www.elsevier.com/locate/gene

Gene 328 (2004) 1–16

Review

Nuclear retinoid receptors and the transcription of retinoid-target genes

Julie Bastien, Cecile Rochette-Egly*

Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM/ULP, UMR 7104, 1 rue Laurent Fries, BP 10142, Illkirch Cedex 67404, France

Received 14 October 2003; accepted 2 December 2003

Received by A.J. van Wijnen

Abstract

The pleiotropic effects of retinoids are mediated by nuclear retinoid receptors (RARs and RXRs) which are ligand-activated transcription

factors. In response to retinoid binding, RAR/RXR heterodimers undergo major conformational changes and orchestrate the transcription of

specific gene networks, through binding to specific DNA response elements and recruiting cofactor complexes that act to modify local

chromatin structure and/or engage the basal transcription machinery. Then the degradation of RARs and RXRs by the ubiquitin–proteasome

controls the magnitude and the duration of the retinoid response. RARs and RXRs also integrate a variety of signaling pathways through

phosphorylation events which cooperate with the ligand for the control of retinoid-target genes transcription. These different modes of

regulation reveal unexpected levels of complexity in the dynamics of retinoid-dependent transcription.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Retinoids; Nuclear receptors; Transcription; Degradation; Kinases; Phosphorylation; Ubiquitin–proteasome

1. Introduction

Vitamin A and its active derivatives referred to as

retinoids are non-steroid hormones which play a critical

role in the development and homeostasis of virtually every

vertebrate tissues through their regulatory effects on cell

differentiation, proliferation and apoptosis (Ross et al.,

2000; Altucci and Gronemeyer, 2001a). It has long been

established that retinoids exert their action by regulating the

expression of specific subsets of genes within target tissues.

However, it is only during the last 15 years that the

understanding for retinoids action rapidly increased, subse-

quently to the cloning of nuclear retinoid receptors and the

0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.gene.2003.12.005

Abbreviations: RAR, retinoic acid receptor; RXR, retinoid X receptor;

RA, retinoic acid; DBD, DNA-binding domain; LBD, ligand-binding

domain; RARE, retinoic acid response element; HAT, histone acetyltrans-

ferase; HMT, histone methyltransferase; HDAC, histone deacetylase; CBP,

CREB-binding protein; Cdk, cyclin-dependent kinase; TBP, TATA-binding

protein; GTF, general transcription factor; MAPK, mitogen-activated

protein kinase; P13K, phosphoinositide 3-kinase; PTEN, phosphatase and

tensin homolog; PPAR, peroxisome proliferator activated receptor; LXR,

liver X receptor; APL, acute promyelocytic leukemia.

* Corresponding author. Tel.: +33-3-88-65-34-59; fax: +1-33-3-88-65-

32-01.

E-mail address: cegly@igbmc.u-strasbg.fr (C. Rochette-Egly).

identification, within the promoters of retinoid-responsive

genes, of elements exhibiting a high affinity for these

receptors (for review, see (Chambon, 1996; Laudet and

Gronemeyer, 2001), and references therein). Then these

nuclear receptors have been shown to work as ligand-

activated transcription activators in a spatiotemporal specific

manner during embryonic development.

During the last decade, the molecular rationale for

retinoid receptors action has been facilitated by the identi-

fication of the DNA- and ligand-binding domains (DBD

and LBD, respectively) (Chambon, 1996), and by the

determination of their crystal structure (Renaud and Moras,

2000). Moreover, a number of studies demonstrated that

they have to contend with repressive chromatin structures

in order to activate gene expression. Indeed, as most target

genes are initially silent and packed in a dense chromatin

structure, liganded retinoid receptors recruit a battery of

intermediary proteins, including coactivators, chromatin

remodellers and modifyers which act in a coordinated

and/or combinatorial manner to decompact chromatin and

direct RNA polymerase II (RNA Pol II) and the general

transcription factors (GTFs) to the promoter (Dilworth and

Chambon, 2001).

Then an important question was what happens after the

retinoid-activated receptors have bound their DNA reponse

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–162

elements and recruited the transcription machinery. Now,

there is increasing evidence that the ubiquitin–proteasome

machinery degrades the retinoid receptors subsequently to

their activation (Zhu et al., 1999; Boudjelal et al., 2000;

Kopf et al., 2000; Gianni et al., 2002a). This degradation

process may either disrupt the transcription initiation com-

plex, allowing elongation to proceed, and/or terminate the

response to retinoids in order to allow rapidly other tran-

scriptional programs.

Finally, the last years have witnessed a new way of

regulation of retinoid receptors. Indeed, ongoing studies

revealed that they can integrate multiple signaling pathways

through their phosphorylation (Rochette-Egly, 2003). Alter-

natively, retinoids cross-talk with a number of signaling

pathways. All these processes converge towards finely

tuned transcriptional control.

This review will describe our current knowledge about

the molecular mechanisms through which retinoid receptors

regulate transcription, ranging from DNA binding, dynam-

ics of ligand binding and chromatin remodeling and finally

to their degradation. It will also focus on how sequential

and/or coordinated phosphorylation events regulate their

functionality.

Fig. 1. Schematic representation of the functional domains and the major phosphor

and the ligand-binding domain (LBD) are schematically represented (not to scale).

respectively, are depicted. The target sequences for phosphorylation are also shown

amino-terminal kinase.

2. Retinoid receptors contain a DNA-binding domain

and two activation functions AF-1 and AF-2

The retinoid signal is transduced by two families of

nuclear receptors, the retinoic acid receptors (RARs) and

the retinoid X receptors (RXRs), which work as RXR/RAR

heterodimers (Kastner et al., 1997; Mark et al., 1999). Each

family consists of three isotypes (a, h and g) encoded by

separate genes (Leid et al., 1992; Mangelsdorf and Evans,

1995; Chambon, 1996). RARs are activated by all-trans

retinoic acid (RA) and its 9-cis isomer, while RXRs are only

activated by 9-cis RA. For each isotype, there are at least

two isoforms that are generated by differential promoter

usage and alternative splicing and differ only in their N-

terminal regions.

