seminars in CELL & DEVELOPMENTAL BIOLOGY, Vol 10, 1999: pp. 205]214Article No. scdb.1999.0302, available online at http:rrwww.idealibrary.com on

MAP kinase-mediated signalling to nucleosomes and

immediate-early gene inductio

Stuart Thomson, Louis C. MahadevanU and

Extracellular signals are transduced into the nucleus througha variety of signalling mechanisms to elicit changes inpatterns of gene expression. This review is focused on theMAP kinase cascades and the part they play in the induction

( )of immediate-early IE genes. We discuss the MAP kinasesand their downstream effectors that are known tophosphorylate substrates in the nucleus. In addition tophosphorylating specific transcription factors, MAP kinasesand their downstream kinases are implicated in elicitingrapidly targeted alterations in the chromatin environment ofspecific genes by modulating the phosphorylation androracetylation of nucleosomal and chromatin proteins.

Key words: acetylation r IE gene induction r MAP kinasesr nucleosomes r phosphorylation

Q1999 Academic Press

Introduction

MECHANISMS RESPONSIBLE FOR programming and reg-ulating gene expression in response to extracellularstimuli have been a focus of major research effortsfor many years. It is now apparent that this is achievedby a variety of different signal transduction mecha-nisms which have the net result of modifying andregulating the transcriptional machinery and chro-matin environment at particular target genes. Forexample, steroid hormones, such as oestrogen and

thyroid hormone, bind to specific intracellular recep-tors causing them to dimerise. Acting through speci-fic DNA regulatory elements, this complex initiates aspecific pattern of gene expression through recruit-

From the Nuclear Signalling Laboratory, The Randall Institute,King’s College London, 26]29 Drury Lane, London WC2B 5RL,UK. UCorresponding author.

Q1999 Academic Press1084-9521r99r020205q10 $30.00r0

205

n

Alison L. Clayton

ment of transcriptional co-activators.1 Certain cy-tokines, such as TNF-a , bind to cell surface receptors

Žand activate the transcription factor NF-k B nuclear.factor kappa-B , through a pathway that involves

phosphorylation of the inhibitor protein Ik-B. 2 Inter-ferons and cytokines bind to cell surface receptorsthat interact with JAK kinases, which in turn phos-phorylate and activate STAT transcription factors thattranslocate to the nucleus to elicit a transcriptionalresponse.3 One of the most extensively studied mech-anisms of signalling into the nucleus involves theMAP kinase cascades. These are activated by diversestimuli that include growth factors, cytokines andstress stimuli as well as many pharmacological sig-nalling agonists.4 This review is focused on the MAPkinase cascades and the part they play in the induc-

Ž .tion of immediate-early IE genes. In addition tophosphorylating specific transcription factors, MAPkinases and their downstream kinases are also impli-cated in eliciting targeted alterations in the chro-matin environment encompassing specific genes, bothdirectly, by phosphorylation of nucleosomal andchromatin proteins, and indirectly, by allowing therecruitment of HATs to specific phosphoepitopes ontranscription factors. In what follows, we first discussIE genes, the upstream regulatory elements and tran-scription factors that control these genes and thenucleosomal response that is thus far indissociablefrom IE gene induction. The three MAP kinase cas-cades are described in brief. However, to keep thefocus of this review on nuclear events, we concentrateon the downstream players in these cascades i.e. theMAP kinases themselves and their downstream effec-tors that are known to phosphorylate substrates inthe nucleus. Finally, we discuss in overview how MAPkinase cascades may be targeted to produce activa-

tion of a small subset of genes, firstly by causingphosphorylation of specific transcription factors andsecondly, by modifying the phosphorylation androracetylation of histones, thereby modulating the chro-matin milieu of these genes.

