Nuclear Hormone Receptors and Gene Transcription
Nuclear receptors encompass a superfamily of conserved receptors that share
structural and functional organization. Nuclear receptors have distinct domains for
binding of lipophilic hormones, including steroids, thyroid hormone, retinoids, and
vitamin D, among others that bind products of lipid metabolism (Lee and Kraus, 2001).
The lipophilic nature of the ligands enables them to readily penetrate the plasma
membrane to enter the cell by simple diffusion (Whitfield et al., 1999). Receptors in this
superfamily play crucial roles in development, homeostasis, and other biological
processes by influencing transcription of their target genes (Whitfield et al., 1999).
The nuclear receptor superfamily can be divided into three groups of receptors.
The first group (class 1) includes the steroid receptors (estrogen receptor, progesterone
receptor, androgen receptor, glucocorticoid receptor, and mineralocorticoid receptor). In
the absence of bound ligand, steroid receptors are coupled to a large multiprotein
complex, including Hsp90, Hsp56, Hsp70, and p23. This complex maintains the
receptors in a conformation able to bind ligand, and sequesters the receptors to the
cytoplasm. In this state, the receptors are unable to influence the rate of transcription of
their cognate promoters (Leo and Chen, 2000). Upon ligand binding, the steroid
receptors dissociate from the heat-shock proteins, homodimerize, and translocate to the
nucleus, where they bind in a head-to-head arrangement, DNA response elements
composed of palindromic repeats of the core 5’-TGTTCT-3’ sequence (Leo and Chen,
2000; Claessens et al., 2001). The unliganded progesterone (especially the A form
described later) and estrogen receptors, unlike the other steroid receptors, are localized in
the nucleus (Hager et al., 2000; Wan et al., 2001). The estrogen receptor further differs
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from the other steroid receptors in that it recognizes a palindromic repeat of the
consensus sequence TGACCT (Claessens et al., 2001; Leo and Chen, 2001). The
mechanisms necessary for the steroid specificity of gene regulation are not completely
understood, but receptor distribution and hormone metabolism can partially explain in
vivo steroid specificity (Claessens et al., 2001).
Steroid hormones are important endocrine messengers. Androgen, estrogen, and
progesterone are important for control of reproduction and sexual development
(Whitfield et al., 1999). In the embryo, the androgen male sex hormones cause the
differentiation of the reproductive organs into the male phenotype. Also, increase in
androgen production during puberty induces the secondary sexual characteristics
(Claessens et al., 2001). The two predominant naturally occurring ligands of the
androgen receptor (AR) are testosterone and dihydrotestosterone (DHT). Estrogen
female sex hormones produce profound effects on the growth, differentiation, and
functioning of many reproductive tissues. However, estrogens also exert actions on other
tissues including bone, liver, brain, and the cardiovascular system (Katzenellenbogen et
al., 2000). The majority of the actions of estrogens seem to be exerted by two estrogen
receptor (ER) subtypes, ER  and ER . The levels and proportion of these two
receptors are variable in different target cells (Katzenellenbogen et al., 2000). The
progesterone receptor (PR) is found in mammalian cells as two variants, PR-A and PR-B.
The A form utilizes an alternative initiation signal and is missing the first 164 amino
acids of the full length B form (Hager et al., 2000; Rowan and O’Malley, 2000).
Progesterone functions to prepare the uterus endometrium for embryo implantation and to
maintain pregnancy in the uterus and ovary. It also suppresses milk protein synthesis in
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the mammary gland during pregnancy and regulates the outgrowth of alveolar structures
in the mammary gland (Rowan and O’Malley, 2000; Wan et al., 2001).
Glucocorticoids and mineralocorticoids are steroid hormones secreted by the
adrenal gland. The mineralocorticoid receptor and its ligand aldosterone control sodium
reabsorption and blood pressure (Whitfield et al., 1999). The actions of the
glucocorticoids (cortisol) include regulation of glucose metabolism (gluconeogenesis),
inhibition of bone formation, and anti-inflammatory and immunosuppressive actions
Whitfield et al., 1999; Wan et al., 2001).