As most nuclear hormone receptors, retinoid receptors

exhibit a modular structure composed of 6 conserved

regions designated A–F (Fig. 1). The highly conserved C

region harbors the DBD which confers sequence specific

DNA recognition. This domain is composed of two zinc-

nucleated modules, two a-helices and a COOH-terminal

extension (CTE) (Zechel et al., 1994a,b). Helix 1 and helix 2

cross at right angles to form the core of the DBD folding

ylation sites of nuclear retinoid receptors. The DNA-binding domain (DBD)

The functional AF-1 and AF-2 domains which lie in the A/B and E regions,

. MAPK, mitogen-activated protein kinase ; MKK, MAPK kinase; JNK, Jun

Fig. 2. (A) High resolution solution structure of the RXRa DNA-binding domain, NMR (mmdbId: 8588). (B) Structure of the RAR/RXR DBD heterodimer in

complex with a DR1 element (mmdbId: 13,928). The interfaces between both partners involve the CII finger surface of the RAR and the T box of the RXRa

partner.

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–16 3

into a single globular domain (Fig. 2A) that has been

determined by nuclear magnetic resonance and crystallo-

graphic studies (Lee et al., 1993).

Region E is the second most conserved region and

corresponds to the LBD. It is functionally complex as it

contains the ligand-binding pocket, the main dimerization

domain and the ligand-dependent transactivation function

(AF-2). The structures of the LBDs of RARs and RXRs are

rather similar as demonstrated by the cristallographic studies

Fig. 3. Comparison of the crystal structures of the apo-RXRa (A, PDB1LBD; R

ligand-binding domains reveals the ligand-induced transconformation.

(Wurtz et al., 1996; Moras and Gronemeyer, 1998; Renaud

and Moras, 2000) (Fig. 3A). The LBDs are formed by 12

conserved alpha-helices and a beta-turn (situated between

H5 and H6) which are folded into a three-layered, antipar-

allel helical sandwich with H4, H5, H8 H9 and H11

sandwiched between H1, H2 and H3 on one side and H6,

H7 and H10 on the other. In this structure, the C-terminal

helix H12, which encompasses the AF-2 activation domain,

points away from the LBD core.

enaud et al., 1995) and holo-RARg (B, PDB2LBD; Bourguet et al., 1995)

Fig. 4. (A) The classical retinoid response element is a direct repeat of the

motif 5V-PuG(G/T)TCA spaced by 1 (DR1), 2 (DR2)or 5 (DR5) base pairs.

The natural retinoid response elements from the promoters of some RA-

target genes are shown. (B) On DR2 and DR5 elements, retinoid receptors

bind as RXR/RAR hetodimers with the RXR partner occupying the 5Vmotif. (C) DR1 elements bind either RXR/RAR hetodimers with the

reverse polarity, or RXR homodimers.

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–164

The heterodimerization surface involves residues from

H7, H9, H10 and H11, as well as loops L8–9 and L9–10

(Bourguet et al., 2000). Helices H9 and H10 contribute to

more than 75% of the total dimerization surface and

constitute the core of the dimer interface. The ligand-

binding pocket comprises hydrophobic residues mainly

from helices H3, H5, H7 and the h-sheet. The precise

contacts with the ligands have been characterized and are

unique for each receptor–cognate ligand pair (Klaholz et al.,

1998; Gehin et al., 1999).

Importantly, the LBD also contains consensus phosphor-

ylation sites (see Fig. 1). Indeed, upon activation of PKA

signaling (Rochette-Egly et al., 1995), the LBD of RARs is

phosphorylated at a conserved serine residue, located be-

tween helices H9 and H10 (Ser 369 for RARa1 and Ser 360

for RARg2). The RXRa LBD can be also phosphorylated

by MAPKs, but at residues (Tyr 248, Ser 265) located in the

Omega loop (between H1 and H3) (Adam-Stitah et al.,

1999; Lee et al., 2000; Matsushima-Nishiwaki et al., 2001;

Adachi et al., 2002).

The N-terminal A/B region harbors a ligand-indepen-

dent transcriptional activation function (AF-1). The three-

dimensional structure of the AF-1 domains of RARs and

RXRs has not been solved yet and structure prediction is

not straightforward. However, the interesting feature of

this domain is that it contains several consensus phos-

phorylation sites (Rochette-Egly, 2003) for proline-depen-

dent kinases which include cyclin-dependent kinases

(CDKs) and MAP kinases (Fig. 1) (Morgan, 1997; Davis,

2000; Pearson et al., 2001). In that context, our laboratory

demonstrated that RARa1 and RARg2 are phosphorylated

in their B region at serines 77 and 68, respectively, by the

cyclin H-dependent kinase cdk7 associated to the general

transcription factor TFIIH (Rochette-Egly et al., 1997;

Bastien et al., 2000). In addition, RARg2 can be phos-

phorylated at the nearby serine residue (Ser66) by

p38MAPK (Gianni et al., 2002a,b). RARh2 is also

phosphorylated at similar residues (K. Drean and C.

Rochette-Egly, unpublished data), but the responsible

kinases remain to be determined. RXRa is phosphorylated

in its A region at Ser22 by a cdk that is distinct from

cdk7 (Adam-Stitah et al., 1999). Importantly, three addi-

tional residues (Ser61, Ser75 and Thr87) can be phos-

phorylated by stress kinases (JNKs) (Adam-Stitah et al.,

1999).

The D region serves as an hinge between the DBD and

the LBD, allowing rotation of the DBD. Therefore it might

allow the DBD and the LBD to adopt different conforma-

tions without creating steric hindrance problems. It also

harbors nuclear localization signals. The F region is absent

in RXRs and its role in RARs, if any, is still unknown.

However, this region is phosphorylated (Rochette-Egly et

al., 1997; Bastien et al., 2000) and it is tempting to speculate

that it may modulate the activation functions AF-1 and/or

AF-2 as in the case of the estrogen receptor (Montano et al.,

1995; Metivier et al., 2002).

