CHAPTER 1
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1. TRANSCRIPTION
REGULATION
2. RETINOID SIGNALING
AIM OF THIS THESIS
Introduction
That retinoids are essential for early embryonic development was indicated
by the abnormalities caused both by excess and lack of retinoids. The cloning
and subsequent characterization of a receptor for the active vitamin A
derivative retinoic acid revealed that the action of retinoids is mediated
by regulation of transcription through retinoic acid receptors (RARs).
These receptors belong to the nuclear hormone receptors superfamily which
can, in a ligand dependent manner, change the expression of genes that
contain binding sites for these transcription factors. It is thought the
ligand dependent changes in target gene expression are responsible for
the action of retinoids in development. During development, gene expression
is thightly regulated, overexpression or lack of expression can result
in severe developmental abnormalities. It is therefore important to understand
how gene expression is regulated in a spatio/temporal fashion.
In the first part of the introduction general constraints on transcriptional
regulation are discussed. Given the importance of retinoids in embryonic
development, studying the mechanisms involved in transcription regulation
by RARs may help to elucidate mechanisms underlying regulated gene expression
during development. The second part of the introduction evaluates the roles
of retinoids and their receptors. First the structure and function of retinoid
receptors is described. This is followed by a description of the processes
that could contribute to cell-specific retinoid signaling, and which may
be important for embryonic development. Finally the roles of retinoids
in the differentiation of embryonic development and
embryonal carcinoma cells is described
1. TRANSCRIPTION
REGULATION
It is very important for an organism to control
gene expression adequately. For example prokaryotes need to regulate the
expression of enzymes involved in growth on a specific carbon source, thereby
preventing the need to express these enzymes permanently. It is even more
important for eukaryotes to regulate gene expression, firstly because non-vertebrate
eukaryotes contain approximately five times more genes, and vertebrates
twenty times more than prokaryotes. Secondly with increasing complexity
during evolution, eukaryotic cells have come to fulfill more specialized
functions, requiring control of the expression of tissue-specific genes.
In mammalian cells, it has been estimated that one third of all genes are
expressed ubiquitously, while the remainder have much more restricted,
cell-type specific, expression patterns, and are therefore likely to fulfill
more specific functions. This requirement for a proper control over gene
expression is emphasized by the developmental abnormalities which are observed
in gene disruption as well as overexpression experiments in mice.
1.1 Repression of transcription
Bird (18) proposed that from evolutionary point
of view it could may be more important to have a good repression mechanism
to keep genes silent than to rely only on the activation of transcription.
Several repression mechanisms are known: (1) The most common transcriptional
repression mechanism is the presence of a repressor which can prevent activation
through various mechanisms; this type of repression mechanism is present
both in pro- and eukaryotes (53). (2) In eukaryotes, in contrast with prokaryotes,
the genetic information is contained in a highly ordered protein-DNA complex
called chromatin which permits transcriptional repression (76). (3) Vertebrates
have an additional repression mechanism to regulate gene-expression: DNA
methylation. The vertebrate genome is highly methylated, only the 5' ends
of transcribed genes generally lack methylation (CpG islands), indicating
that methylation prevents activation (17). All of these repression mechanisms
are only suitable for transcriptional regulation if they can be regulated,
cell-type specifically. The repression mechanisms described above and the
current models concerning transcriptional activation are described below
and their roles in gene regulation during development and in regulating
cell type-specific transcription are discussed.
1.1.1 Role
of chromatin in transcriptional repression
Chromatin is DNA which is highly condensed through
extensive protein-DNA contacts, primarily mediated by histones. Four different
histones: H2A, H2B, H3 and H4 together form an octamer (core histone),
around which 146 bp of DNA can make a 1.75 left handed superhelical turn,
together forming a nucleosome (291). The region between two nucleosomes,
the linker region, can bind several proteins, including histone H1 but
also binds non-histone proteins e.g. members of the family of high mobility
group (HMG) proteins (291). The latter can induce bending of DNA, suggesting
that they have an architectural role in chromatin assembly (96). Binding
of these proteins might cause an inhibition of transcription through an
interaction with TBP as is the case for HMG2 (88), although generally HMG
proteins are thought to be involved in transcriptional activation (264,218,215).
Histone H1 is involved in high order structure formation of core nucleosomes
since it can interact with linker DNA and the exterior part of nucleosomes
of one nucleosome core particle but may also interact with neighboring
nucleosomes. This suggests that Histone H1 is involved in the stabilization
of chromatin, which may explain its negative effect on transcription (12,208).
HMG proteins or H1 compete for binding to the linker region leading to
opposite results: histone H1 binding results in formation of a high order
structure, while HMG1 modulates DNA structure through bending of DNA, which
can prevent high order chromatin structure formation (96). Such high order
structures are described as 30 nm fibers, reflecting the diameter of the
chromatin high order structure (291). Generally, genes packed into nucleosomes
and further into 30 nm fibers, are transcriptionally inactive (76).
To achieve transcriptional activation the nucleosomal
structure must, at least partially, be disrupted (Felsenfeld). Various
groups of proteins modulate the chromatin structure: the Polycomb-group
proteins, the trithorax group, the SWI/SNF complex and the histone acetyltransferases.
The Pc-G genes form a multi-protein complex and are believed to function
by establishing a stable, repressed chromatin structure, thereby preventing
transcriptional activation. The important roles these proteins fulfill
during development (226,268), underscores the importance of transcriptional
repression for gene regulation. The repressive effect of polycomb
is opposed by the trithorax group of activators. Mutations in the
trithorax group gene brahma suppress mutations in polycomb.
Brahma is the Drosophila homolog of the yeast SWI2
gene (129,221), suggesting that the SWI/SNF complex is involved in some
of the functions of the trithorax complex. The SWI/SNF complex is
proposed to function in chromatin destabilization (for review: 129,307).
These yeast proteins were identified by their ability to activate transcription
of various different genes, since mutation of these SWI/SNF proteins caused
repression of transcription. Additional mutations in several loci could
compensate for the suppressive effects of these snf or swi
mutations on transcription (307). Characterization of the mutated genes
revealed that loss of various histone genes could compensate for the repressive
effect of these snf/swi mutations, indicating that the function
of the SWI/SNF complex is to change chromatin structure (108). The SWI/SNF
complex is a large multiprotein complex that is required for activation
by ectopically expressed activators such as GR (226,314). Possibly an interaction
between transcriptional activators and the SWI/SNF complex is required
for transcriptional activation (198,314). Furthermore this complex is present
in the polymerase II holoenzyme, further linking chromatin remodeling with
transcriptional activation (306). The role of SWI/SNF could be to increase
the binding of transcription factors (52,141) or TBP (115) to DNA in an
ATP hydrolysis-dependent, activation domain-independent manner. This would
result in local disruption/remodeling of the nucleosome. Two human SWI/SNF2
homologs have been cloned, BRG1 and hBRM (129). The purification of the
human SWI/SNF complex from several cell lines using antibodies against
BRG1 revealed that this complex is heterogeneous with respect to subunit
composition, varying from 9 to 12 proteins (200). Furthermore both HMG1
and hBRM are differentially expressed in various cell lines (200,201),
indicating that the presence and composition of the SWI/SNF complex is
regulated. Possibly, individual complexes or components thereof fulfill
distinct functions.
The correlation between histone acetylation and
transcriptional activation was observed long time ago, and direct proof
for this model has been obtained by biochemical analysis of histones from
transcriptionally active chromatin (104). The N-terminal tails of histone
H3 and H4 are acetylated on lysine residues, thereby neutralizing the basic
residues in these histone tails. Consequently, the interaction of the Histone
tails with DNA is weakened leading to destabilization of chromatin (309),
thereby making the DNA more accessible for transcription factors (153).
Recently, a histone acetyltransferase (HAT), showing homology to the yeast
Gcn5 protein was purified and cloned from Tetrahymena (30). This protein
is required for transcriptional activation of a subset of genes, and presumably
acts as adaptor/cofactor through interaction with two other coactivators
ADA2 and ADA3 (111). Interestingly, ADA2 which interacts directly with
acidic activators, has some regions of homology to the p300/CBP cofactors
(97). This protein has been reported to act as a cofactor for several classes
of transcriptional activators including CREB, cJUN, cMyb, c-Fos, MyoD,
and nuclear hormone receptors (117), thereby enhancing transcriptional
activation. Recently the mechanism underlying this transcriptional activation
was elucidated by the observation that p300/CBP can interact with a protein
named pCAF for p300/CBP-associated factor which is the human homolog of
Gcn5 which also contains histone acetylation activity (312).
Table 1 Homologous proteins
of yeast and mammalians are involved in transcriptional activation through
acetylation of histones.
|
yeast |
eukaryotes |
| activator |
gcn4 |
c-jun |
| adaptor |
ada2 |
CBP/p300 |
| HAT |
gcn5 |
pCAF |
Furthermore recently it was shown that CBP/p300
itself also contains HAT activity (213,9). The isolation of proteins in
a two hybrid screen using TIF1 (a nuclear receptor interacting protein,
see below) as bait, revealed that multiple proteins are involved in nucleosome
remodeling (150).