S. Thomson et al

The immediate–early genes

This term refers to the set of genes linked directly toreceptors by transduction mechanisms and includethose that are expressed when a cell is stimulated toleave the G phase of the cell cycle and enter G .0 1The expression of these genes requires no proteinsynthesis and is dependent solely on modification offactors already present within the cell, making it agood system for studying the interface between signaltransduction in the cytoplasm and gene induction inthe nucleus. The genes that are expressed, whichinclude members of the c-fos and c-jun families, are

characterised by their rapid and transient expressionin response to extracellular stimuli. In addition,phosphorylation of a number of transcription factorsand two chromatin-associated proteins, HMG-14 andhistone H3, is tightly correlated to IE gene

Figure 1. Schematic summary of the signalling neŽ .see refs 4,10 for more detail . Each MAP kinasdiffering shades of grey in the background. The uc-Raf for the ERK pathway. Upon stimulation withactivation by TPA is less well-understood. Raf pspecificity kinases in the middle tier and these thactivation of JNKrSAPK and p38 is achieved by du

.called JNKKs, RKK, etc. in the middle tier and kinNote that the upstream events associated with thmany candidate effectors have been proposedactivates these two cascades weakly, whereas agentand heat stress as well as the compound anisomextremely strongly. The three MAP kinase subtsubstrates on serine and threonine residues with

Žresidue is followed by a proline examples of suspecific inhibitors of the MAP kinases mentioned

206

induction.5 ] 8 Three broad classes of stimuli elicit thisŽ .type of cellular response: i polypeptide growth fac-

Ž .tors and cytokines such as EGF, FGF, PDGF, etc.; iiŽ .stresses such as UV radiation or heat shock; and iii

pharmacological compounds such as TPA, okadaicacid and anisomycin. Despite their diverse nature, itis a striking observation that all these stimuli arecapable of activating MAP kinase pathways, althoughthe precise extent and latency of activation of each ofthe MAP kinase pathways is distinctive for each stimu-lus.

MAP kinase pathways

At present there are at least three MAP kinase path-ways which are defined according to the MAP kinase

Ž . Ž .that is activated: i the ERK pathway; ii the

twork leading to activation of the MAP kinasese module consists of three tiers depicted withppermost tier is represented by kinases such asEGF, Raf is activated by binding to Ras-GTP; itshosphorylates MEK1 and 2, which are dual-en phosphorylate ERKs in the third tier. The

Žal-specificity kinases called MKKs or SKKs alsoases such as MEKKs 1]4 in the uppermost tier.

ese two cascades are still quite controversial asŽ .see Potential Upstream Activators box . EGFs such as UV radiation, hyperosmotic, chemicalycin activate these two MAP kinase subtypes

ypes can then phosphorylate a wide range ofin a consensus site where the phosphorylated

.bstrates shown in Figure 2 . The two pathway-in the text are also shown.

Ž .JNKrSAPK pathway; and iii the p38 MAP kinaseŽ . 4,9pathway Figure 1 . Mitogenic stimuli, such as EGF,

activate ERKs very rapidly and strongly, and elicitweaker activation of JNKrSAPKs and p38, whereasvarious stress stimuli and anisomycin activate theJNKrSAPK and p38 MAP kinase pathways verystrongly but produce little or no ERK activation. Incontrast, TPA elicits activation mainly or only of theERKs; it is noted that there is some cell-type specific-ity to these patterns of diffferential MAP kinase acti-vation. Nevertheless, the fact that individual stimuliactivate these pathways to precisely controlled andcharacteristic extents could potentially provide anexplanation for the quantitatively regulated transcrip-tion of IE genes seen in response to each stimulus.Quantitative control of IE gene induction is poorlyunderstood at present.

