coregulators and chromatin remodeling in transcriptional control

Coregulators and Chromatin Remodelingin Transcriptional Control

Rakesh Kumar,1,2* Rui-An Wang,1 and Christopher J. Barnes1

1Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas2Department of Biochemistry and Molecular and Biology, The University of Texas M. D. Anderson Cancer Center,Houston, Texas

Despite many years of investigation by numerous investigators, transcriptional regulatory control remains an

intensely investigated and continuously evolving field of research. Transcriptional regulation is dependent not only ontranscription factor activation and chromatin remodeling, but also on a host of transcription factor coregulators-coactivators and corepressors. In addition to transcription factor activation and chromatin changes, there is an

expanding array of additional modifications that titrate transcriptional regulation for the specific conditions of aparticular cell type, organ system, and developmental stage, and such events are likely to be greatly influenced byupstream signaling cascades. Here, we will briefly review the highlights and perspectives of chromatin remodeling andtranscription controls as affected by cofactor availability, cellular energy state, relative ratios of reducing equivalents,

and upstream signaling. We also present the C-terminal binding protein (CtBP) as a novel nuclear receptor (NR)coregulator, which exemplifies the integration of a number of transcriptional regulatory controls. � 2004 Wiley-Liss, Inc.

Key words: signaling; coregulators; transcription

INTRODUCTION

Transcriptional regulatory control is an intenselyinvestigated field of research that is constantlyevolving to accommodate the rapid expansion ofscientific understanding of these processes. Manyearly investigations were dedicated to the identifica-tion and characterization of transcription factorsas regulatable proteins that bind to gene promoterDNA in a sequence-specific, context-dependentmanner. DNA structural rearrangements, nucleoso-mal histone composition and structure, and thenumerous regulatable modifications on histones,such as phosphorylation, methylation, acetylation,and SUMOylation, have emerged as critical elementsin facilitated gene regulation. In addition, multi-layered cell systems tightly control transcriptionalregulation as appropriate for specific stages of devel-opment, cell types, and organ systems. The avail-ability and activation state of transcription factorcoregulators, including coactivators and corepres-sors, also influence ligand-dependent transcrip-tional responses. In addition, cellular energy stateand relative ratios of reducing equivalents (i.e.,NADH andNADPH) have been shown to profoundlyaffect the outcome of transcriptional regulation.Wewill briefly review these areas, highlight recentimportant developments in these areas, and intro-duce data supporting a new regulatory pathway forsignaling-dependent estrogen receptor alpha (ER)transcriptional control.

CHROMATIN REMODELING COMPLEXES

The extreme length of DNA molecules and theextreme spatial constraints in which it is stored,maintained, and utilized necessitate the carefulorganization, packaging, and processing of chroma-tin structure for normal cell function. Chromatinstructure, nucleosomal structure, and histone tailmodifications constitute an intensely investigatedand frequently reviewed field of study [1–3]. Wewillhighlight only a few key elements herein.Thep160/steroid receptor coactivator (SRC) family

is a well-studied family of transcriptional coregula-tory proteins that function through histone tail

MOLECULAR CARCINOGENESIS 41:221–230 (2004)

� 2004 WILEY-LISS, INC.

Abbreviations: ER, estrogen receptor; SRC, steroid receptorcoactivator; ARNT, aryl hydrocarbon receptor nuclear translocator;NR, nuclear receptor; HAT, histone acetyltransferase; BRG1,brahma-related gene 1; SRA, steroid receptor activator; MTA,metastasis-associated-antigens; PELP1, proline-, glutamic acid-,leucine-rich protein 1; MNAR, modulator of non-genomic actionof ER; SMRT, silencing mediator of retinoid and thyroid hormonereceptors; SHARP, SMRT/HDAC1-associated repressor protein; REA,repressor of estrogen receptor activity; NAD, nicotinamide adeninedinucleotide; CtBP, C-terminal binding protein; Pak1, p21-activatedkinase 1; CtIP, CtBP interacting protein.

*Correspondence to: Department of Molecular and CellularOncology, The University of Texas M. D. Anderson Cancer Center,Houston, TX 77030.

Received 24 June 2004; Revised 9 August 2004; Accepted20 August 2004

DOI 10.1002/mc.20056

modifications, altering chromatin structure, andfacilitating transcription initiation. Its members areSRC1, glucocorticoid receptor interacting protein(GRIP1; also known as SRC2, TIF2), and P/CIP (alsoknown as SRC3, AIB1, TRAM1, and RAC3) [4]. TheP160/SRC family members share a common struc-ture frame that includes an N-terminal basic helix-loop-helix domain, a PAS (PER, aryl hydrocarbonreceptor nuclear translocator (ARNT), and SIM)homology domain, a C-terminal transcriptionalactivation domain, and a central region containingthree nuclear receptor (NR) interacting LXXLL(where X is any amino acid) motifs [5]. SRC1 andSRC3 have been shown to have histone acetyltrans-ferase (HAT) activity, which is necessary for theformation of an open chromatin structure [6]. Inaddition, SRC coactivators also interact with generalcoactivators such as the CREB binding protein (CBP)and p300 [7] and thus may help NRs to recruit moreHAT enzymes to the vicinity of target sites uponligand binding. In addition to HAT activity, coacti-vator-mediated methylation of proteins in thetranscription machinery may also contribute totranscriptional regulation by NRs [8]. For example,coactivator-associated arginine methyltransferase1 (CARM1) binds to the carboxyl-terminal region ofp160/SRC coactivators, methylates histone H3, andenhances transcriptional activation by NRs [9].Accumulating evidence suggests that the NR

ER recruits multiprotein complexes that regulatehigher-order chromatin domains intowhichnucleo-somes are organized [10,11]. SW1/SNF, a complexwith ATPase activity, alters nucleosomal structureand is shown to be involved in the transcriptionalregulation of NR [12]. Factors involved in thestructural remodeling of chromatin have also been

shown to mediate hormone-dependent transcrip-tional activation by ER. One example is the coacti-vator brahma-related gene 1 (BRG1), which isrecruited by ER in response to estrogen. Estrogen-mediated stimulation of ER-BRG1 association maycouple BRG1 to specific regions of chromatin in-cluding estrogen-responsive promoters and facilitatethe activity of other recruited factors that alter theacetylation state of chromatin and consequently,modify transcriptional outcome [13].

