Chromatin

changes

during

Raymond Washington

State University,

the cell cycle

Reeves Pullman,

Washington,

USA

Considerable progress has recently been made in elucidating the biochemical mechanisms regulating changes in chromatin structure during all stages of the cell cycle. Although anticipated, the apparently ubiquitous role played by phosphorylationidephosphorylation reactions in modulating these changes is, nonetheless, remarkable.

Current

Opinion

in Cell Biology

1992, 4:413-423

Introduction

Cell cycle

The striking morphological changes that occur during the cell cycle in higher eukaryotes are easily visible under the light microscope and were first described well over a century ago. Only recently, however, has detailed biochemical and structural information emerged supporting the long-held suspicion that at all stages of the mitotic cycle (GJG,, S, Gz and mitosis), eukaryotic chromatin is not only precisely organized, but is *also in a dynamic state of structural, compositional and biochemical change. A most remarkable finding is the extent to which many of these cyclic changes appear to be mediated by post-translational protein modifications, in particular, by reversible phosphorylation/dephosphorylation reactions.

A gratifying convergence of recent data from both genetic and biochemical systems indicates that the cdc2 serine/threonine kinase of the fission yeast Schizosaccbaromycespombe has homologs (e.g. CDC28 of Saccbaromyces cerelisiae and histone Hl kinase of mammals) in all eukalyotes that serve as key regulatory enzymes in controlling the progression of cells through the mitotic cycle. It is beyond the scope of this article to cover the immense literature in this area and interested readers are referred to the excellent review by Forsburg and Nurse [I]. Suffice it to say that in yeast, as well as mammals, active cdc2 kinase (or its homologs) is required at two points in the cell cycle: first, at a point in G1 (called ‘start’ in yeast) that commits the cells to S phase and a cycle of DNA synthesis, and second, at a point in late Gz that commits the cell to mitosis. The enzymatic activity of cdc2 kinase at these two control points is, in turn, regulated by cycle-dependent phosphorylation/dephosphorylation and by formation of heterotypic complexes between the stable kinase and several labile proteins called cyclins and cyclin-like proteins [ 11. For example, a p34cdc%yclin B complex, known as maturation promoting factor (MPF), is thought to regulate mitosis and meiosis in all eukatyotes. In higher eukaryotes there are multiple, structurally distinct, cdc2-like kinases [ 21 and it appears that the G1-S and G2-M transitions in these organisms are controlled by different cdc2 proteins [3] as well as by different cyclins. However, activation of cdc2 kinase activity alone is not always sufficient to initiate mitosis since it now appears that the parallel activation of other protein kinases is also required for the G2-M transition in viuo [4].

172 l’illo protein phosphorylations are thought to modulate chromatin structure and function by altering protein-protein and pro&n-DNA interactions, by activating or inhibiting various enzymatic activities, as well as by changing the subcellular distributions of chromosomal proteins during different phases of the cell cycle (Table 1). In fact, most aspects of the cycle, ranging from control of initiation of DNA synthesis and mitosis down to localized changes in chromatin structure around specific sequences of DNA, are directly or indirectly influenced by protein phosphorylations. This review selectively covers some of the important findings in this area during the past year. It begins with a brief outline of the role played by cdc2 kinase in controlling cell cycle progression; this is followed by a discussion of what is currently known about the mechanisms regulating the reversible nuclear and chromosomal changes occurring at mitosis; the review concludes with a sketch of the dynamic structural changes in chromatin that are known to occur during interphase.

control

by p34CdC2 kinasets)

Abbreviations CK II-casein kinase MPF-maturation

II; NLGnuclear promoting factor;

localization signal; RCCl-regulator

@ Current

Biology

HAM-high of chromatin

mobility group; condensation;

Ltd ISSN 0955-0674

HSF-heat-shock topo-topoisomerase.

factor;

413

414

Nucleus

ahd gene expression

. able 1. Effects of cell-cycle-dependent

nuclear

rotein

Sea urchin

sperm

Lamins

Various

HMC-I PI)

