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EMBO Retinoids 2011: mechanisms, biology andpathology of signaling by retinoic acid andretinoic acid receptorsNeil J. McKenna

Corresponding Author: [email protected]

Department of Molecular and Cellular Biology and Nuclear Receptor Signaling Atlas (NURSA), Baylor College of Medicine, Houston, Texas, USA

Retinoic acid (RA) is one of the principal active metabolites of vitamin A (retinol) which mediates a spectrumof critical physiological and developmental processes.Transcriptional regulation by RA is mediated primarilyby members of the retinoic acid receptor (RAR) subfamily of the nuclear receptor (NR) superfamily oftranscription factors. NRs bind specific genomic DNA sequence motifs and engage coregulators andcomponents of the basal transcription machinery to effect transcriptional regulation at target gene promoters.Disruption of signaling by retinoic acid is thought to underlie the etiology of a number of inflammatory andneoplastic diseases including breast cancer and haematological malignancies. A meeting of internationalresearchers in retinoid signaling was convened in Strasbourg in September 2011 under the auspices of theEuropean Molecular Biology Organization (EMBO). Retinoids 2011 encompassed myriad mechanistic, biologicaland pathological aspects of these hormones and their cognate receptors, as well as setting these advancesin the context of wider current questions on signaling by members of the NR superfamily.

Received October 24th, 2011; Accepted February 10th, 2012; Published February 27th, 2012 | Copyright © 2012, Neil J. McKenna. This is anopen-access article distributed under the terms of the Creative Commons Non-Commercial Attribution License, which permits unrestricted non-commercialuse distribution and reproduction in any medium, provided the original work is properly cited.

Cite this article: Nuclear Receptor Signaling (2012) 10, e003

IntroductionVitamin A, or retinol, is an essential nutrient forvertebrates. Retinoic acid (RA), the major bioactivemetabolite of retinol, is a morphogen with pleiotropic rolesin cell growth and differentiation in embryonicdevelopment and adult physiology [Chambon, 1996; Market al., 2009]. A number of key proteins and enzymescontrol retinoid metabolism and regulate the bioavailabilityof retinoic acid to its target cells. Serum retinol-bindingproteins (RBPs) and cellular retinol binding proteins(CRBPs) transport retinol in the serum and cells; retinoldehydrogenases (RDHs) and retinal dehydrogenases(RALDHs) mediate the biosynthesis of RA from dietaryvitamin A, the latter group of enzymes catalyzing the ratelimiting step in RA biosynthesis; and the cellular RAbinding proteins (CRABPs) mediate the uptake of RA intarget cells.

RA signaling in its target cells occurs primarily throughbinding to members of the retinoic acid receptor (RAR)subfamily of the nuclear receptor (NR) superfamily ofligand-activated transcription factors [Aagaard et al., 2011;Tsai and O'Malley, 1994], namely RARα, RARβ andRARγ. Like other NRs, RARs contain a domain thatmediates interaction with all-trans RA (the ligand bindingdomain, or LBD), a zinc finger-containing DNA bindingdomain (DBD) that binds to RA response elements(RAREs) in target genes; and a dimerization domain thatengages members of the retinoid X receptor (RXR)subfamily in RXR/RAR heterodimers [Rochette-Egly andGermain, 2009]. RA-bound RARs effect transcriptionalregulation by recruitment of coactivators and thesubsequent assembly of a transcriptional preinitiation

complex at target gene promoters [McKenna andO'Malley, 2002]. In contrast, in the absence of RA,unliganded RARs exert repressive effects on target genepromoters by recruitment of corepressor complexes.Central to this model is the engagement by RARs ofepigenetic modifiers of promoter chromatin architecture,creating alternately permissive and restrictiveenvironments for transcriptional initiation [Martens et al.,2011]. In addition to direct genomic effects via RARs, RAis known to engage cellular signaling pathways thatmediate pre-genomic kinase cascades that also ultimatelyeffect regulation of gene expression.

The myriad mechanistic and biological facets of retinoidsignaling pathways, and the pathologies associated withtheir disruption, were the focus of a recent EuropeanMolecular Biology Organization (EMBO)-sponsoredconference held at the Institut de Génétique et de BiologieMoléculaire et Cellulaire (IGBMC) in Strasbourg, France,on September 22-24th, 2011. The conference broughttogether researchers from a wide variety of disciplines,including phylogenetics, cell biology, biochemistry,structural biology, developmental biology and clinicalpractice, who collectively conveyed a keen appreciationof the fundamental roles played by retinoids and RARs,as well as other members of the NR superfamily, inorchestrating programs of transcriptional regulationrequired for the integrity of cellular growth anddifferentiation.

Keynote Lecture: Michael G. RosenfeldMany models of tumor development invoke random DNAtranslocation events which confer selective growth

www.nursa.org NRS | 2012 | Vol. 10 | DOI: 10.1621/nrs.10003 | of 12

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advantage on the cancerous cell. Michael Rosenfeld(UCSD, La Jolla, USA) proposed an alternative modelof tumor translocation in which non-random, induced DNAtranslocation events result in cell proliferation andselection. TMPRSS2 is an androgen-regulated genelocated on human chromosome 21q22.3 that encodesan enzyme of the serine protease family. The humanERG gene, also at 21q22.3, encodes a member of theerythroblast transformation-specific (ETS) family oftranscription factors. Up to 70% of all prostate tumorshave a specific fusion of TMPRSS2 and ERG. Rosenfelddescribed work showing that dihydrotestosterone(DHT)-induced spatial proximity, involving nuclear myosinI-dependent motors, was required for interchromosomaltranslocation between TMPRSS2 and ERG in prostatetumors. The induced double-stranded DNA break siteswere localized to intronic regions of the gene translocationpartners, and were shown to involve the association ofandrogen receptor (AR) with an AID/Gadd45 complex,the recruitment of which was abolished by selective ARmodulators (SARMs). Gadd45/GADD45A is a memberof the Gadd45 family of acidic nuclear proteins, whichalso contains MyD118 and CR6, whose transcript levelsare increased following exposure of cells to stressconditions and treatment with DNA-damaging agents.While Rosenfeld’s observations were made in the contextof tumor translocation events, the invocation of longdistance DNA-DNA interactions in the context of ARaction has exciting implications for the model of regulationof gene expression by this receptor and conceivably othermembers of the NR superfamily.