The second group of nuclear receptors (class II) includes retinoic acid receptors,
thyroid hormone receptors, and the vitamin D3 receptor. These receptors are found
strictly in the nucleus and form heterodimers with the receptor for 9-cis retinoic acid
(RXR). Heterodimer formation with RXR is a necessity because these receptors bind
more efficiently to their target sites in the presence of RXR. The heterodimers bind
constitutively in a head to tail configuration to DNA response elements consisting of
direct repeats of the sequence TGACCT, and in the absence of ligand often exert a
repressive effect upon the activity of their promoters (Leo and Chen, 2000).
The basic molecular mechanism of the retinoic acid receptor proteins is shown
below. Left: The Ligand Binding Domain (LBD) of the receptor is free of hormone and
binds a repressor. The DNA Binding Domain (DBD) of the receptor contacts the DNA
upstream of the transcription start site. Middle: The binding of the hormone induces a
conformational change which results in the expulsion of the repressor. Right: A
coactivator binds to the receptor and switches on transcription.
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A series of receptors bind DNA only when heterodimerized to RXR. In that case, each
member of the dimer takes part in the regulation of the metabolic pathways activated.
Interestingly, the heterodimer can activate the transcription of different genes, whether no
ligand, an agonist, or an antagonist is bound to one receptor or the other. The figure
below is a model for the synergetic activation of the RAR-RXR heterodimer. A: Both
receptors are unliganded. A repressor (bricks) binds to RAR (white) and prevents
transcription. B: RAR is activated (hormone in black). The repressor is replaced by a
coactivator which enables transcription. C: RXR (grey) is activated, but the repressor still
binds to RAR and prevents transcription. D: Both receptors recruit an activator, leading
to a stronger transcriptional response.
The heterodimer RXR alpha-RAR controls the expression of the RAR beta gene, the
level of which seems to be critical for the proliferation of breast tumor cells. The control
of the activation state of RAR alpha and RXR could therefore be a valuable tool to
prevent or treat breast cancer. One of the main purposes in studying these receptors is to
rationally design agonists and antagonists, targeted selectively against RAR or RXR, and
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test them in vitro and in vivo.
The third group consists of orphan receptors, so-called because the endogenous
ligands for these proteins are not currently identified (Leo and Chen, 2000). Orphan
receptors can function as homodimers or monomers (Leo and Chen, 2000). The orphan
receptor CAR- was discovered to bind DNA as a heterodimer with the retinoid-Xreceptor. CAR- stands for constitutive androstane receptor beta. It binds the steroids
androstanol and androstenol, which are naturally occurring androstane metabolites. This
receptor has an unusual steroidal signaling pathway compared to the conventional nuclear
receptor pathway (Forman et al., 1998).
Conventional nuclear receptors function as ligand-regulated, DNA-binding
transcriptional activators. Transcriptional activation by nuclear receptors involves
multiple factors such as cofactors that act in a sequential and combinatorial manner to
reorganize chromatin templates. Generally, unliganded or antagonist-bound receptors are
transcriptionally inactive or promote transcriptional repression, but agonist bound
receptors promote transcriptional activation (Lee and Kraus, 2001). Unlike other nuclear
receptors, the orphan receptor CAR- activates gene transcription in a constitutive
manner. Ligand binding to CAR- results in inactivation of gene transcription.
Therefore, this steroid signaling pathway functions in an opposite manner to that of the
conventional nuclear receptor pathways (Forman et al., 1998).
Important to the understanding of transcriptional activation by nuclear receptors is
the nuclear receptor structure (figure1). Nuclear receptors contain six domains (A-F)
based on regions of conserved sequence and function. The N-terminus contains the
variable A/B region, which includes the ligand-independent activating function-1 (AF-1)
domain. The C region is the DNA binding domain (DBD) region and is the most highly
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conserved and encodes two zinc finger modules. The D region is the hinge domain
located between the DBD and the ligand-binding domain (LBD). The LBD is in region
E, located at the C-terminus. The LBD region is less conserved and mediates ligand
binding, receptor dimerization, and a ligand-dependent transactivation function, AF-2.
