Mol. Endocrinol. 2010 24:2088-2098 originally published online May 19, 2010; , doi: 10.1210/me.2010-0027

Guillermo P. Vicent, A. Silvina Nacht, Roser Zaurín, Cecilia Ballaré, Jaime Clausell and Miguel Beato

Signaling to ChromatinMinireview: Role of Kinases and Chromatin Remodeling in Progesterone

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Minireview: Role of Kinases and ChromatinRemodeling in Progesterone Signaling to Chromatin

Guillermo P. Vicent, A. Silvina Nacht, Roser Zaurín, Cecilia Ballare, Jaime Clausell,and Miguel Beato

Centre de Regulacio Genomica, Universitat Pompeu Fabra, E-08003 Barcelona, Spain

Steroid hormones regulate gene expression by interaction of their receptors with hormone-responsiveelements on DNA or with other transcription factors, but they can also activate cytoplasmic signalingcascades. Rapid activation of Erk by progestins via an interaction of the progesterone receptor (PR)with the estrogen receptor is critical for transcriptional activation of the mouse mammary tumor virus(MMTV) promoter and other progesterone target genes. Erk activation leads to the phosphorylationof PR, activation of mitogen- and stress-activated protein kinase 1, and the recruitment of a complexof the three activated proteins and of P300/CBP-associated factor (PCAF) to a single nucleosome,resulting in the phosphoacetylation of histone H3 and the displacement of heterochromatin protein1!. Hormone-dependent gene expression requires ATP-dependent chromatin remodeling complexes.Two switch/sucrose nonfermentable-like complexes, Brahma-related gene 1-associated factor (BAF)and polybromo-BAF are present in breast cancer cells, but only BAF is recruited to the MMTV promoterand cooperates with PCAF during activation of hormone-responsive promoters. PCAF acety-lates histone H3 at K14, an epigenetic mark recognized by BAF subunits, thus anchoring thecomplex to chromatin. BAF catalyzes localized displacement of histones H2A and H2B, facili-tating access of nuclear factor 1 and additional PR complexes to the hidden hormone-respon-sive elements on the MMTV promoter. The linker histone H1 is a structural component ofchromatin generally regarded as a general repressor of transcription. However, it contributesto a better regulation of the MMTV promoter by favoring a more homogeneous nucleosomepositioning, thus reducing basal transcription and actually enhancing hormone induced tran-scription. During transcriptional activation, H1 is phosphorylated and displaced from thepromoter. The kinase cyclin-dependent kinase 2 is activated after progesterone treatment andcould catalyze progesterone-induced phosphorylation of histone H1 by chromatin remodelingcomplexes. The initial steps of gene induction by progestins involve changes in the chromatinorganization of target promoters that require the activation of several kinase signaling path-ways initiated by membrane anchored PR. Because these pathways also respond to otherexternal signals, they serve to integrate the hormonal response in the global context of thecellular environment. (Molecular Endocrinology 24: 2088 –2098, 2010)

NURSA Molecule Pages: Nuclear Receptors: PR ! GR ! ER"; Corregulators: P/CAF ! BAF57 ! BRM !BRG1 ! SRC-1 ! GRIP1 ! AIB1; Ligands: Progesterone ! Dexamethasone.

The classical picture of steroid hormone action via theirnuclear receptors considered as ligand-regulated tran-

scription factors has undergone dramatic changes in the

past few years. It is becoming increasingly evident that“nuclear” hormone receptors participate in multiple in-teractions within different cellular compartments that are

ISSN Print 0888-8809 ISSN Online 1944-9917Printed in U.S.A.Copyright © 2010 by The Endocrine Societydoi: 10.1210/me.2010-0027 Received January 22, 2010. Accepted April 21, 2010.First Published Online May 19, 2010

Abbreviations: BAF, BRG1-associated factor; BRG1, Brahma-related gene 1; Cdk2, cyclin-dependent kinase 2; ChIP, chromatin immunoprecipitation; DNase, deoxyribonuclease; EGFR,epidermal growth factor receptor; ER, estrogen receptor; FRAP, fluorescence recovery afterphotobleaching; GR, glucocorticoid receptor; HAT, histone acetyl transferase; HDAC, histonedeacetylase; HRE, hormone-responsive element; MMTV, mouse mammary tumor virus; Msk1,mitogen- and stress-activated protein kinase 1; NF1, nuclear factor 1; p, phospho; PR, pro-gesterone receptor; SH, Src homology; SHR, steroid hormone receptor; Src, sarcoma; Stat,signal transducers and activator of transcription; SWI/SNF, switch/sucrose nonfermentable.

