Use of reporter genes for retinoid receptor screening assays having novel retinoid-associated response elements

In accordance with the present invention, there are provided novel cotranscription factors that function to enhance mRNA transcription in cooperation with retinoid receptors. Also provided are novel DNA response elements, DNA constructs and expression vectors containing said constructs useful for providing cell-specific gene expression. Bioassays are also provided that are useful for evaluating whether a compound is a functional ligand (e.g., agonist or antagonist) for retinoid receptor protein(s), or functional engineered or modified forms thereof.

FIELD OF THE INVENTION 
The present invention relates to retinoid associated transcription factor 
proteins, DNA response elements responsive thereto, and various uses 
therefor. 
BACKGROUND OF THE INVENTION 
Retinoids affect DNA transcription in a wide variety of mammalian cells. 
Retinoids exert their effects on transcriptional activity through 
intracellular retinoid receptors, which when complexed as heterodimers 
with a functional ligand bind to a specific retinoid response element, and 
subsequently modulate transcription. Previously, retinoic acid receptors 
(e.g., RAR and RXR) had been found to participate in modulation of nucleic 
acid transcription without requiring the binding of any co-factors, such 
as enhancers, and the like. 
It has been found that RAR and RXR have a high degree of cooperativity in 
binding target DNA. For example, an RAR and RXR heterodimer binds to a DNA 
response element having two 6-nucleotide direct repeat sequences separated 
by a 5-nucleotide spacer sequence (DR-5), and strongly stimulates 
transcriptional activation (Kliewer et al., 1992, Nature 355:446-449). 
Indeed, RXR-TR and RXR-RAR heterodimers have been recently shown to bind 
related response elements (with different spacers), i.e., DR-4 and DR-5 
sites, respectively (see, Perlman et al., 1993, Genes Develop. 
7:1400-1422; and Kurokawa et al., 1993, Genes Develop., 7:1423-1435). It 
has also been found that RXR-RAR heterodimers bind to a related DNA 
response element having two 6-nucleotide direct repeat sequences separated 
by a 2-nucleotide spacer sequence (DR-2) (Rhodes et al., 1993, Genes 
Develop. 7:913-932). 
The mammalian homeobox genes (HOX) encode a family of more than 30 related 
proteins which share the common "homeo box" motif originally identified in 
a Drosophila homeotic complex. Human homeobox gene clusters designated HOX 
A, B, C, and D have been mapped to chromosomes 7, 17, 12, and 2, 
respectively, and retain a linear gene arrangement similar to their 
Drosophila counterparts (Acampora et al., 1989, NAR 17:10385-10402). 
Expression of mammalian homeobox genes is strictly regulated both 
temporally and spatially during embryonic development (see, e.g., 
Wilkinson et al., 1989, Nature 341:405-409). 
Retinoic acid (RA), a natural metabolite of vitamin A has been proposed to 
be both a vertebrate morphogen and a regulator of the HOX gene clusters 
(see, e.g., Eichele, G., 1989, Trends Genet. 5:246-251). Systemic 
treatment of vertebrate embryos with retinoic acid results in severe 
developmental deformities, while local application of retinoic acid to 
chick limb bud produces digit duplication which is accompanied by a change 
of homeobox gene expression. 
In the human embryonal carcinoma cell line NT2/D1 (Andrews et al., 1984, 
Lab. Invest. 50:147-162), homeobox genes are sequentially activated by 
retinoic acid in a graded fashion from the 3' to 5' direction. Activation 
of the 3' HOXB1 gene is not dependent on protein synthesis and thus is a 
candidate for direct regulation by retinoic acid. However, the precise 
molecular link between HOXB1 and retinoic acid signalling and the 
mechanism establishing graded chromosomal expression remains obscure. The 
actions of retinoic acid are mediated by both RARs and RXRs, members of 
retinoid nuclear receptor family (see, e.g., U.S. Pat. No. 5,171,671; 
Gigeure et al., 1987, Nature 330:624-629; Mangelsdorf et al., 1990, Nature 
345:224-229; Mangelsdorf et al., 1992, Genes and Develop. 6:329-344, and 
the like). These receptors have been shown to function via a heterodimer, 
which binds DNA in a sequence specific manner. Binding of DNA serves to 
activate target genes through a retinoic acid responsive element (RARE) in 
a hormone dependent pathway. 