HU”ld” Mouse

SPXKIR SPKK

HMC-14

PTPKR lTPCR

CBF3

Xrnopus

cdc2 Endogenous (metaphasel CAMP-dependent protan klnase rn “dro Endogenous

and transcription

eggs

Endogrnous phospharase T”9, S2.43 p9

Human

SW15

Yeast transcription factor p11oas

PRB ‘olymerases

DNA polymerase

(L

Human

RNA polymerase

II

Various

QRTPRK. LRSPYK

cdc?

cdc2 YSPTSPS LC-doman repeats)

T”4

p34-‘c2

ChIcken

p34cdcz

HUma”

Coupling

NLS. nuclear

locakzatlon

Endogenous synthase klnase-3-lake) Endogenous Icdc2-lake and prow,” klnase A) cdc2

Sill, S”’

group;

klnase, system

@‘CO@”

SPRKR. SPIKE

Leplication fac(orr SV40 large T antigen

iMC. high mobility

DNA banding I” wlro

Dephosphorylarlon

correlated

wth

condensatton of chromatm Phosphorylatlon correlated wtth mhabttlng chromattn condensation Lamma disassembly

Role I” chromatm condensatwnf Reduces DNA binding afflnlty m v,,ro Reduces bandIng to both DNA and nucle~~~mes I” wtro Phosphor&w” reqwed for DNA btndmg m wlro Revers!ble chromatinlnuclear membrane aSSOClatl0” I” w,ro

116-I 1141 1151 129.301

122...25.261

1251 1641 1651

I661 131-1

factors Human

Ocf-1 (OTFl)

Weakens

Reference

Reduces blndmg afflntty for A/l-DNA m wro. role I” rhromatm condensation?

cdc2

T5’

Yeast centromere membrane

cdc2

TPXKK T=d, T’R

Calf

hcogenes c-l””

Endogenous Icdc?-like) Endogenous Endogenous

Human Human

and HMC-17

Proposed phosphorylatlon eflectk)

Kl”JS?

Utwahymma. macronucleaus) Chicken

Histone HS

Pl HMC-14

phosphorylations.

Phosphorylation site

Source

tructural proteins Histone Hl and H2B termin! Histone Hl

Nuclear

protein

Tl,

Endogenour (cdc?-like1

cdc2 Casem kmase II

,4=x, S”’

Endogenous

Tlbl

Endogenous

Reduces

AP-1 site DNA-bandung

1481

affinity I” w1ro Regulation of hlstone H2b gene transcnpt,o”; phosphorylatlon 01 mltosls-specific sws mhlbtts DNA bIndIng Dephosphorylatlon of we5 near the NLS I” C, allows nuclear tra”SlocatlO” Underphosphorylated pRB (I” C,) binds tightly to nuclear structures and transcrlptlonal protew whereas hyperphosphorylated pRB (I” S/M phase) does not Reduces blndang afflnlty for single-stranded DNA I” wtro Induces structural conformatlonal change !n wlro. actlve polymerax multlphosphorylated lnhiblts nuclear

ampon

Phosphorylation at site near the NLS elats maximum amport to the nucleus m WY0 Regulatlo” (mhbltlonl of enzyme actwy and other functmns m wlro Phosphorylatlon reqwd for mteractlon wtth cycl~ns A and B I” wtro

149.671

159.1

lSO.53.54.561

1451 146,471

161**1 I601

1681 I691

signal.

of S phase and M phase

Eukaryotic somatic cells do not normally enter mitosis unless their chromosomes have been replicated in the previous S phase. Precise regulation of both cdc2 kinase [ 51 and cyclin [6] activiues appears to be the primary mechanism responsible for this tight coupling of mitosis and completion of DNA synthesis. A mammalian protein implicated in the suppression of cdc2 kinase activity during S phase is the DNA-binding protein RCCl (prod-

uct of the regulator of cbromutin condensation gene). RCCl protein functions as an inhibitor of premature chromosome condensation and mitosis by preventing the activation of cdc2 kinase activity prior to the completion of DNA synthesis [7]. In human cells it is tightly, but non-covalently, associated with Ran, a nuclear Rasrelated guanine nucleotide-binding protein [8-l. This inactive RCCI-Ran complex is, however, devoid of bound nucleotide. Activated RCCl protein, on the other hand, catalyzes an exchange of guanine nucleotides on Ran