M I N I R E V I E W

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essential to fully understand the response of the cell tovariations in hormone levels. In addition, other mem-brane-bound hormone receptors that have been identifiedand contribute to hormone action are still not well under-stood (1). These multiple interactions serve to integratethe hormonal signal into the network of default programsand external signals impinging on the cell at a given time.It is the outcome of this integration that specifies thenature, intensity, and duration of the cellular response.

Estrogen and progesterone influence a variety of func-tions in different target cells. Many years of attentionhave been focused on the transcriptional effects of thesehormones, because the steroid hormone receptors (SHRs)were seen as ligand-dependent transcription factors.Upon activation by the specific hormone, SHRs wereshown to interact with hormone-responsive elements(HREs) in the promoter/enhancer region of target genes.SHRs can also activate genes lacking HREs by virtue ofinteractions with other sequence-specific transcriptionfactors bound to their target sequences (2).

Important insights into the mechanisms of transcrip-tional regulation were recently obtained with studies onestrogen receptor (ER)-driven gene expression (3, 4). Byusing chromatin immunoprecipitation (ChIP), it wasdemonstrated that, once recruited to target promoters,estradiol-bound ER induces an ordered and cyclical re-cruitment of coactivator complexes, some of which con-tain histone acetyl transferases (HATs), histone methyl-transferases, or ATP-dependent remodeling activities (3,5). After these activating complexes, one observed re-cruitment of components of the ubiquitin-proteasomesystem, displacement of the receptors, and recruitment ofcorepressor complexes, containing histone deacetylases(HDACs). Even CpG methylation, generally associatedwith stable epigenetic silencing of transcription (6),showed cyclical changes critical for transcriptional regu-lation (7, 8). In the mouse mammary tumor virus(MMTV) promoter, we also detect waves of receptorand factors recruitment associated with histone modi-fication, but they are less pronounced and not repeti-tious (9). This is likely due to the fact that hormonalactivation of the viral promoter leads to a single roundof transcription (10).

Although in all hormonally regulated promoters chro-matin is remodeled to facilitate transient interactions withtranscription factors, much still remains to be understoodregarding the role of nuclear organization and the in-volvement of transcription factories in the coordinationof hormonal regulation of gene networks. Both intra-chromosomal and interchromosomal interactions betweenregulatory sequences are likely complex key processesthat involved enzymatic protein modifications, as re-

cently shown for the pS2 locus region (11). Global ChIp-Seq, as recently reported for the ER (12), will be useful tostart analyzing the function of these processes in the con-text of hormonal gene regulation.

In addition to these direct genomic effects, steroid hor-mones induce rapid nongenomic responses similar to thoseinitiated by peptide growth factors (13). For example, estro-gens activate the Src/p21ras/Erk and the PI3K/Akt pathwaysvia direct interaction of the ER" with the sarcoma (Src)homology (SH)2 domain of c-Src and the regulatory subunitof PI3K, respectively (14, 15). Activation of these kinasepathways is essential for estrogen-induced cell proliferationin breast cancer cells. Progestins can also activate these sig-naling cascades, either via an interaction of the progesteronereceptor (PR) with ER", which itself activates c-Src andPI3K, or by direct interaction of PR with the SH3 domain ofc-Src (16–18). The mechanism of this cross talk betweenSHRs and kinase signaling pathways has been a matter ofstudy in the past few years.

Progestin induces phosphorylation of PR-B at severalsites, including a MAPK consensus site, Ser345. Ser345-phosphorylated PR-B receptors strongly associated with thetranscription factor specificity protein 1 to regulate cell cyclerelevant genes (such as p21) and growth-promoting targetgenes, such as epidermal growth factor receptor (EGFR),whose promoters lack canonical progesterone response ele-ment (19). These events are critical for progestin-stimulatedregulation of specificity protein 1 target genes and breastcancer cell proliferation (19). The importance of the Src/MAPK signaling pathway for progesterone-induced tran-scription has been demonstrated with a PR mutant unable toactivate Src signaling. In breast cancer cells expressing thismutant PR, progesterone cannot induce cyclin D1 gene ex-pression and does not stimulate cell cycle progression (20).Complementary studies have been performed with ER usingestrogen-dendrimer conjugates, which because of theircharge and size remain outside the nucleus and can onlyinitiate extranuclear signaling (21). Genome-wide cDNAmicroarray analysis showed that around 25% of E2-regu-lated genes were estrogen-dendrimer conjugates responsive,highlighting the importance of extranuclear ER signalingpathways in regulating patterns of gene expression in breastcancer cells (22).