Since the RARs and RXRs are essentially ubiquitous in their expression, the 
use of co-activators could provide a particularly effective means to 
restrict inducibility of a variety of genes by retinoic acid. Thus, 
retinoic acid induction could be limited to selective cell types in a 
cell-specific manner. For example, it has been found that a RARE in the 
promoter of the Pit-1 gene depends on Pit-1 for inducibility (Rhodes et 
al., 1993, Genes Develop. 7:913-932). How this occurs is unclear but may 
relate to the way in which heterodimers bind to target DNA. 
Prior art bioassays employed for selecting functional ligands that bind 
retinoid receptors and modulate transactivation of protein expression 
employ retinoic acid receptor response elements (RAREs). Up to now these 
assays have only been able to identify ligands that modulate gene 
expression through the binding of a retinoic acid heterodimer to a 
response element having only the direct repeat sequence motif. 
It would be desirable to improve upon prior art methods by developing 
bioassays that enable selection of cell-specific functional ligands (e.g., 
agonists or antagonists) for retinoid receptors. It would also be 
desirable to identify ligands for retinoid receptors that modulate gene 
expression in cooperation with other cell specific transcription factors. 
BRIEF DESCRIPTION OF THE INVENTION 
In accordance with the present invention, there are provided novel 
retinoid-related proteins. Invention retinoid-related proteins are useful, 
in cooperation with an activated retinoid receptor, to enhance 
transactivation of nucleic acid transcription from a suitable 
promoter-containing nucleic acid construct. In addition, invention 
retinoid-related proteins are useful in invention bioassays for 
identifying functional ligands for retinoid receptors. 
In accordance with another embodiment of the present invention, there are 
provided enhanced DNA response elements comprising a retinoic acid 
responsive direct repeat sequence and a co-factor specific binding site. 
Invention response elements are useful, when combined with heterologous 
coding sequences, to promote cell specific transactivation of gene 
expression. Invention response elements are also useful to promote 
reporter gene expression in bioassays for identifying functional ligands 
for retinoid receptors. 
In accordance with yet another embodiment of the invention there are 
provided DNA constructs, recombinant expression vectors, and host cells 
containing such constructs and vectors. Invention DNA constructs comprise 
invention response elements operatively linked to a promoter. Such 
constructs are useful to confer transcriptional activation activity on the 
promoter in the presence of a functional ligand and its associated 
retinoid receptor. 
In accordance with still another embodiment of the present invention, there 
are provided bioassays useful for evaluating whether a compound is a 
functional ligand (e.g., agonist or antagonist) for retinoid receptor 
protein(s), or functional engineered or modified forms thereof.

DETAILED DESCRIPTION OF THE INVENTION 
In accordance with the present invention, there is provided an isolated 
retinoid-inducible-protein (RIP) characterized as binding to a 
RIP-binding-site having substantially the same nucleotide sequence as 
nucleotides 91-99 set forth in SEQ ID NO:1. The RIP, in cooperation with 
an activated retinoid receptor, enhances transactivation of nucleic acid 
transcription from a suitable nucleic acid construct. Such nucleic acid 
constructs comprise a promoter operatively associated with a retinoic 
acid-response element (RARE) and a RIP-binding-site. 
Unexpectedly, it has been found that the association of RARE with a 
RIP-binding-site of the invention substantially enhances transcription 
activity cooperatively induced by an RAR-RXR heterodimer and RIP. It has 
also been found that expression of the endogenous RIP is induced by 
retinoic acid in P19 cells (Andrews et al., 1984, Lab. Invest. 50:147-162, 
incorporated herein by reference), but not in NT2/D1 cells (Andrews et 
al., 1984, supra). 
As used herein, the phrase "isolated" as a modifier of invention proteins 
refers to proteins that have been manipulated, such that they are 
separated from their native in vivo cellular environment and are 
substantially free of other cellular proteins, respectively. Invention 
proteins are useful, for example, in the identification of selective drugs 
or compounds. 