The AF-2 activation domain (AD) contains a short, conserved amphipathic alpha helix
that is necessary for ligand-dependent activation. The F region is not present in all
receptors and its function is not well understood (Bourguet et al., 2000)
Figure 1 Structural and functional organization of nuclear receptors (Bourguet et al., 2000)
The structure of the nuclear receptor ligand binding domain has been
characterized by X-ray crystallography. The LBD is a protein fold comprising 12 alpha
helices and a short beta turn, arranged in three layers to form an antiparallel alpha helical
sandwich. Helices H1-H3 form one face of the LBD, H4, H5, the beta turn, H8, and H9
correspond to the central layer of the domain and helices H6, H7, and H10 constitute the
second face. The top part of the LBD structure is better conserved than the lower part,
which contains the hydrophobic ligand binding pocket (LBP). The structural variability
of the ligand binding pocket among nuclear receptor LBDs can be attributed to affinity
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toward diverse ligands, as the ligands that bind nuclear receptors differ considerably in
size and shape (Bourguet et al., 2000).
Conformational changes take place in the LBD upon binding agonists,
antagonists, or partial agonists. These conformational changes vary depending on which
particular type of molecule is bound and govern the nuclear receptor’s ability or inability
to activate gene transcription. The most striking difference in the conformation of the
LBD structures is the orientation of helix H12, which contains the residues of the AF-2
activation domain core. Upon ligand binding, this helix rotates nearly 180 to pack
tightly against the LBP, serving as a lid closing the entrance to the LBP (Holo-LBD
figure 2b). This brings the AF-2 into position to generate a surface for binding of
coactivators that are necessary for nuclear receptor gene transcription activation. In the
unliganded conformation, this helix points away from the protein core (Apo-LBD figure
2a) (Bourguet et al., 2000).
Antagonist binding inhibits gene transcription. Antagonists, unlike agonists,
have bulky side chains that cannot be accommodated within the agonist binding cavity.
The bulky side chains sterically prevent the positioning of helix H12 in the active
conformation and no coactivator interface is formed (Antagonist-LBD figure 2c). The
antagonists also induce unwinding of the C-terminal part of helix H11. This allows helix
H12 to bind to the coactivator binding region, therefore blocking coactivator binding. In
contrast, agonists stabilize the long H11 helical conformation (Bourguet et al., 2000).
Pure antagonists differ from partial agonists based largely in their steric properties.
Partial agonists do not contain a bulky extension, so they do not sterically preclude the
agonist position of H12. In this respect, they are more like agonists. However, these
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molecules do induce unwinding of helix H11 and in this respect are similar to
antagonists. The partial activity of the ligand in this case is attributed to a poor
stabilization of the active position of H12, so the active conformation of the LBDs is not
stabilized. The position of H12 in this case probably depends on the intracellular
concentration of co-activators and co-repressors (Bourguet et al., 2000). Coactivators are
necessary for nuclear receptor transcriptional activation and corepressors are used by
nuclear receptors to suppress transcription (Leo and Chen, 2000).
Figure 2 Schematic drawing of three different conformational states of nuclear receptor ligand
binding domains. Unliganded= Apo-LBD, agonist bound= Holo-LBD, antagonist bound= antogonistLBD. (Color figure code: coactivator/corepressor binding site is orange, activation helix H12 that
contains residues of the core AF-2 activation domain is red; dimerization site is green) (Bourguet et
al., 2000).
As mentioned previously, conventional unliganded class II nuclear receptors are
able to suppress transcription by recruiting corepressors such as SMRT (silencing
mediator of RAR and TR) and NcoR (nuclear receptor co-repressor) to the promoters of
their target genes. These corepressors can repress the nuclear receptors in the absence of
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ligand through either a Sin3A-dependent or –independent manner by recruiting histone
deacetylases (HDACs). Histone deactylation represses gene transcription by causing
packaging of DNA into nucleosomes. Genes with promoters organized into nucleosomes
are inaccessible to the transcription machinery (Bourguet et al., 2000; Leo and Chen,
2000; Sharma and Zijie, 2001). Corepressors play an important regulatory role by
preventing gene transcription by class II nuclear receptors. Since these receptors are
constantly present on chromatin, they do not interact with a chaperone complex like the
class I steroid receptors, which sequesters the receptors to prevent gene transcription by
unliganded receptors (Hager et al., 2000).