Chromatin

and when GTP is bound to Ran it dissociates from the RCCl complex (8*]. It has, therefore, been suggested that during S phase, activated RCCl might function to prevent premature chromatin condensation by maintaining a high intranuclear concentration of Ran-GTP which, in rum, prevents the synthesis or activation of a mitotic inducer (possibly cdc2 kinase) [8*]. Following completion of DNA synthesis the pool of Ran-GTP could be depleted by inactivation of RCCl and/or by modulating the activity of a Ran-specific GTPase-activating protein [8*). A direct experimental test of this interesting proposal is anticipated in the near future since a gene (BJI) structurally and functionally similar to vertebrate RCCI has been isolated from Drosophila [9] and genes related to both RCCl (piml) and Ran GTPase (spzl > have been isolated from fission yeast [ lO*].

Mitosis: chromatin and topoisomerase

condensation II

-

histone

Hl

It is now widely accepted that members of the Hl/H5 family of ‘linker’ histones are involved in the cooperative condensation of extended, nucleosome-containing chromatin filaments into both 30.nm fibers and higher-order chromosomal structures. Nevertheless, the precise nature of the interactions of these lysine-rich histones with DNA and other chromosomal constituents during chromatin packaging remains largely elusive [ 1 1] An example of the uncertainties surrounding the Hl histones is the continuing debate over the role(s) played by their phosphorylation during chromatin condensation. Early observations that mitotic Hl histones are highly phosphorylated led to the idea that Hl hyperphosphorylation triggers or promotes chromosome condensation (reviewed in [ 111). As Table 1 illustrates, Hl histones are specific substrates for cdc2 kinase and, during mitosis, are multiply phosphorylated on the conserved amino acid motifs Ser/Thr-Pro-X-Basic (where X is any *amino acid) present in their amino- and carboxyl-terminal tails, the protein regions involved in chromatin compaction. It has been suggested that tandem repeats of these COW served motifs, such as those found in sea urchin spennspecific Hl histones, form double B-turn type secondary structures that facilitate preferential binding of the tails of these histones to the minor groove of AT-rich DNA (reviewed in [ 12*]). It is not immediately obvious, however, how phosphorylation of these structural motifs could directly promote chromatin condensation unless the phosphates form strategic salt bridges with the basic residues of other histones or non-histone proteins and thereby act coordinately to atfect compaction. This idea has been advanced by Kharrat and his colleagues [ 131 who have presented evidence that certain phosphorylated synthetic peptides containing these conserved amino acid motifs can induce hypercondensation of AT-rich DNA in r&-o. The in Llivo significance of these observations remains to be determined. An alternative proposal that has often been made is for an ‘indirect role’ of Hl phosphorytation in the control of

changes

during

the cell cycle Reeves

chromatin condensation. In this scheme Hl hyperphosphorylation relieves interphase chromatin constraints by loosening protein-DNA or protein-protein interactions, thereby allowing other mechanisms to modulate chromatin condensation, perhaps acting through the nuclear matrix or scaffold. Two recent complementary reports concerning the in LJI’L~O phospholylation of members of the lysine-rich Hl/H5 family of histones are of interest here. In one case involving the regression of the ‘old’ macronucleus of Tetrdymenu, dephosphotylation, not phosphotylation, of histone Hl was correlated with chromatin condensation [ 141. In the other case, erythroid histone H5 protein was introduced into non-erythroid cells and the phosphotylation of the introduced histone correlated with the inhibition of chromatin condensation [ 151. In neither of these in vivo studies was hyperphosphorylation of lysine-rich histones associated with chromosome condensation, lending support to the idea that other mechanisms are probably involved with chromatin compaction. Also, of considerable interest is the report by Hill et al. [ l6**] on the consequences of phosphorylation of the sperm-specific histones Hl and H2B during different stages of sea urchin spermatogenesis. These workers assessed the effects of multiple histone phosphorylations on chromatin packing by comparing several physical and biochemical properties of sea urchin spermatid (phosphorylated) and sperm (dephosphorylated) chromatin and histones. The results of these experiments led to the conclusion that reversible phosphotylations of the amino- and carboxyl-terminal tails of Hl, and/or the amino-terminal tail of H2B, effectively control intermolecular interactions between adjacent chromatin filaments and hence tight chromatin packing in the highly condensed sperm nucleus [ 16**]. A recent report from Laemmli’s labotatoxy [ 17.1 contains evidence that topoisomerase II (topo II) is required, at least in r&-o, for chromosome assembly and condensation in higher eukaryotes. Mitotic extracts from Xenopus eggs, which normally convert exogenously added nuclei to condensed chromosomes, were first depleted of endogenous topo II by specific immune adsorption. When isolated chicken erythrocyte nuclei, which have a very low content of endogenous topo II, were added to the immunodepleted extracts, nuclear chromatin was not converted to condensed chromosomes, although their condensation became normal upon subsequent addition of the enzyme to the extracts. Dosage experiments involving addition of purified topo II to the depleted extracts provided direct biochemical evidence for the involvement of topo II in chromatin condensation and suggested a structural role for the protein in this process [ 17.1. Hirano and Mitchison [ 181 recently reported an extension of this line of investigation. Using Xenopus egg extracts specific to interphase and mitotic states, these workers demonstrated that the in vitro assembly of naked DNA into higher-order compacted chromatin structures depended both on the stage of the cell cycle from which the egg extracts were derived and on the presence of topo II enzyme in the extracts. A further