RIPs can be isolated so they are substantially free of other cellular 
proteins by, for example, using a nucleic acid having a RIP-binding-site 
sequence to bind the RIP protein, whereby the RIP-binding nucleic acid is 
bound to a solid support. Subsequently, the RIP-nucleic acid (protein-DNA) 
complex can be separated from other proteins, and RIP protein can then be 
eluted from the nucleic acid in substantially isolated form. RIPs have 
been isolated from P19 cells, which are derived from human embryonic 
carcinoma cells. Thus, cells derived from human embryos are a suitable 
source of RIPs. 
As used herein, the phrase "binding" refers to the well-known interaction 
that occurs between DNA-binding proteins (e.g., transcription factors) and 
a particular DNA-binding site. The ability of a given protein to bind to a 
particular DNA-binding site can be assayed by numerous methods well-known 
in the art, such as in gel-shift assays described herein. 
As used herein, the term "RIP-binding-site" (i.e., URE) refers to a 
nucleotide sequence that binds to a RIP at physiological conditions. 
Suitable RIP-binding-sites have substantially the same nucleotide sequence 
as nucleotides 91-99 in SEQ ID NO:1. A particularly preferred 
RIP-binding-site has the same nucleotide sequence as nucleotides 91-102 in 
SEQ ID NO:1. The invention RIP-binding-site, when operatively linked to a 
promoter having a RARE and a coding sequence, functions cooperatively with 
adjacent RAREs to confer full and cell-specific transcriptional activation 
of a desired coding region in response to retinoids. 
As used herein, the phrase "substantially the same nucleotide sequence" 
refers to nucleic acids having sufficient homology to a reference 
polynucleotide, such that it will hybridize to the reference nucleotide 
under moderately stringent hybridization conditions. Alternatively, 
nucleic acids having "substantially the same nucleotide sequence" as the 
reference nucleotide sequence has at least 70% homology with respect to 
the reference nucleotide sequence. Nucleic acids having at least 80%, more 
preferably 90%, yet more preferably 95%, homology to the reference 
nucleotide sequence are also contemplated. 
As used herein, the phrase "moderately stringent hybridization" refers to 
conditions that permit target-DNA to bind a complementary nucleic acid 
that has about 60%, preferably about 75%, more preferably about 85%, 
homology to the target DNA; with greater than about 90% homology to 
target-DNA being especially preferred. Preferably, moderately stringent 
conditions are conditions equivalent to hybridization in 50% formamide, 
5.times.Denhart's solution, 5.times.SSPE, 0.2% SDS at 42.degree. C., 
followed by washing in 0.2.times.SSPE, 0.2% SDS, at 65.degree. C. 
As used herein, the phrase "induced by retinoic acid" refers to the 
intracellular expression of RIP caused by exposure of the cell to retinoic 
acid, or derivatives thereof (e.g., retinoids). It has been found that RIP 
is induced by retinoic acid in P19 cells. 
As used herein, the phrase "cooperative transactivation of transcription," 
or grammatical variations thereof, refers to a well-known mechanism of 
transcription where more than one transcription factor (e.g., co-factors) 
operate in tandem to activate transcription. For example, when RIP binds 
at a RIP-binding-site and an RAR-RXR heterodimer binds at an appropriate 
retinoic acid response element (RARE), these two binding events 
cooperatively and synergistically transactivate transcription to a 
substantial level, relative to transcription levels for each binding event 
alone. 
As used herein, the phrase "activated retinoid receptor" refers to the 
well-known retinoid receptor heterodimer complex that forms when bound by 
a functional ligand (e.g., retinoids, and the like), such that the dimeric 
receptor is able to bind to its appropriate response element and initiate 
mRNA transcription. Suitable retinoid receptors include, for example, RAR 
receptors (e.g., .alpha., .beta., and .gamma.; U.S. Pat. No. 5,171,671, 
incorporated herein by reference) and RXR receptors (e.g., .alpha., 
.beta., and .gamma.). In a particular embodiment, an activated retinoid 
receptor is an RAR-RXR heterodimer. 