Finally, recently TAFII230/250 was
reported to posses HAT activity (194). Moreover evidence has been presented
recently showing a direct interaction between the ADA/GCN5 complex and
the SWI/SNF complex (312), thereby linking the two nucleosome remodeling
complexes. The presence of multiple proteins or protein complexes involved
in the nucleosme disruption/removal process allows cell type-specific modulation
of the rate of transcription. Regulation of the expression of these proteins,
interactions with other proteins of the complex or regulation of the HAT-activity
of these proteins could therefore contribute to the control in gene expression
by modulating chromatin structure.
1.1.2 Role
of methylation in transcription
Large amounts of methylated cytosine (m5C) are
found throughout the mammalian genome. All housekeeping genes, but also
some of the tissue specific genes contain regions of clustered non-methylated
CpG nucleotides, known as CpG islands in the 5' ends of the gene (55).
Methylation, catalyzed by DNA(cytosine-5)methyltransferase, is accompanied
with the inhibition of transcription, presumably by prevention of binding
of transcription factors since it has been reported that E2F, CREB, NF-kB
and cMyc do not bind to methylated DNA, while SP1 can bind to both methylated
DNA and unmethylated CpG islands (27). The latter observation possibly
explains the general involvement of SP1 in regulating the expression of
housekeeping genes. Gene repression through DNA methylation is influenced
by both the level of methylation and the strength of the promoter (23).
Also chromatin structure is different between methylated and non-methylated
DNA and consequently methylation could influence transcription through
changes in the chromatin structure (281).
During early mouse development the pattern of
DNA-methylation is highly regulated (42,236). The sperm genome, in contrast
to the egg genome, is highly methylated. After fertilization, demethylation
occurs, and complete demethylation is observed prior by the 16-cell morula
stage and maintained until the blastocyst stage. After implantation extensive
de novo DNA methylation is taking place, establishing an adult-like
methylation pattern (105). From this stage onward tissue-specific gene
expression is accompanied with programmed demethylation, probably involving
cis-acting elements and thus transcription factors (105). Methylation is
also important for genomic imprinting (27).
1.2. Transcription
activation
Transcriptional activation of RNA polymerase II
promoters rely on two different groups of transcription factors (318).
The general (or basal) transcription factors (GTF) bind (in)directly to
the basal promoter elements near to the transcription start site. Specific
regulatory transcription factors can increase (or repress) the amount of
mRNA produced from particular such promoters when bound to cis-acting elements
present in the promoter or enhancer regions. The GTF alone are not generally
sufficient to activate promoters in vivo, but rely on the activation
domains of enhancer-bound transcription factors for activation. It is thought
that these transcription factors stabilize the formation of the pre-initiation
complex or recruit the GTFs to the transcription start site through their
activation domains (94).
Transcription of polII promoters is directed by
different DNA sequences: the core or basal promoter elements and the cis-regulatory
elements (244). The first class contains three different elements: 1) TATA-box
(consensus TATAa/tAa/t) located approximately 30bp from the transcription
start site. 2) The initiator (INR (consensus YYANt/aYY) encompassing
the transcription start site. 3) Recently a third element has been identified,
the downstream promoter element (DPE), acting synergistically with the
INR sequence in TATA-less promoters (244). Cis-regulatory elements are
found either in the promoter region near the basal promoter elements (proximal
elements) or in enhancers located up to tens of thousands of base pairs
away from the transcription start site (distal elements).
1.2.1 General
transcription factors involved in RNA polymerase II transcription
In vitro transcription experiments using
purified RNA pol II revealed that this polymerase alone is not capable
of initiating
transcription
(243). Addition of various fractions of HeLa nuclear extracts permitted
in vitro transcription in the presence of RNA pol II (185). These
proteins are collectively known as general transcription factors. GTFs
together with pol II form the pre-initiation complex, which can subsequently
initiate transcription. Purification, cloning and biochemical analysis
of the GTFs indicated that each fraction mediates unique functions (216).
In biochemical experiments, it was shown further that the pre-initiation
complex is assembled in vitro in a stepwise fashion (32) as schematically
depicted in Fig. 1. The first step in this process is the binding of TFIID,
consisting of the TATA binding protein (TBP) and the TAFs (TBP-associated
factors) to the TATA box. 2) This is followed by the binding of TFIIB to
TFIID (or TFIID and TFIIA). 3) RNA pol II together with TFIIF is subsequently
recruited to the DAB-complex (TFIID, A,B). 4) Finally, TFIIE and TFIIH
are bound to complete the pre-initiation complex. 5) The pre-initiation
complex can then start transcription: the promoter is melted by TFIIE and
TFIIH, followed by the formation of the first phosphodiester bond. 6) After
release of polII from the promoter-region (promoter clearance), elongation
and re-initiation can occur (216,244). Each subunit fulfills a specific
function in the transcription process as summarized in Table 2.
Table 2 Functions and molecular wheights (KD)
of the various (subunits of the) general transcription factors.
| factor |
subunits (MW) |
function |
| TFIIA |
12, 19, 35 |
antirepression,stabilization of TBP-binding |
| TFIIB |
35 |
RNA-polII/TFIIF recruitment initiation-site selection |
| TFIID |
|
|
| TBP |
38 |
binding to TATA element |
| TAFs |
15-250 |
positive and negative regulation oftranscription by activators,
core promoter recognition |
| TFIIE |
34, 56 |
promoter melting TFIIH recruitment, modulation of TFIIH
function |
| TFIIF |
30, 74 |
RNA polII recruitment |
| TFIIH |
32-89 |
promoter melting, nucleotide excision repair, CTD
kinase activity, promoter clearance |
This stepwise assembly model for pre-initiation
complex (PIC) formation has recently been challenged following the isolation
of a complex from yeast, denoted the holoenzyme (135). The groups of Kornberg
and Young have identified protein complexes required for transcriptional
activation using a biochemical and a genetic approach respectively. These
complexes designated mediator or SRB (suppressor of RNA polymerase B) are
associated with the C-terminal domain (CTD) of RNA POL II, and form a large
complex together with various GTF, largely dependent on the source or isolation
procedure (127,135). Interestingly, the SWI/SNF complex was also recently
reported to be present in the holoenzyme, as evidenced by co-purification
and co-precipitation of the two complexes (306). The presence of the SWI/SNF
complex in the holoenzyme suggests that chromatin modification is an important
step in transcriptional activation. Holoenzyme complexes are also found
in mammalian cells (41,172,217), although again the presence of the GTFs
differed significantly depending on isolation source and/or procedures.
The identification of the holoenzyme clearly challenges the concept of
an ordered step-wise assembly of the pre-initiation complex and leads to
an alternative model whereby TFIID is first bound to the TATA-box (probably
involving activators), followed by a recruitment of the holoenzyme complex
by DNA-bound activators (99) (Fig. 1).
1.2.2 Transcriptional
activators
In order to regulate transcription, transcription
factors must be able to influence processes involved in transcriptional
activation: chromatin remodeling, recruitment of GTFs, enhancement of the
formation or the stability of the PIC. Furthermore activators may also
promote steps following formation of the PIC such as elongation (216).
Most, if not all, of these processes rely on an interaction between the
DNA-bound activator and other proteins that influence the above described
processes.
Transcription factors are modular in nature, consisting
of at least two functionally independent regions: a DNA binding domain
(DBD) and an activation domain (AF, activation function). These are interchangeable
units that when tethered to heterologous AFs or DBDs can activate transcription
(233). Several classes of activation domains have been described sofar:
acidic AFs as found in GAL4, VP16, GCN4, p53; glutamine rich AFs such as
SP1, bicoid and oct2; finally the AFs of CTF (CAAT transcription factor)
and AP2 are classified as proline-rich AFs (195). Experiments using chimeric
activators containing a DBD with only these predominant amino acids of
these different AFs are not sufficient for transcriptional activation (54,266).
However direct proof for structural requirements for activation domains
is scarce. Using circular dichroism, (CD) under acidic conditions, the
presence of a -sheet structure was reported in the activation domain of
GAL4 (167,290). Furthermore, both in CD analysis and NMR experiments AF-1
of the glucocorticoid receptor (presumably acidic in nature), located in
the N-terminal part, was reported to form a -helical structure in a hydrophobic
solvent (56). However the AFs of RAR-AF1 (78), VP16 (210), RelA (254) and
GR-AF1 (56) are all largely unstructured in aqueous solution as determined
by CD and NMR experiments. Detailed mutational analysis using acidic activation
domains indicated however that helical structures are required for activation
(54,92), leading to the proposal that activators adopt a helical structure
in vivo upon interaction with a target (85,265). Recently Shen et
al (263) used fluorescence analysis of the VP16/TBP interaction, thereby
generating the first experimental support for this model (263). Numerous
activators were shown to interact with GTFs such as TBP, TAFs, TFIIB, TFIIF,
TFIIH (32). The significance of these interactions for transcriptional
activation is presently unclear. In vitro transcription experiments
strongly point towards a role for TAFs in activated transcription. It was
observed that recombinant TBP, contrary to TFIID, is unable to support
activated transcription (72). All major TAFs from Drosophila TFIID have
been cloned and several TAFs have been reported to interact with activation
domains (292) Importantly, different classes of activators interact with
distinct TAFs (292). In vitro transcription experiments using TBP
and recombinant TAFs have directly proven the requirement of specific TAFs
for activated transcription, showing that distinct TBP-TAF complexes are
required for different activators (28,29,47,116,251,252). Furthermore,
when two activators are present, synergistic activation can only occur
if the activators are able to contact their target TAF simultaneously (251),
supporting the notion that synergistic activation is the result of simultaneous
contacts between activators and their targets (233). Based on these results
it was proposed that activator-TAF contacts increase the recruitment of
TFIID to promoters (251,252,292).