415

416

Nucleus

and gene expression

discussion of DNA topoisomerases and their biological functions is presented in this issue (Hsieh, pp.396-400). Specialized biochemical mechanisms may also facilitate chromatin decondensation in vivo. In immunodepletion/protein re-addition experiments, Laskey and his colleagues demonstrated an in tlitro requirement for the protein nucleoplasmin to facilitate the first stages of chromatin decondensation of sperm nuclei added to extracts of Xenopus egg cytoplasm [ 191. Since nucleoplasmin is present in somatic cells at only very low levels, if at all, it would not be surprising to find that somatic cells possess proteins that mimic the action of nucleoplasmin and assist in the normal chromatin decondensation of mitotic telophase.

Mitotic group-l

phosphorylation of high mobility non-histone proteins

Non-histone protein high mobility group (HMG)-I (not to be confused with the unrelated HMG-l/-2 chromatin proteins) is a mammalian DNA-binding protein that preferentially binds in zjitro and in viva in the minor groove AT-rich stretches of DNA. 1~ zdtro, the HMG-I protein has been demonstrated to bind to AT-DNA by means of three independent peptide ‘binding domains’, each of which is similar to an 11 amino acid consensus sequence; because of its predicted novel secondary structure, this peptide is called the ‘AT-hook’ motif [20]. As shown in Fig. 1, the backbone of a consensus binding domain peptide has a predicted planar crescent shape that is generally similar in appearance to the AT-binding ligands netropsin (distamycin) and Hoechst 33258 [20]. The predicted tlat, crescent-shaped backbone of a synthetic binding domain peptide has recently been confirmed by solution ‘H-NMR studies (J Evans, MS Nissen, R Reeves, unpublished data). The binding domains of the HMG-I proteins are thought to recognize structure, rather than sequence, in the narrow minor groove of AT-rich DNA [20]. It is now becoming accepted that recognition of DNA structure is also a characteristic feature of many other important DNA-binding proteins [ 12.1. The mammalian HMG-I protein is of considerable biological interest because it seems to play multiple roles in the cell, in some cases apparently acting as a chromatin structural protein, and in others as a candidate gene regulatory molecule. The postulated pleiotropic effects of the HMG-I protein seem in many ways to be analogous to those of the yeast RAF-1 (GRFl) protein that functions either as a chromosome structural protein or as a positive or negative regulator of gene transcriptional activity (references in [21]). The HMG-I protein has been immunolocalized in vitlo to the AT-rich G/Q and C bands of mammalian metaphase chromosomes, suggesting that it may play an important role in the changes in chromosome structure that occur during the cell cycle. On the other hand, many in vitro and in vivo correlations suggest that the protein also participates in such nuclear processes as DNA replica-

tion and gene transcriptional regulation (references in introduction of [22*-l ), probably as pan of a complex with other proteins [ 23.1. In addition, HMG-I has recently been correlated with neoplastic transformation of man-malian cells and may also be a biochemical marker for the progressive metastatic potential of rat prostate cancer cells [24]. 1~ zlitro the human and murine HMG-I proteins are ef-