PIWIL1 is a member of the PIWI subfamily of Argonauteproteins, which play important roles in stem cellself-renewal, RNA silencing and translational regulationin diverse organisms. Sketching a model castingcorepressors as protectors against translocation eventsin cancer, Rosenfeld drew attention to decreased levelsof PIWIL1 in LNCaP prostate cancer cells. Supportingsuch a notion, he described experiments showing thattranslocation events attributable to genotoxic stress wereincreased in the context of PIWIL1 knockdown anddecreased in the context of PIWIL1 overexpression,observations that appeared to apply generally to othertranslocations in addition to TMPRSS2/ERG. Rosenfeldbriefly alluded to a chemical library screen for inhibitorsof translocation using expression of fusion transcripts asa readout and focused on one particular hit, SD70, thatwas shown to bind to the active site of lysine demethylase4C (KDM4C). Rosenfeld proceeded to describe a3-dimensional DNA annealing, selection and ligation(DASL) strategy that had been used to show thatE2-induced binding of estrogen receptor α (ERα) to targetgene enhancers causes activation through induction ofbidirectional non-coding RNA (ncRNA) transcripts andshort range enhancer-promoter looping. In support of this,the SD70 compound, which had earlier been shown toinhibit translocation events, inhibited 17βE2 induction oftarget genes. The principle of DNA breakage eventsplaying a key role in NR-mediated regulation of geneexpression developed into a recurring theme throughoutthe meeting, and would later feature in talks by Pierre

Chambon and Jean-Marc Egly (IGBMC, Strasbourg,France).

While retinoids are well-known inducers of stem celldifferentiation, the cellular mechanisms underlying thisbiology are unclear. Rosenfeld showed that RA treatmentof human stem cell lines, such as Ntera-2, but not stemcell lines from other species, induced RNA PolymeraseIII-regulated DR2 Alu transcripts. These transcripts werestabilized through an interaction with Ago3, a member ofthe Argonaute AGO subfamily, and accumulated incytoplasmic P bodies, where they underwentDicer-dependent processing and mediated degradationof specific stem cell mRNAs and subsequent exit fromthe stem cell state (Figure 1). A biochemical strategy toidentify Ago3-interacting proteins implicated EDC4 in theassembly of a decapping complex and the subsequentdegradation of the stem cell RNAs, leading to inhibitionof stem cell proliferation and progression to a neuronalstem cell fate. Placing his results in a broader context,Rosenfeld speculated that by dint of their sheer numbers– approximately 3500 identified to date – non-codingRNAs were eminently positioned to function as globalmodulators of the regulation of gene expression bymultiple classes of transcription factors.

Figure 1. Role of non-coding RNAs in RA signaling during neuronaldifferentiation. RA induces a RAR, PolIII-dependent activation of DR2Alu repeats in embryonic stem cells, generally adjacent to active PolIItranscription units, and these transcripts are processed and serve to causeAgo3-dependent degradation of stem cell mRNAs harboringcomplementary sequences, with biological effects on signal-dependentexit from the stem cell state.

Special Plenary Lecture: PierreChambonGlucocorticoids are one of the most widely prescribedprescription drug classes and have applications in a broadrange of therapeutic indices, including cancer,osteoporosis and inflammatory disorders such as asthmaand eczema.Their anti-inflammatory properties derive inpart from the ability of agonist-bound GR to effecttransrepression of transcription factors such as AP-1,which are key components of the inflammatory signalingresponse (Figure 2). Despite their clinical success, the


Review EMBO Retinoids 2011

myriad side-effects that accompany their use haverestricted their more widespread use, and the pursuit ofdissociated glucocorticoids, which retain the beneficialanti-inflammatory properties at the expense of undesirableside effects of GR transactivation, is the subject ofintensive investigation in the pharmaceutical industry.Pierre Chambon (IGBMC, Strasbourg, France)described studies into an inflammatory skin condition thatshed light on fundamental mechanisms of transcriptionalregulation by GR and other NRs.

Figure 2. Beneficial and undesirable clinical effects ofglucocorticoids are mediated by distinct transcriptional mechanisms.

See text for details.

Human atopic dermatitis (AD) is a familial skin conditionaffecting up to 20% of children and 3% of adults inWestern society and is characterized by erythema, scalingand epidermal hyperplasia, among other symptoms.Whileglucocorticoid therapy is effective for the disease, chronictreatments using these drugs carry with them debilitatingside-effects. Reviewing previous findings in his laboratory[Li et al., 2009; Li et al., 2006], Chambon described amouse model, in which RXRα and RXRβ were selectivelyablated in epidermal keratinocytes, that developed a skinand systemic syndrome mimicking AD that wasattributable to ectopic expression of the inflammatorycytokine thymic stromal lymphopoietin (TSLP). Undernormal physiological conditions in epidermalkeratinocytes, multiple signaling pathways exertrepressive effects on the TSLP promoter. UnligandedRXRα/VDR and RXRβ/RARγ heterodimers, for example,were shown to bind to VDREs (DR3-d, DR3-f and DR3-g)and a RARE (DR2-b) respectively, in the upstream regionof the TSLP promoter. However, topical treatment withvitamin D3 or its hypocalcemic analog MC903, along withRA, results in the clearance of corepressors andrecruitment assembly of coactivators to the TSLPpromoter, induction of the gene and generation of anAD-like condition.The pivotal role of TSLP in the etiologyof AD led Chambon to speculate whether the beneficialeffects of glucocorticoid therapy in AD might be mediated

in part by repression of TSLP. He demonstrated that theGR agonist fluocinolone acetonide repressed TSLP inepidermal keratinocytes in a GR-dependent manner. Atandem manual and bioinformatically-assisted search ofthe TSLP promoter identified an IR1 (consensus invertedrepeat separated by a single nucleotide) that was shownto bind liganded GR in vivo, along with a cadre ofcorepressors and associated repressive histone writers,including HDAC2 and HDAC3. Referring to this sequenceas an inverted repeat negative GRE (IR nGRE), hedescribed a chromosome conformation capture assay,similar to that discussed earlier by Rosenfeld, whichdemonstrated that binding of GR to the nGRE precludedlooping of the region containing the activating VDRE withthe proximal promoter and subsequent initiation oftranscription. Placing the mechanistic studies in a widerclinical context, Chambon noted not only that side effectsof glucocorticoid-based therapy were attributable incertain cases to nGRE-dependent repression, but thatavailable dissociated glucocorticoids retained the abilityto directly transrepress through IR nGREs. Theseprovocative findings have profound consequences for thedirection of efforts to fine-tune glucocorticoid therapy, andpoint to the possible development of a new class of“improved” dissociated GCs which lack all GR functions,including transactivation and direct transrepressionthrough nGREs, but not tethered transrepression [Surjitet al., 2011].