COACTIVATORS

Transcriptional regulation is dependent not onlyon transcription factor activation and chromatinremodeling, but also on a host of regulatory proteinsreferred to as coactivators and corepressors (Figure 1)[14,15]. Coactivators usually do not bind toDNA butare recruited to the target gene promoters throughprotein-protein interactions with the targeted tran-scription factors and function as linker molecules(sometimes with enzymatic activity) between DNAbinding proteins and protein modifying enzymes,which facilitate local structural alterations [16].For example, overwhelming evidence suggests thatmultiprotein complexes containing coactivators,ER, and other transcriptional regulators assemble inresponse to estrogen binding to ER and subsequentlyactivate transcription [14,17–19].There are many well-characterized coactivators

that have been reviewed extensively elsewhere.However, emerging evidence suggests that thecoactivator repertoire is much larger than originallyanticipated. For example, the TRAP/DRIP complex, alarge multiprotein complex, interacts with the NRsand is thought to connect these receptors with thebasal transcription machinery and thus influence

Figure 1. Overview of the key regulatory components in transcriptional control. [Color figure can be viewed inthe online issue, which is available at www.interscience.wiley.com.]

222 KUMAR ET AL.

NR-mediated transcription [20,21]. Another NRbinding protein, the steroid receptor activator(SRA), is unique among coactivators in that it func-tions as an RNA transcript rather than as a protein[22]. In addition, the E6-associated protein E6-AP, anubiquitin ligase, has been identified as a coactivatorof ER and expression of E6-AP is shown to bederegulated in breast cancers [23,24].Several other new NR coregulatory proteins have

recently been reported. A previously uncharacterizedmolecule, the metastasis-associated-antigens (MTA)1-interacting coactivator (MICoA), was recently iden-tified in a screen of proteins which bind thecorepressor metastasis-associated protein 1 (MTA1)and was shown to be an ER coactivator. MTA1-interacting coactivator cooperates with other ERcoactivators, stimulates ER-transactivation func-tions, and associates with the endogenous ER boundto promoter sequences of target genes [25]. Anothernovel ER coactivator, the ARNT, is an obligatoryheterodimerization partner for the aryl hydrocarbonreceptor (AhR) and hypoxia inducible factor 1a.ARNT also functions as a potent coactivator of ERa-and ERb-dependent transcription [26]. Indeed, thearyl hydrocarbon receptor/ARNT heterodimer isshown to bind directly to ERa and ERb and recruitsunliganded ER and the coactivator p300 directly toestrogen-responsive gene promoters, while the func-tions of liganded ERs are attenuated [27]. These dataprovide a mechanistic explanation for the docu-mented but poorly understood link between expo-sure to dioxin-type environmental contaminantsand adverse estrogen-related actions.Proline-, glutamic acid-, leucine-rich protein 1

(PELP1), another novel steroid receptor coregulator,contains 10 LXXLL motifs for NR interaction andplays an important role in estrogen-mediated tran-scriptional functions [28]. Modulator of non-geno-mic action of ER (MNAR) is another novelcoregulator of ER, which is important in the non-genomic actions of ER via activation of Src/MAPKpathways [29]. Subsequent studies showed thatPELP1 and MNAR are identical proteins and sug-gested that PELP1/MNAR is a novel coactivator of ERthat regulates both the genomic and non-genomicfunctions of ER [30]. Overexpression of PELP1sensitizes cells to estrogen-mediated cell-cycle pro-gression, enhances PELP1 interaction with the re-tinoblastoma (Rb) protein, and upregulates cyclinD1 expression [30]. These data suggest that PELP1may also link cell-cycle regulatory proteins to ER.

COREPRESSORS

As is indicatedby thename, corepressorproteins ingeneral coordinate the inactivation of transcription-ally active complexes through the direct interactionwith DNA-binding transcription factors and thecoordinate recruitment of chromatin modifyingenzymes that may return the nucleosome to an

inactive state. The first corepressors, which wereidentified for NRs, were silencing mediator ofretinoid and thyroid hormone receptors (SMRT)and nuclear hormone receptor corepressor (NCoR)[31]. These two proteins share a similar domainarchitecture and function in a similar fashion [18].Both proteins interact with NRs (often dimers, withone corepressor per dimer) through a C-terminalregion, and nucleate the assembly of multiproteinrepressor complexes through N-terminal repressiondomains,which interactwithchromatin remodelingenzymes such as histone deacetylases.A yeast two-hybrid screen with SMRT as bait led to

the isolation of a novel human protein termedSMRT/HDAC1-associated repressor protein (SHARP).SHARP is a potent transcriptional repressor of NRs[32]. SHARP also binds SRA and suppresses SRA-potentiated steroid receptor transcription activity.In addition, SHARP was reported as a new compo-nent of Notch/RBP-Jk repressor complex, facilitat-ing the transcriptional repression of Notch targetgenes as a component of a histone deacetylaserepressor complex [33]. Interestingly, the expressionof SHARP itself is steroid-inducible, suggesting asimple feedback mechanism for attenuation ofhormonal response [32].Another HDAC-binding, ER-associated corepres-

sor is the repressor of estrogen receptor activity (REA)protein. Although REA was initially described as aREA [34], the mechanism of repression was notclearly defined. Recent data that REA recruits bothclass I and class II HDACs to target promoters andthat its repression is sensitive to the deacetylaseinhibitor trichostatin A strongly supports a modelwhereby REA-mediated ER target promoter inhibi-tion is dependent upon HDAC activity [35].Some NRs, such as ER, undergo distinct intramo-

lecular conformational changes when bound tohormone antagonists which facilitate the physicalinteraction with corepressors [36]. Indeed, hormoneantagonists may function primarily through en-hanced recruitment of corepressor proteins to NRs[37]. Thus, the availability and activity of thesecoregulators will greatly impact the tissue-specificresponses to hormone antagonists.

