DNA (Laybourn & Kadonaga 1991). Doubts raised by the use of templates assembled in vitro may be dispelled by improvement of the assembly systems (Dilworth et al 1987; Kleinschmidt et al 1990; Becker & Wu 1992), or by the use of templates assembled in vivo (Gariglio et al 1979; Brady et al 1982; Batson et al 1992). If activation of transcription with chromatin templates in cell-free systems proves physiologically relevant, then fractionation of the systems should reveal the components and give insight into the mechanisms involved. It will be of particular interest to know whether activators can relieve inhibition by nucleosomes d irectly or whether, as in their effects on transcription of naked DNA templates, additional factors are required (Kclleher et al1990; Flanagan et al 1991; Pugh & Tj ian 1991). The mechanism seems likely to involve displacement of nucleosomes from promoters for the following reasons: First, in many cases such as those of the MMTV LTR and PROS promoters discussed above, activator-binding leads to nucleosome loss. Second, promot ers are very often found in nuclease-sensitive regions under conditions of transcriptional activity. Finally, coiling of DNA around a histone octamer in the nucleosome is incompatible with its assembly in a transcription initiation complex, or even with binding of TFIID, the first and smallest component to enter the complex, which bends the TATA element (Horikoshi et al 1992) and is evidently unable to interact with DNA in a nucleosome. Activators not only bring about nucleosome loss at promoters, but also appear to facilitate assembly of transcription initiation complexes, as j udged from their capacity to stimulate transcription with naked DNA templates. Thus activators may be responsible for a two-step process in which the structure of a promoter is remodeled, one set of proteins being dislodged and another taking their place at the promoter. This inference from biochemical studies coincides with results of genetic analyses described below, which indicate that many, if not all, of the observations made in vitro are pertinent to the gene activation process in vivo. Microinj ection experiments with Xenopus laevis oocytes have further indicated the relevance to regulated transcription in vivo (Perlmann & Wrange 1991).

Initiation of Transcription in Chromatin: Genetic Studies Most genetic studies of chromatin transcription have been performed in yeast because of the tractability of the organism for the purpose and further because of the high conservation of histones and transcription apparatus from yeast to man. Three types of genetic manipulation have proved particularly revealing: alterations of histone expression, mutations in the histones, and mutations in


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other proteins whose effects suggest a role in transcription of chromatin. Strains with an altered histone gene dosage were isolated from screens for suppressors of transposon insertions that inhibit transcription from adjacent promoters (Clark-Adams et al 1988). Specifically, an altered ratio of H2A H2B to H3-H4 dimers causes suppression, presumably through an alteration in the structure of chromatin. A more drastic change in histone levels and chromatin structure has been achieved by placing a single, essential copy of a histone gene under control of a galactose-inducible promoter and then repressing histone synthesis by growth of cells in the presence of glucose (Han et al 1988; Han & Grunstein 1988). Cells completed a round of DNA replication under these conditions, arresting in G2, and exhibited characteristics of chromatin structure consistent with loss of approximately half their nucleosomes. Such nucleosome loss led to activation of repressed genes, such as the PR05 gene in a high concen tration of inorganic phosphate. The level of transcription was about twofold lower than that achieved through normal induction and did not require the presence of the activator-binding sites responsible for normal induction. Similar results were obtained for the effect of nucleosome loss on transcription of other inducible genes such as CYC] and GALl. The levels of transcription were about 20- and 50-fold lower, respectively, than those following induction and, again, activator-binding sites were not required. By contrast, transcription of the HIS3, CUP1, and TRP1 genes was unaffected by nucleosome loss. Several conclusions could be drawn: First, transcription from an inducible promoter may be blocked by a nucleosome under repressing conditions, consistent with the inhibitory effect of a nucleosome on the initiation of transcription in vitro. Second, promoters that do not respond to nucleosome loss may normally be free of nucleosomes, possibly due to activators that confer a high level of basal (constitutive) transcription. Finally, the activation of inducible promoters by nucleosome loss was as much as 50-fold less than that achieved through normal induction, which suggests that activators do not exert their effects through nucleosome displacement alone, but also stimulate the subsequent events of initiation complex formation and transcription, further consistent with the results of biochemical studies described above. Mutagenesis of histones has focused on the amino-terminal regions. These regions constitute distinct protein domains, which are either flexibly linked to the rest of the molecule, or do not adopt an ordered configuration under the conditions of study in vitro. The amino-terminal regions are believed to play some important role since they have been conserved in sequence through evolution and since they are targets of many posttranslational modifications including acetylation, phosphorylation, and methylation. It was therefore surprising to find that the amino-terminal regions are dispensable for both the structure and assembly of the nucleosome in vitro (Whitlock & Stein 1978)