As employed herein, the term "retinoids" refers to naturally occurring 
compounds with vitamin A activity, synthetic analogs, and various 
metabolites thereof. The retinoids are a class of compounds consisting of 
four isoprenoid units joined in head-to-tail manner. Numerous retinoids 
have been identified, as described, for example, by Sporn, Roberts and 
Goodman in the two volume treatise entitled The Retinoids (Academic Press, 
N.Y., 1984), to which the reader is directed for further detail. Exemplary 
retinoids include retinol, retinyl acetate, retinyl hexadecanoate, 
.alpha.-retinyl, 4,14-retroretinol, deoxyretinol, anhydroretinol, 
3,4-didehydroretinol, 15,15-dimethyl retinol, retinyl methyl ether, 
retinyl phosphate, mannosyl retinyl phosphate, retinol thioacetate, 
retinal (retinaldehyde), 3,4-didehydroretinal, retinylidene acetylacetone, 
retinylidene-1,3-cyclopentanedione, retinal oxime, retinaldehyde 
acetylhydrazone, retinoic acid, 4-hydroxyretinoic acid, 4-oxoretinoic 
acid, 5,6-dihydroretinoic acid, 5,6-epoxyretinoic acid, 5,8-epoxyretinoic 
acid, the open-chain C.sub.20 analog of retinoic acid (i.e., 
(all-E-3,7,11,15-tetramethyl-2,4,6,8,10,12,14-hexadecaheptaenoic acid), 
7,8-didehydroretinoic acid, 7,8-dihydroretinoic acid, "Acid" (E, 
E)-3-methyl-5-(2,6,6-trimethyl-2-cyclohexen-1-yl)-2,4-pentanedioic acid), 
"C.sub.17 Acid" 
((E,E,E)-5-methyl-7-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2,4,6-hepatrienoic 
acid), "C.sub.22 Acid" (14'-apo-.gamma., .psi.-carotenoic acid), retinoic 
acid esters (e.g., methyl ester, ethyl ester, etc.), retinoic acid 
ethylamide, retinoic acid 2-hydroxyethylamide, methyl retinone, "C.sub.18 
Ketone" 
6-methyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3,5,7-ocatrien-2-one), and 
the like. 
In accordance with another embodiment of the present invention, there is 
provided an isolated retinoid-activating-protein (RAP) characterized as 
binding to a RAP-binding-site having substantially the same nucleotide 
sequence as nucleotides 101-116 in SEQ ID NO:2. RAP is constitutively 
expressed in P19 and NT2/D1 cells (see, Andrews et al., 1984, Lab. Invest. 
50:147-162), but not in CV-1 cells (ATCC #CCL 70). In addition RAP, in 
cooperation with an activated retinoid receptor, enhances transactivation 
of nucleic acid transcription from a suitable nucleic acid construct. Such 
nucleic acid constructs comprise a promoter operatively associated with a 
RARE and a RAP-binding-site. The association of RARE with a 
RAP-binding-site of the invention substantially enhances transcription 
activity cooperatively induced by an RAR-RXR heterodimer and RAP. 
As used herein, the term "RAP-binding-site" (i.e., DRE) refers to a 
nucleotide sequence that binds RAP at physiological conditions. Invention 
RAP-binding-sites are useful in conjunction with RAREs to provide 
cell-specific transcription initiation. Suitable RAP-binding-sites have 
substantially the same nucleotide sequence as nucleotides 101-116 in SEQ 
ID NO:2. A particularly preferred RAP-binding-site has the same nucleotide 
sequence as nucleotides 86-116 in SEQ ID NO:2. 
In accordance with yet another embodiment of the present invention, there 
is provided an isolated enhanced-response-element comprising: 
(1) a RIP-binding-site having substantially the same nucleotide sequence as 
nucleotides 91-99 in SEQ ID NO:1, operatively linked to 
(2) a direct repeat sequence: 
5'-RGBNNM-(NN)-RGBNNM!.sub.y -3', 
wherein 
each R is independently selected from A or G; 
each B is independently selected from G, C, or T; 
each N is independently selected from A, T, C, or G; and 
each M is independently selected from A or C; with the proviso that at 
least 4 nucleotides of each -RGBNNM- group of nucleotides are identical 
with the nucleotides at comparable positions of the sequence -AGGTCA-, and 
y is at least 1 (up to about 10), preferably up to about 5. 