Phylogeny of retinoid signalingWhile the evolutionary functions of RARs have been wellstudied in chordates, outside this phylum, evidence forroles of RA signaling is scarce and the evolutionary originof the RA pathway itself thus remains elusive. VincentLaudet (IGFL, Lyon, France) highlighted the greatvariability in the NR complement of different organismsin the same subphyla. While vertebrates, he observed,have 50 NR genes, fish have up to 70; similarly,invertebrates share 25 known receptor genes, butnematodes have 250, while sponges have only a pair.Laudet suggested that NR phylogeny, placed in thecontext of metazoan evolution, was needed to detect NRorthologs. Such an approach was used to show that thegenome of the Urbilaterian, the hypothetical last commonancestor of the bilaterian clade, contained about 25 NRgenes, considerably more than anticipated. This numberincluded orthologs of liganded receptors, which werepreviously thought to be specific to vertebrates. Moreover,he suggested that the NR phylogenetic tree, previouslycomprising 6 known subfamilies, could be updated toinclude 3 new subfamilies, namely NR7, NR8 and NR9,which are present in molluscs and annelids, but whichhave been lost in Drosophila, nematodes and vertebrates.Laudet suggested that the ancestral NR was most likelyto be a low affinity fatty acid sensor (Figure 3). Turningto a discussion of the evolution of retinoid signaling, hepointed out that the DNA binding domains of metazoanRARs and RXRs are well conserved, and the RAR/RXRheterodimer was present in ancestral bilaterians. Laudethighlighted the good conservation of residues contactingRA in the LBD of RARs from various metazoans, although



LBDs of early metazoans such as Amphioxus exhibitedmore variability than those of vertebrates. Laudet stressedthe fact that there is a marked difference between thespecies tree and the NR tree: ultraspiracle in locusts, forexample, is more similar to its human ortholog than thatin Drosophila.

Classical models of retinoid signalingClassical retinoid signaling involves the RAR-mediatedtranscriptional activation or repression of target genes inresponse to the presence or absence of RA.The bindingof retinoic acid to RARs results in their formation ofheterodimers with RXRs and their interaction with RAREsin the enhancer regions of target genes, primarily directrepeats separated by one (DR1), two (DR2) or five (DR5)nucleotides. These heterodimers associate withcoactivator complexes and effect long-distancecommunications with the promoters of target genes,culminating in the recruitment of basal transcription factorssuch as TFIIH, the stabilization of a RNA PolymeraseII-nucleated preinitiation complex and the transcription ofthese genes. In contrast, the establishment of repressivecomplexes in the absence of agonist is associated withsilencing of target genes. Interactions of coregulators withNRs are known to involve specific α-helical sequencesreferred to as NR boxes, or LXXLL motifs in the case ofcoactivators [Heery et al., 1997] and CoRNR boxes inthe case of corepressors [Hu and Lazar, 1999]. A solidbody of evidence supports the notion that acetylation andmethylation reactions catalyzed by coregulator-associatedenzymes target a variety of substrates, including histones,and contribute to specific epigenomic signatures acrosstarget promoters that are critical determinants of theirtranscriptional productivity.

Ligand, receptor and coregulator interactions

Small angle X-ray scattering (SAXS) and small angleneutron scattering (SANS) are complementary techniquesused for structural characterization of crystallizedmolecules that yield data in the nanometer range. DinoMoras (IGBMC, Strasbourg, France) discussed a seriesof SAXS and SANS-based crystal structures of RARβ insolution that painted an intimate portrait of this receptor’sassociation with several coregulators. Moras observedthat while the RARβ LBD was stable as a monomer insolution (Figure 4A), the functional form in vitro was aheterodimer with a member of the RXR subfamily. Hedemonstrated that the stoichiometry of the interaction ofMED1/TRAP220 with RARβ was 1 coregulator:1RARβ/RXR heterodimer (Figure 4B) – a ratio that heldtrue for other coregulators such as SRC-1/NCOA1 andTIF2/NCOA2, and that mutation of the RXR moiety hadno effect on the binding of TRAP220/MED1 to theheterodimer. By inducing asymmetry in the complex, DNAsequence and spacing proved to be critical modulatorsof the association of coactivator with receptor, suggestinga structural basis for the well-known effect of promotercontext on receptor and coactivator function [Korzus etal., 1998]. Moras went on to show that in solution, RARhomodimers were asymmetrical, and that ligand alonedid not induce dimerization and that the presence of a

coactivator peptide established an equilibrium betweenthe RARβ monomer and heterodimer. Moreover, in thecontext of a longer domain of SRC-1, the dimeric formwas even more stable, such that a complex comprisingRARβ and a 20kD SRC-1 receptor interaction domain(RID) was principally in the dimeric form. Moras sketcheda model in which binding of coactivator induces aconformational change in the liganded LBD, resulting inincreased stability of the dimeric form, and in whichnegative co-operativity reduces the affinity of the complexfor a second coactivator moiety (Figure 3).

While the centrality of coregulator exchange as a generalmechanism in the dual activation and repressivetranscriptional properties of NRs has been accepted forover a decade [Glass and Rosenfeld, 2000],structure-function studies probing the molecular basis ofRAR-coregulator interactions have been few in number.William Bourguet (Montpelier, France) described anumber of crystallographic studies of RARs whichilluminated the role of specific interaction interfaces onboth receptor and corepressor in mediating genesilencing. Bourguet pointed out that RARs possess astrong basal repression function which RARα antagonistssuch as BMS614 can induce by disrupting the coactivatorrecognition surface of RARα by displacing helix H12.Bourguet presented two structures representing theopposing functionalities of RARα, a repressive form incomplex with the antagonist BMS493 and CoRNR1 ofSMRT/NCOR2 (to which RARα exhibits preferentialbinding), and an active form in complex with the agonistAm580 and an LXXLL motif-containing peptide. Whilebroad similarities are evident between the two structures,they are distinguishable by secondary structures adoptedby residues in helix H12 of the receptor, namely α-helicalin the active form and adopting a predominantly β-strandcharacter in the repressive form (Figure 5). Bourguetproceeded to discuss mammalian two-hybrid dataindicating that residues adopting a β-strand conformationin CORNR1 were essential for corepressor/RARinteractions. Similarly, residues in β-strand 3 (S3) of theRARα LBD were critical for corepressor recruitment: asingle mutation (I396E) prevents corepressor bindingwhile preserving coactivator recruitment in the presenceof agonist, and confers a much higher basal activationfunction than that of the wild type receptor. Bourguetsuggested that by uncoupling the repressive andactivational functions of RARα, the I396E mutant mightbe a valuable molecular tool with which to probe themolecular mechanism of RAR-mediated gene silencingmore deeply.