SHRs are localized predominantly in the nucleus, al-though they actively shuffle between nucleus and cyto-plasm (23). However, distinct pools of functional mem-brane-localized SHR have been described for estrogens(24), progestins (25, 26), or androgens (27). Overall, littleis understood regarding the mechanisms of translocationof SHR to the plasma membrane. A highly conserved nineamino acid motif in the ligand-binding domains of SHRshas been found to mediate their palmitoylation, which

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facilitates caveolin-1 association, subsequent membranelocalization, and steroid signaling (28).

SHRs have been shown to interact with many addi-tional kinase signaling pathways, including CyclinA/Cdk2, JAK/STAT, and EGF receptor. In addition to itsrole in cell cycle regulation, CyclinA/Cdk2 also partici-pates in the control of the transcriptional activity of ste-roid receptors (29). In particular, both ER" and PR areactivated by CyclinA/Cdk2 complex. The complex di-rectly phosphorylates ER", potentiating its transcrip-tional activity (30). In the case of PR, CyclinA/Cdk2 hasbeen reported to increase expression of progesterone tar-get genes (31–33) by a mechanism involving phosphory-lation of the coactivator SRC-1 (32). It seems that in thepromoters regulated by these receptors, CyclinA/Cdk2participates in multiprotein complexes that contain tran-scription factors, corepressors, and coactivators, includ-ing acetyl transferases (34). Interestingly, the ability ofCyclinA/Cdk2 to increase PR activity is independent of itsability to phosphorylate PR (32, 34). Because linker his-tones are main targets of cyclin-dependent kinase 2(Cdk2) (35, 36), one could postulate that activation ofCdk2 by steroid hormones could influence chromatinstructure and gene expression (37–40).

There is also evidence that SHRs interact with the JAK/STAT pathway and can form complexes with variousSTAT family members (41–43). Like PR, signal transduc-ers and activator of transcription (Stat)5a and Stat5b arerequired for normal mammary gland growth and differ-entiation. In breast cancer cells, progestin treatment in-duces translocation of Stat5 to the nucleus, mediated byassociation with PR (42). Moreover, the inhibition oflactogenic hormone induction of the #-casein gene in nor-mal mammary epithelial cells during pregnancy involves anegative cross talk between PR and STAT5a (44). Thereare other examples of PR action via STAT5 binding sites,such as in the mouse Bcl-X gene (45) and in the human11#-HSD2 gene (46).

Several studies have documented a transcriptional and/orproliferative synergy between EGF and progesterone or es-trogen (47, 48). Notably, progesterone up-regulates the ex-pression of EGFR family members on the cell surface (48–51). In addition to increasing the number of high affinityEGFR per cell, progesterone affects the phosphorylationstate of both EGF and c-erbB2 receptors (48). EGF signal-ing can mimic the actions of progestins by inducing changesin PR phosphorylation, nuclear association, and DNA bind-ing (52). Although EGF alone failed to induce appreciablePR transcriptional activity, the actions of ligand-bound PRare dramatically enhanced in the presence of EGF (52).

In this review, we will use progestin regulation of theMMTV promoter to exemplify these issues, and we will

focus on two interconnected fields within the wide area ofhormonal signaling networks. First, cross talk of nuclearreceptors with kinase signaling pathways; and second,chromatin remodeling. We will concentrate on the effectof progesterone on the Src/Erk/mitogen- and stress-acti-vated protein kinase 1 (Msk1) signaling pathway andhow this pathway influences chromatin remodeling andthe transcriptional activity of PR. We will finish with abrief summary of possible roles of linker histone subtypeson gene expression and how they could be influenced bya cross talk of PR with kinase signaling pathways.