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and for yeast cell growth and thus function in vivo (Wallis et al 1983; Schuster et al 1986; Grunstein I 990a). Although amino-terminal regions of the histones can be deleted without loss of viability, deletions and site-directed mutations do give rise to distinctive phenotypes including effects on chromatin structure and transcription. Dele tion of residues 4-30 of H3 increased and deletion of residues 4-28 of H4 diminished transcription of inducible promoters (Durrin et al 1991). Changing lysine residues at positions 5, 8, 12, and 16 of H4, which undergo acetylation, to residues such as arginine, which cannot be modified in this way, also greatly diminished inducible transcription. These observations point to an essential role of the amino-terminal regions in the activation pathway and heighten the significance of correlations between acetylation and transcription of specific gene regions (discussed below). Genetic analyses have also identified nonhistone proteins that may influence transcription through alterations of chromatin structure. Mutations in the negative regulatory proteins SIN1 and SIN2 alleviate defects in transcription of many genes caused by mutations in the positive regulatory proteins SWI l , SWI2, and SWI3. As SIN I is a member o f the HMG I-family of chromosomal proteins, and SIN2 is histone H3, the positive regulatory SWI proteins may function by relieving inhibitory effects of chromatin structure (Peterson & Hershkowitz 1992). Two additional positive regulatory proteins, SNF5 and SNF6, appear to function in concert with the SWI proteins, since mutations in the SWI and SNF genes cause similar phenotypes.

Transcription Elongation in Chromatin Although events leading up to and including the initiation of transcription attract most attention because of their relevance to regulation, the subsequent process of transcription elongation poses challenging questions as well, and some of the answers may shed light on earlier events. Can RNA polymerase traverse a region of DNA coiled in a nucleosome? What alterations or modifications of the structure occur to facilitate the elongation process? What is the fate of the nucleosome - is the histone octamer disrupted or fully dislodged from the DNA? Transcription of chromatin templates in vitro has revealed that RNA polymerases are capable of elongation through nuc1eosomes (Williamson & Felsenfeld 1978; Wasylyk & Chambon 1979, 1980; Lorch et al 1987; Losa & Brown 1987; Knezetic et al 1988; Izban & Luse 1991). This capacity is not unique to eukaryotic polymerases, but extends to prokaryotic polymerases as well. It probably reflects the ease of invading a nucleosome from the ends. Whereas a stretch of DNA cannot readily d issociate from the middle of the nucleosome because of the mechanical constraint of stretches bound on both sides, DNA at the cnds of the nucleosome is held in place only by local


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interaction with the histones. A transcribing polymerase may advance when a turn of DNA double helix is free from the histone surface at an end of a nucleosome and then pause until another turn dissociates and becomes available. The appeal of this mechanism is that it requires no special property of polymerases or feature of nucleosomes beyond what is presently understood. That polymerases do not possess an inherent capacity to dislodge all obstacles from their path is shown by a lack of RNA polymerase II transcription past a bound lac repressor molecule (Deuschle et al 1 990). Whether the nucleo some is in any way d esigned to facilitate polymerase passage remains to be detennined. Although polymerases can extend transcripts through nucleosomes in vitro, the rate may be less than that of chain elongation in vivo. Pausing and tennination occur at the same sites with nucleosomal as with naked DNA templates, but the frequency of pausing is much enhanced (Izban & Luse 1991), as might be expected if the rate of chain elongation were diminished. Many changes have been noted in the chromatin structure of transcribed regions, some of which may facilitate chain elongation. There is no single gene or study that serves to illustrate all these structural changes, but rather the weight of evidence from many examples collected over more than a decade leaves the unmistakable impression that structural changes do occur. As the evidence has been reviewed elsewhere and, for the most part, does not come from recent work, it is summarized only briefly here. Many instances have been reported of more rapid or more extensive cleavage of chromatin by nonspecific nucleases in transcribed regions, compared with nontranscribed controls. While susceptibility to nuclease digestion is virtually always enhanced, the details vary from one gene to another. Such enhanced susceptiblity is not limited to nucleases, but is found with other probes such as DNA methylases as well (Singh & Klar 1992), which is indicative of fundamental changes in the structure of transcribed chromatin. The main types of result from nuclease digestion experiments are as follows:

