Chromatin-remodeling factors: machines that regulate?

346

Chromatin-remodeling factors: machines that regulate? Patrick D Varga-Weisz* and Peter B Beckert Chromatin has shifted into the focus of attention as a key to

understanding the regulation of nuclear processes such as transcription. Protein machines have been described that use the energy of ATP to render chromatin dynamic and hence active, but which may also be involved in chromatin assembly. The discovery of three different

Drosophila nueleosome

remodeling complexes that contain imitation switch (ISWI), an ATPase with a high degree of sequence conservation from

yeast to human, points to a central function of this ATPase in chromatin dynamics.

Chromatin enzymes

remodeling

by ATP-dependent

Nucleosomes are transcriptional repressors in vivo because they impede the access of transcription factors to their target site. An early step in gene activation involves an illdefined alteration of nucleosome structure ('remodeling') at active promoters and enhancers, which allows binding of transcription factors. T h e local change in chromatin structure can frequently be monitored by an increased accessibility to the nuclease DNase I and is referred to as a DNase I hypersensitive site.

Addresses *Marie Curie Research Institute,The Chart, Oxted, Surrey RH80TL, UK *European Molecular Biology Laboratory, Meyerhofstrasse 1,69117 Heidelberg, Germany; e-mail: [email protected] Correspondence: Peter B Becker

Current Opinion in Cell Biology 1996, 10:346-353

http:llbiomednet.com/elecref/0955067401000346 © Current Biology Ltd ISSN 0955-0674

Abbreviations ACF ATP-dependentchromatin assembly and remodeling factor CHRAC chromatinaccessibility complex GR glucocorticoidreceptor ISWI imitationswitch NURF nucleosomeremodeling factor SNF sucrosenon-fermenting SWl switchingmating type

Introduction Packaging is not only a means of storage, but is also important for organization. Usually the important things are the most accessible, while things that are less important may be hidden away. This is true for the eukaryotic nucleus. T h e -50,000-100,000 genes of a human being, encoded in 6×109 base pairs of DNA of a total length of about 2meters, are packaged into a nucleus 6--8 micrometers in diameter. This packaging and organization is achieved by proteins which, together with the DNA, form a complex structure--chromatin. T h e structural unit of chromatin is the nucleosome, about two windings of DNA wrapped around an octamer of histone proteins.

Chromatin is intrinsically involved in the regulation of nuclear processes, especially transcription. Chromatin organization can close off parts of the genome that need to be transcriptionally silenced and can make accessible genes that need to be expressed at an elevated rate. How this is achieved is a focus of current molecular biology studies. This review centers on ATP-hydrolyzing e n z y m e s - - p r o t e i n 'machines' composed of several s u b u n i t s - - t h a t are part of the answer to how chromatin regulation is achieved.

At a subset of genes, in yeast, a large, multisubunit complex is involved in this chromatin remodeling process. Central to its function is an ATPase named switching mating type (SWI)2 or sucrose non-fermenting (SNF)2, which has a domain similar to many DNA-dependent ATPases (for review see [1,2]). Complexes containing ATPases that are closely related to SWI2/SNF2 have been identified in Drosophila and mammalian cells. T h e yeast SWI/SNF complex is not abundant and is not essential for viability. In contrast, another nucleosome-remodeling complex from yeast termed RSC (remodels the structure of chromatin) with similarities to the SWI/SNF complex (see Table 1 and [3°]) is abundant and essential for viability [4°]. Again, an ATPase (STH1) with similarity to the SWI2/SNF2 ATPase is part of the complex. T h e study of chromatin-remodeling activities in extracts of Drosophila embryos yielded three different complexes; they share an ATPase (termed imitation switch [ISWI]) that is similar to the SWI2/SNF2 in its ATPase domain [5] and is highly conserved from yeast to man [6]. Complexes containing the human ISWI homolog (hSNF2L) have been found in extracts of human cells, but have not been further characterized [7,8]. T h e three ISWI-containing complexes from Drosophila termed nucleosome remodeling factor (NURF) [6,9], chromatin accessibility complex (CHRAC) [10"], and ATP-dependent chromatin assembly and remodeling factor (ACF) [11°] (see Table 1) are biochemically and functionally distinct (reviewed in [12], and see below). T h e three complexes have been identified independently by different assays: N U R F was purified as a cofactor that facilitated the interaction of the GAGA transcription factor with chromatin by perturbing a nucleosomal array in the presence of ATE Sub-stoichiometric amounts of N U R F alone (on the assumption that nucleosomes are the substrate for N U R F action) also disrupt nucleosomal arrays as well as mononucleosomes [9]. CHRAC was purified as a factor that caused ATP-dependent accessibility of restriction enzymes to chromatin reconstituted in vitro. T h e restriction enzyme served as a probe for non-targeted global accessibility.