The signalling network leading to activation of theMAP kinases is reviewed extensively elsewhere4,10 andis summarised in Figure 1. Each MAP kinase moduleconsists of three layers. The upstream-most activatingenzymes are kinases such as c-Raf, activated by bind-ing to Ras-GTP, or MEKKs1-4 and others, the mecha-

Žnism of activation of which remain contentious see. ŽFigure 1 for proposed activators ref 10 and refs

.therein . When active, these phosphorylate and acti-vate dual-specificity kinases, such as MEK1 and 2 and

Ž .MKKs3]6 also called SEKs or SKKs . The dual-specificity kinase then phosphorylates the MAP ki-nase itself on threonine and tyrosine residues withinthe motif T-X-Y where X is E for ERKs, P for

ŽJNKrSAPKs and G for p38 MAP kinase reviewed in.ref 4 . MAP kinases phosphorylate a wide range of

substrates on serine or threonine residues within aconsensus site where the phosphorylated residue is

Ž .followed by a proline SrT-P . There is increasingevidence that these kinases are not freely diffusiblewithin the cell but are maintained in a kinase moduleby association with a scaffold protein,11,12 which maybe important for restricting the specificity of sig-nalling.

With the characterisation of these kinases and theirmodes of regulation now well advanced, a majorchallenge is to identify physiologically relevant MAPkinase substrates and to be able to relate their phos-phorylation to the activation of transcription. Thishas been greatly aided by the use of recently identi-

fied inhibitors specific for particular MAP kinase

Ž .pathways reviewed in ref 9 . The ERK pathway isspecifically inhibited by the flavone compoundPD098059,13 which inhibits the activation of MEK1,

Ž .the upstream activator of ERKs see Figure 1 . Re-

20

MAP kinase-mediated signalling to nucleosomes

cently a second MEK inhibitor has been describedwhich inhibits both its activation and activity.14 Thep38 MAP kinase can itself be specifically inhibited by

Ž 15.SB203580 Figure 1, . Studies with these inhibitorsshow that although they are crucial to IE gene induc-tion, alternative MAP kinase pathways may be utiliseddepending on the stimulus used8,16 and further rein-forces the strong correlation between IE gene induc-

Ž8tion and the nucleosomal response , Thomson et al,.unpublished . The main MAP kinase substrates po-

tentially involved in nuclear events can be separatedŽ .into two classes: i transcription factors andror chro-

Ž .matin proteins; and ii downstream SerrThr effectorkinases.

Upstream regulatory elements andtranscription factors controlling the IE genes

Extensive research has built up a clear, although farfrom complete, picture of the upstream regulatoryelements and transcription factors involved in IEgene induction. The recurring theme is one of MAPkinase-mediated transcription factor phosphoryla-

Žtion, co-activator recruitment e.g. p300rCBP,.prCAF and initiation of the transcriptional response

at specific genes.17,18 A comprehensive description ofidentified promoter elements, and transcription fac-tors for all IE genes is beyond the scope of thisarticle. As examples, the regulatory elements andtranscription factors that control the expression of

Žc-fos and c-jun are described in brief, for reviews see.refs 19,18 and references therein .

c-fos

The c-fos promoter consists of three main elements,Ž .the serum response element SRE , the Sis-inducible

Ž .element SIE , and the cAMP response elementŽ . 20CRE . The SRE is constitutively occupied by a

Ž .dimer of the serum response factor SRF proteinŽ .and the ternary complex factor TCF , whose mem-

bers include Elk-1, SAP-1 or SAP-2.21 Similarly theCRE is constitutively occupied by CRE binding pro-

Ž .tein CREB . In contrast, the SIE only becomes occu-pied after growth factor stimulation.

These factors are differentially phosphorylated and

this may explain the quantitative differences observedin c-fos gene expression in response to differentstimuli.22,18 The TCFs are phosphorylated on pro-line-directed sites at their C-terminus by the ERKs inresponse to mitogenic stimuli.18,23 However, TCF can

7

S. Thomson et al

also be phosphorylated in response to stress-relatedstimuli, mediated by p38 MAP kinase.24 Indeedstress-induced c-fos expression is severely inhibited bythe p38 MAP kinase inhibitor SB203580.8 In contrastSRF activity is regulated by an as yet poorly definedpathway involving the Rho family GTPases.25,26 CREBphosphorylation occurs at Ser133 in response to mi-togens, stress and stimuli that elevate cAMP-level.27,28,77 Interestingly, this is not a proline di-rected site, suggesting that it is not a direct target forMAP kinases. The identity of the CREB kinase acti-vated in response to mitogenic or stress-related sti-