Cross Talk of PR with the Src/Erk/MskPathway Participates inTranscriptional Regulation

Traditionally, the genomic and nongenomic actions of ste-roid hormones have been considered as two independentpathways, but we have found that the two pathways con-verge in the modification of structural components of chro-matin. Five minutes after progestin administration to T47Dbreast cancer cells, there is an increase in the activity of thecomponents of the Src/Ras/Erk cascade, which is essentialfor progestin-induced cell proliferation (18). This effect ismediated by a specific interaction between two domains ofthe N-terminal half of PR and the ligand-binding do-main of ER", which in this situation is activated in theabsence of estrogens (16). Activated ER" interacts directlywith the SH2 domain c-Src (15), activating its tyrosine ki-nase activity and consequently initiating the entire MAP ki-nase cascade. Because some of the kinases that phosphory-late core histones (Msk1 and Msk2) and histone H1 (Cdk2)are downstream substrates of Erk, we hypothesized that therapid cytoplasmic effects of steroid hormones could influ-ence their downstream chromatin targets.

Hormone-dependent activation of the MMTV pro-moter is blocked by inhibition of the Erk signaling path-way in breast cancer cells (53). Because Erk phosphory-lates PR at Ser294 in response to progestins (54), it ispossible that the transcriptional inhibition of MMTV in-duction is due to a lack of PR phosphorylation. However,a similar inhibition of MMTV induction was observedwhen interfering with the activation of Msk1, which doesnot compromise PR phosphorylation (53). After 5 min ofhormone addition, activated PR, Erk, and Msk1 form aternary complex, which is selectively recruited to theMMTV promoter nucleosome containing the HREs (Fig.1) (53). We know that the nonphosphorylated PR canalso bind to the exposed HREs on the MMTV promoternucleosome, but the binding is nonproductive and doesnot lead to derepression (53).

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Eukaryotic DNA is packaged into chromatin throughits association with histone proteins. The nucleosomecore particle consists of 146 bp wrapped around a histoneoctamer consisting of two copies each of the core histoneproteins H2A, H2B, H3, and H4. Concomitant with therecruitment of the ternary complex of phospho (p) PR/pErk/pMsk1 to the MMTV promoter, histone H3 be-comes phosphorylated at serine 10 and acetylated at ly-sine 14, only on the nucleosome containing the HREs andnot on adjacent nucleosomes (Fig. 2, middle panel) (53).Phosphoacetylation of histone H3 can be blocked by in-hibiting Erk or Msk1 activation resulting in a markedreduction of MMTV promoter activation by hormone.Blocking H3 phosphoacetylation precludes displacementof a repressive complex containing HP1!, as well as therecruitment of the Brg1-containing chromatin remodel-ing complex, thus preventing displacement of histone H2A/H2B dimers and subsequent promoter activation.

Most reports on the rapid action of PR have focused inthe cell signaling pathways activated by progestins (17,18, 55), but how these pathways are integrated with the

transcriptional function of PR has remained elusive. Wehave shown that some of the kinases activated by proges-tins in the cytoplasm phosphorylate PR and form a com-plex with the activated PR. The complex of activated PRand accompanying kinases is recruited to the target sitesin chromatin where the kinases modify chromatin pro-teins locally as a prerequisite for chromatin remodelingand gene regulation. Thus, we propose that the “non-genomic” and “genomic” pathways of progestin actionconverge on chromatin to enable gene regulation.

Hormone-Induced ATP-DependentChromatin Remodeling NeedsCooperation of Various EnzymaticActivities

Modulation of the structure and dynamics of nucleo-somes is an important regulatory mechanism in all DNA-based processes and is primarily catalyzed by chromatinremodeling complexes. Such complexes can either modify

FIG. 1. Initial steps of PR activation. Progestins bind to cytoplasmic PR/ER complexes, anchored in the cell membrane by palmitoyl residues, andactivate the Src/Ras/Erk pathway, leading to nuclear accumulation of activated pErk. The majority of PR is nuclear and associated with chaperones(Hsps). Upon binding of progestins, PR homodimers dissociate from chaperones, and a fraction of PR is phosphorylated by pErk, which alsophosphorylates Msk1. A “PR-activated complex” composed of pPR/pErk/pMsk1 is formed. Progesterone induction also activates other kinasesignaling pathways as Janus kinase (JAK)/Stat, phosphatidylinositol kinase (PI3K)/serine-threonine kinase (Akt), and Cdk2 (red asterisk).

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histone residues or use the energy of ATP hydrolysis toalter the relationship between histones and DNA (re-viewed in Refs. 56–60). The discovery of ATP-dependent

chromatin remodeling complexes hasbeen a major breakthrough in the un-derstanding of chromatin dynamics.There are two human switch/sucrosenonfermentable (SWI/SNF)-like com-plexes both containing hBRM (humanBrahma) or BRG1 (Brahma-relatedgene 1) as ATPase, as well as a series ofother subunits which differ dependingon the cell type (61). The stoichiometryof the SWI/SNF subunits are tightlyregulated by protein-protein interac-tion between BRG1-associated factor(BAF)155/BAF170 and BAF57 and byproteasome-mediated protein degrada-tion (62).