ENHANCED SUSCEPTIBILITY TO NUCLEASE DIGESTION

1. Cleavage of active genes between nucleosomes by micrococcal nucle ase. Such cleavage results in a ladder of bands upon gel electrophoresis, \ which correspond to fragments that are multiples of the nucleosomal unit size. Fragments from a particular transcribed region are revealed by transfer to nitrocellulose and hybridization with a cloned probe from the region. In the case of the chicken ovalbumin gene, larger fragments are converted to smaller ones at an earlier stage in digestion of chromatin from oviduct, in which the gene is transcribed, than for chromatin from erythrocytes, in which it is not (Bellard et al 1977). Similar observations


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have been. made for immunoglobulin genes expressed in lymphoid cells, but not in other tissues (Weishet et al 1983), These results indicate enhanced susceptibility to digestion of the linkers between nucleosomes following gene activation. 2. Cleavage of active genes within nucleosomes by micrococcal nuclease. Active and inactive ovalbumin and immunoglobulin genes differ only in the kinetics of attack by micrococcal nuclease, giving rise to similar ladders of bands d uring the course of digestion. In contrast, other genes yield markedly different ladders following activation. The Dro sophila heat shock and yeast galactose-inducible genes exhibit a loss of definition (smearing) of the band pattern, which is indicative of enhanced susceptibility to digestion within the nucleosome (Lohr 1983, 1984; Wu et al 1979). The chicken ovalbumin gene also yields a diffuse band pattern in the transcriptionally active state (Bellard et al 1982). The chicken j3-g10bin gene in erythrocytes gives rise to a well defined ladder of bands, but with a shift in size upon activation from multiples of the nucleosomal repeat to multiples of the repeat minus100 base pairs (Sun et al 1986). This shift could result from enhanced susceptibility of 50 base pairs of DNA at either end of the nucleosome to exonucleolytic attack (trimming) by micrococcal nuclease, which might in tum result from loss of histones H2A and H2B, associated with some 40 base pairs of DNA at either end of the core length of DNA in the nucleosome (Richmond et al 1984). Both loss of a defined band pattern and shift in size of the products of digestion are found not only in regions traversed by RNA polymerase, but also upstream of the start site of transcription, which suggests that the alterations of chromatin structure are not necessarily a consequence of polymerase passage, but might precede and facilitate the process. 3. Cleavage of active genes within nucleosomes by DNase I. Early evidence of an altered chromatin structure of active genes came from d igestion with DNase I, which attacks sites within nucleosomes more readily than does micrococcal nuclease. Upon extensive DNase I digestion, active genes were more rapidly converted to an acid-soluble form than inactive genes (Weintraub & Groudine 1976). It has since been shown that such enhanced susceptibility to attack by DNase I within nucleosomes extends over very large domains, about 80, 000 and 24,000 base pairs in the case of the chicken ovalbumin and lysozyme genes, respectively (Lawson et al 1982; Fritton et al 1988). 4. Altered distributions of nucleosomes on active genes. Sites of cleavage by nucleases in chromatin may be mapped by indirect end-labeling (Wu 1980; Nedospasov & Georgiev 1980), and such mapping frequently reveals regions of roughly the nucleosome core length protected from micrococcal nuclease digestion, alternating with smaller regions exposed


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to digestion. There have been many reports of altered patterns of micrococcal nuclease digestion upon gene activation, ranging from slight to complete loss of protection, limited to part of the transcribed region or including the entire region and even extending beyond (for example, Samal & Worcel 1981; Udvardy & Schedl 1984; Cartwright & Elgin 1986; Benezra et al 1986; Fedor & Kornberg 1989). Mapping experiments have frequently revealed enhanced accessibility to DNase I digestion as well (for example Cartwright & Elgin 1986), including, in one case, exposure of sites in the centers of presumed nucleosome core particles, ascribed to nUcleosome-splitting (Lee & Garrard 1991).

Does enhanced susceptibility to nuclease digestion reflect a minor alteration in nucleosome structure, more extensive disruption, or even complete loss of histones from the DNA? Diminished levels of HI have been reported for active gene regions (see for example Karpov et al 1984; Tazi & Bird 1990; Kamakaka & Thomas 1990), and removal of HI can facilitate transcription in vitro (Wolffe & Brown 1988; Wolffe 1989; Shimamura et al 1989). HI is not, however, completely absent from transcribed genes (Mendelson et al 1986; Ericsson et al 1990) and may differ only in its manner of association between active and inactive regions (Nacheva et al 1989). A deficiency in H2A and H2B has been reported for transcribed sequences (Baer & Rhodes 1983) and inferred from results of nuclease digestion (see above), but these histones and H3 and H4 remain associated with active genes, although possibly in an altered manner (Solomon et al 1988; Nacheva et al 1989). Diminished levels of histones and other alterations could reflect the dynamic character of chromatin undergoing transcription (Bjorkroth et al 1988), with nucleosomes disrupted in the vicinity of RNA polymerase and reassembled following polymerase passage. Indeed, H2A and H2B are added in a separate step after H3 and H4 in nucleosome assembly systems (Dilworth et al 1987; Kleinschmidt et al 1990), and HI is presumably incorporated at a still later stage, thus possibly accounting for the diminished levels of these histones in transcribed regions. The question remains whether histone loss precedes and facilitates transcription or is simply a consequence of the process.