Chromatin-remodeling factors Varga-Weisz and Becker

347

Table 1 A survey of known energy-dependent nudeosome remodeling machines. Factor

SWI/SNF

Organism

ATPase

Saccharomyces SWI2/SNF2 cerevisiae

Size

No. of peptides associated with activity

In vivo function

2MDa

11

Transcription of specific genes; remodels chromatin at promoters in vivo

Comments

References

[27,28",29 ° 30,31,32 °]

Brahma complex

Drosophila melanogaster

BRM (brahma)

2 MDa

?

Essential for cell viability

Drosophila homolog of SWI/SNF complex

[26 °]

Mammalian SWI/SNF

Homo sapiens

BRG1

-2MDa

?

hBRM

-2 MDa

Human homolog of SWI/SNF complex Complex is similar to the one containing BRG1

[7,8,43]

H. sapiens

9-12, subunit heterogeneity 9-12

RSC

S. cerevisiae

STH1

-1 MDa

15

Essential for mitotic growth

Sthl p, Rsc6p, Rsc8p and Sfhlp of RSC are similar to Swi2/Snf2, Swp73p, Swi3 and Snf5 of the SWI/SNF complex

[3*,4 °]

NURF

D. melanogaster

ISWI

500 kD*t

4

?

ATPase stimulated by nucleosomes, not free DNA Contains p55

[6,9,13,20]

CHRAC

D. melanogaster

ISWI

670 kD*

5

?

Contains topoisomerase II Nucleosome spacing factor

[10 °]

ACF

D. melanogaster

ISWI

220 kDt

4

?

Nucleosome-spacing factor

[11 °]

?

[7,8]

*Molecular mass determined by gel filtration, tMolecular mass determined by glycerol gradient sedimentation.

While the three ISWI-containing Drosophila remodeling machines all increase the access to nucleosomal DNA in specific assays, they apparently achieve this by different mechanisms. N U R F destabilizes nucleosomal arrays [9], whereas CHRAC and ACF are involved in the contrary reaction, the creation of regular nucleosomal arrays [10",11°]. T h e common denominator of all three factors seems to be that they all target nucleosomes, which points to a role of the shared subunit, ISWI, in this process. However, not all subunits of the complexes have been identified to date, which leaves the possibility that additional ATPases may be involved in the various functions. In any case, unraveling the role and function of ISWI in diverse molecular environments and its physical and functional interactions with other subunits may shed light on the mechanisms of nucleosome remodeling.

subunit is also found in other complexes involved in histone metabolism: p55 is a subunit of the Drosophila chromatin-assembly factor dCAF-1 [14,15], and is homologous to a human protein that is part of a histone deacetylase complex [14,16] and also a component of the human CAF-1 [15]. In the context of human CAF-1, p55 interacts with histone H4 [15]. Closely related proteins in yeast and humans are subunits of histone H4 acetyltransferases, which are involved in chromatin assembly. Histone acetylation is targeted to the highly conserved, amino-terminal tails that extend beyond the globular histone octamer structure [17"]. Thus, p55-1ike proteins may be involved in targeting the catalytic subunits of various subunits to the histones [18,19]. There are hints that the histone amino termini may be recognized by remodeling machines: removal or extensive crosslinking of the histone tails will prevent the nucleosome-stimulated ATPase activity of N U R F [20]. It will be interesting to determine whether p55 in N U R F directly targets histone tails. If a nucleosome-remodeling factor bound both the nucleosome-associated DNA as well as the histones themselves, an ATP-driven conformational change in the factor could translate into a change in nucleosome structure. Alternatively, p55 may be involved in sequestering a histone that has been released after nucleosome disruption.