METASTASIS-ASSOCIATED ANTIGENS

MTA represent a group of structurally relatedproteins encoded by the same or different genes.Currently there are three separately encoded mem-bers and six reported forms (MTA1, MTA1s, MTA-ZG29p, MTA2, MTA3, and MTA3L) [38,39]. MTA1was originally cloned by differential screening of thecDNA library from mouse metastatic tumor by Tohet al., and so named because of its elevated expres-sion in tumors with increased metastatic potential[40]. Except for MTA1s, which is localized in thecytoplasm, all other members are localized inthe nucleus, and are components of a chromatin

COREGULATORS IN TRANSCRIPTIONAL CONTROL 223

structure-remodeling complex termed Mi-2D orNuRD [39,41,42]. Themajor functional componentsof these complexes are the histone deacetylasesubunits. By deacetylation of the histones, the chro-matin structure becomes more condense and tran-scriptional activity is reduced. This, in turn, mayexplain the generally noticed transcriptional repres-sion by the MTA proteins. However, these MTAmembers are mutually exclusive, i.e., they formdistinct complexes in which only one MTAmoleculeispresent [27,43]. Thismayconstitute thebasis for thefunctional specialization of different MTA proteins.To date, significant data accumulated have shown

the positive correlations between MTA1 expressionlevel and tumor metastasis [38,40,44–51], includingcarcinomas from breast, pancreas, stomach, esopha-gus, liver, colon, and ovaries, etc. Overexpression ofMTA1 results in increased migration, increased an-chorage independent growth, and increased growthof the tumor xenograft [49,52,53]. Furthermore,MTA1 and its smaller, alternatively spliced formMTA1s both repress ER’s nuclear functions [54,55].The effect of MTA1 might be mediated by eitherrecruiting HDAC to the ER target site [52], or byinteracting with MAT1, a component of the cyclin-dependent kinase complex that phosphorylates ERand inhibits the ER transactivation function [54].MTA1s, on the other hand, sequesters and redirectsER to the cytoplasm.The expression level ofMTA1s isinversely correlated with the nuclear ER status [56].The sequestration of ER andMTA1s-mediated repres-sion of the genomic function of ER is potentiated byMTA1s phosphorylation at Ser 321 by Casein kinase1-gamma 2 [57]. Thus, the overexpression of MTA1and its short form MTA1s may contribute tohormone independent growth in breast cancer.There is no data yet to show a connection between

the expression of MTA2 and tumorigenesis. Phylo-genetically, themta2 gene evolved earlier than bothmta1 andmta3, and its rolemaymore possibly be of ahousekeeping gene [58]. Interestingly, MTA3 hasbeen shown to modulate the metastatic potentialof breast cancer via influencing the expression ofSnail, a master regulator of epithelial-to-mesenchy-mal transition (EMT) [59]. Further ER-initiatedsignaling upregulates MTA3 expression, leading torepression of Snail, and consequently, upregulationof E-cadherin. Recent data suggest that ER influencesMTA3 promoter via dynamic changes in levels ofnuclear coregulators, i.e., AIB1 and PELP1/MNAR. Inaddition, modulation of ER function by corepressors(i.e.,MTA1 andMTA1s) suppresses ER recruitment toMTA3 chromatin, upregulation of Snail, and promo-tion of epithelial-to-mesenchymal transition [60].

CELLULAR REDOX ANDTRANSCRIPTIONAL REGULATION

There is a growing interest within the researchcommunity in the influence of oxidant signals,

reactive oxygen intermediates, and cellular redoxstate on cell physiology, function, and viability. Inprevious years, this area of research received attentiondue to the association between the deregulation ofreactive oxygen and cell redox state and humandiseases, including cardiovascular disease, aging, andcancer. The growing understanding of how theseredox-based mechanisms influence normal physiol-ogy and disease has been the subject of manyexcellent recent reviews [61–63]. Elements of thiscomplex system can directly impact gene transcrip-tion. One example of this influence is throughnicotinamide adenine dinucleotide (NAD), a wide-spread small biological molecule that participates innumerous cellular reactions including transcriptionalcontrol [64].NADis synthesized fromtryptophanandfunctions both as a cofactor and substrate [65,66].NAD is converted to NADH through catabolic reac-tions inglycolysis andothermetabolic cycles.BecauseNAD influences many important cellular reactions,theratioofNADtoNADHis tightlycontrolled inmostcells and fluctuates in response to changes inmetabolism [67,68]. However, this ratio can becometilted towards the oxidized (NAD) or reduced (NADH)state, leading to cell stress, changes in gene expres-sion, and eventually cell death.The activity of two important transcriptional

regulators has been reported to be influenced byNAD. The first is the Sir2p repressor protein, which isrequired for nucleolar silencing and was recentlyshown to be a critical mediator of calorie restriction-induced increases in longevity [69–71]. Sir2p iscritical for silencing of the telomere and rDNA lociand this activity requires NAD as a cofactor [72].Sir2p also silences other important loci, includinggenes thought to be involved with regulation of thecircadian clock [73]. Sir2p-interacting transcrip-tional regulators clockandNPAS2are alsodependenton NAD cofactors [74], with enhanced repressoractivity when bound to the reduced NADH andinhibition of repressor activity when bound to NAD.Another NAD-dependent master coregulatory

protein is the C-terminal binding protein (CtBP), aubiquitous corepressor with numerous interactingproteins (Figure 2) [75]. CtBP binds both NAD andNADH, but its corepressor functions are three ordersof magnitude higher when bound to the latter [76].NADH binding changes the CtBP three-dimensionalconfirmation [77], resulting in a shift in protein-protein interactions and increased corepressor activ-ity [76,78]. NADH-bound CtBP is also a bettersubstrate for phosphorylation and inactivation ofCtBP by the signaling kinase p21-activated kinase 1(Pak1) (described below). However, NADH can alsoreduce CtBP interaction with other regulatory mole-cules, as occurs with HDM2 [79]. CtBP-HDM2 inter-action is onemechanismof p53 protein suppression.Under conditions of oxidative stress, NADH levelsrise, resulting in dissociation of CtBP from HDM2