ficient substrates for cdc2 kinase phosphorylation and in rklo the same sites of iu vitro protein modification are phosphotylated in a &ell cycle-dependent fashion [22-•,25,26]. The sites of cdc2 phosphorylation are on threonine residues at the amino-terminal ends of two of the three DNA-binding domains present in the human protein [22 l ,26]. As illustrated by computerderived molecular models (Fig. 2), phosphotylation of the amino terminal threonine of a binding-domain peptide by cdc2 kinase is predicted to significantly decrease the tightness of minor groove binding, because the negative charge of the phosphothreonine is repulsed by the phosphodiester backbones of the DNA substrate, a prediction confirmed by experimentation [ 26). Furthermore, phospholylation by purified cdc2 kinase of intact recom binant human HMG-I at the same two sites that are modified in tirfo reduces the itI !litro DNA-binding attinity of the whole HMG-I protein by about 20.fold at physiological salt concentrations [22-l. As with the unphosphorylated DNA-binding domains of HMG-I, the unphosphoxylated tails of histone Hl proteins also preferentially bind to the minor groove of AT-rich DNA (reviewed in [12*] ). A considerable body of evidence suggests that if either histone Hl [ 271 and/or intact nucleosomes [ 281 bind to gene promoter/enhancer sequences, transcription of the associated gene by RNA polymerase is likely to be repressed. It is thus tempting to speculate that one of the i)z l+lto functions of HMG-I might be to act as an ‘antirepressor’ molecule [ 27,281 that can compete with inhibitov histone Hl and/or nucleosomes for binding to AT-rich gene regulatory sequences and establish an open or accessible chromatin structure in these critical DNA regions. Once such an ‘open’ chromatin structure has been established by HMG-I binding, this configuration would probably be perpetuated from one interphase to the next as both HMG-I and histone Hl change their phosphorylation levels, and hence their relative strengths of AT-DNA binding, in a coordinated manner during the cell cycle [22-l.

Mitotic disassembly/assembly envelope

of the nuclear

The nuclear envelope consists of two distinct membrane layers, perforated by nuclear pore complexes, and lined internally by the lamina, which is in intimate physical contact with chromatin (see the review by Dessev in this issue, pp.430-435). A number of different laboratories have recently demonstrated both in zu’tro and in z~it~o that the transient depolymerization and breakdown of

Chromatin

(a)

(b)

changes

during

the cell cycle

Reeves

NH, + :I

I HA Hc

H,N=C\N~

’

N-CH,

,dH, ‘3-L / H,N+ + -NH,

1. Comparison of the predicted planar crescent-shaped structure of (a) consensus DNA-blnding domain peptide of high mobility group-l proteins with those of the minor groove ATDNA binding ligands (b) netropsin and (cl Hoechst 33258 (reproduced from 1201 with permission).

Fig. N

the nuclear lamina at mitosis is the direct result of phosphorylation by activated cdc2 kinase [29,30]. Nonetheless, it is important not to confuse lamina disassembl) with nuclear envelope breakdown. While lamina depolymerization is almost certain to be required for envelope breakdown, it is not sufficient for such disassembly in all cell types. Other ;ts yet unidentified factors must also be involved in this process. Reassembly of the nuclear envelope at the end of mitosis occurs in several discrete stages and there is evidence of different pathways in different cell types. A significant advance in our understanding of the stages involved has recently been reported by Pfaller et al. [31**]. Using purified nuclear membrane vesicles and cytosolic fractions from Xenopus eggs these workers developed a simple assay for studying the reversible, receptor-mediated binding of membrane vesicles to chromatin. Using

phosphatase and kinase inhibitors they demonstrated that membrane-chromatin association is regulated by a cell cycle-dependent phosphatase/kinase system. In interphase, the balance in the system favors dephosphorylation, possibly of a membrane receptor, which then mediates chromatin binding. At mitosis the putative membrane receptor is phosphorylated, causing release of the membranes from chromatin. Surprisingly, purified MPF (cdc2 kinase plus cyclin B) does not directly cause membranes to dissociate from chromatin. Rather, the reversible binding of membranes to chromatin at mitosis appears to be regulated indirectly by cdc2 kinase via its action on a ‘downstream’ phosphatase/kinase system that subsequently modulates the phosphorylation state of a nuclear membrane component [31m*]. It would not be surprising if additional examples of such indirect regulation by cdc2 kinase are soon discovered.