3. First step: retinoid receptors binding to responsive

elements located in the regulatory sequences of target

genes

In the absence of ligand, retinoid receptors are found

primarily in the nucleus. They bind as asymetric, oriented

RAR/RXR heterodimers to specific DNA sequences or RA

response elements (RAREs) composed typically of two

direct repeats of a core hexameric motif, PuG(G/T)TCA

(Leid et al., 1992; Mangelsdorf and Evans, 1995) (Fig. 4A).

The classical RARE is a 5-bp-spaced direct repeat (referred

to as DR5). However, RAR/RXR heterodimers also bind to

direct repeats separated by 1 bp (DR1) or 2 bp (DR2). RXRs

also bind to DR1 as RXR/RXR homodimers.

RAREs have been identified in the promoters of a large

number of retinoid-target genes implicated in a wide variety

of functions (Fig. 4A). The classical DR5 elements are

found in the promoters of the RARh gene itself (de The

et al., 1990), CYP26 (Loudig et al., 2000), and several Hox

and HNF genes (Dupe et al., 1997; Qian et al., 2000). DR2

elements were identified in the CRBPI and CRABPII

promoters (Smith et al., 1991; Durand et al., 1992). The

only natural DR1 element binding RXR homodimers has

been found in the rat CRBPII promoter (Mangelsdorf et al.,

1991).

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–16 5

On DR2 and DR5 elements (Fig. 4B), RXR occupies the

5Vhexameric motif, whereas the RAR partner occupies the 3Vmotif (Chambon, 1996; Laudet and Gronemeyer, 2001). In

contrast, on DR1 elements (Fig. 4C), the polarity is re-

versed, with the RAR DBD binding upstream and the RXR

DBD downstream (5V-RAR/RXR-3V), switching the activity

of the heterodimer from an activator to a repressor of

retinoid-responsive genes.

The cristallographic structure of RXR homodimers and

RAR/RXR heterodimers in complex with DNA has been

solved (Rastinejad et al., 2000; Khorasanizadeh and Rasti-

nejad, 2001; Rastinejad, 2001) (Fig. 2B). Each DBD interacts

with the DNAmajor groove at the level of an half-site through

the P box of the first helix containing three exposed residues

responsible for discrimination between different half-sites

sequences. Then, they arrange in head to tail, with coopera-

tive contacts between the DBDs, leading to a mutual rein-

forcement of protein–protein and protein–DNA interactions.

Depending on the DR spacing, different regions of the DBD

of each receptor are used to create the dimerization interface,

in order to achieve the required binding to the response

elements. The heterodimeric DBD interface that is responsi-

ble for the binding of RXR/RAR heterodimers to DR5

elements involves the D box of the RXR second zinc finger,

and the tip of the RAR first zinc finger. However, when the

heterodimers bind with the reverse polarity to DR1 elements

(Fig. 2B), they associate through the second zinc finger of

RAR and the so-called T box (within the CTE) of RXR. The

same type of dimerisation interface (T box of the downstream

partner and second zinc finger of the upstream one) is

responsible for the cooperative binding of RXR homodimers

to DR1 elements. This implies that the DBDs must be

rotationally flexible with respect to the LBD dimerization

interface and that the DNA curvature is different.

In conclusion, the DBDs of each heterodimerization

partner dictate the specificity of RAREs recognition and

contribute through their dimerisation to increase DNA

binding efficiency.

4. Second step: ligand binding, coactivators recruitment

and chromatin decompaction

When genes are silent, DNA is packaged into a highly

organized and compact nucleoprotein structure known as

chromatin which impedes all the transcription steps. The

basic unit of chromatin is the nucleosome which consists of

DNA wrapped around a protein core containing two copies

each of four histone proteins. Protruding from the nucleo-

somes are the N-terminal «tails» of the core histones whose

interaction with DNA can be modulated upon covalent

modifications (acetylation, phosphorylation, methylation,

etc).

According to the current model of gene regulation by

retinoids established by Dilworth et al (Dilworth and

Chambon, 2001), in a context of chromatin where the

nucleosomes do not impede the binding of RAR/RXR

heterodimers to their DNA recognition sequences, unli-

ganded and DNA-bound retinoid receptors repress transcrip-

tion (Fig. 5A) through the recruitment of the corepressors

NCoR and SMRT (Glass and Rosenfeld, 2000; Aranda and

Pascual, 2001). A single corepressor molecule interacts

through two conserved box motifs, with the LBDs of both

heterodimeric partners. In fact, the corepressors reside in or

recruit high molecular weight complexes endowed with

histone deacetylase activity (HDACs) which increase the

interaction of the N-terminal histone tails with the nucleo-

somal DNA.

Thus, to activate gene expression, retinoid receptors will

have to contend with the repressive chromatin structures in

order to allow the recruitment of the transcription machin-

ery. In this regard, the ligand-induced conformational

changes in the receptors will cause the dissociation of

corepressors and the coordinated and/or combinatorial re-

cruitment of coactivators which upon association with larger

complexes with chromatin modifying and remodeling ac-

tivities will decompact repressive chromatin and facilitate

the positioning of the transcriptional machinery at the

promoter (Fig. 5B and C).

Indeed, ligand binding induces conformational changes

in the LBD (Bourguet et al., 1995; Renaud et al., 1995;

Wurtz et al., 1996; Moras and Gronemeyer, 1998; Egea et

al., 2001), including repositioning of helix H11 in the

continuity of H10. The most striking effect is the swinging

of helix 12 which moves in a «mouse trap model», being

tightly packed against H3 and H4 (Fig. 3B). H12 makes

direct contacts with the ligand and seals the «lid»of the

ligand-binding pocket, further stabilizing ligand binding.

Simultaneously, the Omega loop flips over underneath H6,

carrying along the N-terminal part of H3. According to

recent studies, ligand binding would be facilitated by

cellular retinoic acid-binding protein II (CRABPII) which

upon shuttling into the nucleus and interaction with RAR/

RXR heterodimers (Delva et al., 1999) channels retinoic

acid to RARs (Budhu and Noy, 2002). This interaction of

RARs with CRABPII is stabilized by cyclin D3 (Despouy et

al., 2003).