There is evidence for a role of theSWI/SNF complex in glucocorticoidgene regulation in yeast (63) and in an-imal cells (64). Indeed, hSWI/SNFseems to be required for chromatin re-modeling initiated by glucocorticoids(65); however, the situation appears tobe different for progestins, althoughthey act through the same HREs as glu-cocorticoids (65). The genome-widebinding of glucocorticoid receptor (GR)occurs mainly at constitutive or hor-mone-induced deoxyribonuclease(DNase) I hypersensitive sites, some ofwhich require Brg1-containing SWI/SNF complex, whereas others are Brg1independent (66). The H2A.Z histonevariant is highly enriched at both con-stitutive and inducible DNase I hyper-sensitive sites.

The MMTV long terminal repeat re-gion encompasses a promoter that inaddition to five degenerated HREs alsocontains a binding site for nuclearfactor 1 (NF1), located immediatelydownstream of the HREs. In chroma-tin, the MMTV-LTR is organized intopositioned nucleosomes (67), with anucleosome located over the five HREsand the NF1 binding site (68). On thispromoter nucleosome, the binding sitefor NF1 is not accessible (69). Onlytwo of the five HREs, the strong palin-dromic HRE1 and the weak half-palin-

drome HRE4, can be bound by hormone receptors. Thecentral HREs, in particular the palindromic HRE2 and

FIG. 2. Model for the role of “PR activated complex” in chromatin. Top, In the uninducedstate, an HP1!-containing repressive complex is bound to the promoter, keeping it silent.Middle, Upon hormone induction, activated PR complexes bind BAF and P300/CRE-bindingprotein-binding protein-associated factor (PCAF) and recruit them to the exposed HRE1 onnucleosome B. This is followed by H3 phosphoacetylation and displacement of the repressivecomplex and histone H1. Bottom, The BAF complex, stabilized by PCAF-dependent H3K14acetylation, catalyzes ATP-dependent H2A/H2B displacement and NF1 binding. This facilitatesbinding to the HREs 2 and 3 of further PR molecules with associated BAF, followed by othercoactivators and the basal transcriptional machinery, including RNA polymerase II, leading topromoter activation. PBAF, Polybromo-associated BAF; Nuc, nucleosome.

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the half-palindrome HRE3, are not accessible for recep-tor binding (70). After hormone induction in vivo, allHREs and the binding site for NF1 are occupied simul-taneously on the surface of a nucleosome-like struc-ture, and a functional synergism is observed betweenPR and NF1 (68).

Progesterone treatment of T47D-MTVL cells carryinga single integrated copy of the MMTV-Luc transgeneleads to recruitment of PR, SWI/SNF, and SNF2 h-relatedcomplexes to the MMTV promoter, accompanied by se-lective displacement of histones H2A and H2B from nu-cleosome B (71, 72). Thus, these two remodeling ATPasescould be part of the complexes responsible for the changesin chromatin sensitivity to nucleases detected 30 min afterhormone exposure (68). Recently, the acidic N terminusof the Swi3p subunit of yeast SWI/SNF was identified asa novel H2A/H2B-binding domain required for ATP-de-pendent H2A/H2B dimer displacement (73).

The ySWI/SNF complex could displace H2A and H2Bfrom well-positioned MMTV promoter nucleosomes butnot from a mouse ribosomal promoter nucleosome wellpositioned on DNA fragments of the same length (71).Thus, the nucleotide sequence seems to contain topolog-ical information that determines not only nucleosome po-sitioning but also the outcome of the remodeling process.

After hormone activation in breast cancer cells, theSWI/SNF complex named BAF is recruited to the MMTVpromoter, whereas the closely related PBAF complex isnot (Fig. 2) (74). The BAF250a/ARID1A subunit presentin BAF is essential for SWI/SNF-dependent transcrip-tional activation of the MMTV promoter and is a neces-sary facilitator of BRG1-mediated chromatin remodeling(75). Interestingly, BAF250 functions as an E3 ubiquitinligase adapter for histone H2BK120 (76). This new find-ing could have important implications for gene activa-tion, because H2BK120 ubiquitination could affect SWI/SNF chromatin remodeling (76).