417

418

Nucleus

ahd

rrene

exDression

Fig. 2. (a) Computer derived model of an unphosphorylated high mobility group (HMC)-I binding-domain peptide (dark color) [201, binding to the narrow minor groove of AT-rich DNA (light color). (b) Computer-derived model of a cdc2 kinase-phosphorylated HMG-I bindlngdomain peptide binding to DNA. Phosphorylation of the amino-terminal end of a binding-domain peptide is predicted to significantly loosen the tightness of minor groove binding because of negative charge repulsion of the modified threonine by the phosphodiester backbones of the DNA substrate, a prediction confirmed by experimentation [261. The computer modelling was performed by LT Holth.

Interphase: chromatin

cycle-dependent compartments

alterations

to

Following mitosis, the extremely compacted chromatin of metaphase chromosomes unfolds and disperses in interphase. The resulting chromatin arrangement in the interphase nucleus is not random, however, and appears to be highly organized in a cell-type specific manner. Topologically independent chromatin domains, attached by specific DNA sequences to a protein-based nuclear matrix core or scaffold, appear to be fundamental to most aspects of interphase nuclear organization (reviewed in [32,33] >. Recent advances in molecular biology, combined with high-resolution in situ hybridization and immunolocalization techniques, have made it possible to precisely visualize the spatial localizations of entire single chromosomes, selected chromosome domains and even individual transcribing genes in interphase nuclei (reviewed in [34]).

The structure and composition of the highly organized compartments of interphase chromatin are, nonetheless, dynamically changing throughout the cell cycle, with some of the alterations relating to DNA replication and others not. For example, it has long been known that the subcellular localization and/or concentrations of many chromatin proteins related to S phase, such as the proliferating cell nuclear antigen (also known as DNA polymerase 6 auxiliary factor) and histones, change markedly during the cell cycle. Advances in ,nultiparameter flow cytometry [35] have allowed precise simultaneous measurements of these changes for several proliferation-associated chromatin proteins during the mammalian cell cycle without having to resort to the use of synchronous cell populations. Recently, Pfeffer and Vidali [36] have identified a novel nuclear antigen (M-2), apparently not directly associated with DNA replication, which is related to cell-cycle-dependent alterations in chromatin structure. Likewise, Earn-

Chromatin

shaw and his colleagues [ 371, using immunofluorescence techniques, have identified a set of related proteins (PIKA proteins) that localize to a large structural compartment in the nucleus of a variety of human cell types. The morphology of this compartment changes dramatically during the cell cycle, but these changes do not appear to be related to DNA replication, heterogeneous nuclear RNA storage, or mRNA processing.

Cell cycle

changes

in chromatin

fine structure

Specific sequences or stretches of DNA are known to exhibit cycle-dependent alterations in interphase chromatin fine structure (as determined by localized alterations in DNase I digestion sensitivity) in connection with changes in replication or gene transcription. For example, in S. ceretu3ae, replication-dependent changes have been observed in the line structure of two different autonomously replicating sequences located near either a histone 1-14 gene or the 77U’l gene [381. These reversible, cell cycledependent changes in chromatin structure are restricted to the autonomously replicating regions and are dependent on DNA replication [ 381.

Inducible structure

changes

in interphase

chromatin

fine

The placement of nucleosomes over critical regions of DNA, such as gene enhancer/promoter elements, is known to inhibit the transcriptional activity of both po& merase II [27] and polymerase III [39] genes in uitro and probably also in z@vo(reviewed in [40] ). The assumption is that nucleosome occlusion of &acting gene regulatory elements prevents their availability for interaction with trans-acting factors necessary for transcription. How activator proteins gain access to their recognition sequences in chromatin is largely unknown. Replication has often been proposed as a mechanism for displacing nucleosomes to allow for factor binding. However, new DNA synthesis is not necessary for the activation of many inducible eukatyotic genes thus implicating tram-a&vating factors in direct nucleosome displacement [41 I. The rapid activation of mouse mammary tumor virus transcription by hormone-bound glucocorticoid receptor, for example, results in disruption of a nucleosome that is specifically positioned on the promoter without the necessity of prior DNA replication [41]. Likewise, in yeast the PH02/PHO4 proteins induce gene transcription by specific nucleosome disruption without a requirement for DNA synthesis (reviewed in [ 421). These examples of direct nucleosome and chromatin disruption by activator proteins may be exceptional situations. In both cases the chromatin changes are mediated by accurately positioned nucleosomes on the cognate promoters, and such positioning may be necessary for exposing the speciIic DNA sequences bound by the activator proteins on either the