Ž .muli remains somewhat contentious see later . Re-cently CREB phosphorylation has been identified as acritical event in EGF-, but not serum-induced c-fosexpression.29

c-jun

The c-jun promoter is more complicated than that ofc-fos , containing at least five different regulatory ele-ments. There are two AP-1 sites, an NF-jun site, twoSp-1 sites, a CCAAT box and a related-to-serum

Ž .response factor RSRF or MEF-2 binding site site, allconstitutively occupied by their respective binding

Ž .proteins refs 30,31 and references therein . Themost extensively studied of these regulatory elementsare the AP-1 sites. These are occupied by the AP-1transcription factor complex, which itself is a homoor heterodimer composed of members of the JunrFosand ATF families of transcription factors.20 In re-sponse to mitogenic andror stress stimuli, these pro-teins are phosphorylated on proline-directed sites,suggesting direct phosphorylation by MAP kinases.Indeed, c-Jun is a known in vivo substrate ofJNKrSAPK1, being phosphorylated on Ser 63 and73.32 It has also been shown that the MEF-2C tran-scription factor, which is phosphorylated by p38 MAPkinase, may be involved in controlling the c-jun genevia the RSRF site.

In addition to activating transcription factors di-rectly by phosphorylating them, MAP kinases mayalso play a role in modifying the chromatin environ-ment of the targeted genes. This is achieved bothindirectly via recruitment of Histone Acetyltrans-

Ž .ferase HAT complexes to phosphorylated transcrip-

Ž .tion factors discussed later , or by causing phospho-

rylation, mediated by kinases downstream of the MAPkinases, of the nucleosomal proteins histone H3 andHMG-14. These latter events are referred to here asthe nucleosomal response.

208

The nucleosomal response

There is considerable evidence that IE gene induc-tion is associated with alterations in nucleosome andchromatin structure. For example, within 5 min ofserum stimulation the c-fos gene exhibits increasedsensitivity to DNase1.33,34 It has also been shown thatc-fos nucleosomes undergo a defined and rapid con-formational change concomitant with induction35 ex-posing a previously buried cysteine residue in histoneH3.36 The precise mechanisms involved in switchingchromatin from inactive to active conformations arestill not fully understood. However, one possiblemechanism involves the post-translational modifica-tion of chromatin-associated proteins. Indeed histoneH4 hyperacetylation has been linked to growth fac-tor- and stress-induced activation of SRF-regulatedreporter genes.26 In addition, phosphorylation of nu-cleosomal core histone H3 and the nucleosome-bound high-mobility group protein HMG-14 is thusfar indissociable from IE gene induction.5,6,8,37,38

Histone H3

Phosphorylation of histone H3 occurs on Ser 105 inthe highly conserved amino-terminal tail, a domainthat is also subject to reversible acetylation at lysines9, 14, 18 and 23. The phosphorylation of H3 at theG rG transition is restricted to a minute fraction of0 1

H3 which is also highly susceptible to hyperacetyla-tion,6 suggesting that there is a high level ofbiochemical compartmentalisation within chromatin,and that the enzymes mediating the mitogen-stimu-lated phosphorylation and hyperacetylation of his-tone H3 are targeted to the same subset of nucleo-somes. In contrast, histone H3 on condensed chro-mosomes is very highly phosphorylated on Ser 10during mitosis.39 ] 41 It seems hard to reconcile thefact that the same modification is involved in appar-ently opposite processes, i.e. chromosome condensa-tion and gene transcription. It has been proposedthat H3 phosphorylation might facilitate the access oftrans-acting factors to their target DNA sequences bycausing the decondensation or relaxation of limitedregions of chromatin. These trans-acting factors couldbe chromosome condensation factors or transcrip-