An isolated "enhanced-response-element" having a RIP-binding-site (also 
referred to herein as a "RIP-associated response element"), refers to a 
nucleotide sequence motif that, when operatively linked to a promoter, 
confers transcriptional activation on the promoter in the presence of RIP 
and a functional ligand bound to its associated retinoid receptor (e.g., 
an RAR-RXR heterodimer). RIP-associated DNA response elements contain 
several functionally separable components, two of which are a 
RIP-binding-site and a "direct repeat" sequence also referred to herein as 
a "retinoic acid response element" (RARE). 
The RIP-binding-site binds RIP, and the RARE binds an activated RAR-RXR 
heterodimer. It has been found through mobility shift analysis as well as 
co-transfection assays that the binding activity of RIP and the RAR-RXR 
heterodimer is cell-specific. In addition, RIP-associated response 
elements of the invention have been found to activate either the HOXB1 
promoter or heterologous promoters (e.g., thymidine kinase promoter) from 
both the 5' or 3' direction. Thus, it is also contemplated to operatively 
link RIP-associated response elements with heterologous promoters so as to 
confer cell-specific retinoid-responsive recombinant expression of desired 
proteins. 
As used herein, the term "operatively linked" refers to the functional 
relationship of DNA with regulatory and effector sequences of nucleotides, 
such as promoters, enhancers, transcriptional and translational stop 
sites, and other signal sequences. For example, operative linkage of DNA 
to a promoter refers to the physical and functional relationship between 
the DNA and promoter such that the transcription of such DNA is initiated 
from the promoter by an RNA polymerase that specifically recognizes, binds 
to and transcribes the DNA. 
Suitable half-sites having the -RGBNNM- motif for use in the invention 
"RIP-associated response element" include, for example, half-sites 
selected from -AGGGCA-, -AGTTCA-, -AGGTAA-, -AGGTCA-, -GGTTCA-, -GGGTTA-, 
-GGGTGA-, -AGGTGA-, or -GGGTCA-. A particularly preferred first -RGBNNM- 
group in the invention RIP-associated response element is -AGGGCA-. A 
particularly preferred second -RGBNNM- group in the invention 
RIP-associated response element is -AGTTCA-. The presently most preferred 
RARE for use in the invention "RIP-associated response element" is 
-AGGGCA-TC-AGTTCA- (SEQ ID NO:4). 
In accordance with yet another embodiment of the present invention, there 
is provided an isolated enhanced-response-element comprising: 
(1) a RAP-binding-site having substantially the same nucleotide sequence as 
nucleotides 101-116 in SEQ ID NO:2, operatively linked to 
(2) a direct repeat sequence, as described above. 
An isolated "enhanced-response-element" having a RAP-binding-site (also 
referred to herein as a "RAP-associated response element"), refers to a 
nucleotide sequence motif that, when operatively linked to a promoter, 
confers transcriptional activation on the promoter in the presence of RAP 
and a functional ligand bound to its associated retinoid receptor (e.g., 
an RAR-RXR heterodimer). RAP-associated DNA response elements contain a 
RAP-binding-site and a "direct repeat" sequence also referred to herein as 
a "retinoic acid response element" (RARE) . 
A particularly preferred first -RGBNNM- group for use in the invention 
"RAP-associated response element" is -AGGTAA-. A particularly preferred 
second -RGBNNM- group in the invention RAP-associated response element is 
-AGGTCA-. The presently most preferred RARE for use in the invention 
"RAP-associated response element" is -AGGTAA-TT-AGGTCA- (SEQ ID NO:5). 
In accordance with another embodiment of the invention, there is provided 
an isolated DNA construct comprising: 
an enhanced-response-element operatively linked to a promoter, so as to 
confer transcriptional activation activity on said promoter in the 
presence of a functional ligand and its associated retinoid receptor, 
wherein said enhanced-response-element is selected from a RIP-associated 
response element or a RAP-associated response element, as described above. 
As used herein, "DNA construct" refers to a segment of DNA that confers the 
ability to controllably induce nucleic acid transcription on a particular 
stretch of DNA. Invention DNA constructs are designed to be recombinantly 
interchangeable with numerous heterologous DNA fragments so that protein 
expression occurs when the construct is contacted with an activated 
retinoid receptor and either a RIP or RAP protein. 
As used herein, the phrase "transcriptional activation activity" refers to 
the well-known ability of promoters to initiate the transcription of a 
coding strand into mRNA. 