The ligand-induced conformational changes favor the

interactions between RAR and RXR and therefore increase

their DNA affinity (Rastinejad et al., 2000; Depoix et al.,

2001). They also cause corepressor release and create a new

hydrophobic cleft formed between H3, H4 and H12 which

constitutes a surface where coactivators can bind (Fig. 5B).

According to recent studies, the surfaces involved in core-

pressor and coactivator binding partially overlap and the

ligand-induced repositioning of H12 would result in a

switch from a corepressor to a coactivator-binding surface

(Nagy et al., 1999; Perissi et al., 1999; Glass and Rosenfeld,

2000). Within RAR/RXR heterodimers bound at DR5

elements, though both liganded partners are theoretically

able to recruit coactivators, RXR is «subordinated» to its

RAR partner (Roy et al., 1995; Willy and Mangelsdorf,

Fig. 5. Three-step mechanism of retinoid receptor action. (A) In the absence of ligand, retinoid receptors bound to response elements located in the promoter of

target genes are associated with histone deacetylase-containing (HDAC) complexes tethered through corepressors and repress transcription. (B) Upon ligand

binding, the corepressors dissociate, allowing the recruitment of coactivators associated with complexes displaying histone acetyltransferase (HAT),

methyltransferase, kinase or ATP-dependent remodeling (SWI/SNF) activities that decompact repressive chromatin. (C) In the third step, the coactivators

dissociate and the SMCC mediator complex assembles. Then the mediator expedites entry of the RNA Pol II and the general transcription factors to the

promoter, resulting in transcription initiation.

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–166

1999). This phenomenon has been attributed to the fact that

liganded RXR cannot dissociate corepressors and therefore

coactivators cannot be recruited (Germain et al., 2002).

However, in the presence of both RAR and RXR ligands,

there is synergy originating from the RAR agonist-induced

dissociation of corepressors and the subsequent cooperative

binding of coactivators to the two partners. Most impor-

tantly, the two partners synergize with each other for

transcription, not only through their AF-2 domains but also

through their AF-1 domains (Gianni et al., 2003), very

likely via their cooperation for the recruitment of coactiva-

tors (Bommer et al., 2002).

The first identified family of ligand-recruited retinoid

receptors coactivators is the SRC/p160 family which

includes SRC-1/NCoA-1, TIF-2/GRIP-1/NCoA-2/SRC-2

and pCIP/ACTR/AlB1/TRAM1/RAC3/SRC-3 (Chen,

2000; McKenna and O’Malley, 2002). Other coactivators,

p300/CBP (Vo and Goodman, 2001) and CARM-1 (Chen et

al., 1999a; Kouzarides, 2002), have been characterized, but

they are structurally and functionally distinguishable from

the SRC/p160 family. A recurring structural feature of all

these coactivators is a highly conserved alpha-helical

LxxLL motif (where L is leucine and x is any amino acid)

from a single to several copies, which is implicated in their

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–16 7

ligand-dependent recruitment by the AF-2 domain of reti-

noid receptors. Cocrystal structures indicate that two LxxLL

motifs from a single p160 coactivator molecule interact with

the AF-2 domains of both partners. However, one molecule

of coactivator can be cooperatively recruited by each

member of the heterodimer. Most importantly, the coacti-

vators also contain domains which interact with other

coactivators. Accordingly, TIF2 possesses one domain

interacting with CBP/p300 and a second one which has

been recently shown to interact with CARM-1 (McKenna

and O’Malley, 2002).

p300/CBP and, to a lesser extent, the members of the

p160 family locally modify chromatin structure through

their histone acetyltransferase (HAT) activity which acety-

lates lysine residues located at the N-terminal tails of

histones, thereby weakening the interaction of the N-termi-

nal tails with the nucleosome DNA (Fig. 5B). Other

coactivators such as CARM-1 act through their histone

methyltransferase (HMT) activity which upon methylation

of specific arginine or lysine residues also change histone–

DNA and histone–histone contacts. Then the opening of the

chromatin environment is achieved by the recruitment

through pCIP and CBP, of larger complexes with histone

acetyltransferase (PCAF) or histone methyltransferase ac-

tivities (Kouzarides, 2000; Roth et al., 2001; Zhang and

Reinberg, 2001). Note that the efficiency of histone acety-

lation and methylation is regulated upon phosphorylation of

the nearby serines residues by associated kinases (Cheung et

al., 2000; Lo et al., 2001). Altogether, these histone mod-

ifications create tags or binding sites that form an «histone

code» read by a specialized bromodomain present in the

chromatin modifiers (Jeanmougin et al., 1997; Strahl and

Allis, 2000; Berger, 2002). This code would coordinate the

recruitment of additional HATs or HMTs for further chro-

matin decompaction. It would also allow the recruitment of

ATP-dependent chromatin remodelers (SWI/SNF) which

use the energy of ATP hydrolysis to reposition nucleosomes

at the promoter through sliding them in cis or displacing

them in trans, allowing the formation of nucleosome-free or

nucleosome-spaced regions (Kingston and Narlikar, 1999;

Vignali et al., 2000; Narlikar et al., 2002).

5. Third step: recruitment of the transcriptional

machinery

Once repressive chromatin has been decondensed, it has

been proposed that a coregulators exchange occurs, in order

to allow the RARE-bound heterodimers to participate in the

entry of RNA-Pol II and GTFs into the preinitiation com-

plex (Chen et al., 1999b; Malik and Roeder, 2000). The

current working hypothesis is that the p160 coactivators

dissociate, subsequent to their acetylation which decreases

their ability to interact with the receptors (Chen et al.,

1999b), or to their degradation by the proteasome (Yan et

al., 2003). Then the retinoid receptors become able to recruit

the transcription machinery via their association with the so-

called SMCC (Srb and Mediator protein containing com-

plex) mediator complex (Malik and Roeder, 2000; Dilworth

and Chambon, 2001; Woychik and Hampsey, 2002). The

subunit of the mediator complex that is responsible for

interaction with the AF-2 domain of liganded retinoid

receptors was identified as DRIP205 which is identical to

TRAP220 and contains a LxxLL nuclear receptor box motif.