HISTONE LOSS

If histones persist in transcribed regions but in an altered structure, might this be a consequence of modifications of the histone molecules? There is much circumstantial evidence for the involvement of histone acetylation in transcription (Turner 1991). Acetylation of lysine residues in the amino-terminal regions of the histones, especially H3 and H4, is more abundant in chromatin enriched for transcribed genes (Allegra et al

HISTONE MODIFICATION


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1987; Johnson et al 1987; Ip et al 1988) and in transcriptionally active nuclei (Lin et al 1989). Antibodies against acetylated H4 (Hebbes et al 1988) or acetylated synthetic peptides (Turner & Fellows 1989; Lin et al 1989) have been used to demonstrate a preponderance of transcribed sequences in acetylated chromatin and in nuclei undergoing transcription. It was recently shown with such antibodies that H4 acetylated at residues 5 or 8 is present at many sites in the transcriptionally active arms of Drosophila polytene chromosomes, but not in the inactive heterochromatin of the chromocenter; H4 acetylated at residue 12, on the other hand, is depleted in the chromosome arms and more abundant in centric heterochromatin (Turner et al 1992). Although the correlation between histone acetylation and transcription seems well established, its significance remains obscure. Three findings call into question the idea that acetylation is directly responsible for the disruption of nucleosomes for transcription. First, histone acetylation has no substantial effect on core particle structure or stability (Simpson 1978; Ausio & van Holde 1986; Marvin et al 1990). Second, histone hyperacetylation, brought about by cell growth in the presence of the deacetylase inhibitor butyric acid, antagonizes rather than enhances the displacement of a nucleosome from the MMTV L TR promoter in the course of glucocorticoid hormone induction of transcription (Bresnick et al 1990). Finally, histone acetylation is correlated not only with transcription, but also with DNA replication (Jackson et al 1976; Allis et al 1985; Lin et al 1989), and the possibility must be considered that acetylation is only involved in chromatin assembly, which may occur following transcription, if nucleosomes are displaced in the process. Possible relationships of other histone modifications to transcription are less well established. Some attention has been given to ubiquitination, primarily a modification of H2A, in the extreme carboxy-terminal region of the molecule (Goldknopf et al 1975), This modification is correlated with the cell cycle, appearing in interphase but not in mitotic cells (Matsui et al 1979; Mueller et al 1985), and may be catalyzed by a cell cycle gene (Goebl et al 1988). The correlation with transcription is less clear, even doubtful. Nucleosomes derived from active gene regions appear to be enriched in ubiquitinated H2A in some cases (Barsoum & Varshavsky 1986) and deficient in this modified histone in others (Huang et al 1986). Mutations in yeast eliminating the site that is ubiquitinated in higher organisms resulted in no distinctive phenotype (Swerdlow et al 1990). While there is no formal requirement for topoisomerases as swivels for transcription or necessary involvement of DNA supercoiling, transient changes affecting supercoiling probably occur during transcription, and if uncompensated, may affect the rate of the process. These transient changes include unwinding of the DNA double helix in advance of and

DNA SUPERCOILING

ACTIVE CHROMATIN

58 1

rewinding behind the site of RNA synthesis, thus generating positive and negative supercoiling, respectively (Wu et al

1988), as well as nucleosome

disruption and reassembly , introducing negative and positive supercoiling, respectively. The transient nature of such changes has been demonstrated by topological analysis of yeast plasmids undergoing transcription (Pederson & Morse

1990). Evidence that topoisomerases are important to keep changes in


supercoiling in balance and prevent topological stress that would interfere with transcription includes the following: Mutations and other manipulations of yeast topoisomerase genes can cause the accumulation of positive or

1987; B rill & 1988; Giaever & Wang 1988). Moreover, the topoisomerase 2

negative supercoils in consequence of transcription (Brill et al Stemglanz

inhibitor novobiocin blocks the induction of transcription by heat shock in

Drosophila, and also prevents the restoration of an inactive chromatin structure during recovery from heat shock (Han et al 1985). Finally, topoisomerase 1 is associated with transcriptionally active loci in Drosophila polytene chro mosomes (Reischmann et al 1984), sites of topoisomerase 1 binding have been identified in nuclease-sensitive regions in Tetrahymena (Bonven et al 1985), and sites of topoisomerase 1 and 2 action have been mapped in regions flanking transcriptionally active genes in Dictyostelium and Drosophila (Ness et al 1988; Udvardy et al 1 985).