Yeast experiments in which TAFII expression
was depleted, raise questions about the importance for TAFs in activated
transcription since transcription can generally occur in these mutant strains
(6,196,299). However other targets were shown to be required for transcriptional
activation in yeast, namely the SRB/mediator proteins, components of the
holoenzyme (127,135). The holoenzyme was further reported to activate transcription
in the absence of TAFs (10) showing that, in yeast, an upstream-bound activator
can recruit the holoenzyme to a promoter without an obvious need for TAFs.
Furthermore, artificial recruitment of a component of the holoenzyme or
TBP to a DBD will overcome the requirement for activators to support transcription
(10,43,130). Whether the requirement of TAFs for activated transcription
in Drosophila but not in yeast is a reflection of biochemical (involvement
of TAFs) versus genetic approach (involvement of holoenzyme) or the consequence
of species-specific differences in transcriptional activation, remains
to be seen. Both models however argue that formation of a functional PIC
by recruitment of TBP through the TAFs and/or the holoenzyme by DNA bound
activators is an important step in transcriptional activation (99,244)
as schematically depicted in Fig.1).
1.2.3 Transcriptional
repressors
Repression of transcription can occur through
competition for binding sites between activators and repressors. The repressive
effect of COUP-TF on transcriptional activation by nuclear hormone receptors
is a good example of such competition for binding sites leading to the
prevention of binding of NRs, and thereby preventing transcription (234).
A different mechanism for repression is mutual inhibition of transcription
between two classes of transcriptional activators. The best characterized
example is the mutual repression between the nuclear hormone receptors
and AP1 (fos/jun). Both classes of proteins can activate transcription
through their response elements, when however a promoter contains a binding
site for one class of transcription factors and both are present they can
prevent the other from activation (250). Similar mutual repression of transcription
was also reported for MyoD/AP1 and GR, AR, PR/NFB. The molecular mechanism
involved in this mutual inhibition is largely unknown but may depend on
direct interaction between the two classes of transcription factors. Such
interactions may lead to prevention of binding to DNA, by masking activation
domains and thereby preventing interactions with cofactors required for
PIC formation. This mutual inhibition of transcription may also be independent
on direct interactions between the two proteins; in this case competition
between the two types of transcription factors for a limiting amount of
an essential cofactor, could lead to a titration of this cofactor by the
unbound transcription factor, and thereby prevent activation of the DNA-bound
activator. This repression mechanism is known as squelching (233). Interestingly
p300/CBP has been reported to act as cofactor for all the above described
transcription factors (117), thus making squelching a likely mechanism.
A third mechanism for repression is active repression.
In this situation, both the activator and repressor are bound to DNA, but
the activator is unable to support activation as a result of the binding
of the repressor to DNA. This repression mechanism is dependent on the
presence of a repression domain. Several structurally unrelated repression
domains have been identified in WT1, Krüppel, even-skipped, the thyroid
hormone receptor (TR), and the retinoic acid receptor (53). Such repression
modules, like activators, can interact with components of the basal transcription
machinery. In contrast to the case with activators this interaction does
not lead to formation of the PIC. Evidence has been presented recently
that transcriptional regulators interact with different targets in the
basal transcription machinery when they act as repressors than when they
act as activators. Krüppel can activate transcription as a monomer
through an interaction with TFIIB, but when Krüppel acts as a repressor
of transcription, it does not interact with TFIIB, but rather with TFIIE
(253). The thyroid hormone receptor is a repressor in the absence of T3,
while it can activate transcription in the presence of this hormone. Again,
functionally different interactions occur between TR and TFIIB, depending
on the presence or absence of ligand (8,81). The functional consequences
of these different interactions are presently unclear but such interactions
may prevent or promote subsequent binding to other GTFs, depending on whether
the transcription factor in question acts as a repressor or an activator.
1.2.4 Cell
specific transcriptional activation by GTFs
Transcriptional activation is dependent on the
presence of DNA-bound activators. Thus, cell-specific transcription is
primarily mediated by cell-specific expression and activity of the activators
and repressors that can bind to the promoter/enhancer region (see below).
However the basal promoter elements and the factors that bind these sequences
also contribute to cell-specific transcription.
None of the models presented above describing
transcriptional activation by DNA-bound activators give insight into cell-specific
gene regulation. Cell type-specific expression of GTFs or holoenzyme components
could influence the rate of transcription in a cell-specific fashion. The
first evidence for cell-specific expression of GTFs was obtained by Brou
et al (28) who purified multiple TFIID complexes with distinct TAF compositions.
These complexes mediated transcriptional activation of different classes
of activators (28,29), suggesting that differential TAF expression can
be involved in cell-specific transcriptional regulation. In agreement with
this it has been shown that overexpression of TAFII28 causes
a cell-type specific increase in RXR AF-2 dependent transcriptional activation
(186). Cloning of a B cell-specific TAF provided direct support for the
notion that TAFs are involved in cell-type specific gene regulation (63),
although no activator has yet been shown to interact with and activate
via this B cell-specific TAF.
Data are accumulating suggesting a role for core
promoter elements in cell type specific gene regulation (209,244,292).
Critical determinants are the presence of a TATA box and/or the identity
of the INR sequence. Mutation in the core promoter can result in a change
in response to transcriptional activators (58). Cell specific expression
of several genes including FcR1b (73), and TdT (86) has been reported to
require specific INR sequences. Furthermore the regulation of the Drosophila
proximal and distal alcohol dehydrogenase gene promoters during development,
was strictly dependent on the INR sequences of the two promoters (102).
Together these data indicate that promoter architecture can influence gene
expression in a cell-specific manner.
back
2. RETINOID
SIGNALING
2.1.1 Identification
of retinoids
Long before the description of vitamin A, the
ancient Egyptian medical practicians already had noticed that liver extracts
could cure some eye diseases. From the work of Wald (298) we know that
vision is dependent on the presence of retinal. Since liver is the richest
source of Vitamin A, this obviously explains why liver extracts could cure
these eye diseases (308). Vitamin A, a fat soluble factor essential for
growth and survival, was first described by McCollum and Davis (189). Subsequently
this factor was purified and shown to be retinol. In clinical and experimental
approaches it was shown that, vitamin A and its biologically active metabolites
known as retinoids, are essential for vision, embryonic development, cell
differentiation and homeostasis (270). These findings were emphasized by
the observations that both excess as well as lack of retinoids can result
in abnormal development (168).
2.1.2 Identification
and molecular cloning of the retinoid receptors
The mechanism underlying the action of retinoids
became apparent after the cloning of a retinoid receptor. The receptor
for retinoic acid (RA) was cloned on the basis of its homology within the
DNA binding domain of other nuclear hormone receptors (NR). This particular
receptor was shown to be a retinoic acid receptor (RAR) in experiments
using chimeric receptors that contain the DBD of GR or ER. These chimerae
could RA-dependently activate GRE- or ERE-containing promoters (90,227).
Subsequently two additional subtypes were cloned and named RAR and RAR
(26,137). Most of these RAR subtypes were cloned from various mammals,
amphibians and chicken (176). Sequence comparison of the different RAR
subtypes of various species revealed that conservation between homologous
subtypes is larger than homology between the different subtypes in one
organism (158). This interspecies sequence conservation indicated that
the different receptor subtypes fulfill distinct functions in retinoid
signaling (158). The complexity of retinoid signaling was further emphasized
by the identification of various isoforms in all three RARs, which are
the result of different promoter usage and/or alternative splicing (91,121,161,200,321).
The various isoforms all differ in their N-terminal region, which has been
shown to contain a transcriptional activation domain (77,202). By screening
cDNA libraries for new orphan receptors (receptors for which the ligand
is unknown), Mangelsdorf et al (173) isolated a novel retinoid receptor
denoted RXR. This receptor could be activated with RA, albeit at higher
concentrations. No binding of RA to RXR was found, indicating that RXR
is a receptor for a novel retinoid. Subsequently, two groups showed independently
that 9-cis RA, a RA isomer, binds with high affinity to this receptor and
could activate RXR more efficiently than RA (106,163). Three subtypes were
also cloned for RXR, and some isoforms were identified. The availability
of several retinoid receptor subtypes that may generate distinct isoforms
may be an important characteristic for RA-dependent gene regulation. Transient
transfection experiments have demonstrated a receptor subtype- and isoform-dependent
regulation of RA responses of various RA-responsive promoters (201).
2.2
Structure and function of RAR and RXR
The retinoid receptors belong to the nuclear hormone
receptor superfamily. This family includes receptors for glucocorticoids
(cortisone), estrogens, mineralocorticoids (aldosterone), androgens
(testosterone), progesterone, fatty acids, thyroid
hormone, vitamin D, ecdysone, (13,177) and a large number of receptors
for which no ligand has been identified so far, the orphan receptors (74,178,234).