T h e identification of the N U R F subunit p55 [13] testifies to the modular nature of remodeling complexes as this

T h e feature that distinguishes CHRAC and ACF from N U R F is the fact that they catalyse the formation of

Surprisingly, CHRAC is also able to introduce regularity into an irregular nucleosomal array by acting as an energy-dependent nucleosome-spacing factor (see below) [10"]. ACF was purified as an ATP-dependent nucleosome spacing factor in a fractionated system. However, ACF is also involved in transcription factor-mediated chromatin disruption, similarly to N U R E and can also remove nucleosomes from densely packaged chromatin [11"]. Mechanism

of nucleosome

remodeling

348

Nucleusand gene expression

regular nucleosomal arrays from irregular chromatin. In this reaction, one molecule of the remodeling factor acts on many nucleosomes ([11°]; P Varga-Weisz and P Becker, unpublished data). Figure 1 shows two non-exclusive working hypotheses of how this may be achieved. T h e first scenario assumes that CHRAC and ACF promote the mobility of nucleosomes on DNA. It takes into account the ATP-dependent mobility of nucleosomes reconstituted in Drosophila embryo extracts [21,22] as well as observations of nucleosome repositioning by ACF [I1"]. An enhancement of nucleosome mobility will lead to increased access of a restriction enzyme or other factors to chromatin (for example, the nucleosome slides off the site). On the other hand, nucleosome mobility may explain the establishment of regular nucleosomal arrays (Figure la). If an ordered nucleosomal array was energetically favorable over irregular chromatin, perhaps due to improved folding of the fiber into higher order structures, a 'mobilization' of nucleosomes would accelerate the speed with which the energetically favorable regularity is reached. Once regularity is established, the nucleosome may still be subject to dynamic transitions, but will oscillate around the preferred site. T h e second scenario involves a 'nucleosome maturation' step, which may involve removal of unbound DNA segments within nucleosomes, removal of histone chaperones or a completion of nucleosome assembly (Figure lb). This scenario is consistent with the greater nuclease sensitivity of chromatin that has been assembled without ATP. Under these conditions micrococcal nuclease--which does not cleave nucleosomal DNA well--releases subnucleosomesized DNA [10",11",23}. T h e observation of increased nucleosome assembly in the presence of ACF and histone chaperone (as opposed to chaperone alone), even at early stages of chromatin assembly, is also in favor of the latter scenario [11"]. Nucleosome mobility could result from a partial or even complete nucleosome disassembly/reassembly cycle but one can also envision mechanisms that leave the histone octamer intact. Van Holde and Yager [24] suggest that the thermal twisting of DNA, especially at the edges of a nucleosome (the entry/exit site of the DNA around the nucleosome), can lead to a dissociation of the histone octamer from DNA in mononucleosomes or the sliding of nucleosomes along DNA within an array of nucleosomes. How such twisting of the DNA may cause nucleosome translocation is illustrated in Figure 2. A nucleosome-remodeling factor may accelerate this process by using the energy of ATP to twist the DNA at the edges of the particle. T h e main function of remodeling factors may be, therefore, to affect the dynamic transitions of histone-DNA interactions, which are already intrinsic to the nucleosome per se [25].