Whether other subunits interact with the N-terminal AF-1

domain of RARs and RXRs, as described for the glucocor-

ticoid receptor (Hittelman et al., 1999), remains to be

determined.

Then the mediator expedites entry of the transcriptional

machinery to the promoter (Fig. 5C) through its interaction

(via other subunits) with the RNA Pol II holoenzyme

(Woychik and Hampsey, 2002). This process also involves

the six GTFs (Orphanides et al., 1996). The large multi-

subunit TFIID, which binds to the promoter through its

TBP, possesses associated factors or TAFIIs developing

kinase and acetylase activities (TAFII250). This will in-

crease chromatin remodeling at the promoter to permit tight

binding of the basal transcriptional machinery. Some TAFIIs

(May et al., 1996; Lavigne et al., 1999) and TFIIH also

interact with retinoid receptors (Rochette-Egly et al., 1997),

thus increasing the efficiency of the preinitiation complex

assembly (see below). Finally, the recruitment of GTFs is

also enhanced by p300/CBP and by components of the SWI/

SNF complex associated to the RNA Pol II holoenzyme

(Adelman and Lis, 2002; Orphanides and Reinberg, 2002).

Once transcription has been initiated, RNA Pol II traffics

along the gene to be transcribed. This process involves

chromatin remodeling and modifying activities endowed by

subunits of the elongation factors that track with elongating

RNA Pol II (Orphanides and Reinberg, 2000). Finally, the

equilibrium will shift in favour of histone tail deacetylation,

and methylation at residues leading to the rapid conversion

of chromatin to a repressed conformation.

In summary, in a context of chromatin where the nucle-

osomes do not impede the binding of RAR/RXR hetero-

dimers to their DNA recognition sequences (Dilworth and

Chambon, 2001), liganded RAR/RXR heterodimers recruit

first coactivators and HAT complexes resulting in histone

acetylation. Then ATP-dependent remodeling complexes are

recruited, leading to the displacement of impeding nucleo-

somes within the proximal promoter region, thus facilitating

access of the general transcription machinery to the pro-

moter. However, it cannot be excluded that the reverse order

of events, which is the recruitment of ATP-dependent

remodelers before HATs, could occur in a context of highly

condensed chromatin (Cosma, 2002). Therefore, the relative

timing and order of recruitment of chromatin modifyers and

remodellers would depend upon the nature of the promoter

and the chromatin structure in which it resides (Fry and

Peterson, 2001) in order to allow the most efficient solution

(Aalfs and Kingston, 2000; Cosma, 2002), each player

helping the other and each step facilitating another one.

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–168

The key is that the appropriate end stage, e.g. a properly

decondensed chromatin with a functional preinitiation com-

plex posed for transcription, be attained in a timely manner.

6. Control of RAR/RXR transactivation by the

ubiquitin–proteasome system

In recent years, it has become evident that the transcrip-

tional activity of retinoid receptors, as that of most tran-

scription factors, is also regulated by the ubiquitin–

proteasome pathway. Paradoxically, both the proteolytic

and non-proteolytic activities of this system appear to

modulate transcription at different levels (Ferdous et al.,

2001; Salghetti et al., 2001; Tansey, 2001; Conaway et al.,

2002; Muratani and Tansey, 2003).

One main role of the ubiquitin–proteasome system is to

degrade transcriptional activators. In this process, following

a signal, the substrate protein is multi-ubiquitylated at a

lysine group and then targeted for destruction by the 26S

proteasome. The 19S subcomplex of the proteasome recog-

nizes the multi-ubiquitylated substrate, removes the ubiq-

uitin groups, unfolds the substrate and feeds the resulting

unstructured chain into the 20S catalytic core of the protea-

some where it is degraded (DeMartino and Slaughter, 1999).

It has been recently demonstrated that, within RAR/RXR

heterodimers bound at response elements, both partners are

degraded by the proteasome in response to retinoids (Zhu et

al., 1999; Boudjelal et al., 2000; Kopf et al., 2000; Osburn et

al., 2001; Tanaka et al., 2001; Gianni et al., 2002a, 2003).

This process involves the ubiquitylation of RARs (Zhu et

al., 1999; Kopf et al., 2000) and the recruitment of the

proteasome at the AF-2 domain through SUG-1 (Gianni et

al., 2002a) which is one of the six ATPases in the base of the

19S regulatory complex of the 26S proteasome. It has been

proposed that this degradation process would provide a

mechanism to control the magnitude and the duration of

retinoid-mediated transcription.

The importance of the ubiquitin–proteasome system in

retinoid receptors transactivation came from the particular

case of the RARg isotype (Gianni et al., 2002a). Indeed,

blocking either the ubiquitin or the proteasome systems

abrogates not only the degradation of RARg, but also

RARg-mediated transcription. The paradoxal mechanism

of how the ubiquitin–proteasome system regulates RARg/

RXR transcriptional activity is currently unknown but

several lines of evidence indicate that the ubiquitin ligases

(Imhof and McDonnell, 1996; McKenna et al., 1998), as

well as some components of the proteasome system, such as

SUG-1 (vom Baur et al., 1996), are able to bind retinoid

receptors. In addition, ubiquitin ligases belong to complexes

that are integral components of the mammalian mediator

complex associated to the Pol II transcription machinery

(Brower et al., 2002; Conaway et al., 2002) and SUG-1 also

interact with the general transcription factor TFIIH (Fraser

et al., 1997; Weeda et al., 1997; Sandrock and Egly, 2001).

Finally, the 19S subcomplex of the proteasome has been

shown to associate with transcription activators (Gonzalez et

al., 2002) and to participate to elongation (Ferdous et al.,

2001). Therefore, the ubiquitin–proteasome machineries

may play a dual role, controlling on the one hand the

functionality of RARg/RXR heterodimers through helping

the recruitment of the transcription machinery (Lin et al.,

2002), and on the other hand the ubiquitylation and the

subsequent degradation of the heterodimers. Such a dual

role may regulate the dynamic assembly/disassembly of

retinoid receptors to the promoter of the target genes, as

recently demonstrated for other nuclear receptors such as the

estrogen and androgen receptors (Freeman and Yamamoto,

2002; Kang et al., 2002; Reid et al., 2003).