TFIIH is a conserved multi-subunit complex involvedprimarily in transcriptional initiation and elongation andis comprised of a 7-subunit core, associated with a3-subunit CDK-activating kinase (CAK) module. Mutationsin genes encoding subunits of TFIIH involved in nucleotideexcision repair (NER) have been characterized in a varietyof genetic diseases, including xeroderma pigmentosum(involving XPB/TCRR3), trichothiodystrophy (involvingXPD/TTD/ERCC2) and Cockayne syndrome (involvingXPG/ERCC5). These diseases share a spectrum of



Figure 3. Evolution of retinoid signaling. See text for details.

Figure 4. Stable forms of RARα in solution and in vivo. A. The RARα LBD exists as a stable monomeric form in solution. B. The functional formof RARα is as a heterodimer with RXR. The stoichiometry of its interaction with its heterodimeric partner and coactivator is 1:1:1 RAR:RXR:coactivatorpeptide.

common symptoms involving the skin (photosensitivity,skin cancer), development (growth and skeletalretardation) and neurological deficits. In addition tophosphorylating RNA Polymerase II, TFIIH has beenshown to phosphorylate the N-terminal domains of severalnuclear receptors, including RARα. Jean-Marc Egly(Illkirch, France) showed data demonstrating thatresidues in XPD played an essential role in anchoring thebinding of the CAK moiety to the core TFIIH. Egly went

on to show that transfection of wild type XPD into theHD2 cell line, which contains a XPD R683G mutation,restored RARα phosphorylation concomitant withrestoring the responsiveness of these cells to all-transretinoic acid, indicating a critical functional link betweenTFIIH phosphorylation of the receptor and itstranscriptional function. Moreover, a RARα containing aphosphomimetic mutation at one of the serine residuesdiscussed in Bruno Kieffer’s talk (S77E) was



Figure 5. A secondary-structure switch controls constitutive gene repression by RAR. The constitutive recruitment of corepressor (CoR) byRAR is conferred by an extended β-strand (S3) that forms an antiparallel β-sheet with CoR residues (strand β1).This specific interaction allows dockingof helix α1 of the CoR into the coregulator binding groove of RAR otherwise masked by helix H12. Upon agonist binding, a β-strand-to-α-helix transitionallows for helix H11 formation which in turn provokes CoR release, repositioning of the activation helix H12 and coactivator (CoA) recruitment. Forclarity, only the regions involved in the interaction between RAR, CoR and CoA are depicted.

transcriptionally-competent in HD2 cells. Egly next useda ChIP-based approach to clarify the mechanistic basisof the involvement of NER factors in transcription. Hedescribed the recruitment of NER proteins at target genepromoters and showed that transient siRNA-mediatedknockdown of NER factors disrupted patterns of histonemodifications (H3K9/14) and DNA methylation(H3K4/H3K9) at RAR-regulated promoters. RecallingRosenfeld’s earlier talk, Egly showed that XPG mutationsprevented the establishment of a chromatin loop betweenthe promoter and the terminator during RAR activation,suggesting that these factors were required for relaxingDNA and establishing conditions favorable for acetylationand demethylation. Concluding, he speculated that thepathological consequences of mutations in NER proteinsin genetic disorders could be ascribed in large part totheir role in establishing transcriptionally-permissiveenvironments at promoters regulated by a broad rangeof transcription factors.

In fibroblasts undergoing in vitro differentiation intoadipocytes, the CRABP family member CRABPI is subjectto alternate up- and downregulation by thyroid hormone(T3). Li-Na Wei (University of Minnesota, Minneapolis,USA) discussed recent work in her laboratoryinvestigating the role of coregulators in mediating thisbidirectional regulation. During the initial phase ofadipogenesis, CRABPI is upregulated in aTRAP220/MED1-dependent manner, in a mechanisminvolving chromatin looping and nucleosome sliding. Ina subsequent second phase of differentiation, CRABPIis downregulated by T3 in a RIP140/NRIP1-dependentmanner. Wei demonstrated that knockdown of RIP140during this phase results in T3 activation of the CrabpIpromoter and that its re-expression restored RIP140repression. Contrasting the loose nucleosomalorganization of the CrabpI promoter during Phase I withits condensed conformation in Phase II, Wei suggested

that the bidirectional regulation of the CrabpI promoterwas a function of its higher order chromatin organization.Wei went on to attribute the induction of RIP140 in thelatter stages of adipocyte differentiation to thedownregulation of a microRNA, miR-33, which targetsthe 3’-UTR of RIP140. Finally, post-translationalmodification of RIP140 was shown to play a role inmediating its interaction with TRα. Wei showed thatRIP140 was subject to acetylation during adipogenesis,and that acetylation of K158 and K287 were required forrepression of CrabpI and efficient interaction with TRα.

The identification and characterization of RAR family DNAbinding events in response to retinoid signaling in modelsystems is an area of active investigation. Irwin Davidson(Illkirch, France) described recent genome-wide locationanalysis of RARα in mouse embryonic fibroblasts (MEFs)and embryonic stem (ES) cells [Delacroix et al., 2010].Using a stably-transfected FLAG-tagged RARα-basedstrategy, he demonstrated that there were uniquegenomic occupancy sites in MEFs versus ES cells and,surprisingly, that there were relatively few RAR-occupiedsites – those that were identified were DR1, DR2 or DR5RAREs.

Davidson progressed to describe ChIP-Seq analysis ofRAR genomic DNA binding events during the RA-induceddevelopment of neural progenitors from embryonic stemcells via embryoid body cultures, a well-characterizedmodel system for retinoid signaling. Comparing hislaboratory’s dataset with a similar study in F9 cells[Lalevee et al., 2011], he found that there were bothoverlapping and distinct cistromic signatures for RARαin both model systems. The most commonly occurringRAREs were DR0, DR2, DR5 and IR0 in addition to anovel DR8 motif which was a linear composite of DR2and DR0 elements. In vitro analysis demonstrated thatDR0 and IR0 elements bound RAR/RXR heterodimers



and that IR0s and DR8s, but not DR0s, acted asindependent RAREs on minimal promoters. ParallelmRNA-seq analysis showed that only a small subset ofRAR-occupied genes were actually regulated by retinoicacid in embryoid bodies, and that a high frequency of theinduced genes were in fact DR0s – which begs thequestion: what is RAR co-operating with if DR0 is not anindependent RAR binding site? Most likely, elements inneighboring DNA sequences play a role, and future workwill investigate this possibility.