In fact almost all members of this large superfamily of transcription factors
(over 150) were cloned on the basis of their very conserved DNA-binding
domain. Sequence comparisons between the various steroid hormone receptors,
revealed both conserved and non-conserved regions. On the basis of the
sequence homology these receptors were subdivided into six regions denoted
the A-F domains (75,95). The DBD region (C-region) is most conserved, the
ligand binding domain (E-region) showed a moderate sequence conservation,
while the N-terminal region was least conserved between the various members
of this family. Functional analysis of GR and ER indicated that the E-region
apart from its ligand binding domain also contains a dimerization domain,
and a hormone dependent transcriptional activation function (TAF, later
renamed AF). Furthermore a ligand-independent AF was found in the N-terminal
part of these receptors (AB-region), which is able to activate transcription
independently of ligand when linked to a (heterologous) DBD. The D-region
forms a flexible linker between the DNA and hormone binding domains, and
may contain a nuclear localization signal (154). The F-region, for which
no specific function has yet been described is not conserved and is absent
in some receptors. The structure and function of the various domains is
schematically depicted in Fig. 2. The 
nuclear
receptor superfamily has been divided into four classes (177,273), on the
basis of their DNA binding properties. Class I comprises the classical
steroid hormone receptors that bind to inverted repeats as homodimers.
The second class of receptors bind to direct repeats with variable spacing,
all requiring RXR as dimerization partner. Class III and IV are dimeric
and monomeric (orphan) receptors that bind to direct repeats or extended
half sites respectively (Fig. 3).
2.2.1
Retinoic acid response elements (RAREs)
Like classical steroid hormone receptors, RAR
and RXR are capable of activating transcription of genes that contain hormone
response elements (HRE). Early experiments indicated that both RAR and
RXR could activate a synthetic thyroid hormone response element: a palindromic
half site (TREpal 173,286). Characterization of the sequences upstream
from RAR2 resulted in the identification of the first natural RA-responsive
promoter, showing that RAR-dependent activation occurs preferentially through
direct repeats consisting of two half-site with (a/gGTTCA) separated by
5 nucleotides (DR5; 60,275). The cloning and characterization of other
RA-responsive promoters indicated that a consensus RARE consists of two
half-sites (a/gGg/tTCA) separated by 2 or 5 basepairs, although palindromic
RAREs and more complex RAREs, containing multiple degenerated half sites
are also found (176). RXR response elements (RXRE) generally contain two
half sites separated by 1 basepair (174).
The cardiac myosin heavy chain (MHC) alpha gene
promoter TRE appeared, in contrast with the TRE pal, to be T3 responsive
but unresponsive to RA. Examination of this TRE revealed that it consists
of a direct repeat separated by 4 bp (DR4) (287). Binding and transactivation
assays using direct repeats with variable spacers led to the postulation
of 3-4-5 rule, as direct repeats with these spacers appeared to be selectively
for VDR, TR, and RAR, respectively (287). This was thereafter extended
to a 1-2-3-4-5 rule to define preferred binding sites for the various class
II receptors as schematically depicted in Fig. 4 (174).
Binding experiments revealed that for high affinity
binding of VDR, TR or RAR to their cognate HRE, nuclear extracts were required
(93,164,199), suggesting a requirement for accessory factors in DNA binding.
Through the screening of an expression library for a RAR interacting protein
(315), by the direct biochemical purification of an accessory factor from
HeLa nuclear extracts (159) and by other methods several groups identified
RXR as heterodimeric partner for RAR and other nuclear hormone receptors
(131,159,179,315, 322).
Mutant RARs or RXRs with altered DNA binding specificity
(recognizing GRE half sites only), as well as by chemical cross linking
experiments, were used to show that polarity of binding to REs occurs (140,223),
resulting in preferential half site occupancy by particular receptor types.
On DR3, DR4 or DR5 RXR is occupies the 5' halfsite whereas VDR, TR and
RAR respectively occupy the 3' half site. On the DR1, polarity is reversed
(141), leading to the inability of the RAR/RXR heterodimer to activate.
These findings suggest that RAR must occupy the 3'half site to allow activation.
Recent experiments indicated that the DR1-mediated repression by the RAR/RXR
heterodimer is mediated by the binding of the co-repressor N-Cor to RAR
(142).
In the above described situation, RXR acts as
a dimerization partner, not requiring functional activation domains, and
presumably acts as a silent partner (151,178). However with other receptor
combinations such as NGF1B/RXR (83,223) PPAR/RXR (125,133) LXR/RXR (305)
and FXR/RXR (84) transcriptional activation is more dependent on 9-cis
RA binding to RXR. Finally, recent experiments using receptor-specific
synthetic ligands indicated that in the RAR/RXR heterodimer, transcriptional
activation is also positively influenced by binding of ligand to RXR, although
binding of RAR is a requirement for activity by the RAR/RXR heterodimer
(46,146).
2.2.2 Structure
of the DNA binding domain
Sequence comparison of the first cloned nuclear
hormone receptors revealed strict conservation of cysteine residues within
the receptor. Based on homology with other DBDs it was proposed that two
separate zinc fingers are formed (75,95). Determination of NMR structure
of GR (103), ER (260), RAR (134) and RXR (152) revealed a conservation
of the overall three-dimensional fold. Instead of forming two separate
zinc finger subdomains one compact domain is formed, encompassing two -helical
strands folded perpendicularly to each other. The crystal structure of
the ER-DBD (261) and the GR-DBD bound to DNA (167) revealed that the amino
acids determined by swapping experiments between GR and ER to be required
for DNA binding (P-box), are located in the so called recognition helix.
This helix is positioned in the major groove of the response element where
it makes base contacts. The region previously determined to be involved
in dimerization (D-box) located between the first two cysteines of the
second zinc finger makes contact between two monomers when bound to a GRE
(325). The structure of RAR and GR in solution are similar. Whereas the
first binds to direct repeats with variable spacing, the latter binds only
to palindromes separated by three basepairs, indicating that when bound
to DNA the D-boxes of the RAR/RXR heterodimers can not contact each other.
Since RAR, TR, and VDR each require RXR for binding to DNA, it is likely
that different mechanisms account for cooperative DNA binding when compared
to GR homodimer binding to a GRE. Several extensive studies (140,169,319,320)
have addressed this question, showing that cooperative binding is dependent
on: 1) RXR occupying the 5' halfsite, 2) the D-box of RXR, 3) distinct
regions of the N-terminal part of the first zinc finger of RAR or TR. From
these experiments, it was concluded that binding site preference is determined
by the DBDs and not by the LBDs, involving cooperative binding on preferred
sites and stearic hindrance on less favorable REs. Furthermore a C-terminal
extension named the T/A box, adjoining the zinc-finger structure was reported
to be involved in protein-DNA interactions of TR with a DR4 (140,320) and
RAR to a DR2 (but not to a DR5). This region is also involved in homodimerization
of RXR (320). Structure determination of RXR by NMR (152) revealed the
presence of an additional helix immediately after the second zinc finger.
This helix was required both for protein DNA and protein-protein interactions
which are prerequisite for cooperative binding of RXR homodimers to RXREs.
The crystal structure of the RXR/TR heterodimer bound to a DR4 (235) showed
perfectly that the regions which were suggested from biochemical experiments
to be involved in DNA binding and co-operativity indeed fulfill important
structural functions involving protein-protein and protein/DNA interactions.
The C-terminal extension of TR makes extensive contacts with the minor
groove of the 3' half site and the spacer between the half sites, and may,
through stearic hindrance, prevent formation of RXR/TR heterodimers on
response elements with a smaller spacing.
Whether the C-terminal extension is also required
for RXR/RAR binding to a DR5 remains to be seen. The presence of two independent
dimerization interfaces (DBD and LBD) together with the proposed capacity
to rotate over 180o of the DBD with respect to the LBD when
bound to either a DR or a inverted repeat may further allow the RAR/RXR
heterodimers to bind to inverted or everted repeats with variable spacing.
2.2.3 The
structure of the ligand-binding domain
The ligand-binding domain of nuclear hormone receptors
is a multifunctional domain, involved in ligand binding (156,280), nuclear
localization (154), homo- or hetero-dimerization (225) and hormone-dependent
transactivation (76) and hormone-reversible repression (8). Deletion and
mutational analysis indicated that these functions are at least partially
interconnected, probably relying on a highly ordered structure (see Fig.
2). The notion that C-terminally truncated receptors lack the ability to
transactivate led to the identification of an activation domain in the
C-terminus of all transcriptionally active members of the NR family (11,57,70,164,249).