It must be noted that the same conclusions could not be

made for the other RAR isotype RARa, since inhibition of

the proteasome by specific inhibitors did not abrogate, but

amplified RARa-mediated transcription (Gianni and Roch-

ette-Egly, unpublished observations), as described for the

glucocorticoid receptor (Wallace and Cidlowski, 2001).

Why the two isotypes RARa and RARg are not regulated

similarly will require further investigations. Finally, the

ubiquitin–proteasome system also targets histones and other

coregulators (Wang et al., 2002; Yan et al., 2003), therefore

increasing the complexity of retinoid-dependent transcrip-

tional control (Muratani and Tansey, 2003).

7. Regulation of RAR/RXR-mediated transcription

through phosphorylation

RARs and RXRs are substrates for a multitude of kinases

(see Figs. 1 and 6) (Rochette-Egly, 2003). Importantly,

subsequent to their interaction with TFIIH, RARs (RARa

and RARg) are phosphorylated in their N-terminal A/B

region by the cdk7 subunit of TFIIH which has a cyclin-

dependent kinase activity (Rochette-Egly et al., 1997; Bas-

tien et al., 2000). This phosphorylation process which has

been extensively studied, especially in the case of RARa,

plays a critical role in the retinoid response. Indeed, upon

mutations in a TFIIH subunit resulting in an incorrect

positioning of the cdk7 kinase relative to its substrate,

RARa is underphosphorylated and retinoid-dependent tran-

scription is decreased (Keriel et al., 2002). If phosphoryla-

tion of the AF-1 domain by TFIIH occurs when the GTFs

are recruited at the promoter, the hypothesis that the

phosphorylation of the AF-1 domain helps the ligand-

dependent recruitment of coactivators and chromatin mod-

ifyers would be rather elusive. Instead, phosphorylation

could facilitate the recruitment of components of the tran-

scription machinery and therefore stabilize the formation of

the NR transcription complex. Future efforts will undoubt-

edly reveal new «coregulators» interacting with the phos-

phorylated motif of RARa and therefore regulating RARa-

mediated transcription. However, it is not excluded that

phosphorylation might rather facilitate the dissociation of

Fig. 6. Signaling pathways activating MAP kinases (Erks, JNKs and p38), PI3K, Akt, PKA and PKC are involved in the control of retinoid-mediated

transcription. Nuclear retinoid receptors are targeted by phosphorylations in response to signaling pathways. These phosphorylations may modulate their

potentiality to recruit cofactors associated with the trascription machinery. The coactivators are also phosphorylated, modulating their activity and/or their

potentiality to recruit the chromatin modifying (HAT, HMT) and remodeling complexes. Finally, histones, the general transcription factors and the RNA

Pol II can be also phosphorylated. RTK, receptor tyrosine kinase; PI3K, phosphatidylinositol 3-kinase; Erk, extracellular signal-related kinase; MAPK,

mitogen-activated protein kinase; MEKK, MAPK kinase kinase; ASK1, apoptosis-stimulating kinase 1; AC, Adenylate cyclase; JNK, Jun amino-terminal

kinase.

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–16 9

RARa from transcription inhibitors or help the dissociation

of RARa from the transcription machinery in order to allow

elongation to proceed. Note that, in contrast to other

transcription activators, phosphorylation of the AF-1 do-

main of RARa does not influence the ubiquitylation and the

proteasomal degradation of RARa (Kopf et al., 2000).

It must be pointed out that in the particular case of the

RARg isotype, phosphorylation by TFIIH, though neces-

sary (Bastien et al., 2000), is not sufficient. Indeed, RARg

needs to be also phosphorylated at an additional nearby

residue by p38MAPK, subsequently to its activation by

retinoids (Gianni et al., 2002a,b). Phosphorylation of these

two residues is required for both the transactivation and the

degradation of RARg.

The critical role of RARg phosphorylation has been

further dissected in our group, by using F9 cells which

represent a cell-autonomous system for analyzing retinoid

signaling (for review, see Rochette-Egly and Chambon,

2001). In these cells, the retinoid signal is transduced by

RARg/RXR heterodimers and therefore the various RA

responses are abolished in RARg null cells. Taking advan-

tage that the RA responses can be restored upon reexpres-

sion of the receptor to wild-type levels, the same strategy

has been used with RARg mutated at the phosphorylation

sites located in the N-terminal AF-1 domain. It has been

demonstrated that the integrity of these phosphorylation

sites is indispensable to the activation of a subset of RA-

target genes, for RARg degradation and for RA-induced F9

cell differentiation (Taneja et al., 1997; Kopf et al., 2000;

Gianni et al., 2002a).

Though it is assumed that phosphorylation by both

TFIIH and p38MAPK is crucial for both the transcriptional

activity and the degradation of RARg, the paradoxal mech-

anism of how phosphorylation regulates these two processes

is currently unknown. Nevertheless, some speculative mod-

els can be proposed (see Fig. 7). As phosphorylation of the

Fig. 7. Recapitulation of the effects of phosphorylations on RARg-mediated transcription of target genes. The fraction of liganded RARg2 that is bound to

cognate response elements as heterodimers with RXRa is phosphorylated by the cdk7 subunit of TFIIH at one serine residue located in the AF-1 domain. Then

the RA-induced activity of p38MAPK leads to the phosphorylation of the second serine residue. Phosphorylation of these two serines is a checkpoint

controlling on the one hand transcription and on the other hand the degradation of RARg by the 26S proteasome. How? One can propose that phosphorylation

helps the association and/or the dissociation of coregulators. Indeed, phosphorylation may allow the recruitment of ubiquitin ligase complexes which will

regulate both the transcription and the degradation of RARg through their association to the transcription machinery and through signaling RARg

ubiquitylation, respectively. However, it is not excluded that phosphorylation induces the dissociation of inhibitors, therefore making the nearby lysine residue

available for ubiquitin ligases recruited at the AF-2 domain. In that context, it has been suggested that oligo-ubiquitylation would modulate transcription, while

poly-ubiquitylation signals the switch to the degradation by the 26S proteasome.