Intrinsic transcriptional activity of nuclear receptors is low, so these receptors
function primarily as a nucleation site for the assembly of coactivator complexes at
promoters assembled into chromatin (Kim et al., 2001). Coactivators bind to nuclear
receptors that are occupied by the appropriate agonist but generally not to ligand-free
nuclear receptors or receptors occupied by antagonists (Bourguet et al., 2000). However,
CAR- is able to constitutively activate gene transcription because it recruits coactivators
in the absence of binding ligand. CAR- LBD must be able to adopt the active
conformation in the absence of ligand and ligand binding shifts the LBD to the inactive
conformation. Forman et al. refer to the CAR- ligands as inverse agonists (Forman et
al., 1998).
At least two classes of cofactors have been identified that function to assist
nuclear receptors. The first are nucleosome remodeling complexes such as SWI/SNF that
use energy generated by ATP hydrolysis to alter nucleosome structure and increase
access of the transcriptional machinery to the promoter on the DNA template. The other
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class includes coactivators, which are a diverse group of molecules and complexes that
have distinct roles in the transcription process. In general, one or more of these roles are
played by coactivators: 1) they function as bridging factors to recruit other cofactors to
DNA-bound nuclear receptors, 2) they acetylate nucleosomal histones and various
transcription factors at the promoters of hormone target genes, and 3) they function as
bridging factors between the DNA bound receptors and the basal transcriptional
machinery (Kim et al., 2001; Lee and Kraus, 2001).
The SRC (steroid receptor coactivator)/p160 family of 160 kilodalton proteins
contains three structurally and functionally related members unified under the
nomenclature SRC1, SRC2, and SRC3 (Lee and Kraus, 2001; Kim et al., 2001).
These molecules function primarily as coactivators for nuclear receptors. The SRC
proteins bind directly to liganded (or unliganded in the case of CAR-) nuclear receptors
via the receptor interacting domain (RID). The RID contains three motifs of the
conserved sequence Leu-X-X-Leu-Leu (referred to as NR boxes). These motifs form
amphipathic alpha helices with the leucine residues comprising a hydrophobic surface on
one face of the helix. The hydrophobic surface of the helix is able to interact with the
AF-2 domain of the liganded receptor via the hydrophobic groove. The hydrophobic
groove of the receptor is composed of residues from receptor helices 3,4,5, and 12, and is
produced by the conformational change induced by agonist binding (McInerney et al.,
1998; Bourguet et al., 2000; Leo and Chen, 2000; Kim et al., 2001). The interaction of
receptors with coactivators helps explain nuclear hormone receptor specificity of gene
regulation (Claessens et al., 2001). However, this mechanism of interaction fails to
explain differences in the specific NR boxes required for transcriptional actions by
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different nuclear receptors. There are NR box preferences displayed by nuclear receptors
and there are at least two layers of specificity to the NR box preference code. The first
involves a differential requirement for the number of LXXLL containing helices utilized
by the different receptors. For example, the estrogen receptor utilizes the second NR box
and this single LXXLL containing helix is sufficient for binding SRC proteins by this
receptor. However, receptors binding as heterodimers with RXR (class II) and the
progesterone receptor require two NR boxes for binding SRC proteins. The second layer
of specificity concerns the requirements of specific residues adjacent to the LXXLL core
motif for function of a particular nuclear receptor. These receptor specific differences are
dictated by flanking carboxy-terminal residues with different residues modulating
specific interactions with the LBD of different receptors. The structural features,
duplication and spacing between LXXLL motifs have evolved to serve roles that are
likely to permit both receptor specificity and ligand specific assembly of coactivator
complexes required for nuclear receptor gene activation (McInerney et al., 1998).