This core motif (xE; represents hydrophobic residues) was proposed to form
an amphipatic -helix, which can when tethered as a multimeric sequence
to a heterologous DBD activate transcription only weakly, contrary to the
strong activation of the complete LBD (11,70,160). With the characterization
of apo-RXR crystals the presence of this amphipatic helix (helix 12) was
confirmed (21). The structure consists of 12 helices, two -strands separated
by loops, together forming an anti-parallel -helical sandwich. RXR has
been crystallized as a dimer, and this enabled the determination of the
region involved in homodimerization. The dimer interface is presumably
formed by helix 10 (21). The availability of crystals of ligand-bound RAR
(holo), led to the idea that binding of ligand results in marked allosteric
changes (237), which was already proposed on the basis of tryptic digestion
experiments (124). Direct proof for this awaits crystallization of apo-RAR
or holo-RXR. However, since the overall fold of apo-RXR, holo-RAR
and holo-TR (297) are similar, the proposed mechanisms are thought to be
relevant (40,311). The overall structure of holo-RAR is more compact than
that of apo-RxR. Most notably helix 12 containing the AF2 core folds back
towards the core of the LBD. The ligand-binding pocket is formed by 24
amino acids from 5 different helices, the -turn and two loops, together
forming a hydrophobic pocket. Ligand binding is proposed to occur by electrostatic
guidance through the cavity and ligand is positioned by hydrophobic interactions,
which leads to conformational changes whereby helix 12 seals the ligand-binding
pocket. In agreement with this idea, several amino acids of this helix
directly contact the ligand (40,237).
The pronounced changes in structure between apo-
and holo-receptor may explain ligand-induced transactivation. The altered
structure prevents repressor from binding to the LBD and simultaneously
permits coactivators to interact with the LBD (see below). Whether these
interactions require only the AF-2 core or other regions of the receptor
remains to be determined.
2.2.4 Receptor-interacting
proteins
Evidence for the presence of intermediary proteins,
adaptors, or cofactors for NRs came from transient transfection experiments
(16,20,192,279). Overexpression of a given activator can decrease the activity
of a different activator belonging to the same class of activators but
not those from another class. On the basis of these experiments, it was
proposed that overexpression of a transcriptionally active protein titrates
out a limiting component that is required for activated transcription probably
through an interaction with the basal transcription machinery, a process
known as squelching (233). To act as a cofactor for NRs several criteria
must be fulfilled: 1) ligand-dependent interaction with the receptor 2)
activation of transcription when tethered to DNA either artificially through
a heterologous DBD or through the receptor itself (3) relief from squelching
caused by overexpression of the liganded receptor (4) influence processes
involved in transcriptional activation and (5) targeted disruption should
lead to a decrease in activation by the liganded receptor. Using various
techniques, relying on the ability to interact with cofactors in the presence
but not in the absence of ligand, several cofactors have been identified:
TRIP1/SUG1 (155,294), TIF1 (149), RIP140 (34), SRC-1 (215) and TIF2 (293).
Recently it was shown that p300/CBP (39,120) can also act as cofactors
for NR. Interaction between liganded receptor and cofactor was found to
be dependent on the presence of a functional AF-2 core domain. This could
mean that this specific region is directly involved in this interaction
although the ligand-induced conformational changes in receptor structure
may also allow interactions to occur that are otherwise prevented. Furthermore,
RARs have been reported to interact with several viral activators, including
E1A (15,79,138) and BLZF (62,228), resulting in activation or repression,
respectively. Finally, interactions have been reported with GTFs. Liganded
RXR and TBP can interact (258), and furthermore interactions of TR with
different regions of TFIIB (8,81), depending on the presence or absence
of ligand, as well as interactions with TBP (82) have been observed.
Conversely, proteins that only interact with apo-LBD
have also been identified using the yeast two hybrid system: NCoR (nuclear
receptor co-repressor) (112) and SMRT (silencing mediator for RAR and TR)
(44). Interestingly, these proteins were only found to interact with unliganded
TR and RAR but not with other NRs. These repressors contain a repressor
domain, demonstrated by their ability to repress transcription when tethered
to a promoter through a heterologous DBD. Addition of ligand results in
loss of binding. The interaction between corepressors and receptors is
dependent on two regions of the receptor: the hinge region (44,112) and
the AF2 core region (helix 12) (44,316), linking repression and activation.
This may indicate
2.3 CELL-TYPE
SPECIFIC TRANSCRIPTIONAL ACTIVATION BY RA
Transcriptional activation by RA is dependent
on the binding of a heterodimeric receptor complex to a response element.
The presence of a response element in a promoter does not directly imply
that such a promoter can be activated under all circumstances in all cell
lines. Generally, multiple factors regulate promoter activity in concert.
Factors influencing the RA response are (1) ligand availability (2) receptor
expression (3) receptor binding to DNA (4) receptor activity (5) promoter
architecture (6) post transcriptional modifications as schematically depicted
in Fig 5. Below we describe the individual steps involved in the RA response.
Each step allows control over retinoid-response, relying on a differential
expression and/or activity of factors involved in the response. This differential
expression and/or activity can lead to cell-type specific activation of
RA-target genes and consequently to a differential response to retinoids.
2.3
CELL-TYPE SPECIFIC TRANSCRIPTIONAL ACTIVATION BY RA
2.3.1
Availability of ligand
Control over presence and levels of biologically
active retinoids is required since both excess as well as lack of vitamin
A lead to developmental abnormalities and diverse effects on different
organs (190). This control likely involves both biosynthesis and metabolic
degradation of retinoids through highly regulated enzymatic process. Multiple
mechanisms compensate for dietary fluctuations of retinoid intake, keeping
the amount of retinol in the serum constant (219). Retinol is transported
by the retinol binding protein (RBP), which delivers the retinoid to the
cell. In the cell, two groups of retinoid-binding proteins may be present:
the cellular retinol-binding proteins (CRBPI,II) and the cellular retinoic
acid-binding proteins (CRABPI,II) that bind retinol and retinoic acid,
respectively. CRBP is thought to be involved in the synthesis of RA from
retinol, and CRABP is proposed to sequester RA (204). This sequestration
of the ligands may influence metabolism or availability of the ligands
(204). This suggests that these binding proteins together regulate the
availability of biologically active retinoids. Further support for this
hypothesis came from the spatio/temporal expression patterns of the retinoid-binding
proteins during mouse development (65,66,248). Recent gene disruption experiments
however have raised doubt concerning these functions since mice lacking
both CRABPI and CRABPII are apparently normal (147).
The conversion of retinol to retinal is thought
to be the rate limiting step in the synthesis of biologically active retinoids.
Several enzymes including alcohol dehydrogenase (ADH) I, ADH IV, and microsomal
short-chain ADH can convert retinol to retinal. These processes may occur
in the cytoplasm or in microsomes, each relying on different enzymes. Three
microsomal retinol dehydrogenases (RoDH) have been cloned, belonging to
the family of short chain dehydrogenases/reductases (37,38,39). Expression
pattern of these enzymes in different tissues is distinct, suggesting that
these enzymes may regulate the biosynthesis of active retinoids in a cell-type
specific fashion. Furthermore, also the ADH I and ADH IV gene expression
is spatially/temporally restricted during embryonic development (5,295).
Formation of RA from retinal is catalyzed by retinal dehydrogenase (RalDH).
To date two different isoforms of RalDH have been cloned (302,324), and
although mRNA is expressed in many tissues, considerable variations in
expression levels were observed (302). A detailed in situ hybridization
analysis of RALDH-2 gene expression (207) during embryogenesis suggest
a tissue-specific involvement of retinoid synthesis in retinoid signaling
since expression of this enzyme is detected in close proximity to a region
(Hensen's node) which is thought to contain high concentrations of active
retinoids (48).
Active retinoids such as atRA and 9-cis RA are
thought to be converted to inactive polar compounds. The first step involves
enzymatic 4-hydroxylation of atRA catalyzed by a cytochrome P450. Recently
a hydroxylase (p450RAI) that specifically converts atRA to 4-OHRA has been
cloned from zebrafish (304). The expression of this enzyme is upregulated
by RA, and is developmentally regulated in zebrafish. We reported previously
that 4-oxoRA is a very potent retinoid in modulating positional specification
in Xenopus embryos, being even more potent than atRA. Furthermore, 4-oxoRA
can bind to RARs and activates RARs but not RXRs (229). Recently, 4-oxoretinol
and 4-oxoretinal have been detected in F9-EC cells and Xenopus embryos
respectively and have been shown to be biologically active retinoids which
can bind to RARs (1,19). Together, these observations suggest that oxidation
at the 4 position of retinoids does not necessarily lead to formation of
an inactivate ligand, possibly the various retinoids fulfill distinct functions
in vivo.
The differential expression of the proteins involved
in RA synthesis (ADH, RoDH, RalDH), transport (RBP, CRBP), storage (CRABP)
and metabolism (P450s) strongly support the view that the availability
of retinoids is actively regulated during embryonic development and in
adult life. The observation that an enzyme involved in the break-down of
biologically active retinoids is upregulated by RA, while expression of
RalDH-2 is down-regulated upon RA-treatment during embryogenesis, is indicative
for the regulation of the availability of RA. Recent experiments by us
and others have shown that RA metabolism and sensitivity to retinoids with
respect to growth inhibition, coincide in some cell lines (278,289), supporting
the view that the availability of retinoids is actively regulated.
2.3.2 Receptor
expression
Expression of retinoid receptors during mouse
development has been studied extensively by in situ hybridization,
showing both specific and overlapping expression patterns of individual
receptors (65,66,247,248). Distribution of the RAR transcript, especially
RAR1, is ubiquitous during development and in adult life, indicating that
RAR fulfills a general function. Expression of RAR is much more restricted
and has been suggested to be involved in the central nervous system development,
differentiation of certain epithelia and in cell death. RAR transcripts
are present during various stages of skin development suggesting roles
in squamous differentiation during development and in adult life. Furthermore
RAR may be involved in chondrogenesis and morphogenesis. Expression of
RXRs during development (67,175) indicate that every tissue expresses at
least one RXR transcript at every stage of development. RXR is ubiquitously
expressed, RXR transcripts are also detected in all tissues during early
development, followed by a higher expression in several squamous epithelia
during late development. Expression of RXR is mainly restricted to the
myogenic lineage. Expression of the retinoid receptors has also been studied
in organs and tissues, tumors and cell lines from various species (89).