224 KUMAR ET AL.

and increased p53 activity. CtBP may then be free tointeract with other, NADH-dependent binding part-ners and influence transcription through other,possibly HDAC-dependent mechanisms.We recently reported the phosphorylation and

functional regulation of CtBP by the Pak1 [78]. Pak1is activated by growth factor receptor tyrosinekinases via Rac1 or Cdc42, by non-receptor tyrosinekinases, and by lipids [80,81]. A wide range ofbiological activities has been shown to result fromPak1 phosphorylation of downstream substrates,including cell cytoskeletal reorganization leadingto increased motility, enhanced cell survival, andcross talk with signaling pathways that influencegene expression [80].We foundPak1 phosphorylatesCtBP selectively on Ser158 within a putative regula-tory loop, triggering CtBP cellular redistribution andblocking CtBP corepressor functions [78]. In thepresence of NADH, Pak1 super-phosphorylated CtBPand inhibited CtBP dehydrogenase activity, suggest-ing that preferential phosphorylation of active CtBPmay alter secondary structures and influence pro-tein-protein interaction, enzymatic and corepressorfunctions [78]. Pak1 regulation of CtBP represents anew transcriptional regulatory pathway whereby asignaling kinase can inactivate a corepressor andstimulate dissociation of a corepressor regulatorycomplex from an endogenous gene promoter. Over-all, thesemechanisms of signaling- andNAD-depen-

dent regulationof repressor andcorepressor functionprovide direct routes for cell metabolism, reactiveoxygen balance, and cell redox state to directlyinfluence gene transcription and cell physiology.

CtBP—A NEW, REGULATED ERINTERACTING COREPRESSOR

Wewould like to briefly introduce for the first timeevidence for the direct interaction of the regulatablecorepressor, CtBP, with ER. The CtBP facilitates generegulation during development and oncogenesis[82]. CtBP specifically associates with a variety ofDNA-binding regulatory proteins with roles incancer [83–86], primarily via a conserved PXDLSCtBP interaction motif (where X is any amino acid)in CtBP interacting proteins. Through its protein-protein interactions, CtBP is thought to act as amolecular bridgebetweenDNAbindingproteins andenzymes associated with transcriptional repressionsuch as histone deacetylases [82]. CtBP was recentlyshown to bind to NADH, resulting in a proteinconformational change, enhanced repressor bind-ing, and increased corepressor activity [77,87].While examining the human CtBP amino acid

sequence, we noted that CtBP contains two NR-interacting LXXLL motifs (where X is any aminoacid). Buildings upon our recent reports of directPak1 phosphorylation and regulation of ER [88,89],we decided to investigate whether CtBP might

Figure 2. The transcriptional corepressor C-terminal binding protein (CtBP) influences cell homeostasis via itsmultiple interactions. CtBP protein-protein interaction and corepressor activity are activity regulated by upstreamsignaling molecules, covalent modification, and cellular redox state.


interact with ER and thus provide an additionalmechanism by which Pak1 and its upstream regula-tors might also regulate ER function.As shown in Figure 3, in vitro transcribed and

translated ER bound to GST-CtBP (Figure 3A). Weperformed the reverse experiment and found thatin vitro transcribed and translated CtBP was able tobind specifically to the AF2 domain of the purifiedGST-tagged ER protein (Figure 3B). This ER bindingpattern was consistent with that seen by othercorepressor proteins such as metastasis-associatedantigen 1 (discussed above). This in vitro bindingpattern was confirmed in vivo by immunoprecipita-tion of endogenous CtBP fromMCF-7 cells, followedby polyacrylamide gel electrophoresis and detectionof co-immunoprecipitated ER by Western blotanalysis (Figure 3C). As further confirmation ofin vivo protein-protein interaction, we determinedwhether ER and CtBP colocalized in MCF-7 cells byimmunofluorescent protein detection followed byconfocal microscopic analysis (Figure 3D). Resultsindicated that even in steroid-free media, there issome colocalization between ER (green) and CtBP(red), as indicated by the yellow fluorescence result-ing from overlapping red and green fluorescence.However, estrogen treatment of cells dramatically

increased the nuclear concentration of ER and thedegree of colocalization. This increased colocaliza-tion was blocked by pretreatment with the pureantiestrogen ICI 182780 prior to estrogen treatment(Figure 3D). Thus, CtBP-ER interaction might actu-ally represent a second mechanism by which signal-ing pathways incorporating activated Pak1 mightregulate ER transcriptional regulation.As shown in Figure 2, CtBP also interacts with

a number of other established ER regulatory pro-teins, either directly or indirectly through the CtBPobligatory heterodimer protein CtIP (CtBP Interact-ing Protein). These knownCtBP- or CtIP-interacting,ER regulatory proteins include retinoblastoma [90],BRCA1 [91], the NR corepressor RIP140 [92], and arecentlydescribedNRcorepressor, ligand-dependentcorepressor (LCoR) [91]. In addition, CtBP has beenshown to be part of the NuRD repressor complex[93,94]. NuRD recruitment has recently been shownto be a critical component in the cyclical activationof ER target promoters [95]. Also, CtBP functions,at least, in part through mediating protein-proteininteractions and recruiting HDACs to target promo-ters in transcriptional regulatory processes (reviewedin Ref. [82]). Thus, CtBP may be an uninvestigated,important element in ER transcriptional regulation.

Figure 3. CtBP, a potential estrogen receptor a (ER) interactingcorepressor. (A) In vitro interaction of a purified GST-CtBP fusionprotein [78] with in vitro transcribed and translated, 35S labeled ER(performed as previously described [78]). (B) In vitro interaction ofthe purified GST-AF2 ER domain fusion protein [52] with in vitrotranscribed and translated, 35S labeled CtBP. (C) Endogenous ER wasimmunoprecipitated from human breast cancer MCF-7 cells aspreviously described [52] with a monoclonal antibody directedagainst endogenous CtBP (BD Transduction Laboratories, San Diego,

CA). (D) Immunofluorescent localization of CtBP (red) and ER (green)in MCF-7 cells grown in 17b-estradiol-free media (left), stimulatedwith 10�9 M 17b-estradiol (Sigma Chemical Co., St. Louis, MO) for10 min (center), or pretreated with 10�8 M of the pure antiestrogenICI 182 708 (AstraZeneca, Macclesfield, UK) for 30 min prior toestrogen treatment. Colocalization between red and green fluores-cence is indicated by yellow. [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]