changes

during

the cell cycle

Reeves

419

outward-facing surface of a nucleosome or in a linker region between nucleosomes [ 421. It seems doubtful that the precise nucleosome positioning discussed above occurs in all eukaryotic promoter regions, and other mechanisms probably exist for remodeling the interphase chromatin of rapidly induced genes. Indirect support for this idea comes from the recent studies of in vitro GAL4 binding by Kingston’s laboratory [43-l, indicating that this activator protein can interact with its recognition sequence regardless of location in a nucleosome, although this interaction occurs with much lower aIfinity than in free solution. Additional in vitro activator binding/gene transcription studies by this same group, using GAL4 fusion proteins containing activation domains derived from different sources demonstrated that it was the acid activation domains and not the GAL4 DNA-binding domain, of such fusion proteins that stimulate transcription by enhancing the ability of basal transcription factors to compete with nucleosomes for occupancy of a promoter [ 441.

Phosphorylation of replication transcription factors

and

It has been known for some time that the DNA replication functions of the large T antigen of SV40 virus and the replication factor A (RF-A) of yeast and humans are controlled by reversible phospholylations. More recently it has also been found that the single-stranded DNA-binding affinity, and therefore probably the biological activity, of human DNA polymerase OL,a principal chromosome replication enzyme, is likewise cell cycle-controlled by cdc2 kinase phosphorylation [45]. Although the structure of active chromatin will not be discussed here (see the review by Fedor in this issue, pp.4364431, a brief glance at Table 1 shows that the phosphotylation levels of RNA polymerase II and a number of gene transcription factors vary during the ceU cycle and that such phosphorylations are postulated to affect the in zkjo biological activity of these proteins. For example, detailed analyses of mouse RNA polymerase II has shown that active polymerases are phosphotylated in villa on multiple sites in the repetitive carboxyl-terminal domain of the largest subunit of the enzyme by an endogenous cdc2-like activity and that the phosphorylation level of this subunit varies during the cycle [46]. Further analysis indicated that cdc2 kinase phosphorylation causes a conformational change in the carboxyl-terminal domain of the largest polymerase subunit in vitro, suggesting that such structural changes might modulate the activity of the enzyme in tklo [47]. Earlier studies indicated that several oncogene products and/or transcription factors, such as c-Fos, c-h&b and cAbl, are phosphorylated in a ceU cycle-regulated manner. As noted in Table 1, the in vitro DNA-binding affinities of several additional transcription factors, including c-Jun [48] and Ott-1 [49], are now also known to be specifitally reduced or inhibited by phosphorylation. Modulation of transcription factor DNA-binding alfinity by phos-

.

420

Nucleus

and nene exoression

Cyclic phosphorylation of the tumor suppressor proteins pRB and ~53 by cdc2 kinase is known to markedly affect their affinity of interaction with other chromatin proteins and is thereby postulated to alter their biological functions (reviewed in [ 501). The pRE3anti-oncogene protein, which regulates progression through the G,/S phase of the cell cycle [51], is phosphoxylated on multiple sites by cdc2 kinase from S to M phases and is then dephosphorylated in G, [ 521. The unphosphorylated RE3protein in G1 cells is tightly associated with chromatin whereas the G/S phosphorylation of pRB markedly reduces this binding affinity [ 53,541. A number of chromatin proteins have been identified that specifically interact with the unphospholylated, but not the phosphorylated, forms of RB including several gene transcription factors [55,56], cyclin A [ 551 and the c-Myc protein [57]. These observations have led to the hypothesis that the unphosphorylated RI3 protein suppresses cell proliferation by tightly binding critical growth-promoting regulatory proteins and that phosphotylation of RB at the G,/S transition inactivates this RB function and allows cells to enter S phase. This idea has received strong support from experiments in which various phosphorylated forms of the RI3 protein were microinjected into synchronized human Saos-2 osteosarcoma cells [ 511 and the phosphorylated form of the protein was correlated with the entrance of cells into S phase.