As used herein, the phrase "functional ligands" refers to any compound 
capable of binding to a retinoid receptor such that the pharmacological 
activity of the receptor is activated or inhibited. Compounds contemplated 
for screening as functional ligands in accordance with the invention 
bioassays include retinoid or retinoid-like ligands, as well as compounds 
which bear no particular structural or biological relatedness to 
retinoids. Suitable compounds may be obtained from well-known sources, 
e.g., from peptide libraries, chemical libraries, bacterial and yeast 
broths, plants, and the like. 
Examples of compounds which bear no particular structural or biological 
relatedness to retinoids, but which are contemplated for screening in 
accordance with the bioassays of the present invention, include any 
compound that is an antagonist (i.e., is capable of blocking the action of 
retinoid receptors), or an agonist (i.e., is capable of promoting the 
action of retinoid receptors), such as, for example, alkaloids and other 
heterocyclic organic compounds, and the like. 
As used herein, a promoter region refers to a segment of DNA that controls 
transcription of DNA to which it is operatively linked. The promoter 
region includes specific sequences that are sufficient for RNA polymerase 
recognition, binding and transcription initiation. This portion of the 
promoter region is referred to as the promoter. In addition, the promoter 
region includes sequences that modulate this recognition, binding and 
transcription initiation activity of RNA polymerase. These sequences may 
be cis acting or may be responsive to trans acting factors. Promoters, 
depending upon the nature of the regulation, may be constitutive or 
regulated. Exemplary heterologous promoters contemplated for use in the 
practice of the present invention include the SV40 early promoter, the 
cytomegalovirus (CMV) promoter, the mouse mammary tumor virus (MMTV) 
steroid-inducible promoter, the Herpes simplex virus thymidine kinase (TK) 
promoter, the Drosophila alcohol dehydrogenase promoter, Moloney murine 
leukemia virus (MMLV) promoter, and the like. 
Invention DNA constructs, containing invention response elements, may 
optionally further comprise a gene which encodes a protein, such as a 
reporter protein. As used herein, the phrase "reporter protein" refers to 
a protein whose expression can be detected in a variety of well-known 
protein expression assays. Particularly preferred reporter proteins for 
use herein include, for example, proteins selected from luciferase, 
chloramphenicol acetyl transferase (CAT), .beta.-galactosidase, or the 
like. 
As used herein, an invention "recombinant expression vector" (or plasmid) 
refers to discrete elements that are used to introduce heterologous DNA 
into cells for either expression or replication thereof. Selection and use 
of such vehicles is well within the skill of the artisan. 
An expression vector includes elements capable of expressing DNAs that are 
operatively linked with regulatory sequences (such as promoter regions) 
that are capable of regulating expression of such DNA fragments. Thus, an 
expression vector refers to a recombinant DNA or RNA construct, such as a 
plasmid, a phage, recombinant virus or other vector that, upon 
introduction into an appropriate host cell, results in expression of the 
cloned DNA. Appropriate expression vectors are those that are replicable 
in eukaryotic cells and/or prokaryotic cells, including those that remain 
episomal or those which integrate into the host cell genome. 
Exemplary eukaryotic plasmid expression vectors include eukaryotic 
cassettes, such as the pSV-2 gpt system (Mulligan et al., 1979, Nature 
277:108-114) and the expression cloning vector described by Genetics 
Institute (1985, Science 228:810-815). These plasmid vectors, when 
modified to contain an invention DNA construct, are able to provide at 
least some expression of the protein of interest in response to a 
retinoid, or the like. 
Other plasmid base vectors which contain regulatory elements that can be 
operatively linked to the invention response elements are cytomegalovirus 
(CMV) promoter-based vectors such as pcDNA1 (Invitrogen, San Diego, 
Calif.), MMTV promoter-based vectors such as pMAMNeo (Clontech, Palo Alto, 
Calif.) and pMSG (Pharmacia, Piscataway, N.J.), and SV40 promoter-based 
vectors such as pSV.beta. (Clontech, Palo Alto, Calif.). 
In accordance with another embodiment of the invention, there are also 
provided host cells transformed with invention expression vector(s). 