Targeting of n u c l e o s o m e r e m o d e l i n g T h e Drosophila SWI2 homolog BRM and the Drosophila ISWI protein are expressed at relatively high levels

in nuclei throughout the developing organism (at least 100,000 molecules of BRM or ISWI protein per nucleus at their peak stages of expression, corresponding to one BRM or ISWI per 20 nucleosomes [6,26"]). By contrast, the yeast S W I / S N F complex is not abundant [27], which provokes the question of how remodeling activities are targeted to relevant sites, if at all? Two opposing scenarios are, first, that transcription factors have domains that specifically interact with the chromatin remodelers and thereby target them to the site of action and, alternatively, that transcription factors may take advantage of a global action of the chromatin remodeler and preserve an otherwise transient state of, for example, nucleosome disruption. There is evidence for both scenarios. T h e S W I / S N F complex is able to remodel nucleosomes within an array in a catalytic and reversible manner in the absence of targeting to a specific nucleosome [28",29"]. Owen-Hughes et al. [28"] found that the transient disruption of nucleosomes in an array caused by the SWI/SNF complex reverted to inaccessible chromatin unless binding of a transcription factor stabilized the disrupted state. Only the synergism between the remodeling machine and the sequence-specific DNA-binding protein led to a persistent alteration of a nucleosome as characterized by DNase I hypersensitivity. In this scenario the specificity of the transcription factor is the sole determinant of the persistent change in chromatin structure. A similar scenario may also apply to the ISWI-containing factors as N U R F appears to be rather promiscuous with respect to the transcription factors it can synergize with in vitro. There is no evidence so far for direct interaction between a remodeling machine and a transcription factor. However, there are examples of targeted nucleosome remodeling by the SWI/SNF complex. Binding of the glucocorticoid receptor (GR) to a mononucleosome in vitro specifically enhanced nucleosome disruption by a mammalian SWI/SNF preparation [30]. GR binds to nucleosomal DNA with almost the same affinity as to naked DNA and per se does not disrupt the nucleosome upon binding. Together with previous studies that showed a co-precipitation of GR with SWI/SNF components [31], this argues for a direct targeting through protein-protein interactions. Furthermore, the targeting of a nucleosomeremodeling activity, most probably the SWI/SNF complex, to a promoter by the human heat-shock factor resulted in the localized remodeling of nucleosomes downstream of a heat-shock promoter [32"]. It is likely that chromatin reorganization in vivo is a multistep process involving several factors. This idea is consistent with the observation of several groups that the interaction of a transcription factor with chromatin leading to a local disruption (or taking advantage of a pre-existing accessible site) is followed by a wider rearrangement of chromatin structure [33",34,35"]. T h e initial ATP-dependent disruption or mobilization of nucleosomes would be

Chromatin-remodeling factors Varga-Weisz and Becker

349

Figure 1

(a)

CHRACAcForS~7

( ~

ATP ADP + Pi

(b)

or

ADP + Pi

Current Opinion in Cell Biology

(a) A hypothetical scenario for CHRAC or ACF action involving nucleosome mobility. The formation of nucleosomes in the absence of CHRAC or ACF and ATP creates arrays of nucleosomes with irregular spacing (which may be the result of the strong binding of the histone octamer to DNA, which 'freezes' the octamer to the position at which it initially formed). CHRAC and ACF use the energy of ATP to allow them to move, possibly by sliding along the DNA. If more regular positioning is thermodynamicallymore favorable compared with the previous irregular positioning, the nucleosome mobility will accelerate the adoption of the preferred state. (b) A different scenario assumes that CHRAC and ACF are necessary for a 'maturation' step of newly formed nucleosomes. This may involve, for example,the removal of 'bubbles' of unbound or 'incorrectly' bound DNA within the nucteosome, or removal of still-associated histone chaperones (which are necessary to deliver the highly charged histones to the DNA), or the mediation of complete and correct assembly of the nucleosomes.