226 KUMAR ET AL.

CYCLICAL MODEL OF TRANSCRIPTION INITIATION

Transcriptional regulation byNRs has long served asa model system with which to investigate ligand-dependent receptor activation, translocation to sites ofactivity,multi-proteincomplex recruitment, initiationof transcription, and finally return to an inactivatedstate. Our current understanding of transcriptionalactivation involves the precisely coordinated recruit-ment of the basal transcriptional machinery, chro-matin remodeling to facilitate transcription, andpositioning of activation complexes that enhancebasal transcription. Previous models of this processemphasized an exchange of repressor complexes foractivationcomplexesaspartof transcriptioninitiation.However, recent reports have revealed that transcrip-tional activation is more likely a cyclical process thatrequires both activating and repressive epigeneticprocesses [95–97]. Through the evaluation of theregulation of the ER target gene pS2, Metivier et al.[95] recently described integrated chromatin remodel-ing and the kinetic evaluation the cyclical recruitmentof 46 different transcriptional regulators to thepromoter. What emerged was a concept of a ‘‘tran-scriptional clock;’’ essentially, the sequential andcombinatorial assembly of a transcriptionally produc-tive complex on a promoter through the systematicrecruitment and derecruitment of specific transcrip-tional regulators and chromatin modifiers. Once agiven protein binds to a promoter, it may rapidlydissociate and bind again, continuing unproductivelyuntil another eventoccurswhichpushes theprocess tothe next stage to the initiation cycle.The authors propose three distinct cycles [95]. The

first stage is an initial, unproductive process thatcommits to activation of the promoter following ERbinding through the recruitment of the ATP-depen-dent SWI/SNF complex. As part of nucleosomalremodeling, histone methyltransferases and HATsare recruited prior to binding of the basal transcrip-tional machinery. Interestingly, ERa then becomestargeted to the proteasome by a subset of the 20Sproteasome subunit, the APIS complex. The tran-scriptionally productive cycle can then initiate, withthe recruitment of helicase enzymes, HMTs andHATs, which bring about additional nucleosomalremodeling and transcriptional initiation. Followinginitiation, ER is again targeted to the proteasomealong with the recruitment of repressive SWI/SNFcomplexes, which remodel the nucleasome and setthe stage for additional rounds of transcription.Finally, basal transcriptional complexes are dis-placed by the recruitment of the NuRD repressorcomplex, which restores restrictive chromatin struc-ture and blocks productive cycling.

NUCLEAR ARCHITECTUREAND TRANSCRIPTIONAL CONTROL

There is a growing realization that transcription,replication, and DNA repair require both temporal as

well as spatial coordination. Indeed, there is likely apivotal role for architecturally associated nuclearmicroenvironments in the regulation of transcrip-tional complex assembly and the proper and orderedtargeting of the transcriptional machinery to specificpromoter and enhancer regions. This emergingfield ofinvestigation received a recent, insightful review [98]and will only be briefly discussed here. Much of thework in this area has focused onDNA replication, witha common theme that the nuclear substructurefunctions as an architectural platform for replicationproteins to organize into focal thresholds that areobservable by microscopy. It now appears that similarregulatorymechanismsmay facilitatemost subnuclearcompartmental organization, targeting, and fidelity[99]. Key components of the basal transcription com-plex are functionally compartmentalized [100,101].For the targeting of some (if not many) transcriptionfactors, both nuclear import and nuclear matrixtargeting are critical to proper function. Sequence-specific targetingplaces transcription factors in specificpositions to interact with (and serve as scaffolds for)coregulatory proteins that in turnmediate the organi-zation of chromatin remodeling complexes and thetranscriptionalmachinery.Althoughmuch remains tobe defined in the area of nuclear matrix targeting,insight into this additional transcriptional regulatorycomponent will likely have an impact on our under-standing of the importance of nuclear microenviron-ments in the normal and diseased state.

CONCLUSIONS

Functions ofNRs aremodulated throughanumberof concurrent events in response to membrane- orcytoplasm-initiated signals. Such signals feed intothe NRs through components of the classical hor-moneor growth factor signalingpathways.However,deregulation of growth factor signals could alsoinfluence this process by affecting the phosphoryla-tion status of critical coregulatory molecules. Addi-tionally, signaling pathways also phosphorylate NRsdirectly and change their ability to interact withother coregulatory proteins, or influence the recruit-ment of coregulators. Although investigations in thelast decade has been very useful in providing us withan evolving, detailed understanding of chromatinremodeling and transcription control, it is clear thatfor amajor gain,weneed to validate these findings torelevant whole animal setting.

ACKNOWLEDGMENTS

The work in the Kumar laboratory is supportedby the NIH grants CA80066, CA65746, CA90970(to RK). We apologize to several of our colleagues fornot citing their references due to space limitations.

REFERENCES

1. Kinyamu HK, Archer TK. Modifying chromatin to per-mit steroid hormone receptor-dependent transcription.Biochim Biophys Acta 2004;1677:30–45.


2. van Holde K, Yager T. Models for chromatin remodeling: Acritical comparison. Biochem Cell Biol 2003;81:169–172.

3. Maison C, Almouzni G. HP1 and the dynamics of hetero-chromatin maintenance. Nat Rev Mol Cell Biol 2004;5:296–304.

4. Torchia J, Glass C, Rosenfeld MG. Co-activators and co-repressors in the integration of transcriptional responses.Curr Opin Cell Biol 1998;10:373–383.

5. McKenna NJ, Lanz RB, O’Malley BW. Nuclear receptorcoregulators: Cellular and molecular biology. Endocr Rev1999;20:321–344.

6. Spencer TE, Jenster G, Burcin MM, et al. Steroid receptorcoactivator-1 is a histone acetyltransferase. Nature 1997;389:194–198.

7. Chakravarti D, LaMorte VJ, Nelson MC, et al. Role of CBP/P300 in nuclear receptor signalling. Nature 1996;383:99–103.

8. ChenD,MaH,HongH,et al. Regulationof transcriptionbyaprotein methyltransferase. Science 1999;284:2174–2177.

9. Chen D, Huang SM, Stallcup MR. Synergistic, p160coactivator-dependent enhancement of estrogen receptorfunction by CARM1 and p300. J Biol Chem 2000;275:40810–40816.