Generally cells express RAR and/or RAR and at least one RXR ( or ). The
expression of RAR2, 2, 2 is generally ligand-dependent.
Another interesting aspect of cell-type specific
regulation of retinoid receptor expression is the role of translation-regulation
as demonstrated for RAR2 mRNA. The presence of a specific upstream open
reading frame (ORF4) in the untranslated leader of the RAR2 mRNA allows
tissue-specific and developmentally regulated inhibition of translation
in heart and adult brain but not in other tissues in transgenic mice carrying
a RARb2 promoter leader LacZ construct (238,326). These experiments clearly
demonstrate that tissue-specific regulation can occur at the translational
level.
Collectively, these studies indicate that receptors
show a spatially and temporally restricted expression during development
followed by cell-type specific expression pattern in adult life. The differential
expression patterns are indicative for distinct functions for the individual
receptors. Transient transfection experiments using various RA-responsive
promoters indicate that distinct receptor subtypes can regulate RARE-containing
promoters differentially (114,144,201), thus supporting the hypothesis
that differential receptor expression could result in cell-specific promoter-activation.
Hallenbeck et al (100) have presented evidence indicating cell type
and receptor specific functions for retinoid receptor activation, showing
similar activation of the CRBPII RXRE by RXR and RXR in P19-EC cells but
not in 3T3 cells, where RXR was much more potent. This differential activation
appeared to be dependent on the AB-region and the hinge-region, suggesting
that cell-type specific AF-1 activity is contributes to cell-specific activation
of this RXRE.
2.3.3 Receptor
binding to DNA
In eukaryotic cells DNA is generally packed in
chromatin. Transcription of such highly organized histone/DNA complexes
is dependent on (partial) disruption of the chromatin (309). This can occur
by at least two classes of proteins: the histone acetylating proteins and
the SWI/SNF complex that modify nucleosomal conformation, thereby allowing
binding of (general) transcription factors (309). Distinct mammalian SWI/SNF
complexes possibly fulfilling distinct functions have been identified and
are thought to remodel chromatin by an unknown mechanism (300,301). The
observation that the SWI/SNF complex is a component of the polII holoenzyme
(306) has linked (basal) transcription directly to nucleosome remodeling.
Further, SWI3, a component of the SWI/SNF complex, has been reported to
be required for transcriptional activation by GR, possibly involving a
direct interaction between GR and SWI3 (314). In mammalian cells also,
GR and RAR activation was increased by cotransfection of hBrm or hBrg1,
the human homologs of yeast SWI2/SNF2 (35,198). These observations together
suggest that a triad interaction between activator, SWI/SNF complex and
GTF might be required for transcriptional activation of nucleosome-assembled
DNA (Struhl). A different mechanism involved in transcriptional activation
of chromatin-DNA is acetylation of histone tails, leading to an altered
nucleosomal conformation. This histone modification is mediated by proteins
containing histone acetyltransferase (HAT) activity (309). The recent finding
that p300, a previously identified cofactors for RAR, itself has contains
histone acetyl transferase activity and binds a protein pCAF that also
has HAT-activity, directly links histone acetylation to transcriptional
activation (222). TIF-1 is also thought to be involved in nucleosome reorganization
(150).
Any component within the nucleosome as well as
any factors involved in the nucleosome assembly and remodeling can be a
target for cell-specific regulation of transcription. Interestingly, the
composition of the nucleosome changes dramatically during development.
In the sea urchin, expression of different histones is tightly regulated.
The composition of chromatin, in particular the linker proteins, which
are involved in the formation of high order nucleosomal structures also
changes markedly, during early stages of Xenopus development. In Xenopus
oocytes linker histone B4 is most prominent, while after fertilization
histone B4 is replaced by histone H1, which is translated from maternal
RNA, allowing the embryo to repress oocyte-specific genes (208,221). Interestingly,
the linker histone H1o gene is regulated by retinoic acid through
a RARE present in its promoter region (22,171). This suggests that RA can
modulate nucleosome subunit composition and thereby the rate of transcription
through transcriptional regulation of this a-typical, differentiation-specific
histone (64). The presence of and requirement for a proper control over
histone acetylation/ deacetylation during early embryonal development has
been shown in experiments in Xenopus embryos using inhibitors of
deacetylation (3). Such histone modifications could be developmentally
regulated via differential expression of proteins containing HAT activity
or by regulation of HAT activity. Both the SWI/SNF- and the HAT-dependent
nucleosome remodeling activity is dependent on the binding of transcription
factors to DNA.
Wong et al (310) have shown that TR/RXR
heterodimers can bind to a TRE within chromatin, thereby enhancing silencing
of the TRE-containing gene, while addition of T3 resulted in a local disruption
of chromatin structure, independent of transcriptional activation (310).
Possibly, co-repressor and co-activators contribute to repression and chromatin
disruption.
Retinoic acid responsive promoters identified
sofar generally contain two or more common half site hexamer motifs (a/gGg/tTCA)
or variants thereof. Four different types of RAREs have been identified:
direct repeats with variable spacing (DR1, DR2, DR5), palindromic response
elements, everted repeats, and complex promoters containing multiple degenerate
half site sequences (89,176).
The affinity of receptors for different elements
varies markedly. In parallel with the binding affinities, the responsiveness
to RA is also binding-site dependent. For example, the RAR2 promoter RARE
is a strong binding site for RAR/RXR, and is activated highly efficiently
by RA in P19-EC cells. The F-crystallin promoter RARE, an everted repeat,
is only marginally upregulated by retinoic acid in the same cell line and
is a weak binding site for RAR/RXR (283). The lamininB1 promoter, containing
multiple half-sites, as well as several promoters containing palindromic
response elements such as growth hormone or the osteocalcin gene are much
less responsive than promoters that contain a DR5-type RARE (89,176). Furthermore
specificity could be the result of the (direct repeat) sequences: RXR has
a distinct halfsite sequence preference, gGGGTCA (274) compared with RAR
(agAGGTCA)(140) as determined by binding site selection experiments. In
natural RAREs found in RA-responsive promoters, although matching well
to these defined consensus sites, differences are apparent which clearly
contribute in determining binding affinities and transcriptional activation
capacity of receptors (169,170). Furthermore spacer sequence differences
can contribute to binding efficiency (161,170). Together these findings
indicate that binding specificity towards specific RAREs is clearly an
important determinant of (cell-specific) transcriptional activation.
RXR is not only a heterodimerization partner for
RAR but is also involved in the binding of various (orphan) receptors to
DNA, including VDR, TR, COUP-TF, PPAR, NGF1B/NURR1, FXR, LXR (178). Some
of these orphans can recognize RAREs with different functional consequences,
leading either to activation or repression of such promoters. When RXR
is bound to a RARE together with an "orphan"receptor, these heterodimers
could respond either to 9-cis RA or to the ligand of the heterodimeric
partner of RXR such as fatty acids and prostaglandins in case of PPAR,
or farnesoids in case of FXR (151,177,178). Such heterodimers can also
activate putative RAREs without a ligand for the orphan receptor, in the
presence of retinoids, which is best illustrated by the binding to and
activation by a 9-cis RA liganded NGF1B/RXR heterodimer on a DR5 (83,224).
Furthermore several (orphan) receptors can bind to halfsites (e.g. NGF1B,
ELP/SF-1/Ftz-F1) as monomers or to direct repeats as homodimers (e.g. RXR,
COUP-TF, HNF4)(178). Binding of these orphan receptors could interfere
functionally with the retinoid response. For example COUP-TF can bind to
various retinoic acid response elements either as homodimer or as heterodimer,
causing a decrease in RA-dependent activation of transcription (51,132,285)
. On the contrary, overexpression of NGF1B (224) or Dax1 (317) or HNF-4
(203) can modulate RA-responsive promoters positively. Together these transfection
experiments indicate that expression of orphan receptors that can bind
to response elements may influence RA-dependent activation of target genes.
The expression of some of these orphans has been reported to be developmentally
regulated (74,119,234) arguing that in vivo retinoid response could
also be influenced by expression of these orphans. Support for this hypothesis
has been presented for the Oct-4 promoter and the RAR2 promoter, which
are negatively regulated by COUP or COUP-related orphans and positively
by RAR/RXR heterodimers (14,255, Chap. 8).
Differential responses by RAR/RXR heterodimers
can also be dependent on the spacing between the binding sites (174). When
bound to a DR1 cause repression, while the RAR/RXR heterodimer can activate
transcription on a DR2 or DR5. Recently a molecular basis for this observation
has been presented, indicating that N-CoR is involved. Upon ligand binding,
N-CoR is released from RAR when bound to a DR5 while this is not the case
when it is bound to a DR1 (142). Northern blot analysis indicated that
cell-specific expression is found for NCoR (112) but not for SMRT (44),
which may explain the cell-specific RAR/RXR-dependent activation of a DR1-containing
promoter. Together, these experiments indicate that binding and transactivation
potential of the RAR/RXR heterodimer is modulated by the accessability
of the response element, the sequence of the response element, the combination
of (orphan) receptors that bind to the response element, and the orientation
of the receptor heterodimer. Cell-type dependent expression of (orphan)
receptors that bind to different RAREs or of proteins that modulate binding
affinity could influence the RA-response cell-specifically.