followed by wider chromatin disruption mediated by the bound factor and/or other chromatin-remodeling activities, for example histone acetyhransferases. T h e acetylation of histone tails has long been suspected to be involved in chromatin reconfiguration and transcriptional activation, mainly because of correlative findings. Recently, a convergence of genetics and biochemistry identified certain transcriptional co-activators as histone acetyltransferases, whereas certain co-repressors were found to be histone

deacetylases (reviewed in [36]). Genetic studies in yeast provide evidence for a potentially functional interaction of the SWI[SNF complex and a histone acetyltransferase complex ([37°-40"], reviewed in [41])

Function of the diverse nucleosome remodeling complexes T h e yeast SWI/SNF complex is involved in the transcriptional activation of a relatively small set of inducible

350

Nucleus and gene expression

Figure 2

Thermal twist or

CHRAC/ACF +ATP

A model of how nucleosome mobility may be achieved. Twisting of DNA, either because of thermal movement or because of a nucleosome-remodeling factor, creates a 'bubble' of unbound DNA, a one-phosphate displacement, which causes a transient removal of a 'cell' of DNA from the histone octamer. The migration of this bubble through the entire nucleosome will lead to movemenl of the DNA by one base pair with respect to the histone octamer. The small ovals represent backbone residues contacted by the histones (gray hooks). Figure adapted from [24].

genes. Less clear are the in vivo functions of those complexes that contain the SWI2/SNF2 homologs in Drosophila and mammalian cells (BRM and BRG1 respectively, see Table 1). In both Drosophila and F9 murine carcinoma cells, those ATPases are essential for cell viability [26",42°]. Here SWI2/SNF2 homologs are involved--either directly or indirectly - - in cell proliferation or cell cycle regulation. T h e y may, at least in part, cooperate with the retinoblastoma protein, which regulates progression from G 1 to S phase but which also has a critical role in the development and differentiation of certain cell types [43]. T h e functions of the Drosophila ISWI-containing complexes in vivo are unknown. It~ vitro, ATP-dependent nucleosome remodeling by N U R F is required for transcrip-

tional activation by a synthetic G A L 4 - H S F transcription factor in chromatin reconstituted by a Drosophila embryo extract [35°]. Nucleosome remodeling by N U R F enabled the transcription factor to function in chromatin, but was not required for subsequent preinitiation complex formation and transcription. T h e fact that two other chromatin remodelers (CHRAC and yeast SWI/SNF) were not functional in this assay supports the notion that other remodeling activities may be dedicated to different promoter or enhancer configurations, different stages of transcription such as elongation (see for example, [32°,44,45]), or to functions separate from transcription such as DNA replication or DNA repair. Chromatin remodeling or disruption may not only be involved in direct transcriptional activation, but may

Chromatin-remodeling factors Varga-Weisz and Becker

create an epigenetic mark that leads to inheritance of gene activity states through replication (see for example [46]). Finally, chromatin remodelers may be involved in the domain-wide alteration of chromatin structure (the 'higher-order' folding of the chromatin fiber), which could target genes to nuclear domains of high transcriptional activity [47]. T h e in vivo function of CHRAC and ACF may be in chromatin assembly. These complexes could function in 'chromatin repair' after or even during transcription; the process of transcription perturbs chromatin structure (see for example [48-50]) and such on-going repair may be necessary for chromosome integrity. Another possibility is that the ATPase ISWI is involved in the targeting or transport of molecules into chromatin or specific chromatin domains: CHRAC contains topoisomerase II and may be a dedicated complex that efficiently shuttles topoisomerase II into chromatin [10"]. Classical topoisomerase II activity does not seem to be involved in the creation of chromatin accessibility and remodeling of nucleosomal arrays (P Varga-Weisz, P Becker inpublished data). However, topoisomerase II needs to be effectively incorporated into chromatin because it is vital for sister chromatid separation, chromosome untangling after DNA replication, and chromosome condensation and decondensation during mitosis [51].