Alternative models of retinoid signalingAlternative pre-genomic (or “non-genomic”) models ofNR signaling invoke the interface between receptors andthe cellular kinase cascade-mediated signaling pathwaysthat are initiated by the binding of peptide hormones andgrowth factors to cell membrane-bound G-protein coupledreceptors. Many proteins involved in these pathwayscontain Src-homology (SH) domains which havewell-characterized roles in signal transduction andtumorigenesis. Work in the laboratory of CécileRochette-Egly (Illkirch, France) previouslydemonstrated the identification of SORBS3/vinexin-β, amultiple SH3 domain-containing protein involved incell-cell adhesion and cytoskeletal function, as a RARγcorepressor. The interaction between the two proteinswas shown to be mediated by the third SH3 domain(SH3.3) of vinexin-β and was uncoupled byphosphorylation of RARγ at two serine residues in itsAF-1 domain.

In the first of the meeting’s short talks, Bruno Kiefferdescribed recent work in the Rochette-Egly laboratory onthe solution structure of the vinexin-β SH3.3 domain incomplex with a proline-rich domain (PRM) of RARγ thatmediates the interaction with vinexin-β. Analysis of thestructure, in addition to biophysical interrogation of theeffect of serine phosphorylation on the interactionsurfaces of the two moieties, indicated that thephosphorylation of PRM modulated the conformations ofthe interacting partners by reducing their type IIpolyproline (PPII) character, an important conformationin disordered states of proteins. Kieffer concluded bysuggesting that the interaction between the SH3.3 andPRM domains was modulated by a conformer selectionmechanism.

Pre-genomic activation of kinase signaling cascades byretinoids was also the subject of a short talk by AleksandrPiskunov (IGBMC, Strasbourg, France) of theRochette-Egly laboratory. Members of the Gq family ofheterotrimeric G proteins activate β-isoforms ofphospholipase C which in turn catalyzes the hydrolysisof phosphatidylinositol phosphate to diacylglycerol andinositol triphosphate, resulting in protein kinase Cactivation and mobilization of cellular calcium.

Piskunov showed that RAR agonists induce thep38/MAPK/MSK1 pathway, and went on to describe aninteraction between Gαq, a member of the Gq family, andRARα in lipid rafts, which are dynamic microdomainswithin the cell membrane lipid bilayer that assemble

specific populations of transmembrane signaling proteins.Moreover, knockdown of either Gαq or p38 significantlyattenuated RA induction of a reporter gene.

Recapitulating the phosphorylation of RARα serine 77by kinase signaling cascades alluded to in Jean-MarcEgly’s talk, Piskunov demonstrated that mutation of theseresidues abrogated induction of the RARβ gene by RA,providing further evidence for the contribution of cellularkinase signaling pathways to retinoid signaling.

Continuing the theme of non-classical pathways of retinoidfunction, Noa Noy (Case Western Reserve University,Cleveland, USA) described a specific cell-surface RBPreceptor, STRA6, a multitransmembrane domain andSH2-binding domain protein that is widely expressed inembryonic development and in adult organ systems, andpreviously characterized as a RA-induced gene. Mutationsin STRA6 contribute to a spectrum of congenitalmalformations collectively known as Matthew Woodsyndrome [Pasutto et al., 2007], and which includeanophthalmia, congenital heart defects and mentalretardation.

Noy pointed out that ectopic overexpression of RBPcontributes to insulin resistance in animal models, asomewhat surprising observation given that RAsuppresses obesity and improves insulin resistance. Aclue to this apparent paradox is the crosstalk of STRA6with the JAK/STAT pathway, a kinase cascade thatmediates cellular response to peptide hormones includinginterleukins, interferons, leptin and insulin. RBP-retinolbinding to STRA6 was shown to induce recruitment ofJAK2 and STAT5 by STRA6, as well as triggeringphosphorylation of all three proteins. Moreover,RBP-retinol induced STAT5, SOCS3 and PPARγexpression in adipocytes via a mechanism independentof RA.

In addition to its effects on transcription, Noydemonstrated that RBP-retinol abrogated activation ofthe insulin receptor in a STRA6-, STAT5- andSOCS3-dependent manner.

Finally, Noy showed that transient recruitment of cellularretinol binding protein I (CRBPI) to STRA6 uponretinol-RBP binding was required for retinol signaling,and that STRA6 signaling was abolished in CRBPImutants. Her data sketch the outline of a negativefeedback loop model in which retinol, signaling thoughRBP, induces SOCS3 and PPARγ via the JAK/STATpathway, resulting in lipid accumulation (via PPARγ) anddownregulation of glucose uptake through antagonismof insulin-insulin receptor signaling (via SOCS3).

The contribution of CRBP1 to retinoid signaling outlinedin Noy’s talk is interesting in the context of data presentedby Eduardo Farias (New York, USA), who demonstratedthat CRBP-1 impaired c-myc-induced tumorigenesis. Hisdata indicated opposing effects of RAR isotypes, suchthat RARα and RARβ drive cell cycle arrest anddifferentiation, whereas RARγ had a repressive effect onCRBP expression, which is silenced in a wide range of



tumor types. He suggested that RARα/β agonists andRARγ antagonists could provide novel avenues fortreatment of breast cancer.

Developmental biology of retinoidsignalingThe functions of RA and RARs in developmental biologyis a complex interplay of factors ranging fromtissue-selective distribution of RA and its biosyntheticprecursors, expression of RAR isoforms, and thefunctional interactions of RARs and RXRs withtranscriptional coregulators to effect co-ordinatedprograms of gene activation and repression. Retinoldehydrogenases (RDH) comprise a family of enzymesthat generate all-trans retinaldehyde from all-trans retinolduring the initial step of retinoic acid biosynthesis.Downstream of the generation of retinaldehyde substratesby RDH enzymes is the conversion of these to retinoicacid by tissue-specific enzymes of the retinaldehydedehydrogenase (Raldh) family, which is a key componentin the establishment of morphogenetic gradients ofretinoic acid which play demonstrably crucial roles in earlysomitogenesis and neurogenesis.