tion factors.41

HMG-14

HMG-14 is rapidly phosphorylated on serine 6 by a

nsSodninHIblasfrsemrtpg

mea8rrMpd

T

K

M

M

M

P

M

M

R

is

th

ucleosomal kinase that is activated in response totimulation with diverse IE gene-inducing agents.7

er 6 is adjacent to the nucleosomal binding domainf HMG-14 42 and its phosphorylation weakens, butoes not abolish the binding of HMG-14 to isolateducleosomes in vitro.7,43 There are two specific bind-g sites for HMG-14, or the closely related proteinMG-17, within the last 30 bp of nucleosomal DNA.44

t is noteworthy that although HMG-17 has previouslyeen regarded as interchangeable with HMG-14 44 itcks serine 6 and is not phosphorylated in circum-

tances studied so far.7 This implies distinguishableunctions for these proteins, in which context it iselevant that they are reported to bind to nucleo-omes as mutually exclusive homodimers.45,46 Thexact cellular functions of these proteins and theirode of action are still not fully understood, but

esults from many experiments are consistent withhese proteins acting as architectural proteins47 and aotential function in mediating transcriptional elon-ation has been suggested.48

Although no definitive function for the nucleoso-al response has yet been described, it has been

stablished through the use of inhibitors describedŽbove that MAP kinases play a crucial causal role ref

. Ž ., Thomson et al, unpublished . The exact kinase sesponsible are unknown, but as the sites of phospho-

ylation are not proline-directed it is certainly not theAP kinases themselves that phosphorylate these

roteins. It follows that one or more of the kinasesownstream of the MAP kinases must be involved.

able 1. Properties and potential substrates of known effecto

inase MW‡ Upstream KinaseŽ .kDa activating domains

kinases

APKAP-K1 90 ERK DoubleŽ .RSK

APKAP-K2 50 p38 Single

APKAP-K3 43 p38 Single

RAK 54 p38 Single

NK 47 ERK, p38 Single

SK 90 ERK, p38 Double

SK-B 70 ERK, p38 DoubleUCellular localisation refers to the distribution of the kinasetranslocation after activation. See text for details.†Nuclear localisation of these kinases has only been determie localisation of the endogenous kinase is available.‡For simplicity, the MW of the human kinase is indicated. T

20

MAP kinase-mediated signalling to nucleosomes

Effector kinases downstream of the MAPkinases

MAP kinase pathways mediate the phosphorylation ofCREB, HMG-14 and H3 but these are not directsubstrates for MAP kinases. This suggests that kinasesdownstream of the MAP kinases are responsible forphosphorylating these, and possibly other, nuclearsubstrates. Kinases which are known downstream ef-fectors of the MAP kinases are shown schematicallyin Figure 2 and a summary of their properties andpotential substrates are listed in Table 1. These ki-nases are activated by either the ERK or the p38 MAPkinase pathways or, in the case of the more recentlydiscovered MNK and MSK kinases, by both pathways.There are no known effector kinases for theJNKrSAPK pathway at the present time.

The first effector kinase to be identified was MAP-Ž .KAP-K1 MAP kinase activated protein kinase , al-

ternatively known as RSK or pp90rsk.49 To date threeisoforms have been identified and termed MAPKAP-K1a, b and c, or RSK1, 2 and 3 and all can beactivated by ERK in vitro. It was subsequently shownthat they are also activated in vivo by agents that onlyactivate the ERK pathway and that this activation was

50

inhibited by PD098059. Two kinases closely relatedto MAPKAP-K1 have been identified and termedMAPKAP-K2 51 and MAPKAP-K352, respectively.These kinases are only activated by stimuli which

r kinases of the MAP kinase pathways

Cellular Suggested ReferencesUlocalisation substrates

wCytoplasm CREB 29,49,53,56,62,x63,64

w xNuclear CREB, HSP27 50,51,54,55

w x? HSP27 52

w x? HSP27 57

w x? eIF4e 59,60

w xNuclear† CREB 28

w xNuclear† CREB 61

in an inactive state. These kinases differ as to whether there

ned when the kinase is transiently overexpressed. No data on

here are differences in MV between species homologues.