10. Collingwood TN, Urnov FD, Wolffe AP. Nuclear receptors:Coactivators, corepressors, and chromatin remodeling inthe control of transcription. J Mol Endocrinol 1999;23:255–275.

11. Fryer CJ, Archer TK. Chromatin remodeling by theglucocorticoid receptor requires the BRG1complex. Nature1998;393:88–91.

12. Yoshinaga SK, Peterson CL, Herskowitz I, Yamamoto KR.Roles of SWI1, SWI2, and SWI3 proteins for transcriptionalenhancement by steroid receptors. Science 1992;258:1598–1604.

13. DiRenzo J, Shang Y, Phelan M, et al. BRG-1 is recruited toestrogen-responsive promoters and cooperates with fac-tors involved in histone acetylation. Mol Cell Biol 2000;20:7541–7549.

14. McKenna NJ, Lanz RB, O’Malley BW. Nuclear receptorcoregulators: Cellular and molecular biology. Endocr Rev1999;20:321–344.

15. Glass CK, Rose DW, Rosenfeld MG. Nuclear receptorcoactivators. Curr Opin Cell Biol 1997;9:222–232.

16. Ma H, Hong H, Huang SM, et al. Multiple signal input andoutput domains of the 160-kilodalton nuclear receptorcoactivator proteins. Mol Cell Biol 1999;19:6164–6173.

17. Couse JF, Korach KS. Estrogen receptor null mice: Whathave we learned and where will they lead us? EndocrinolRev 1999;20:358–417.

18. Levin ER. Bidirectional signaling between the estrogenreceptor and the epidermal growth factor receptor. MolEndocrinol 2003;17:309–317.

19. Schiff R, Massarweh S, Shou J, Osborne CK. Breast cancerendocrine resistance: How growth factor signaling andestrogen receptor coregulators modulate response. ClinCancer Res 2003;9:47S–454S.

20. Ito M, Yuan CX, Malik S, et al. Identity between TRAP andSMCC complexes indicates novel pathways for the func-tion of nuclear receptors and diverse mammalian activa-tors. Mol Cell 1999;3:361–370.

21. Freedman LP. Increasing the complexity of coactivation innuclear receptor signaling. Cell 1999;97:5–8.

22. Lanz RB, McKenna NJ, Onate SA, et al. A steroid receptorcoactivator, SRA, functions as an RNA and is present in anSRC-1 complex. Cell 1999;97:17–27.

23. Nawaz Z, Lonard DM, Smith CL, et al. The Angelmansyndrome-associated protein, E6-AP, is a coactivator forthe nuclear hormone receptor superfamily. Mol Cell Biol1999;19:1182–1189.

24. Sivaraman L, Nawaz Z, Medina D, et al. The dual functionsteroid receptor coactivator/ubiquitin protein-ligase inte-

grator E6-AP is overexpressed in mouse mammarytumorigenesis. BreastCancer Res Treat 2000;62:185–195.

25. Mishra SK, Mazumdar A, Vadlamudi RK, et al. MICoA, anovel metastasis-associated protein 1 (MTA1) interactingprotein coactivator, regulates estrogen receptor-atransac-tivation functions. J Biol Chem 2003;278:19209–19219.

26. Brunnberg S, Pettersson K, Rydin E, et al. The basic helix-loop-helix-PAS protein ARNT functions as a potentcoactivator of estrogen receptor-dependent transcription.PNAS USA 2003;100:6517–6522.

27. Ohtake F, Takeyama KI, Matsumoto T, et al. Modulation ofoestrogen receptor signaling by association with theactivated dioxin receptor. Nature 2003;423:545–550.

28. Vadlamudi RK, Wang RA, Mazumdar A, et al. Molecularcloning and characterization of PELP1, a novel humancoregulator of estrogen receptor alpha. J Biol Chem 2001;276:38272–38279.

29. WongCW,McNally C, Nickbarg E, et al. Estrogen receptor-interacting protein thatmodulates its nongenomic activity-crosstalk with Src/Erk phosphorylation cascade. PNAS USA2002;99:14783–14788.

30. Balasenthil S, Vadlamudi RK. Functional interactions be-tween the estrogen receptor coactivator PELP1 andretinoblastoma protein. J Biol Chem 2003;278:22119–22127.

31. Davie JK, Dent SY. Histone modifications in corepressorfunctions. Curr Topics Dev Biol 2004;59:145–163.

32. Shi Y, Downes M, Xie W. Sharp, an inducible cofactor thatintegrates nuclear receptor repression and activation.Genes Dev 2001;15:1140–1151.

33. Oswald F, Kostezka U, Astrahantseff K. SHARP is a novelcomponent of the Notch/RBP-J signaling pathway. EMBO J2002;21:5417–5426.

34. Montano MM, Ekena K, Delage-Mourroux R, Chang W,Martini P, Katzenellenbogen BS. An estrogen receptor-selective coregulator that potentiates the effectiveness ofantiestrogens and represses the activity of estrogens. PNASUSA 1999;96:6947–6952.

35. Kurtev V, Margueron R, Kroboth K, Orgis E, Cavailles V,Seiser C. Transcriptional regulation by the repressor ofestrogen receptor activity via recruitment of histonedeacetylases. J Biol Chem 2004;279:24834–24843.

36. Jordan VC. Antiestrogens and selective estrogen receptormodulators as multifunctional medicines 1 receptor inter-actions. J Med Chem 2002;46:883–908.

37. Jordan VC. Selective estrogen receptor modulation: Con-cept and consequences in cancer. Cancer Cell 2004;5:207–213.

38. Kumar R,Wang RA, Bagheri-Yarmand R. Emerging roles ofMTA family members in human cancers. Semin Oncol2003;5:30–37.

39. Kumar R. Another tie that binds the MTA family to breastcancer. Cell 2003;113:142–143.

40. Toh Y, Pencil SD, Nicolson GL. A novel candidatemetastasis-associated gene,mta1, differentially expressedin highly metastatic mammary adenocarcinoma cell lines:cDNA cloning, expression, and protein analyses. J BiolChem 1999;269:22958–22963.