2.3.4 Receptor
activity
RARs as well as RXRs each contain two activation
functions (AF) (77,202). AF-1, located in the AB-region, activates transcription
ligand-independently, while the ligand-binding domain contains the ligand-dependent
AF-2. These activation domains contribute differentially to transcriptional
activation of RA-responsive promoters, depending on promoter architecture,
and the type of RARE present. Furthermore, the activity of the various
activation domains is cell-type specific (68,76,80), showing different
activities in various cell lines. Finally, the various activation domains
may act synergistically, dependent on the presence of the two different
AFs in one receptor for maximal activation (heterosynergism) (201,202),
or of one AF that activates on multiple binding sites containing reporters
(homosynergism)(80). The mechanism underlying cell-type and promoter-dependent
differences in activity of the activation domains are largely unknown,
but phosphorylation could possibly be involved (for review on phosphorylation:
107), as all RARs have indeed been reported to be phosphor-proteins (87,240,241).
Phosphorylation sites have however sofar not been mapped although mutational
analysis of AF-1 of RAR is indicative for a role of phosphorylation in
the activity of the RAR AF-1 (78). Preliminary in vivo phosphorylation
experiments further indicate that this region is phosphorylated, and mutation
of the putative phosphorylatable residues resulted in a cell-type specific
effect on the activity of RAR AF-1 (Folkers et al in preparation, Chapter
4). In agreement with this hypothesis, the expression of PKC isotypes is
regulated by RA in F9-EC cells (228). Both PKA and PKC can phosphorylate
RARs in vitro and/or in vivo, leading to activation by PKA (113,242) and
PKC (313) although others reported a PKC-dependent decrease in RAR activity
(277), this might involve a cell-specific effect of phosphorylation on
RAR activity. Finally, dephosphorylation by protein phosphatases 1 and
2A negatively modulates the activity of RARs through inhibition of DNA
binding by the RAR/RXR heterodimers (157). Phosphorylation sites for other
members of the nuclear hormone receptor have been identified and in some
cases shown to contribute to the activity of the receptors (303), but no
direct evidence is available for a role for phosphorylation in the cell-specific
activity of a activation domain. Interestingly different phosphorylation
patterns of ER were observed by different groups; these could be the result
of cell-specific phosphorylation (see 303).
Furthermore, the presence and amounts of available
cofactors or corepressors present as well as their ability to interact
with these receptors could also contribute to the activity of the activation
domain. Expression levels of several of the recently identified cofactors
in various cell lines support the involvement of these cofactors in the
cell-specific modulation of RA-response (Chap 8). We and others have shown
before that the adenovirus E1A 13S protein can act as a cofactor for RARs
(15,39) through a direct interaction with RAR (79). This viral cofactor
can modulate the RA-response of RAR-AF2, or of the RAR/RXR heterodimer
on a natural or on artificial promoters, in a cell type-specific fashion
(80, Chap. 6) underscoring the involvement of cofactors in cell-type specific
regulation of RA-response.
2.3.5
Promoter architecture modulates RA-response
The above described mechanisms involving retinoid
signalling can lead to cell-specific transcription of RA-responsive promoters.
Here we describe a few examples of promoters that are cell-type specifically
regulated by retinoids through their RARE.
In agreement with expression of RAR2 which has
been reported to be highly restricted in vivo (65,66,248) the promoter,
which is strictly dependent on RA for activation, is also cell-type dependently
activated (15,39,323). The molecular basis for this cell-specific regulation
is largely unknown. Recently we have identified several factors that could
be involved in cell-specific modulation of the RA-response of this promoter
such as the ratios between the expression levels of RAR/RXR and orphan
receptors, the presence and levels of cofactors, and the presence of different
initiator binding proteins (Chap.8). The observed differential expression
levels of these factors in different cell lines could be involved in the
cell-specific RA-response.
Additional evidence for cell-specific promoter
regulation of RA-responsive promoters came from transient transfection
experiments of the mRAR2 promoter, the mCRBPII promoter and a RXRE-tk reporter
in various cell lines (203). Some cell lines, permit activation of the
RAR2 promoter but not of the CRBPII promoter. Furthermore, cotransfection
of receptors had no effect in one cell line, while it dramatically increased
the activity of these promoters in another cell line, strongly arguing
that promoter activation is regulated in a cell-type specific manner. These
authors further suggested that the presence of the orphan receptors ARP-1
and HNF-4 can prevent RA-dependent activation of the CRBPII promoter.
The presence of a RARE in a promoter shown to
bind RAR/RXR in vitro, and to be upregulated by retinoids (after
cotransfection of receptors) does not necessarily mean that this promoter
is regulated in vivo by this element. Generally, a promoter or enhancer
contains (overlapping) binding sites for numerous transcription factors
that can either co-operate or interfere with each other, thereby creating
a additional levels of regulation. Here we describe a few examples of promoters
of known RA-target genes, that are cell-type specifically or developmentally
regulated by more complex mechanism relying for their activity on other
transcription factors besides the RAR/RXR heterodimer.
Hoxb-1
Analysis of transgenes carrying various mutant
Hoxb-1-LacZ gene constructs, revealed the presence of two enhancers 3'
of the Hoxb-1 gene, the first containing a functional RARE (180,230) that
is required for establishing some of the aspects of the early Hoxb-1 expression
in the neuroectoderm. The other enhancer is involved in the Hoxb-1 expression
in mesodermal tissue (180). Furthermore in the 5' region of the Hoxb-1
gene an enhancer was identified, which appeared to be involved in expression
in rhombomeres 3-5. This region also contains a repressor sequence restricting
expression to r4, these two elements together are responsible for the restricted
Hoxb-1 expression in rhombomere (r)4 of the hindbrain. This negative regulatory
region contains a RARE, mutation of this sequence leads to expression in
adjacent rhombomeres (272). Recently evidence has been presented for the
presence of an autoregulatory loop regulating the expression in r4 through
the enhancer present in the 5' region of the gene (231). Taken together,
using transgenic mice carrying Hoxb-1/LacZ constructs it was determined
that spatio/temporal expression of Hoxb-1 is dependent on RAREs that are
regulating expression, both positively and negatively. Furthermore cooperativity
with other transcription factors is required for proper expression of Hoxb1.
Recently targeted disruption of the RARE present in the 3' region of the
Hoxa-1 gene strongly supports the involvement of this RARE and consequently
retinoids in Hox gene expression (69). The elements involved in Hoxb-1
expression in P19-EC cells were identified (212,213) and found to be similar
to the above described elements as identified in transgenes, underscoring
the ability to study some aspects of regulation of spatio/temporal expression
in EC cells.
Major histocompatibility complex class I gene
Developmental regulation of MHCI expression is
dependent on two conserved upstream regions: region I contains binding
sites for NFkB like factors whereas region II contains a binding site for
NRs (59). The factor binding to this region was cloned and later appeared
to be RXR (101) and RAR. Activation of the MHC class I gene in NTera-2
cells by retinoic acid was dependent on both enhancer regions (262). Binding
of p50/p65 heterodimers and RAR/RXR heterodimers to region I and region
II respectively was shown to be enhanced by RA treatment of NTera-2 cells,
accompanied with an increase in enhancer activity. Cotransfection of these
transcription factors can increase the activity of the MHC promoter through
their respective enhancer regions. These data indicate that the two different
families of transcription factors together regulate the RA-dependent increase
in MHC class I expression (262).
AP1
Another level of complexity is the modulation
of the activity of the RA response by AP1 through transcriptional interference.
Analysis of the osteocalcin gene (256) has led to the hypothesis that AP1
and RARs could bind to overlapping composite sequences, whereby binding
of one class of transcription factors prevents binding of the other, thus
causing reciprocal modulation of the responses. First indications for cross-talk
between these two transcription factor families on promoters containing
a simple binding site came from the studies involving the stromelysin gene
and the collagenase gene promoters (145,206). Expression of these gene
products is upregulated by PMA through an AP-1 site, and inhibited by RA.
In transient transfection experiments it was shown that the AP-1 site is
required for activation by PMA and repression of transcription by RA, not
involving binding of RAR to the AP-1 site (257). The mechanism underlying
the cross talk between AP1 and RAR/RXR is unknown but may involve direct
interactions (250) or competition for limiting cofactors that are required
for both classes of transcription factors such as p300/CBP (120).
PIT-1
Pit-1 is a POU domain-containing transcription
factor that is cell-specifically expressed and involved in pituitary development
(245). Analysis of the Pit gene has revealed that an enhancer, located
10 kb upstream from the transcription start site, is essential for its
expression. This enhancer is dependent on the presence of multiple binding
sites, including Pit autoregulatory sites, a D3/RA response element and
most interestingly a RARE that is dependent on the presence of Pit-1 (239).