Tissue-specificity of RA biosynthesis: retinoldehydrogenases (RDHs) and retinaldehydedehydrogenases (RALDHs)

Pascal Dollé (IGBMC, Strasbourg, France) discussedCre-Lox conditional knockout model-based studies onthe role of one member of the Rdh family of enzymes,Rdh10, in early embryogenesis, and the insights theseafford into tissue-specific principles in retinoid signaling.While Rdh10 null mutants are early embryonic lethal(E10.5-E12.5), retinoid signaling as reported by aRARE-Lacz reporter transgene, is significantlycompromised (Figure 6), but not fully abolished – areflection of the existence of other members of thisenzyme family possibly acting during tumorigenesis.Rdh10-/- mutant embryos display a number ofdevelopmental defects, including abnormal patterning inthe posterior hindbrain and compaction of the cervicalsomitic region. Defects in Rdh10-/- mutants were also thesubject of a short talk by Gregg Duester (La Jolla, USA),who noted impaired forelimb development as well asdefects in early somitic development related to ectopicFgf8 expression in the trunk at early stages ofdevelopment. Reiterating the tissue-specific nature ofRaldh action, Duester noted that while RA signaling wascompromised in the somitic mesoderm, it was intact inthe neuroectoderm.

Dollé next used rescue experiments involving RA orretinaldehyde transplacental supplementation of pregnantmice to determine whether Rdh10 activity was requiredfor generating tissue-specific distributions of substratesfor embryonic RALDH. He found that RA rescue did notresult in viable Rdh10-/- mutant embryos, and that thesemutants displayed organogenesis defects characteristicof RA deficiency in the heart (reduced myocardialcompact zone, epicardial detachment), large vessels(pulmonary artery atresia) and lungs (absence of left lung,

hypoplastic right lung). In contrast, retinaldehydesupplementation rescued most developmental defectsand resulted in viable, fertile mutants, suggesting that byfailing to recapitulate the precise tissue-specific gradientsof RA required during development, RA supplementationis unable to rescue mutants lacking Rdh10.

Figure 6. Highly diminished RARE-lacZ reporter transgene activityin an early mouse embryo with a targeted Rdh10 gene disruption.See text for details.

Dollé concluded with data implicating a member of theRaldh family, Raldh3, in development of the inner earvestibular system. Raldh3 is expressed in discrete areasof the inner ear, including the spiral ganglia of the cochleaand specific regions of the vestibular sensory epithelium,such as the utricle and saccule. Reflecting the role ofthese structures in hearing and balance, Raldh-/- mutantmice exhibit an abnormal spontaneous circling behaviorand motor skill deficiencies, as well as impaireddevelopment of inner ear vestibular structures. Inparticular, there is an absence in these mice of otoconia,mineralized structures in the utricle and saccule thatprovide mass loads for detection of the gravity and linearacceleration. Dollé concluded by speculating thatRA-based therapeutics might be profitably used to offsetthe otoconial degeneration that contributes to a varietyof senescent diseases.

Retinoid signaling in somitic development andneurogenesis

In vertebrate species, the vertebral column, skeletalmuscle and dermis are derived from embryonic structurescalled somites, which are formed by the periodic,bilaterally symmetrical process of mesodermalsegmentation.The fibroblast growth factor (FGF) pathwayestablishes a traveling posterior to anterior gradient ofactivity in the presomitic mesoderm such that thesegmentation transcriptional program, involving genessuch as Mesp2, is triggered only in cells exposed tosufficiently low levels of FGF. Opposed to the FGFgradient is a RA gradient, and the antagonistic effects ofsignaling pathways regulated by these ligands establishes



a determination front for segmentation. Raldh2/Aldh1a2catalyzes the synthesis of RA from retinaldehyde insomitic and rostral presomitic mesoderm; its inhibition bydisulfiram in the chick, or by gene targeting in the mouse,results in left-right asymmetry of somitogenesis [Vermotet al., 2005; Vermot and Pourquie, 2005]. OlivierPourquié (IGBMC, Strasbourg, France) drew attentionto defects in somitogenesis, including delayed somiteformation on the right side, in a mouse model containinga mutation in Rere/Atrophin2, previously characterizedas a coregulator for members of the COUP-TF subfamilyof NRs [Wang and Tsai, 2008]. In support of a role forAtrophin 2 in retinoid signaling, developmental defects inthe Rere/Atrophin2 mutant were strikingly phenocopiedin the Raldh2 mutants. Pourquié proceeded to show datafrom a RARE-LacZ mouse crossed into the Atrophin 2mutant that Atrophin 2 was required for the in vivoresponse to RA, and that Atrophin 2 potentiated thetranscriptional response to RA in F9 cells. Supportingbiochemical and in vivo data demonstrated that a complexcomprising Atrophin 2, p300 and COUP-TFII/Nr2f2 wasrecruited to RA-responsive genes, and that knockdownof COUP-TFII and Atrophin 2 abrogated RA signaling.Pourquié demonstrated that RARs were expressed in thepre-somitic mesoderm in mouse E8.5 embryos, and thatasymmetric expression of COUP-TFII overlapped withthe RA response domain in the right pre-somiticmesoderm. Finally, Pourquié discussed multi-dimensionalprotein identification technology (MUDPIT) analysis ofAtrophin 2-associated proteins which demonstrated theexistence in the Atrophin 2 interaction network of HDAC1,HDAC2, p300 and a protein named STRAP(serine/threonine kinase receptor-associated protein),mutations in which result in somite desynchronizationdefects.

Kate Storey (University of Dundee, Dundee, UK)continued the developmental theme with a discussion ofneurogenesis, which has several parallels withsomitogenesis in the context of retinoid signaling, notablythe functional antagonism between retinoid- andFGF-regulated pathways. During neurogenesis, retinoidsignaling, driven in part by Wnt promotion of retinoidbiosynthesis represses FGF action, resolves mesodermaland neural cell fates and promotes commitment to theneural cell lineage. Genes involved in neurogenesiswhose expression is subject to mutually opposedregulation by FGF (repression) and retinoids (induction(activation) include neurogenin (Ngn)1 and Ngn2, NeuroMand Crabp1, although these represent only a smallfraction of the major re-organization of transcriptomeswhich takes place along the neural axis duringneurogenesis. The FGF/retinoid antagonistic paradigmso clearly characterized in this context is not a generalrule however, and Storey went on to discuss how retinoidsignaling during embryonic stem (ES) cell differentiationcan, under certain conditions, promote FGF/Erk signaling,at least transiently. Specifically, RA initially induces arapid increase in Erk1/2 signaling via induction of Fgf8,which ChIP data from the laboratory of Gregg Duester(La Jolla, USA) implicate as the result of direct bindingof RAR to the Fgf8 promoter. As differentiation progresses

however, there is a net repression of ERk1/2 signaling,most likely via repression of Fgf4.