Histone acetylation is a highly dynamic posttransla-tional modification that plays an important role in geneexpression. High doses of histone deacetylase inhibitors,butyrate or TSA, lead to intense hyperacetylation of corehistones and inhibit hormone induction of the MMTVpromoter (77, 78) without altering nucleosome position-ing (78). However, low doses of the inhibitors activate theMMTV promoter in the absence of hormone and generatea DNAse I-hypersensitive site similar to that observedafter hormone treatment (77). This suggests that partialacetylation of histones, other chromatin proteins, orother factors can generate a chromatin structure similarto that induced by hormone.

Histone acetylation at the MMTV promoter in re-sponse to hormone shows an initial increase followed by

an eventual net deacetylation of histone H4 (79). Thehistone deacetylases HDAC1 and HDAC3 are bound tothe MMTV promoter before transcription activation, andtheir levels fluctuates after hormone treatment (79). Thehistone acetyltransferase PCAF is required for progestininduction of target genes and catalyzes the acetylation ofhistone H3 at K14. This epigenetic mark interacts withthe BAF subunits anchoring the complex to chromatin(Fig. 2) (74). Thus, for full activation of the MMTV pro-moter, cooperation between the two chromatin remodel-ers BAF and PCAF is needed.

Once the BAF complex is recruited to the MMTV pro-moter, via interaction with PR and with acetylatedH3K14, it displaces histones H2A and H2B, thus facili-tating NF1 binding (Fig. 2) (74). The presence of NF1 atthe promoter favors binding of PR and associated BAFmolecules to the previously hidden HREs 2 and 3, not viaprotein-protein interaction but by exposing these HREson the surface of an H3/H4 tetramer (80). These sites arenot essential for ATP-dependent H2A/H2B displacementor NF1 binding but are critical for complete PR loadingand MMTV promoter activation (80).

Fluorescence photobleaching experiments in livingcells containing a tandem of 200 MMTV promoters driv-ing reporter genes have provided direct evidence that thehormone-bound GR undergoes rapid exchange betweenchromatin and the nucleoplasmic compartment with ahalf-life of less than a minute (81). This rapid exchangehas also been reported for the ER and PR (82–84), fur-ther supporting the model of a highly dynamic turnover oftranscription complexes at promoters. In agreement withthe difficulties in visualizing a footprint of GR on theMMTV promoter in vivo (85) is the formulation of a“hit-and-run” model of receptor action (81). This rapidturnover measured by fluorescence recovery after photo-bleaching (FRAP) contrasts with the results obtained byChIP, which reveal cyclical waves of receptor associationto promoters with periods of 10–90 min (3–5). It is likelythat the 20-min cycles of receptors detected by ChIP donot reflect the behavior of individual receptor moleculesbut instead indicate changes in the configuration of thepromoter that favor factor binding. It is also possible thatmany of the receptor interactions detected by FRAP aretransient and nonproductive and that more stable bindingrequires interaction with other transcription factors orother chromatin components. An example of this behav-ior has been recently demonstrated by FRAP for GR andHMGB1 (86). Conversely, GR appears to bind to preex-isting regions of open chromatin, as detected by DNase Ihypersensitivity (66). Because the pattern of higher orderchromatin architecture that generates DNase I sensitivityis likely established during cell differentiation, cell iden-

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tity could play a major role in the determination of tissue-selective receptor function (66).

PR and NF1 Synergize on the MMTVPromoter Wrapped Around andH3/H4 Tetramer

A tetramer of histones H3 and H4 would be a feasiblestructure of the MMTV “remodeled nucleosome” afterhormone induction. H3/H4 tetramers position MMTVpromoter sequences in a similar way to histone octamers,but NF1 can bind to a H3/H4 tetramer particle with rel-atively high affinity (87). MMTV sequences positionedaround an H3/H4 tetramer can bind PR and NF1 simul-taneously reminiscent to what is observed in cells carryinga single copy of the MMTV promoter integrated in chro-matin (68). Binding of PR to the MMTV tetramer particleis enhanced if NF1 is prebound, indicating that binding iscooperative. The nature of this synergism is unknown,but one possibility is that the deformation of the DNAimposed by NF1 binding debilitates the interaction ofDNA with the H3/H4 tetramer particle, consequently fa-cilitating access of PR to the essential HREs 2 and 3,which in the octamer particle are oriented with their ma-jor grooves pointed toward the histones (80). This modelprovides a direct molecular mechanism for the observedfunctional synergism between NF1 and PR during induc-tion of the MMTV promoter (88).