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–1610

two residues is also required for retinoid-induced ubiquity-

lation of RARg (Gianni et al., 2002a), one can hypothesize

that phosphorylation signals the recruitment of ubiquitin–

ligase complexes which would play a dual role. Indeed,

such complexes might control on the one hand transcription,

through their association with the transcription machinery

(see above), and on the other hand RARg degradation

through its ubiquitylation. However, it is not excluded that

phosphorylation induces the dissociation of inhibitors,

therefore making the nearby lysine residue available for

ubiquitylation by the ubiquitin ligases recruited at the AF-2

domain. Note in that context that oligo-ubiquitylation could

modulate the activity of transcription activators (Salghetti et

al., 2001), while poly-ubiquitin chains signal the switch to

the degradation by the proteasome (Gonzalez et al., 2002).

In conclusion, it is tempting to speculate that, in the case

of RARg, phosphorylation would be a checkpoint, control-

ling on the one hand transcription and signaling and on the

other hand the degradation of RARg. Future efforts will

undoubtedly reveal new «coregulators» (ubiquitin ligases,

WW domains, F-box proteins) interacting with the phos-

phorylated or unphosphorylated AF-1 domain of RARg and

therefore regulating RARg functionality.

Interestingly, RARa and RARg transcriptional activities

can be also modulated upon phosphorylation by other

kinases in response to a variety of signals. Indeed, phos-

phorylation by PKA at serine 369, between H9 and H10,

modulates positively the transcriptional activity of RARa

(Rochette-Egly et al., 1995), very likely through helping

coregulators binding and/or DNA binding of RAR/RXR

heterodimers. Finally, phosphorylation of RARa DBD

(Delmotte et al., 1999) that occurs upon activation of PKC

signaling favors dimerization and subsequently DNA bind-

ing of RARa/RXR heterodimers.

Recently, the role of RXRa phosphorylation has been

also studied. Accordingly, phosphorylation of Ser22 located

in the N-terminal AF-1 domain is required for the activation

of a subset of target genes and for the antiproliferative effect

of retinoids (Bastien et al., 2002). Recently, it has been

found that three additional residues (Ser61, Ser75 and

Thr87) located in the same N-terminal domain can be also

phosphorylated in response to retinoids (Gianni et al., 2003)

and that this phosphorylation modulates the synergy be-

tween both heterodimeric partners for maximal transcrip-

tional activity, very likely through helping the recruitment of

coregulators. In contrast, MAPK-mediated phosphorylation

of the serine residue located in the Omega loop of RXRa

between helices H1 and H3 and close to helix 12, according

to the three-dimensional structure, impairs the transcription-

al activity of RAR/RXR heterodimers (Lee et al., 2000;

Matsushima-Nishiwaki et al., 2001). It has been proposed

that phosphorylation of this residue would create conforma-

tional changes within the LBD, disrupting the interactions

with coregulators and therefore decreasing transcription.

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–16 11

However, according to recent studies, it would make RXRa

more resistant to proteolytic degradation, therefore exerting

dominant negative inhibition (Matsushima-Nishiwaki et al.,

2001).

It must be noted that the various signal transduction

pathways also cross-talk with retinoid receptors transactiva-

tion through the phosphorylation of the coactivators and

corepressors (see Fig. 6). The phosphorylation of corepres-

sors such as SMRT correlates with an inhibition of their

interaction with RARs and their redistribution from the

nucleus to the cytoplasm (Hong and Privalsky, 2000). In

contrast, the phosphorylation of p300/CBP, pCIP, SRC-1

and TIF-2 by a variety of kinases, including MAPKs or

PKA (Fig. 7), rather enhances their enzymatic activity as

well as their efficiency to interact with retinoid receptors

and/or the HAT complexes (Font de Mora and Brown, 2000;

Rowan et al., 2000a,b; Lopez et al., 2001; Vo and Goodman,

2001). Histones are also phosphorylated, increasing mark-

edly the efficiency of HATs and HMTs to acetylate or

methylate the nearby lysine residues. The same observation

has been made for the general transcription factors (GTFs)

and RNA Pol II (Orphanides and Reinberg, 2002). All these

phosphorylation processes converge towards the formation

of an efficient transcription initiation complex and a con-

trolled maximal response.

8. Retinoid receptors cross-talk with other signaling

pathways

Due to the ability of RXRs to serve as heterodimeric

partners not only to RARs, but also to several other nuclear

receptors (PPARs, LXR) (Willy and Mangelsdorf, 1999), it

is evident that retinoids can also control, the transcription of

a wider set of hormone-responsive genes (Leid et al., 1992;

Mangelsdorf and Evans, 1995; Chambon, 1996). Moreover,

one has to consider that, as is true for many other genes, the

promoters of retinoid-target genes contain, in addition to the

cognate response elements (RAREs), other regulatory

sequences which associate together with several transcrip-

tion activators in enhanceosomes. As an example, RARs

cooperate with SF1 and Sp1/Sp3 for the transactivation of

the Oct-3/4 and CYP26 promoters, respectively (Barnea and

Bergman, 2000; Loudig et al., 2000). Similarly, in the

presence of cytokines, STAT5 cooperates with RARs to

achieve maximum transcription of some RA-target genes (Si

and Collins, 2002). Such synergistic effects very likely

result from the cooperative recruitment of coregulators,

increasing chromatin remodeling and/or entry of the tran-

scription machinery to the promoter. This would explain

why, in vivo, the mechanisms of regulation differ from one

gene to the other and depend on the cell type (Nagpal et al.,

1992; Folkers et al., 1993).

However, retinoids are also able to antagonize the

activation of a subset of heterologous genes. The best

example is that of the AP-1-regulated genes (Shaulian and

Karin, 2002). The repressive effect of retinoids on AP-1

activity has been correlated with their antitumor activity and

the importance of this cross-talk for growth control is

increasingly recognized (Altucci and Gronemeyer, 2001b).