gen are fused to the amino terminus of Escheri&a coli P-galactosidase into Vero or hepatoma cells, Rihs and his colleagues [60] demonstrated that efficient nuclear import of the fusion protein is dependent on both the presence of the T-antigen NE and a protein casein kinase II (CK 11) phosphorylation site (T antigen Serll l/SerllZ) located at some distance from the NIS. While the NL5 determined the specificity of nuclear import, the adjacent CK II site, which is phosphorylated in uiuo, was necessary for the maximal rate of nuclear transport of the fusion protein [60]. In marked contrast, phosphorylation of another nearby T-antigen residue, Thr124, by purified cdc2 kinase, markedly reduced the maximal extent of nuclear accumulation of the fusion proteins while negligibly affecting their rate of import [6I-*I. A search of the literature showed that a number of other nuclear proteins contain potential and confirmed CKII and cdc2 kinase phospho@ation sites located near NLS sequences. Based on these observations, Jans et al. [6I**] made the provocative suggestion that these three elements combined (CK II and cdc2 sites located near a NIS) represent a functional unit, under the dual control of CKII and cdc2 enzymes, that regulates the rate and extent of nuclear protein import in eukaryotes. In this regard, the recent elegant immunofluorescence staining studies of Pines and Hunter [ 62.1, which showed that cyclins A and Bl are differentially located in the nucleus and cytoplasm of HeLa cells depending on the cell cycle phase, raise the intriguing question of whether these remarkable nucleocytoplasmic redistributions of the cyclin proteins are likewise modulated by differential protein phosphorylations.

Nuclear

Caveat

phorylation suggests a general mechanism for rapidly and reversibly controlling gene expression in rkjo, particularly for those genes whose activity is regulated by signal transduction pathways involving phosphorylation cascades.

import+!xport

of phosphoproteins

Transport of certain chromatin proteins into and out of the nucleus is known to be reversibly controlled by phosphorylation (Table 1) and appears to be an efficient common way to regulate gene expression or DNA replication (reviewed in [ 581 and by Davis, this issue, pp.424-429). A well characterized example is SW15, a cell cycle-specific transcription factor for the HO endonuclease gene in the yeast S. cer-ellisiue. This protein is expressed in the S, G2 and M phases, during which time it remains cytoplasmic, and only enters the nucleus during G1, when it acts on the HO gene. SW15 is phosphol)llated when in the cytoplasm and dephosphorylated when it is in the nucleus. Recent studies by Moll et al. [ 59.1 have conclusively demonstrated that the cell cycle redistribution of SW15 is dependent on the phosphorylation by CDC28 kinase (the .S.cerezvkiae homolog of cdc2 kinase) of serine residues located within or close to the nuclear localization signal (NE) of the protein. The mechanisms regulating viral SV40 T antigen entry into the nucleus illustrate particularly well the complex ways in which phospholylations can regulate nuclear import of chromatin proteins (Table 1). By microinjecting recombinant proteins in which short fragments of T-anti-

emptor

Reversible phosphorylation of nuclear proteins has long been suspected to be one of the major processes modulating chromatin structure during the cell cycle. Recent work, some of which is highlighted here, emphasizes and reinforces this idea. This view, however, is probably an oversimplification of the true in tdzjo mechanics regulating chromatin srructure. Indeed, using evidence from in ~dtro experiments, Burgoyne and colleagues [63] have suggested the converse, namely that previous changes in chromatin substrate conformation are the most important factors controlling the types of protein phosphorylations occurring in the mammalian nucleus.

Acknowledgements This work was supported in part by grants from the National Science Foundation (DCH-8904408) and the National Institutes of Health (ROlGM+6352). Special thanks are owed to lx~rel Thorlacius Holth for the computer models of the HMG-I peptides binding to DNA I would also like to thank AMichael Friedman, Debra Hoover, Nancy Magnuson. Mark Nisscn, and members of the Reeves lahordtory for ideas, comments and critical reading of the manuscript.

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