Invention expression vectors are introduced into suitable host cells to 
produce transformed cell lines that express a desired protein (such as a 
reporter protein). The resulting cell lines can then be produced in 
quantity for reproducible quantitative analysis of the effects of 
functional ligands on retinoid receptor function via invention 
RIP-associated and RAP-associated response elements. The transfected 
mammalian cells may also be used in the methods of drug screening provided 
herein. 
Suitable host cells in which DNA or RNA may be introduced include both 
eukaryotic and prokaryotic cells. Preferred eukaryotic cells are those 
that can be transiently or stably transfected and also express the DNA and 
RNA. Such cells may be identified empirically or selected from among those 
known to be readily transfected or transduced. Suitable prokaryotic cells 
are well-known in the art, and are those that are useful for preparing 
large quantities (clones) of invention expression vectors. 
Exemplary eukaryotic cells for introducing invention expression vectors 
include, e.g., P19 cells and NT2/D1 cells (which are derived from human 
embryo carcinomas), COS cells, mouse L cells, Chinese hamster ovary (CHO) 
cells, human embryonic kidney cells, African green monkey cells, HEK 293 
(ATCC accession #CRL 1573; U.S. Pat. No. 5,024,939), Ltk.sup.- cells 
(ATCC accession #CCL1.3), COS-7 cells (ATCC under accession #CRL 1651), 
and DG44 cells (dhfr.sup.- CHO cells; see, e.g., Urlaub et al. (1986) 
Cell. Molec. Genet. 12: 555). Presently preferred cells include P19 cells 
and NT2/D1 cells. 
For invention bioassays in which a RIP-associated response element is 
employed to activate transcription of the reporter gene, P19 cells are 
preferred. For invention bioassays in which a RAP-associated response 
element is employed to activate transcription of the reporter gene, both 
P19 cells and NT2/D1 cells are preferred. 
Suitable means for introducing (transforming) vectors into host cells to 
produce transduced recombinant cells (i.e., a cell containing a 
recombinant heterologous nucleic acid) are well-known in the art (see, for 
review, Friedmann, 1989, Science 244:1275-1281; Mulligan, 1993, Science 
260:926-932, each of which are incorporated herein by reference in their 
entirety). Exemplary methods of transduction include, e.g., infection 
employing viral vectors (see, e.g., U.S. Patent 4,405,712 and 4,650,764), 
calcium phosphate transfection (U.S. Pat. Nos. 4,399,216 and 4,634,665), 
dextran sulfate transfection, electroporation, lipofection (see, e.g., 
U.S. Pat. Nos. 4,394,448 and 4,619,794), cytofection, particle bead 
bombardment, and the like. 
In accordance with a still further embodiment of the invention, there is 
provided a bioassay for evaluating whether a compound is a functional 
ligand for retinoid receptor protein(s), or functional engineered or 
modified forms thereof, said bioassay comprising: 
(a) culturing cells which contain: 
retinoid receptor protein(s), or functional engineered or modified forms 
thereof, 
a protein selected from RIP or RAP, 
DNA which encodes an enhanced-response-element operatively linked to a 
reporter gene, wherein said enhanced-response-element is selected from a 
RIP-associated response element or a RAP-associated response element, 
wherein said culturing is conducted in the presence of at least one 
compound whose ability to function as a ligand for said retinoid receptor 
protein, or functional engineered or modified forms thereof, is sought to 
be determined; and 
(b) assaying for evidence of transcription of said reporter gene in said 
cells. 
As used herein, the phrase "functional engineered or modified forms" of 
retinoid receptors refers to non-naturally occurring receptors, such as 
recombinant retinoid receptors. produced by well-known methods. For 
example, the production of functional recombinant steroid/thyroid nuclear 
receptors by interchanging functional ligand-binding and DNA-binding 
domains is well-known to those of skill in the art (e.g., Evans et al., 
1988, Science 240:889-895; U.S. Pat. No. 5,171,671, and the like). Thus, 
any recombinant nuclear receptor containing a functional domain, 
preferably a DNA-binding domain, derived from a retinoid receptor is 
contemplated for use herein. 
As used herein, the phrase "heterologous DNA" refers to a genetically 
engineered DNA not already possessed by the recipient (e.g., exogenous or 
non-endogenous). The heterologous DNA is introduced into the cells as part 
of an invention expression vector by any of a variety of well-known 
methods, such as calcium-phosphate transfection, viral-vector infection, 
and the like. 