CONCLUSIONS AND PERSPECTIVES Chromatin structure is a factor at all levels of the gene activation and transcription process: decondensation of large chromosomal domains is evidently a prerequisite for transcription; some activator proteins appear to bring about the formation of nucleosome-free regions containing binding sites for additional transcription factors and for RNA polymerases; and the chromatin structure of transcribed regions may be altered in consequence of polymerase passage. Recent findings have shed light on all these aspects , and important new avenues of research are available. Conclusions that may be drawn at present include the following:

1.

The structure of the nucleosome is based o n a protein superhelix in which the histone dimers H2A-H2B and H3-H4, rather than individual histone molecules, are the folding units .

2.

The amino-terminal regions o f the histones represent distinct structural domains , either disordered or flexibly linked to the carboxy-terminal portions of the molecules.

3.

Preference of the histone octamer for interaction with certain DNA sequences leads to nonrandom locations of nucleosomes assembled in

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vitro. The largest difference in affinity of the octamer for sequences so far tested is about 4.

1 03 .

The way in which sequence specific DNA-binding proteins gain access to sites in chromatin is an enigma since such sites are often effective in regulation regardless of their location in the vicinity of a promoter, and thus regardless of whether the site is inward-facing on the surface


of a nuc1eosome or outward-facing towards solution.

S.

Nucleosome positioning may account for the exposure of activator protein-binding sites at some promoters .

6.

A nucleosome assembled on a promoter blocks the initiation of transcription .

7.

Activator proteins appear to relieve inhibition of transcription by

8.

The amino-terminal regions of H3 and H4 are involved in the gene

nuc1eosomes. activation process and may afford points of attack on the otherwise compact structure of the nucleosome. Enhanced acetylation of these regions is correlated with elevated levels of transcription .

9.

Nonhistone proteins have been identified that may assist activators in binding to specific sites in chromatin and alleviating inhibition by

histones. 10.

RNA polymerases can elongate transcripts through regions of DNA

1 1.

Enhanced susceptibility of transcribed regions to nuclease digestion may

12.

Enhanced susceptibility to nuclease digestion in more extended regions

coiled in nucleosomes. Histones may be dislodged in the process. reflect the transient displacement of histones by RNA polymerases . surrounding active genes may result from uncoiling of higher order chromatin structure .

1 3.

Locus control regions appear to trigger the unfolding of higher order structure , and other special sequences define the boundaries of unfolded domains .

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Histone exchange, chromatin structure and the regulation of transcription.

Transcription complex disruption caused by a transition in chromatin structure.

Chromatin potentiates transcription.

Chromatin and nucleosome structure.

Nucleosomal Barrier to Transcription: Structural Determinants and Changes in Chromatin Structure.

Coordinated effects of sequence variation on DNA binding, chromatin structure, and transcription.

The sequence-specific transcription factor c-Jun targets Cockayne syndrome protein B to regulate transcription and chromatin structure.

Chromatin architecture underpinning transcription elongation.

Effect of histone acetylation on structure and in vitro transcription of chromatin.

Regulation of alternative splicing through coupling with transcription and chromatin structure.

SWI2 and SNF5 activate transcription in yeast by altering chromatin structure.

Genetic Architecture of Transcription and Chromatin Regulation.

Chromatin transcription with mercurated nucleotides.

Chromatin structure and gene expression.

Chromosomal proteins and chromatin structure.

DNA methylation and chromatin structure.

Quaternary structure of chromatin.

Yeast chromatin subunit structure.

Transcription-dependent generation of a specialized chromatin structure at the TCRβ locus.

Effector bottleneck: microbial reprogramming of parasitized host cell transcription by epigenetic remodeling of chromatin structure.

Low molecular weight peptides controlling transcription are present in the calf thymus chromatin structure.

Transcription-induced nucleosome 'splitting': an underlying structure for DNase I sensitive chromatin.

Nucleolin: dual roles in rDNA chromatin transcription.

Chromatin remodeling: from transcription to cancer.