Norbert Ghyselinck (IGBMC, Strasbourg, France)concluded the developmental strand with a discussion ofspecific and overlapping roles of RARs during male germcell differentiation. Dietary vitamin A deficiency inducestestis degeneration via arrest of spermatogoniaproliferation, which can be reversed by vitamin A or RAsupplementation, indicating that germ cell differentiationdepends on RA signals. Ghyselinck showed that RARγis expressed at specific stages during spermatogenesis,specifically in single cell spermatogonia, as well as inaligned A spermatogonia in later stages (Figure 7).Cre-lox-mediated ablation of RARγ or RARα individuallyin Sertoli cells is sufficient to induce testis degenerationthrough loss of germ cells, although the specificdevelopmental deficit in each mutant appears to occur atdifferent points during spermatogenesis. RARα and RARγappear to be functionally redundant to some extent in thiscontext since expression of Stra8, a RA-inducible germcell marker, which is undetectable in RARγ-deficient mice,can be restored by administration of the RARα agonistBMS753. Observing that ablation of RXRα, RXRβ andRXRγ in spermatogonia results in a loss of germ cellssimilar to that in RARγ and RARα, Ghyselinck proposeda model in which RARα and RARγ functioncell-autonomously as heterodimers with RXRs.

Retinoid signaling and diseaseConsistent with their promotion of exit from the cell cycleand progression towards differentiated cell fates, retinoidshave anti-proliferative and apoptotic effects and havefound application in a number of inflammatory conditionsincluding skin disorders, as well as diseases such asbreast cancer and leukemia. Moreover, the functionalintegrity of RARs is often subverted in gene translocationevents commonly encountered in leukemias, resulting inectopic repression of transcriptional programs involvedin myeloid cell differentiation [Duong and Rochette-Egly,2011]. Harnessing retinoid signaling pathways has metwith mixed success as a cancer therapeutic strategy,providing an impetus for more detailed models of retinoidaction in cancer cells and the exploration of alternativestrategies, including epigenomics, for intervention inretinoid pathways opposing tumor progression.

Breast cancer

Sylvie Mader (Montreal, Canada) described recentefforts in her laboratory focused on RALDHs astherapeutic leverage points in breast cancer. RALDH3 isthe predominant RALDH family member in normal breasttissue, as well as immortalized and breast cancerepithelial cell lines. Mader noted that the majority of breastcancer cell lines tested have low RA synthetic capacity,which correlates with low levels of RALDH3 expressionat both the mRNA and protein levels, a patternrecapitulated in luminal breast tumors. In contrast,RALDH3 was highly expressed in normal breast tissueand mediated retinoic acid biosynthesis in humanimmortalized mammary gland 184B5 cells. Given these



Figure 7. RARγ is expressed in Aal and A1 spermatogonia. External views of seminiferous tubules of wild-type, sexually mature, mice that weredouble-immunostained with anti-RARγ (red nuclear signal) and anti-GFRA1 (green membrane signal) antibodies in panel A, with anti-RARγ (red nuclearsignal) and ZBTB16 (green nuclear signal) antibodies in panel B, and with anti-RARγ (red nuclear signal) and anti-KIT (green membrane signal)antibodies in panel C. Legend: As, Aal, and A1 indicate A single, A aligned and A1 spermatogonia, respectively. Bar: 50 µm.

observations, Mader next asked whether ectopic RALDHexpression might inhibit growth and proliferation of breastcancer cell lines and luminal tumors. RALDH3 wascapable of inducing G0/G1 arrest in tumorigenic SKBR3cells, inhibited colony formation in MCF-7 breast cancercells and retarded tumor growth in MCF-7 xenograftmodels. At the transcriptional level, Mader demonstrateda remarkable overlap between patterns of geneexpression in luminal cells exposed to retinoic acid orexpressing RALDH3, resulting in the activation ofluminal-specific transcription factors and ofanti-proliferative and pro-apoptotic gene networks. The17β-estradiol/ER proliferative paradigm is well establishedin models of epithelial breast cancer, and Mader’s closingremarks illuminated the dynamic antagonism that existsbetween this signaling conduit and that of retinoids. Sheobserved that RALDH3 expression is blocked by17β-estradiol in an ERα-dependent manner, while thiseffect is reversed by anti-estrogens such as Fulvestrantand Tamoxifen.

A number of models exist for the development ofresistance by breast tumors to treatment with Tamoxifen,one of which invokes the role of a cancer stem cellpopulation in the generation of novelchemotherapeutically-resistant cell lineages in tumors.Maria del Mar Vivanco (Bilbao, Spain) described recentwork in her laboratory which sheds light on the role oftumor stem cells using a Tamoxifen-resistant MCF-7 cellline. These cells resemble progenitor cells in theirexpression of high levels of Sox2, a member of theSRY-related HMG-box family of transcription factorsrequired for the maintenance of cancer stem cells. Ectopicoverexpression of Sox2 was shown to confer increasedresistance of cells to the anti-proliferative effects ofTamoxifen, and clinically, Sox2 was more highlyexpressed in recurrent tumors than in primary tumors.

While most of the speakers reiterated thepro-differentiative, antiproliferative effects of retinoidsignaling, Vincent Giguère (Montreal, Canada) soundeda dissonant note by discussing data casting RA and RARβas cancer promoters. RARβ null mice exhibit delayed

mammary gland development and tumorigenesis isdelayed in a MMTV-ErbB2 model in a RARβ nullbackground. Giguère cited downregulation of theCxc12/SDF-1 pathway in RARβ null mice as a possiblemechanistic basis for RARβ’s promotion of tumorigenesis.While provocative, Giguère’s results did not exclude thepossibility that there is developmental overcompensationfor germline loss of RARβ from other RAR isoforms, apossibility given the overlapping functions of members ofthis subfamily, and a conditional mammary glandknockout of RARβ would more clearly address the natureof its role in breast cancer.

Leukemias

Hematopoiesis is the continuous formation of all bloodcell types from hematopoietic stem cells residing in thebone marrow. Cell fates during hematopoiesis are afunction of both extracellular signaling networks mediatedby soluble growth factors and their receptors, as well asspecific transcriptomic fingerprints triggered in individualcell populations in response to these signaling events. Inmyeloid leukemia, the mechanisms that normally promotedifferentiation into the diverse myeloid lineages aredisrupted, leading to pathological accumulation ofimmature hematopoietic cell types.