Role of Histone H1

Histone H1 is the prototype of the “linker histones,”which are in contact with the linker DNA that joins con-secutive nucleosomes (89). Histone H1 participates in nu-cleosome positioning, nucleosome spacing, and in thehigher-order structure of chromatin. H1-containing chro-matin is more resistant to nuclease digestion and showsstrong inhibition of nucleosome sliding (90). Conse-quently, H1 is seen as a structural component related tochromatin compaction and inaccessibility to transcrip-tion factors or RNA polymerase. In addition, H1 seems tobe actively involved in the regulation of gene expression,as it inhibits chromatin remodeling by the ySWI/SNFcomplex (91). However, the repressive role of histone H1on gene expression is controversial. Although it has beenreported that MMTV promoter chromatin is depleted ofhistone H1 after hormonal induction (92), overexpres-sion of histone H1 in cultured cells enhances hormonaltrans-activation of the promoter (93).

Histone H1 in mammals consists of a family of closelyrelated, single-gene encoded proteins, including five so-matic subtypes (from H1.1 to H1.5) and a terminally

differentiated expressed isoform (H1.0). Because knock-down of individual somatic H1 subtypes in mouse has nomarked phenotype (94), they have been assumed to behighly redundant. However, inducible knockdown of in-dividual somatic H1 subtypes in breast cancer cells al-tered a different subset of genes, and the majority of themare down-regulated upon H1 depletion (95). This arguesagainst a general repressive role of linker histones andsuggests some nonredundant effects on gene expression.Indeed, depletion of individual subtypes had different ef-fects on cell survival. H1.2 depletion specifically causescell cycle arrest due to repression of key cell cycle genes,whereas H1.4 depleted cells eventually die of necrosis.Moreover, although H1.2 accounts for approximatelyonly 20% of the total H1 content in T47D breast cancercells, its depletion causes a general decrease in nucleo-some spacing (95) that is not compensated by the overex-pression of other subtypes. This suggests that individualsubtypes have a selective effect on chromatin structure.Thus, specific phenotypes are observed in breast cancercells depleted of individual histone H1 subtypes, support-ing the idea that distinct roles do exist for the linker his-tone variants in these cells and proposes that theoreticallythe same situation may also occur in other cell types.

A recent study using Atomic Force Microscope showedthat different H1 subtypes exhibit a different affinity forchromatin and differ in their capacity to condense chro-matin (96). H1 subtypes can be classified as weak con-densers (H1.1 and H1.2), intermediate condensers(H1.3), and strong condensers (H1.0, H1.4, H1.5, andH1x). The variable C-terminal domain is required fornucleosome spacing by H1.4 and is likely responsible forthe chromatin condensation properties of the various sub-types, as shown using chimeras between H1.4 and H1.2(96). Moreover, linker histones do not preclude ATP-dependent remodeling of minichromosomes by yeastSWI/SNF or Drosophila NURF when tested in minichro-mosomes, a dynamic system that mimics the situation invivo. Thus, linker histone subtypes can be considered asdifferential organizers of chromatin, rather than generalrepressors (96).

Asymmetric binding of histone H1 to chromatin-orga-nized MMTV promoter sequences compacts the nucleo-somal structure (97) and leads to repression of basal tran-scription and reduced binding of NF1 (10). In contrast,H1 containing MMTV chromatin binds PR with higheraffinity and is transcribed more efficiently in the presenceof PR and NF1 than chromatin free of linker histone (10,97). Thus, histone H1 represses hormone independenttranscription and enhances the synergism between PRand NF1, resulting in tighter hormonal regulation (10).

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This positive effect of H1 is likely due to a more ho-mogeneous nucleosome positioning over the MMTV pro-moter (10). Therefore, H1 plays a key role during theinitial hormonal activation of the MMTV promoter innative chromatin by favoring a better nucleosome posi-tioning and a higher binding of PR. However, for tran-scription to take place, H1 has to be phosphorylated anddisplaced from the promoter (10). Phosphorylation islikely mediated by a protein kinase recruited to the pro-moter by activated PR. In view of the reported activationof Cdk2 after progesterone treatment (33), and due to itsability to phosphorylate histone H1 (37), this kinase is agood potential candidate for catalyzing progesterone-in-duced phosphorylation of histone H1. One could envis-age a more general role for the phosphorylation of histoneH1 by Cdk2, because this modification disrupts the inter-action between H1 and HP1" promoting decondensationof chromatin independent of H3K9 methylation (36). Incombination with the in vitro results, these considerationsjustify the inclusion of histone H1 and its phosphoryla-tion in a hypothetical model of MMTV activation (Fig. 2).Additional experiments will reveal the nature of the PR-containing complexes responsible for phosphorylationand displacement of histone H1 subtypes from progestin-regulated promoters.