However, the mechanistic basis of the anti-AP-1 activity of

retinoid receptors remains still elusive, despite the proposal

of several distinct mechanisms. Competition for limiting

amounts of a common coactivator (Kamei et al., 1996) has

been proposed, as well as the inhibition of the JNKs

pathway (Caelles et al., 1997; Lee et al., 1999), disruption

of the Jun–Fos dimerization (Zhou et al., 1999) or exclusion

of some components (kinases, CBP) from the AP-1 com-

plexes (Benkoussa et al., 2002). Another example of repres-

sion by retinoid receptors has been demonstrated for

oncogenic h-catenin-mediated gene transcription (Easwaran

et al., 1999; Xiao et al., 2003) but whether liganded retinoid

receptors compete with h-catenin or induce its proteasomal

degradation is still elusive. Finally, retinoids have been

recently demonstrated to antagonize the activation of Smads

regulated genes that occurs in response to TGF-h, very

likely through the activation of a phosphatase, thereby

reducing the levels of phosphorylated Smads (Cao et al.,

2003). However, Pendaries et al. (2003) reported that, in the

presence of antagonists, RARg can potentiate the activation

of TGF-h target genes, via a direct interaction with Smads.

Such observations are of prime importance since it would

dictate whether retinoid agonists or antagonists should be

used in association with TGF-h in the treatment of abnormal

wound healing.

Finally, retinoids have been shown recently to inhibit the

PI3K/Akt pathway. Indeed, our group demonstrated recently

that retinoids target both the PI3K and the phosphatase

PTEN through transcriptional processes involving RAR/

RXR heterodimers (J. Bastien et al., manuscript submitted).

According to another recent study (del Rincon et al., 2003),

retinoids antagonize the mitogenic IGF-IR/Akt pathway

through the dephosphorylation and degradation of the

IRS-1 adaptor protein. This will undoubtedly have conse-

quences on the phosphorylation and therefore on the activity

of the Akt targets, therefore increasing the complexity of the

antiproliferative and differentiative action of retinoids.

9. Conclusion and perspectives

Retinoids are essential for the control of normal cell

differentiation and proliferation. Therefore they are morph-

ogens and essential regulators of embryogenesis. In adults,

they are required for the proper functioning of a number of

organs. All these functions involve the transcriptional con-

trol of a large number of genes by RXR/RAR heterodimers,

as gene-ablation experiments generate embryonic develop-

ment defects and abrogate the differentiative and antiproli-

ferative effects of RA.

According to the current model described in the present

review, retinoid-dependent gene-specific transcription is

J. Bastien, C. Rochette-Egly / Gene 328 (2004) 1–1612

orchestrated by nuclear receptors which upon DNA and

retinoid binding trigger a cascade of dynamic events result-

ing in an appropriately remodelled chromatin template with

a functional preinitiation complex positioned for transcrip-

tion. At the final end, retinoid receptors are degraded by the

ubiquitin–proteasome pathway.

Though such a model improved efficiently our under-

standing of retinoids action, it is evident that the mecha-

nisms by which retinoid receptors regulate gene

transcription in vivo must be more complex and a number

of questions remain still unanswered. For instance, it is still

unclear how RARs phosphorylation controls the dynamic

recruitment and disassembly of complexes formed at the

promoters of retinoid-target genes during the initiation of

transcription. In addition, considering the complexity of the

promoters of the various retinoid-responsive genes, with

respect to response elements for other transcription activa-

tors, the nucleosomal architecture, the diversity of the cell

type specific coregulators and of the physiological signals, it

is evident that gene transcription is subject to multiple cell

specific combinatorial regulations.

Considering the importance of the regulatory networks

governing the normal transcription of a given gene, the

deregulation of any factor or of the signal transduction

pathways would contribute to the development of diseases

or tumoral processes. As an example, a number of cancers,

especially lung cancers (Picard et al., 1999), are character-

ized by the loss of RARh2 expression due to the hyper-

methylation of its promoter and its subsequent silencing.

Though the gene programs that are specifically regulated by

RARh are unknown, this receptor has been considered as a

tumor suppressor (Altucci and Gronemeyer, 2001b). Anoth-

er example is given by the PML-RARa and PLZF-RARa

fusion proteins (for review, see Altucci and Gronemeyer,

2001b) which, through aberrant recruitment of corepressors,

silence RA-target genes leading to a block of differentiation

of promyelocytic cells in acute promyelocytic leukemia

(APL).

Interestingly, alterations in the phosphorylation of RARs

may also account for some diseases or cancers. Indeed, a

defect in the phosphorylation of RARs has been observed in

cells from xeroderma pigmentosum patients (Keriel et al.,

2002) due to a decreased interaction with the transcription

factor TFIIH which harbours the kinase cdk7. This phos-

phorylation defect would account in part for the develop-

ment abnormalities of these patients (Egly, 2001). In

addition, as alterations in the phosphorylation of RARs

result in aberrant turnover and transactivation (Taneja et

al., 1997; Matsushima-Nishiwaki et al., 2001; Gianni et al.,

2002b), one can postulate that the retinoid resistance of

some cancer cells would result from aberrant kinome

signaling (Blume-Jensen and Hunter, 2001; Tari et al.,

2002).

Future research should focalize at understanding how

retinoid-mediated transcription is finely tuned in the context

of complex natural promoters. In addition, more studies

concerning the regulation of retinoid receptors activity

through phosphorylation should provide new insights in

the developmental processes and in cancer mechanisms.

Acknowledgements

We are particularly grateful to Gaetan Bour and Emilie

Gaillard for enthusiastic discussions and for critics. Many

thanks also to all the past members of the group for their

contribution to the work. We are also very grateful to Prof.

P. Chambon for constant support. Our studies mentioned in

the text have been supported by funds from the Centre

National de la Recherche Scientifique (CNRS), the Institut

National de la Sante et de la Recherche Medicale

(INSERM), the Hopital Universitaire de Strasbourg, the

Association pour la Recherche sur le Cancer, and Bristol-

Myers Squibb. JB was supported by the Ministere de la

Recherche et de l’Enseignement Superieur and by the Ligue

Nationale contre le Cancer.

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