41. Xue Y, Wong J, Moreno GT, et al. NURD, a novel complexwith both ATP-dependent chromatin-remodeling andhistone deacetylase activities. Mol Cell 1998;2:851–861.

42. Zhang Y, Ng HH, Erdjument-Bromage H, et al. Analysis ofthe NuRD subunits reveals a histone deacetylase corecomplex and a connection with DNA methylation. GenesDev 1999;13:1924–1935.

43. Yao YL, Yang WM. The metastasis-associated proteins 1and 2 form distinct protein complexes with histonedeacetylase activity. J Biol Chem2003;278:42560–42568.

44. TohY,Oki E,OdaS, et al.Overexpressionof theMTA1genein gastrointestinal carcinomas: Correlation with invasionand metastasis. Int J Cancer 1997;74:459–463.

228 KUMAR ET AL.

45. Toh Y, Kuwano H, Mori M, Nicolson GL, Sugimachi K.Overexpression of metastasis-associated MTA1 mRNA ininvasive oesophageal carcinomas. Br J Cancer 1999;79:1723–1726.

46. Sasaki H, Yukiue H, Kobayashi Y, et al. Expression of theMTA1 mRNA in thymoma patients. Cancer Lett 2001;74:159–163.

47. Moon WS, Chang K, Tarnawski AS. Overexpression ofmetastatic tumor antigen 1 in hepatocellular carcinoma:Relationship to vascular invasion and estrogen receptor-a.Hum Pathol 2004;35:424–429.

48. Hofer MD, Kuefer R, Varambally S, et al. The role ofmetastasis-associated protein 1 in prostate cancer progres-sion. Cancer Res 2004;64:825–829.

49. Hofer MD, Menke A, Genze F, et al. Expression of MTA1promotes motility and invasiveness of PANC-1 pancreaticcarcinoma cells. Br J Cancer 2004;90:455–462.

50. Yi S, Guangqi H, Guoli H. The association of the expressionofMTA1, nm23H1with the invasion, metastasis of ovariancarcinoma. Chin Med Sci J 2003;18:87–92.

51. Sasaki H,Moriyama S, NakashimaY, et al. Expression of theMTA1mRNA in advanced lung cancer. Lung Cancer 2002;35:149–154.

52. Mazumdar A, Wang RA, Mishra SK, et al. Transcriptionalrepression of oestrogen receptor by metastasis-associatedprotein 1 corepressor. Nat Cell Biol 2001;3:30–37.

53. Mahoney MG, Simpson A, Jost M, et al. Metastasis-associated protein (MTA)1 enhances migration, invasion,and anchorage-independent survival of immortalizedhuman keratinocytes. Oncogene 2002;21:2161–2170.

54. Talukder AH, Mishra SK, Mandal M, et al. MTA1 interactswith MAT1, a cyclin-dependent kinase-activating kinasecomplex ring finger factor, and regulates estrogen receptortransactivation functions. J Biol Chem 2003;278:11676–11685.

55. Talukder AH, Gururaj A, Mishra SK, Vadlamudi RK, KumarR. Metastasis-associated protein 1 (MTA1) interacts withNRIF3, an estrogen-inducible nuclear receptor coregulator.Mol Cell Biol 2004;24:6581–6591.

56. Kumar R, Wang RA, Mazumdar A, et al. A naturallyoccurring MTA1 variant sequesters oestrogen receptor-alpha in the cytoplasm. Nature 2002;418:654–657.

57. Mishra SK, Talukder AH, Gururaj AE, et al. Metastatictumor antigen 1 short form (MTA1s) associates withcasein kinase I-2, an estrogen-responsive kinase. Onco-gene 2004;23:4422–4429.

58. Bowen NJ, Fujita N, Kajita M, Wade PA. Mi-2/NuRD:Multiple complexes for many purposes. Biochim BiophysActa 2004;1677:52–57.

59. Fujita N, Jaye DL, Kajita M, et al. MTA3, a Mi-2/NuRDcomplex subunit, regulates an invasive growth pathway inbreast cancer. Cell 2003;113:207–219.

60. Mishra SK, Talukder AH, Gururaj AE, et al. Upstreamdeterminants of estrogen receptor-alpha regulation ofMTA3 pathway. J Biol Chem 2004;279:32709–32715.

61. Finkel T. Oxidant signals andoxidative stress. CurrOpinCellBiol 2003;15:247–254.

62. Barnham KJ, Masters CL, Bush AI. Neurodegenerativediseases and oxidative stress. Nat Rev Drug Discov 2004;3:205–214.

63. Papp E, Nardai G, Soti C, Csermely P. Molecular chaper-ones, stress proteins, and redox homeostasis. Biofactors2003;17:249–257.

64. Lin SJ, Guarente L. Nicotinamide adenine dinucleotide, ametabolic regulator of transcription, longevity, and dis-ease. Curr Opin Cell Biol 2003;15:241–246.

65. Grubmeyer CT, Gross JW, Rajavel M. Energy couplingthrough molecular discrimination: Nicotinate phosphor-ibosyltransferase. Methods Enzymol 1999;308:28–48.

66. Anderson RM, Bitterman KJ, Wood JG, et al. Manipulationof a nuclear NADþ salvage pathway delays aging without

altering steady-state NADþ levels. J Biol Chem 2002;277:18881–18890.

67. GaikwadA, LongDJ II, Stringer JL, Jaiswal AK. In vivo role ofNAD(P)H: Quinone oxidoreductase 1 (NQO1) in theregulation of intracellular redox state and accumulationof abdominal adipose tissue. J Biol Chem 2001;276:22559–22564.

68. RamasamyR, TruebloodN, Schaefer S.Metabolic effects ofaldose reductase inhibition during low-flow ischemia andreperfusion. Am J Physiol 1998;275:H195–H203.

69. Imai S, Armstrong CM, Kaeberlein M, Guarente L.Transcriptional silencing and longevity protein Sir2 is anNAD-dependent histone deacetylase. Nature 2000;403:795–800.