PR vs. GR

There are similarities between PR and GR during hor-mone-regulated events. Briefly, both receptors recognizethe same DNA sequence (98), are phosphorylated by Erkkinases after hormone induction (99, 100), show interac-tion with SWI/SNF (65), recruit the SWI/SNF complex tothe target chromatin, are dependent on HATs for its ac-tivity, and associate with several common coactivatorsand corepressors (101, 102). However, although GR ex-hibits rapid nongenomic effects, it activates signalingpathways different from those activated by PR. GR acti-vates p38 and JNK MAPKs through a PKC-dependentpathway and is also implicated in the inhibition of EGFRsignaling in lung epithelial cells (103, 104), whereas thispathways have not been reported to be activated by PR.Despite the fact that both PR and GR share the sameDNA binding sequence, clear differences in their interac-tions with the HRE region of the MMTV promoter havebeen observed (105). Thus, the binding of the receptors togenome loci depends not only on the DNA sequence.Rather, other factors, such as chromatin state, cell type-specific transcription factors, and signaling pathways,may determine the specific genomic interactions of bothreceptors.

Both GR and PR depend on HATs for their activity,but they utilize overlapping but not identical sets of co-activators. Progesterone promotes the recruitment by PRof SRC-1/SRC-3, whereas dexamethasone enhances GRinteraction mainly with SRC-2 and SRC-3 (106). Thus,the SRC family members could play an important butreceptor-specific role in the orchestration of downstreamevents at target promoters (106).

Conclusions

The initial steps of gene induction by progestins involvemainly changes in chromatin organization of target pro-moters that require the activation of kinase signalingpathways initiated by membrane-bound PR. These ki-nases eventually phosphorylate the receptor to which theybind. The receptor complexes containing the kinases andhistone modifying enzymes are recruited to the target pro-moters where they modify the protruding core histonetails and the linker histones. These modifications lead tothe displacement of linker histones and a repressive com-plex containing HP1!, likely by specialized ATP-depen-dent remodeling complexes. In a second step, other spe-cialized ATP-dependent remodelers displace histoneH2A/H2B dimers from the promoter nucleosome, en-abling synergistic access of other transcription factors andadditional receptor complexes to previously hidden bind-ing sites on the surface of a histone H3/H4 tetramer par-ticle. It is only after completion of these initial chromatinremodeling steps that complexes containing mediator andRNA-polymerase with associated basal transcription fac-tors are recruited and further steps in transcription initi-ation, elongation, RNA splicing, etc. can take place. Itremains to be seen how these findings can be reconciledwith the rapid exchange of GR-green fluorescent pro-tein observed in living cells carrying a cluster of some200 MMTV reporters (81). The activation of theMMTV promoter seems to be a very dynamic process,where PR, as a master key regulator, directs the rapidexchange of chromatin remodeling complexes on chro-matin. One possibility is that the majority of the GRbinding events observed in the MMTV cluster in vivoreflect nonproductive or abortive binding of the vari-ous PR-containing complexes. Thus, a fully productiveinteraction would only occur when the different com-plexes are recruited in appropriate combinationsand/or in the correct sequence.

Acknowledgments

Address all correspondence and requests for reprints to: GuillermoP. Vicent, Centre de Regulacio Genomica, Universitat Pompeu

Mol Endocrinol, November 2010, 24(11):2088–2098 mend.endojournals.org 2095

Fabra, Parc de Recerca Biomedica, Dr. Aiguader 88, E-08003Barcelona, Spain. E-mail: guillermo.vicent@crg.es; or miguel.beato@crg.es.

This work was supported by grants from the EuropeanUnion (High-throughput Epigenetic Regulatory OrganisationIn Chromatin integrated project), the Departament d!InnovacioUniversitat I Empresas, the Ministerio de Educacion y CienciaGrants BMC 2003-02902 CSD2006-00049, and the Fondo deInvestigacion Sanitaria Grants PI0411605 and CP04/00087.G.P.V. was a recipient of a fellowship of the Ramon y CajalProgram.

Disclosure Summary: The authors have nothing to disclose.

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