Henk Stunnenberg (Nijmegen,The Netherlands)opened with an overview of the European Union-fundedBLUEPRINT of Hematopoietic Epigenomes, a teamscience endeavor that aims to generate genomic DNAsequence, epigenomic and transcriptomic datasets fromabout 60 distinct types of primary hematopoietic cells andblood cancer subtypes. Oncofusions involving AML andPML proteins are well characterized in leukemias: thefusion protein, promyelocytic leukemia (PML)-retinoicacid receptor-α (RARA), initiates acute promyelocyticleukemia (APL) through both a block to differentiationand increased self-renewal of leukemic progenitor cells.Similarly, the fusion of AML1 and the transcriptionalcorepressor ETO (RUNX1T1) through a t(8;21)translocation is a frequent genetic lesion in acute myeloidleukemia (AML). Stunnenberg posed the questions: wheredo these oncofusion proteins bind, do they alter the



epigenomic environment where they bind, and docommon mechanisms exist by which they achieve theseends? Stunnenberg described a comparison of AML1and AML1/ETO sites in SKNO/Kasumi cells and AMLpatient blasts, and demonstrated a redistribution of targetsfrom gene promoters in the case of AML1 towards intronsand intergenic regions for AML1/ETO. Stunnenbergpointed out that AML1/ETO bound preferentially tomultimerized sites, and speculated that this was due tothe oligomerization properties of AML1, previously shownto be required for the interaction of ETO withtranscriptional corepressors. Members of the erythroblasttransformation-specific (ETS) family of transcriptionfactors are key regulators of embryonic development, cellproliferation, differentiation, angiogenesis, inflammation,and apoptosis. Stunnenberg described an overlapbetween binding sites for ERG, a member of the ETSfamily, and AML1-ETO in U937 cells, and highlighted thefact that elevated expression of ERG in AML wasassociated with poor prognosis in the disease. Addressingthe epigenomic consequences of AML1-ETO,Stunnenberg demonstrated that AML1/ETO binding inU937 cells was associated with local hypoacetylation,consistent with the association of ETO with corepressors.Drawing parallels between the AML1/ETO andPML/RARα paradigms, he described extensive overlapbetween the binding sites of the two fusion proteins. Heconcluded by speculating that a common mechanism fordisease progression was the recruitment of the oncofusionproteins to promoters of ETS family-regulated genesencoding proteins required for myeloid differentiation,and the subsequent epigenomic silencing of these genesvia a mechanism involving association of corepressorswith the fusion proteins.

In addition to the well-characterized functions oftranscription factors and coregulators, there is anincreasing recognition of miRNAs as key players inorchestrating specific transcriptional endpoints duringhematopoiesis. Clara Nervi (University of Rome, Rome,Italy) focused on the role of one such miRNA, miR-223,in normal hematopoietic development as well as in thedeficits in differentiation characteristic of the progressionof leukemic cells, and the interface between this miRNAand retinoid signaling. Administration of RA in HL60 cellsresults in the association of miR-223 with the miRNAprocessing ribonuclease Dicer1, a key step in theprocessing of miRNAs. Nervi showed that ectopicexpression of miR-223 potentiated granulocyticdifferentiation of ALP-NB4 cells, and that the AML1/ETOoncofusion targeted miR-223 for heterochromaticsilencing.

Further evidence for the functional interaction of miR-223and AML1/ETO in hematopoietic pathologies wasprovided by the similar myeloproliferative disorders inmiR-223 knockout and AM1/ETO transgenic mice. Nerviprovided evidence that one of the targets of miR-223 inthis context was NFIA: treatment of HL60 cells with RAresulted in silencing of the NFIA promoter accompaniedby recruitment of miR-223. Moreover, NFIA wasdownregulated in HL60 cells following overexpression of

miR-223. Progressing to the clinical setting, Nervidemonstrated that expression of miR-223 in primaryblasts from AML patients restored myeloid differentiation.

Failure of tumors to respond to retinoid therapy is acommon clinical observation, suggesting that establishingan environment more receptive to retinoid signaling mightbe a profitable approach in treatment of the disease. Inthis context, Christine Chomienne (Hôpital St Louis,Paris, France) discussed epigenetic strategies toenhance RAR promoter permissiveness as a therapeuticstrategy in leukemogenesis. She demonstrated thatnon-responsiveness of thyroid cancer cells to RA wasassociated with reduced histone acetylation and increasedDNA methylation of the RARβ promoter.The combinationof RA with a histone deacetylase inhibitor resulted inincreased expression of RARβ in these cells. In theclinical setting, the combination of RA with histonedeacetylases such as valproic acid in non-APL leukemiaswas associated with complete remission in older patients,albeit with a modest response rate.

Hugues deThé (Hôpital St Louis, Paris, France)concluded the meeting with a discussion of data on themolecular mechanisms of RA and arsenic trioxide (As2O3)therapy in APL, a combination therapy that has becomestandard of care for the disease, with an emphasis onthe PML-RARα oncofusion. Both RA and As2O3 degradePML-RARα, and synergize in the treatment of APL inmice, radically extending their life span. This synergyarises from the fact that they engage parallelproteasome-dependent PML/RARα degradationpathways, As2O3 targeting the PML moiety of theoncofusion for sumoylation and RA targeting the RARportion of the molecule in a manner evoking the targetingof ERα for degradation by Fulvestrant. deThé highlightedthe role of a CC motif in the B2 box region of PML,pointing out that amino acid variability in this region ofthe molecule, secondary to mis-sense mutations in thePML gene, was a critical determinant of theresponsiveness to As2O3 treatment. deThé invoked aderepression model for PML-RARα-targeted therapy,suggesting that loss of PML-RAR was the motive forcebehind the reversion to a default myeloid differentiationpathway.

ConclusionsThis brief, focused meeting reflected the ongoing effortsof a highly active clinical and translational global researchcommunity and the contributions that it continues to makenot only in the context of retinoid signaling, but to thebroader picture of nuclear receptor signaling. It reiteratedthe myriad critical junctures along the axis of retinoidsignaling – retinoic acid biosynthesis, the interactionbetween ligand and receptor, or the action of RARcoregulators, has been shown to have profoundconsequences both in early embryonic development andin a variety of human pathologies. In addition to thesebetter characterized components, compelling evidencewas made for non-coding RNAs, DNA looping andexcision repair mechanisms, as well as epigenomicprogramming of promoters, as emerging mechanistic



principles not only in retinoid signaling, but in the widercontext of signal transduction by the NR superfamily.Finally, the field has shown itself to be highly active in thetranslation of mechanistic principles of retinoid signalinginto strategies that are showing promise in the treatmentof myriad neoplastic and inflammatory conditions, andone can justifiably anticipate further exciting advances inthe mechanism, biology and pathology of retinoidsignaling in the coming years.

AcknowledgementsThe author thanks the European Molecular Biology Organization (EMBO)and the meeting organizers, Cécile Rochette-Egly, Christine Chomienneand Vincent Laudet for their kind invitation to write a review of the meeting.

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