9

S. Thomson et al

Figure 2. Schematic representation of substrates of the ERK, JNKrSAPK and p38 MAP kinasesubtypes. Immediate substrates phosphorylated on proline-directed sites fall into two classes,

Ž .transcription factors Ternary complex factors: Elk, SAP1, SAP2; c-Jun; ATF-2; MEF-2C, etc. andŽdownstream effector SerrThr kinases MAPKAP K1-3; MNK1,2; MSK1,2; PRAK; shown in greater

.detail in Table 1 . These latter kinases can then phosphorylate other transcription factors such asCREB or nucleosomal proteins such as HMG-14 and histone H3 on sites which are not

Ž .proline-directed phosphorylation sites indicated by broken arrows in the figure . It is importantto note that there are no downstream kinases presently reported for the JNKrSAPKs; they are

best characterised as kinases for transcription facand effector kinases which can be accessed by m

Ž .in white. For technical reasons see text , some oand remain to be unequivocally proven.

activate the p38 MAP kinase pathway, and this activa-tion is inhibited by SB203580.8,52

A mutation in the kinase domain of MAPKAP-K1bŽ .RSK2 has recently been linked to a human disease

Ž . 53called Coffin]Lowry syndrome CLS . Studies witha fibroblast cell line derived from a CLS patient haveindicated that of the three isoforms, only MAPKAP-

Ž .K1b RSK2 is inactive, and that the cells are defectivein CREB phosphorylation and c-fos gene expressioninduced by EGF, but not by serum or UV radiation.29

On this basis, these authors propose that CREB mayŽ .be a physiological substrate for MAPKAP-K1b RSK2 ,

at least in response to EGF and that this event isŽcrucial for regulating c-fos gene expression see up-

.date at end .Both MAPKAP-K2 and K3 have been shown to

phosphorylate HSP25r27 in vivo and in addition,

MAPKAP-K2 was suggested to be the CREB kinaseactivated in response to FGF and stress in SK-N-MCcells.50 Recently another function for MAPKAP-K2has been suggested based on the observation that

21

tors. Transcription factors, nucleosomal proteinsore than one MAP kinase pathway are indicatedf the relationships indicated are still contentious

inactive MAPKAP-K2 is localised within the nucleusand that it is translocated to the cytoplasm uponactivation.54,55 In addition, MAPKAP-K2 was shown tocomplex with inactive p38 MAP kinase within thenucleus and proposed to mediate its rapid transloca-tion to the cytoplasm after activation.54 It is impor-tant to note that this mode of regulation is entirelydifferent from that envisaged for the ERK pathway,whereby ERK and its effector MAPKAP-K1rRSK areactivated in the cytoplasm and then rapidly translo-cate to the nucleus.56

Another kinase activated solely by the p38 MAPkinase pathway was cloned from human cells by ho-mology to the MAPKAP kinases57 and termed p38-

Ž .regulated activated kinase PRAK . A mouse homo-logue, termed MAPKAP-K5, has also been cloned.58

This kinase has also been shown to phosphorylate

HSP27 in vitro and in vivo on functionally relevantsites;57 no other substrates have yet been identified.Although highly related and sharing at least one invivo substrate, more information will be required as

0

precipitation experiments using antibodies specific

to cellular localisation and substrate specificity ofMAPKAP-K1, -K2 and -K3 and PRAK before theirroles in cellular function are fully identified.