Some SRC family members may possess a weak intrinsic histone acetyl
transferase activity that has been shown to activate gene transcription but this activity is
not detected universally for the SRC family (Kim et al., 2001). Instead, SRC proteins
appear to function in transcription activation by recruiting p300/CBP to liganded
receptors bound at gene promoters (figure 3). P300 and CBP, CREB (cAMP response
element-binding protein) binding protein, are large, highly related multifunctional
coactivators that share many structural and functional attributes and are referred to
collectively as p300/CBP. It functions as a coactivator for many DNA-binding
transcriptional activator proteins, including nuclear receptors. P300 and CBP have
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intrinsic acetyltransferase activity and an SRC interaction domain. They also have a
histone interacting bromodomain for direct interaction with chromatin. Also, they
contain a region for interaction with many different transcription related factors including
RNA polymerase II complexes. P300/CBP recruits the RNA polymerase II machinery to
the promoters of the nuclear receptor targeted genes (figure 3). The intrinsic
acetyltransferase activity is capable of acetylating free or nucleosomal histones, as well
as SRC family members and some transcriptional activator proteins. These specific
acetylation events can contribute to receptor-mediated transcription (Lee and Kraus,
2001). Post-remodeling acetylation of nucleosomal histones by p300 has been shown to
facilitate the transfer of histone H2A-H2B dimers from nucleosomes to NAP-1, a histone
chaperone. The resulting altered nucleosome structure may help to maintain an open
chromatin conformation that is conducive to the formation of a stable transcription preinitiation complex and transcription initiation (Kim et al., 2001; Lee and Kraus, 2001).
Figure 3 Representation of interactions between 1. nuclear receptors (estrogen receptor) and SRC
proteins and 2. SRC proteins and p300/CBP and 3. p300/CBP and the RNA pol II machinery (Kim et
al., 2001).
In addition to the nuclear receptor coactivator SRC family, a second type of
complex TRAP/DRIP, mediates transcription stimulator effects of nuclear receptors on
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the basal transcriptional machinery. TRAP (thyroid hormone receptor associated protein)
and DRIP (vitamin D receptor interacting protein) as their names suggest, interact with
class II steroid hormones. These complexes lack p300/CBP or SRC proteins and are
recruited to the nuclear receptor by the DRIP205/TRAP220 subunits and bind via a single
LXXLL motif. The SRC proteins and TRAAP220/DRIP205 bind in a mutually exclusive
manner to the nuclear receptor (Leo and Chen; 2000; Lee and Kraus, 2001; Kang et al.,
2002).
There may be a two step model involved in which the SRC and p300/CBP
proteins are recruited to the receptor first to mediate histone acetylation and
decondensation of the chromatin, thereby allowing access of the large DRIP/TRAP
complex which takes over and establishes the link from the nuclear receptor to the basal
transcriptional machinery to activate target gene transcription (Leo and Chen, 2000; Kang
et al., 2002). Recently, the TRAP complex was also discovered to interact with the class
I estrogen receptors  and  through the TRAP220 subunit and enhance the estrogen
receptor function (Kang et al., 2002).
Transcriptional activation of a gene by nuclear receptors must be silenced.
Histone deacetylases (HDAC) are important for attenuation of hormone mediated
transcriptional responses. Recruitment of corepressors with associated histone
deacetylases to nuclear receptors (class II) may be a conversion point from transcriptional
activation to repression. However, receptors belonging to class I, such as the androgen
receptor cannot silence transcription through a similar mechanism. However, Sharma
and Sun have demonstrated that androgen receptor mediated transcription repression is
mediated through histone deacetylase recruiting. The DNA binding domain of AR binds
5’TG3’ interacting factor (TGIF), which interacts with the transcription corepressor
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Sin3A, which recruits HDAC1 to the repression complex (Sharma and Zijie, 2001).
However, acetylation may also attenuate nuclear receptor transcription. P300/CBP
mediated acetylation of SRC-3 was shown to cause the disruption of receptor-coactivator
complexes, resulting in an attenuation of hormone-mediated transcriptional responses
(Kim et al., 2001). The unique nuclear receptor CAR- inactivates gene transcription by
binding one of its endogenous ligands (androstanol and androstenol), which promotes
coactivator release from the receptor (Forman et al., 1998).