The latter element is involved in the cell-specific effects of RA. RAR/RXR
can weakly bind to this element, however stronger binding (weakly cooperative)
was observed upon addition of Pit-1 and RAR, suggesting that Pit-1 is a
coregulator for RAR acting similarly as RXR. Given the highly restricted
expression of Pit-1 this heterodimerization provides an excellent mechanism
to allow cell-type specific activation of the Pit enhancer by RA.
Together these data indicate that various other
transcription factors can modulate the RA-response of RARE-containing promoters,
both in a positive or negative fashion. Cell-specific expression of these
modulating transcription factors allows cell-type specific regulation of
the activity of these promoters. After transcription various processes
such as RNA processing, transport, stability and translation efficiency
modulate the formation of protein. Various post-translational modifications
such as phosphorylation, glycosylation, sequestration or degradation can
subsequently modulate the activity of the translated protein (33). In principle
RA could influence all these processes through modulation of the expression
or activity of splicing factors, translation factors, kinases or phosphatases.
2.4 RA
AND DEVELOPMENT
Experiments in various species have indicated
that excess as well as absence of retinoids during embryonic development
can lead to a variety of developmental abnormalities. Teratogenic defects
are found in include the central nervous system, developing craniofacial,
heart, ear and eye (168,190,197), as well as vertebrae and limb structures
(50). There is a relatively short period during early development at which
exogenous retinoids can exert drastic teratogenic effects on development
of the anterior/posterior (A/P) axis. Durston et al have found that
early RA treatment has a strong posteriorizing effect on Xenopus embryos
leading to loss of anterior structures such as forebrain at the expense
of hindbrain structures (71). In mice, excess RA can also cause posterior
transformations in the hindbrain and of vertebrae and ribs (126,181). These
experiments and experiments on limb development (282) have led to the proposal
that retinoic acid is acts as a morphogen, a diffusible substance present
in a concentration gradient that could provide positional information for
patterning the embryo (276). This means that the posterior part of the
embryo should contain a higher concentration RA than the anterior part.
Support for this hypothesis came from experiments performed to measure
endogenous retinoids in the organizer, the region that determines the site
of gastrular invagination, and later defines the posterior end of the embryo.
Using cell lines containing a RA-responsive promoter, it was shown that
this specific region in Xenopus (blastopore lip), in mouse (node) and in
chick (Hensen's node) is the major source for RA in the early embryo (48,49,110).
Furthermore, the concentration RA increased during gastrulation indicating
that retinoids could act as a morphogen during gastrulation (48). Finally,
indirect evidence has also been presented suggesting the presence of a
RA-gradient during spinal cord development, of development (188,296). Whether
RA diffuses from the organizer region or whether high levels of retinoids
present in this region induce formation of a gradient of other factors
is not clear. Alternatively, an A/P dependent response to retinoids could
be determined by the time cells have been exposed to retinoids present
in the organizer prior to invagination during gastrulation, whereby cells
that first invaginate receive the lowest dose. Using reporter constructs
containing RAREs it was shown that retinoid signalling is occurrs near
the organizer at the posterior part of the embryo (191,246), indicating
that retinoids could modulate the A/P exis by activation of target-gene
expression. The Hox genes are possible targets for RA-mediated determination
of positional information (181). The vertebrate homeotic genes are located
on 4 homologous chromosomal clusters and are the homologs of the Drosophila
HOM-C genes. These clusters show a colinearity in the spatial sequence
of anterior expression boundaries and in the temporal order of their activation
with respect to the location of the gene in the cluster: 3' genes are expressed
first and show the most anterior expression boundaries, whereas the genes
located more 5' in the cluster are expressed only in posterior regions
and are activated later in development. Furthermore colinearity also applies
to sensitivity for RA treatment in a time- and concentration-dependent
manner: 3' genes are activated earlier in development and require the lowest
RA concentration for activation (61,267). This argues that RA, present
in the organizer, could define a certain Hox expression pattern (Hox-code),
which can be modified by exogenously added retinoids altering this hox-code
and causing homeotic transformations (126). As described above, at least
the expression of the most 3' Hox genes have been shown to be directly
regulated by RA through RAREs present in the enhancer regions of these
genes. Whether the more 5' genes also contain RAREs or whether other mechanisms
account for their RA-regulated expression remains to be seen.
Further proof for a direct involvement of retinoids
in Hox gene regulation (see: 50) came from the observed similar axial homeotic
transformations found when RA (126) is in excess or when a Hox gene is
overexpressed (136), leading to a more posterior identity of the skeleton.
Contrary, loss-of-function mutants for both RAR (165) or RAR double mutants
(166) and Hox genes (136) result similarly in more anterior transformation.
Together these experiments strongly implicate retinoids as important regulators
in determination of positional information through regulation of Hox
gene expression
2.5 THE
EC CELL MODELSYSTEM
P19 embryonal carcinoma (EC) cells are pluripotent
cell lines that can differentiate in vitro into cell types resembling
cells from all three germ layers. Furthermore, when injected into mouse
blastocysts, EC cells can participate in normal development although abnormalities
were seen in embryos with large contribution of EC-cells (183,184). The
similarities between inner cell mass cells and EC cells that resemble primitive
ectodermal cells (184), with respect to differentiation capacity in
vitro and in vivo make this cell line a valuable model system
for early embryonal development. Like other EC cells, P19-EC cells can
differentiate upon RA treatment, leading to the formation of endodermal
cells. Culturing these cells after an aggregation step lead to the formation
of endodermal and mesodermal cells at low RA concentrations while mainly
neurons and glial cells are formed at high concentrations (7,187). These
RA-concentration dependent differences in differentiation are accompanied
by a change in expression of various genes (98), some of which have been
implicated in early embryonal differentiation processes such as mesoderm
induction (269). The above described RA-induced differentiation is dependent
on the presence of functional RARs since a RA-resistant P19-EC cell derived
cell line, RAC65, contains a truncated (dominant negative) RAR (232,139).
Introduction of a functional RAR into this cell line restores its ability
to differentiate upon RA-treatment (139). Furthermore, gene disruption
experiments in F9-EC cells strongly suggest that RARs are required for
RA-dependent differentiation (24,25).
AIM OF THIS
THESIS
The role of retinoic acid in EC cell differentiation
is well documented and known to involve an increase the length of the G1
phase of the cell cycle, repression of growth, change in morphology of
the cells, differentiation, and apoptosis. These processes are accompanied
by RA-induced changes in gene expression. Most if not all of these effects
are directly or indirectly the consequence of changes in gene expression
caused by transcriptional activation of retinoic acid receptors by RA.
To understand how RARs exert their effect it is important to know how these
receptors activate transcription, and how the level of transcription is
regulated in a cell-type specific manor.
In Chapter 2, we show that RAR and RAR contain
two distinct autonomous transcriptional activation domains (AFs). The activity
of the individual AFs is cell-type specific: the ability of the AFs to
modulate transcription is dependent on the cell context. In Chapter 3 we
further characterize AF1, the transcriptional activation function present
in the AB-region of RAR, and it is shown that this activator belongs to
the acidic class of transcriptional activators. Further, we present evidence
for a role of phosphorylation in the cell-specific activity of this activator
(Chapter 3 and 4). The cell-specific activity of AF-2 is regulated differently.
Previous experiments performed in this laboratory have indicated that E1A
could function as a cofactor for RARs. We show here (Chapter 5) that the
viral protein E1A 13S can act as a cofactor in RA-dependent transcriptional
activation through a direct interaction with RAR. E1A can also interact
with TBP (Boyer, Horikoshi, Lee) and TAF110 (Mazzarelli) thereby connecting
RAR with the basal transcription machinery. This led to the proposal that
cell-specific activation by AF-2 can be the consequence of differential
cofactor expression. Chapter 6 describes that E1A 13S can partially blunt
the cell-specificity of the activity of AF-2, and documents evidence for
a role of cofactors in synergistic activation of transcription. This involvement
of cofactors in cell-specific transcription is further investigated by
characterising the cell-specific differences in expression of some of the
recently cloned cofactors (Chapter 8).
The multiple functions of retinoids and the presence
of multiple receptors each containing two autonomous activation domains
further strengthened the idea that each receptor fulfills a specific function
in EC cell differentiation. Overexpression of RAR or RAR in a RA-resistant
EC cell line RAC65 resulted in RA-dependent differentiation for the RAR
overexpressing cells and growth repression accompanied with cell death
for the RAR transfectants (Chapter7). Additional experiments further indicated
that P19-EC cells that express high levels of RAR eventually undergo apoptosis.
This Suggests that RAR is not involved in the differentiation process but
primarily involved in growth regulation and apoptosis. Given the role for
RAR we investigated the regulation of the RAR2 promoter. This promoter
is activated by RA in a strictly cell-type specific manner, being highly
active in EC cells but not in non-EC cells. In chapter 8 it is shown that
several cell-specific proteins including cofactors, orphan receptors and
initiator-binding proteins contribute to this cell-specific activation
of the RAR promoter and possibly also of promoters of other RA-target genes.
Together these experiments underscore the cell-specific
constraints on RA-signaling as an important mechanism for regulating the
effects of RA in a cell-specific fashion. Finally in chapter 9 the role
of (cell-specific) RA-signaling is discussed in relation to EC cell differentiation
and early embryonal development.
back
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