Recently, two related but distinct kinases have beenidentified which are activated in vivo by both theERK and p38 MAP kinase pathways. These kinasesare therefore able to integrate both mitogenic andstress-related signals into a common response. Assuch, they have been referred to as MAP kinase

Ž . 59,60signal-integrating kinase MNK , and mitogen-Ž . 28and stress-activated protein kinase MSK . In addi-

tion another kinase, termed RSK-B, has been identi-fied which also appears to be regulated by both MAPkinase pathways.61

MNK1 was identified independently by two groupsusing either a novel in situ phosphorylation screen ofa HeLa cell cDNA expression library,59 or a moreclassical yeast two hybrid screen.60 The second groupalso identified an additional isoform, MNK2. MNKwas shown to phosphorylate eukaryotic initiation fac-

Ž .tor 4e eIF 4e on a functionally relevant site in vitro,suggesting a role in the regulation of translation.60

Ž .28The MSK kinases MSK1 and MSK2 were identi-fied through homology-searching using the N-termi-nal domain of MAPKAP-K1. They are activated byboth the ERK and p38 MAP kinase pathways. LikeMAPKAP-K1,62 these kinases contain two kinase do-mains, suggesting a novel form of regulation.63,64 Inaddition MSK was shown to be a nuclear kinase, andto remain nuclear after activation.28 This identifies itas a good candidate kinase for the phosphorylationof nuclear substrates and indeed, this study providedevidence that it was the main CREB kinase activatedin response to various stimuli in a number of differ-

Ž .ent cell lines see update at end .The recently identified RSK-B kinase also appears

to be regulated by both the ERK and p38 MAP kinasepathways.61 However, present information regardingthe activation of this kinase relies heavily on transientoverexpression of MAP kinase activators and awaitsverification in vivo.

Although no definitive phosphorylation consensussite has been established for these kinases, it wouldappear that they share common substrates, at least in

Ž .vitro, e.g. HSP25rHSP27 and CREB Table 1 . Al-though limited information is available, it has been

suggested that they require basic residues at positions-2 andror -3 relative to the phosphorylated Ser.28

Note that both H3 and HMG-14 are phosphorylatedat sites encompassing such a consensus.

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MAP kinase-mediated signalling to nucleosomes

Targeted nucleosome modifications and IEgene induction

The MAP kinase-mediated mechanisms describedprovide a means for sending signals to IE genesprimed for rapid response via phosphorylation ofpre-associated sequence-specific transcription factors.Recent research indicates possible mechanistic linksbetween these signal-dependent phosphorylationevents and previously observed changes in chromatinstructure that correlate with IE gene activation. Forexample, it is reported that phosphorylation of cer-tain transcription factors, such as c-Jun, allows re-cruitment of the CBPrp300 co-activator, a proteinthat not only provides a link to the basal transcriptionmachinery 65 but is itself a histone acetyl-transferase.66,67 CBP interacts with a variety of tran-scription factors including CREB, c-Jun, c-Fos and

Ž .SAP-1 see ref 68 for a review . CBP itself can recruitfurther HAT activity by its association with prCAF.The MAP kinase-dependent recruitment of co-activa-tors with HAT activity to sequence-specific regulatoryelements provides a mechanism for localised alter-ations in histone acetylation levels which can con-tribute to targeted changes in nucleosome structure,raising the possibility that targeted histone acetyla-tion is involved in IE gene induction.

Immunoprecipitation experiments using anti-bodies that recognise highly acetylated forms of allthe core histones show that there is a general level ofacetylation associated with active genes.69 ] 71 Additio-nally, immunofluorescence studies using site-specificacetyl H4 antibodies show that particular acetylatedisoforms are differentially distributed in chromo-somes, indicating that acetylation of particular lysinesmay have distinct functions.72,73 In a pioneering study,Allfrey and colleagues showed that c-fos nucleosomeswould bind to organomercurial columns via an ex-posed histone H3 cysteine residue only when thegene was active, and that these nucleosomes con-tained hyperacetylated H3 and H4.36,35 Furthermore,column-bound nucleosomes exhibited higher rates ofacetate turnover than unbound nucleosomes.74 How-ever, these experiments did not locate the modifica-tion to specific regions of the c-fos gene. By immuno-

for tri- and tetra-acetyl H4, hyperacetylation of his-tone H4 has recently been implicated in the signal-dependent activation of the SRE in the c-fos pro-moter.26 Activation of the chromosomal c-fos gene, or