Conclusions An important lesson from the discovery of factors such as N U R E CHRAC and ACF is that processes involved in chromatin assembly and processes involved in making chromatin more accessible, for example, to allow transcription, are most likely to be intimately interconnected. T h e existence of a whole battery of muhiprotein complexes that bring about dynamic transitions of chromatin structure points to an important layer of regulation of nuclear processes that is waiting to be fully unraveled.

351

is required for cell cycle progression. Mol Ce//Biol 1997, 17:3323-3334. Sfhlp, which is very similar to SNF5 (a component of the SWI/SNF complex), is part of the RSC (remodels the structure o1 chromatin) complex. Sfhlp is essential for viability as a temperature-sensitive sfhl allele arrests cells in the G2/M phase of the cell cycle. SFH1 and SNF5 are not functionally redundant. There is biochemical and genetic evidence for an interaction between Sfhlp and Sthlp. Sfhlp phosphorylation is regulated during the cell cycle. 4. •

Cairns BR, Lorch Y, Li Y, Zhang M, Lacomis L, ErdlumentBromage H, Tempst P, Du J, Laurent B, Kornberg RD: RSC, an essential, abundant chromatin-remodeling complex. Ceil 1996, 87:1249-1260. RSC is an abundant nucleosome remodeling complex in yeast. It resembles the SWI/SNF complex in many respects, but several subunits are essential for yeast growth. 5.

Elfring LK, Deuring R, McCallum CM, Peterson CL, T~-nkun JW: Identification and characterization of Drosophila relatives of the yeast transcriptional activator SNF2/SWI2. Mo/Ce//Bio/ 1994, 14:2225-2234.

6.

Tsukiyama T, Daniel C, Tamkun J, Wu C: ISWl, a member of the SWl2/SNF2 ATPase family, encodes the 140 kD subunit of the nucleosome remodeling factor. Ceil 1995, 83:1021-1026.

7.

Wang W, Cbtb J, Xue Y, Zhou S, Khavari PA, Biggar SR, Muchardt C, Kalpana GV, Goff SP, Yaniv Met aL: Purification and biochemical heterogeneity of the mammalian SWI/SNF complex. EMBO J 1996, 15:5370-5382.

6.

9.

Wang W, Xue Y, Zhou S, Kuo A, Cairns BR, Crabtree GR: Diversity and specialization of mammalian SWl/SNF complexes. Genes Dev 1996, 10:211 '7-2130. Tsukiyama T, Wu C: Purification of an ATP-dependent nucleosome remodeling factor. Cell 1995, 83:1011-1020.

10. •

Varga-Weisz PD, Wilm M, Bonte E, Dumas K, Mann M, Becker PB: Chromatin-remodelling factor CHRAC contains the ATPases ISWl and topoisomerase II. Nature 388:598-602. The 670 kDa chromatin-remodeling complex CHRAC uses ATP to render chromatin generally more accessible, but at the same time functions as a nucleosome-spacing factor. Among its five subunits are the ATPases ISWI and topoisomerase II. 11. •

Ito T, Bulger M, Pazin MJ, Kobayashi R, Kadonaga JT: ACF, an ISWl-containing and ATP-utilizing chromatin assembly and remodeling factor. Cell 1997, 90:145-155. ACF, an ATP-utilizing chromatin assembly and remodeling factor, contains ISWI and three additional polypeptides. In a chromatin assembly system consisting of DNA, histones, and a histone chaperone, ACF functions as a catalytic nucleosoms-spacing factor. ACF may also mediate promoter-specific nucleosome reconfiguration by Gai4-VP16 in an ATP-dependent manner.

Acknowledgements

12.

Patrick Varga-Weisz was supported by a grant from the Deutsche Forschungsgemeinschaft to Peter Becker and is currently supported by Marie Curie Cancer Care, Great Britain. We thank all the authors who provided us with unpublished material and Colin Goding from the Marie Curie Research Institute, for comments that improved the manuscript.

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14.

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15.

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Cao Y, Cairns BR, Kornberg RD, Laurent BC: Sfhlp, a component of a novel chromatln-remodeling complex,

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