There has been quite a bit of research devoted to understanding nuclear receptor
gene transcription. Protein-protein interaction assays, such as the yeast two-hybrid
screen and in vitro binding assays with recombinant proteins have been used to detect
associations between nuclear receptors and coactivators. In these experiments, the LBDs
of receptors were used as bait to screen cDNA libraries to identify coactivators by their
ability to bind the LBDs (Leo and Chen, 2000). This process was followed by transient
transfection analysis and microinjection of immuno-neutralized antibodies to analyze the
potential coactivator activities of the identified proteins (Lee and Kraus, 2001). The
injected antibodies were used to abolish the function of particular coactivators to help
establish their relevance to nuclear receptor gene transcription activation (Leo and Chen,
2000). Mutagenesis studies have been used to determine which resides in the N-terminal
portion of the ligand binding domain determine ligand binding specificity (Wan et al.,
2001). Site directed mutagenesis studies have also been used to provide evidence for the
requirement of the LXXLL motif for mediating interaction between coactivators and
liganded nuclear receptors (Leo and Chen, 2000). X-ray crystallographic analysis of
nuclear receptor domains has allowed determination of the structure of liganded and
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nonliganded LBDs and LBD conformational changes associated with ligand binding
(Bourguet et al., 2000). This technique has allowed visualization of the contact surfaces
between nuclear receptors and coactivators (Whitfield et al., 1999).
Recent experiments have been directed at determining nuclear receptor and
coactivator mediated transcription in the context of chromatin. Approaches used include
real time imaging in living cells, in vitro microinjection of hormone responsive reporter
genes into Xenopus oocytes, in vitro chromatin assembly and transcription assays, and
CHIP assays (Lee and Kraus, 2001). Real time imaging in living cells is possible with
green fluorescent protein (GFP) from the jellyfish Aequorea victoria. Fusion of receptors
or coactivators to GFP is used to monitor protein localization and trafficking (Hager et
al., 2000; Lee and Kraus, 2001). For example, this procedure has been used to monitor
the differential subcellular localization between the two highly related GR and PR
receptors (Wan et al., 2001).
Microinjection of Xenopus oocytes has been used to analyze various aspects of
transcriptional regulation by the TR and GR, including receptor-mediated chromatin
remodeling and the requirements of various coactivator domains of p300. Single
stranded DNA templates microinjected into the nuclei of Xenopus oocytes are rapidly
replicated and assembled into chromatin. Transcriptional regulatory proteins can be
introduced through the coinjection of mRNAs encoding specific factors, and transcription
from the microinjected templates can be monitored by primer extension analysis (Lee and
Kraus, 2001).
In vitro chromatin assembly and transcriptional experiments use chromatin
assembly extract containing receptor binding sites derived from Drosophila embryos,
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with added transcript activator proteins (purified receptor) and coactivators coupled with
in vitro transcription systems. The RNA products that are produced are analyzed and
quantified. This method has helped elucidate the mechanisms underlying nuclear
receptor mediated transcription (Lee and Kraus, 2001). Specifically, this procedure has
been used to demonstrate that the interaction among ER alpha and the SRC proteins and
p300/CBP and the RNA polymerase II machinery as well as histone acetylation are
required for ER alpha mediated transcription (Kim et al., 2001).
A method for the analysis of protein-DNA interactions in the chromatin
environment of a cell is chromatin immunoprecipitation assays (CHIP). This assay
involves the use of formaldehyde to cross-link protein-DNA complexes in intact cells,
followed by the selective immunoprecipitation of these complexes with specific
antibodies. The complexes contain fragments of genomic DNA that can be identified
with quantitative PCR or DNA hybridization techniques. In this way, the gene segments
bound by specific proteins can be identified. These assays have been used to analyze
nuclear receptor-dependent changes in histone acetylation, coactivator recruitment, and
the phosphorylation state of RNA polymerase II at target promoters in vivo (Lee and
Kraus, 2001).
Nuclear receptors activate gene transcription by binding as dimers to DNA
binding domains within the chromatin environment of the cell. Transcription activation
by classic nuclear receptors is mediated by ligand binding, resulting in transfer of the
receptors to the nucleus (class I) or release of corepressors (class II). The nuclear
receptors then recruit coactivators, which function to remodel the nuclear chromatin to
expose gene promoters to allow binding of the RNA polymerase II basal transcriptional
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machinery. A unique nuclear receptor, CAR- was identified that functions in a manner
that is opposite to the classic receptors. CAR- binds coactivators in the absence of
ligand binding to promote transcription constitutively and ligand binding causes release
of the coactivators, resulting in transcriptional silencing. The regulation of nuclear
receptor transcriptional activation is complicated and it is likely that other unique
activation pathways will be identified as researchers continue to study the methods of
nuclear receptor gene transcription activation.
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