S. Thomson et al

a stably-integrated chromatinised SRF-controlled re-porter gene, were shown to require signals that causea rapid increase in the hyperacetylation of H4 atreporter gene chromatin.26 In contrast to these re-sults, O’Neill and Turner showed that immunopre-cipitation experiments using a mixture of site-specificacetyl H4 antibodies did not detect an increase in theacetylation of c-fos chromatin from HL-60 cells afterTPA treatment.75 The reasons for these differencesare unclear, but could be attributed to differences inthe antibodies used, regions of the gene analysed and

Žthe sensitivity of the detection methods PCR analysis.compared with slot blot hybridisation analysis . Nev-

ertheless, it is important to note first that all currentevidence points to targeted histone acetylation beingclearly achievable and strongly implicated in geneinduction, and second that the hyperacetylation andphosphorylation of histone H3 have been shown tobe highly targeted to the same small subset of H3molecules. Given that the fraction of H3 that isphosphorylated is also extremely sensitive to hyper-acetylation,6 it is conceivable that both these modifi-cations, being elicited via MAP kinase cascades, arelinked and may be crucial for IE gene induction.6

In the experiments of Allfrey and colleagues hy-peracetylation per se was found not to be sufficientfor retention of nucleosomes on organomercurialcolumns, indicating that other structural transitionsof the nucleosome were required. HMG-14 binds tothe nucleosome close to the globular domain of

76 Žhistone H3 and its reversible phosphorylation and.possibly that of H3 may be involved in the transient

conformational change that allows binding to thesecolumns via the H3 cysteine sulphydryl group. Invivo, these structural transitions may reduce the con-straints on transcription imposed by the nucleosome,although this is difficult to prove. The precise molec-ular function of H3 and HMG-14 phosphorylation inIE gene induction is still far from clear. Identificationof the specific kinases involved, their mode of regula-tion and identification by CHIP assays of specificgene or promoter DNA sequences associated withmodified nucleosomes should provide further in-sights.

Update on ligand-stimulated histone H3

kinases

Recent work from the laboratories of David Allis andŽ .Paolo Sassone-Corsi suggest that MAPKAP-K1b Rsk2

may represent the ligand-stimulated histone H3 ki-

212

Žnase described in meeting reports: CSH Symposiumin Quantitative Biology 1999, vol. 63, pp. 469]481;

.Cell 1999, vol. 96, pp. 759]767 . This is based both onin vitro kinase assays as well as on the observation thatCoffin-Lowry fibroblasts, defective in Rsk2, are alsodefective for inducible histone H3 phosphorylation;mitotic H3 phosphorylation on condensed chromo-somes is unaffected. This would form a potentialroute by which ERK activation could result in H3phosphorylation because Rsks lie immediately down-stream of ERKs. However, it does not accomodate theobservation that histone H3 is also phosphorylatedvia a p38-dependent pathway; Rsks are not activatedby p38. Work in our laboratory identifies MSK1 as anextremely efficient kinase for both HMG-14 and his-

Žtone H3 in vitro S.T., A.L.C., Catherine A. Hazzalin,Sally Rose, Michael J Barratt and L.C.M.; submitted

.for publication In direct comparisons, this kinase ismuch more efficient that Rsk1 or Rsk2 as an H3 andHMG-14 kinase. This kinase lies immediately down-stream of both ERK and p38 MAP kinases and fulfilsthe requirement that the histone H3 kinase must beaccessible via both pathways. Furthermore, experi-ments with inhibitors on intact cells suggest thatMSK1 is a strong candidate for the H3 kinase in vivo.Thus, there is emerging evidence supporting rolesfor both Rsk2 and MSK1 as kinases potentially medi-ating the nucleosomal response.

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