70. Landry J, Sutton A, Tafrov ST, et al. The silencing proteinSIR2 and its homologs are NAD-dependent proteindeacetylases. PNAS USA 2000;97:5807–5811.

71. Smith JS, Brachmann CB, Celic I, et al. A phylogeneti-cally conserved NADþ-dependent protein deacetylaseactivity in the Sir2 protein family. PNAS USA 2000;97:6658–6663.

72. Sandmeier JJ, Celic I, Boeke JD, Smith JS. Telomeric andrDNA silencing in Saccharomyces cerevisiae are dependenton a nuclear NAD(þ) salvage pathway. Genetics 2002;160:877–889.

73. McMurray MA, Gottschling DE. An age-induced switchto a hyper-recombinational state. Science 2003;301:1908–1911.

74. Rutter J, ReickM,Wu LC,McKnight SL. Regulation of clockand NPAS2 DNA binding by the redox state of NADcofactors. Science 2001;293:510–514.

75. Chinnadurai G. CtBP, an unconventional transcriptionalcorepressor in development and oncogenesis. Mol Cell2002;9:213–224.

76. Zhang Q, Piston DW, Goodman RH. Regulation of core-pressor function by nuclear NADH. Science 2002;295:1895–1897.

77. Kumar V, Carlson JE, Ohgi KA, et al. Transcriptioncorepressor CtBP is an NAD(þ)-regulated dehydrogenase.Mol Cell 2002;10:857–869.

78. Barnes CJ, Vadlamudi RK, Mishra SK, et al. Functionalinactivation of a transcriptional corepressor by a signalingkinase. Nat Struct Biol 2003;10:622–628.

79. Mirnezami AH, Campbell SJ, Darley M, et al. Hdm2recruits a hypoxia-sensitive corepressor to negativelyregulate p53-dependent transcription. Curr Biol 2003;13:1234–1239.

80. Kumar R, Vadlamudi RK. Emerging functions of p21-activated kinases in human cancer cells. J Cell Physiol 2002;193:133–144.

81. Bokoch GM. Biology of the p21-activated kinases. Ann RevBiochem 2003;72:743–782.

82. Chinnadurai G. CtBP, an unconventional transcriptionalcorepressor in development and oncogenesis. Mol Cell2002;9:213–224.

83. Sewalt RG, Gunster MJ, van der Vlag J, et al. C-terminalbinding protein is a transcriptional repressor that interactswith a specific class of vertebrate Polycomb proteins. MolCell Biol 1999;19:777–787.

84. Schaeper U, Subramanian T, Lim L, et al. Interactionbetween a cellular protein that binds to the C-terminalregionof adenovirus E1A (CtBP) anda novel cellular proteinis disrupted by E1A through a conserved PLDLSmotif. J BiolChem 1998;273:8549–8552.

85. Yu X, Baer R. Nuclear localization and cell-cycle-specificexpression of CtIP, a protein that associates with theBRCA1 tumor suppressor. J Biol Chem 2000;275:18541–18549.

86. Feloni AR, Smith EJ, Nevins JR. A mechanism for Rb/p130-mediated transcription repression involving recruitment ofthe CtBP corepressor. PNAS USA 1999;96:9574–9579.


87. Zhang Q, Piston DW, Goodman RH. Regulation ofcorepressor function by nuclear NADH. Science 2002;295:1895–1897.

88. Wang RA, Mazumdar A, Vadlamudi RK, Kumar R. P21-activated kinase-1 phosphorylates and transactivatesestrogen receptor-alpha and promotes hyperplasia inmammary epithelium. EMBO J 2002;21:5437–5447.

89. Balasenthil S, Barnes CJ, Rayala SK, Kumar R. Estrogenreceptor activation at serine 305 is sufficient to upregulatecyclin D1 in breast cancer cells. FEBS Lett 2004;567:243–247.

90. Meloni AR, Smith EJ, Nevins JR. Amechanism for Rb/p130-mediated transcription repression involving recruitment ofthe CtBP corepressor. PNAS USA 1999;96:9574–9579.

91. Yu X, Wu LC, Bowcock AM, et al. The C-terminal (BRCT)domains of BRCA1 interact in vivo with CtIP, a proteinimplicated in the CtBP pathway of transcriptional repres-sion. J Biol Chem 1998;273:25388–25392.

92. Vo N, Fjeld C, Goodman RH. Acetylation of nuclearhormone receptor-interacting protein RIP140 regulatesbinding of the transcriptional corepressor CtBP. Mol CellBiol 2001;21:6181–6188.

93. Fernande I, Bastien Y, Wa T, et al. Ligand-dependentnuclear receptor corepressor LCoR functions by histonedeacetylase-dependent and -independent mechanisms.Mol Cell 2003;11:139–150.

94. Shi Y, Sawada J, Sui G, et al. Coordinated histonemodifications mediated by a CtBP co-repressor complex.Nature 2003;422:735–738.

95. Metivier R, Penot G, Hubner MR, et al. Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruit-ment of cofactors on a natural target promoter. Cell 2003;115:751–763.

96. Perissi V, Aggarwal A, Glass CK, et al. A corepressor/coactivator exchange complex required for transcriptionalactivation by nuclear receptors and other regulatedtranscription factors. Cell 2004;116:511–526.

97. AnW, Kim J, Roeder RG. Ordered cooperative functions ofPRMT1, p300, and CARM1 in transcriptional activation byp53. Cell 2004;117:735–748.

98. Stein GS, Zaidi SK, Braastad CD, et al. Functionalarchitecture of the nucleus: Organizing the regulatorymachinery for gene expression, replication, and repair.Trends Cell Biol 2003;11:584–592.

99. Stein GS, van Wijnen AJ, Stein JL, et al. Intranucleartrafficking of transcription factors: Implications for biolo-gical control. J Cell Sci 2000;113:2527–2533.

100. Misteli T. Cell biology of transcription and pre-mRNAsplicing: Nuclear architecturemeets nuclear function. J CellSci 2000;113:1841–1849.

101. Gasser SM. Visualizing chromatin dynamics in interphasenuclei. Science 2002;296:1412–1416.

230 KUMAR ET AL.

coregulators and chromatin remodeling in transcriptional control

Documents