As used herein, the phrase "RAR-RXR heterodimer complex" refers to a 
protein complex between any one of the RAR receptors (e.g., .alpha., 
.beta., and .gamma.; see U.S. Pat. No. 5,171,671) and any one of the RXR 
receptors (e.g., .alpha., .beta., and .gamma.). The heterodimer, when 
functioning to transactivate gene expression in cooperation with either 
RIP or RAP, binds to RAREs of the DR-2 class (i.e., a 6 nucleotide direct 
repeat sequence having a spacer of 2 nucleotides between repeats), such as 
those provided in SEQ ID NO:1 (nucleotides 30-43) and SEQ ID NO:2 
(nucleotides 41-54). 
As used herein, the phrase "assaying for evidence of transcription" refers 
to well-known methods for detecting the various products of transcription, 
such as mRNA or the corresponding amino acid sequence. Exemplary methods 
for detecting evidence of transcription include, for example, the 
cis/trans assay described in U.S. Pat. Nos. 5,171,671 and 4,981,784, (each 
of which are incorporated herein by reference), and the like. 
In yet another embodiment of the invention, there is provided a bioassay 
for detecting compounds that are antagonists for retinoid receptor(s) or 
functional modified forms thereof, said bioassay comprising: 
(a) culturing test cells in culture medium containing: 
increasing concentrations of at least one compound whose ability to inhibit 
the transcription activation activity of retinoid receptor agonists is 
sought to be determined, and 
a fixed concentration of at least one agonist for said retinoid receptor(s) 
or functional modified forms thereof, 
wherein said test cells contain: 
retinoid receptor(s) or functional modified forms thereof, 
a protein selected from RIP or RAP, 
DNA which encodes an enhanced-response-element operatively linked to a 
reporter gene, wherein said enhanced-response-element is selected from a 
RIP-associated response element and a RAP-associated response element, and 
thereafter 
(b) determining the amount of transcription of said reporter gene in said 
cells as a function of the concentration of said compound in said culture 
medium, thereby indicating the ability of said compound to inhibit 
activation of transcription by retinoid receptor agonists. 
The invention assay is particularly useful for identifying compounds that 
inhibit ligand-binding to retinoid receptors, or inhibit activated 
retinoid receptor-binding to DNA response elements, or inhibit activated 
receptor-binding to cofactors required for transcription (e.g., RIP or 
RAP). 
The phrase "agonists of retinoid receptors" refers to compounds that are 
able to form a complex with retinoid receptors and bind a respective DNA 
response element, so that the receptor-ligand complex is able to 
participate in transactivation or transrepression of nucleic acid 
transcription. 
The phrase "antagonists of retinoid receptors" refers to compounds that are 
able to inhibit agonist activity, such that functional receptor binding to 
a particular DNA response element is inhibited. Antagonists can act 
mechanistically by either inhibiting ligand-binding to a respective 
receptor, or by inhibiting an activated ligand-receptor complex from 
binding to its respective DNA response element, or by inhibiting an 
activated ligand-receptor complex from binding to a cofactor required for 
the activation of transcription. 
As used herein, the phrase "inhibit activation of transcription" refers to 
blocking the well known process whereby mRNA is transcribed from a 
respective cDNA coding sequence. The amount of mRNA transcription can be 
detected by a variety of methods well-known in the art, such as detecting 
levels of reporter protein expression, detecting directly the level of 
mRNA transcribed, and the like. 
In accordance with yet a further embodiment of the invention, there is 
provided a method for testing the activity of a test compound as an 
agonist for a retinoid receptor, said method comprising: 
(a) culturing host cells containing an invention expression vector in the 
presence of an intracellular retinoid receptor, an intracellular protein 
selected from RIP or RAP, and in the further presence, or in the absence, 
of the test compound; and thereafter 
(b) selecting test compounds that increase the amount of reporter protein 
expression relative to expression levels in the absence of said test 
compound. 
Intracellular retinoid receptors and RIP or RAP proteins can be obtained by 
selecting a host cell that endogenously expresses either of these 
proteins. Retinoid receptors, RIP and RAP proteins can also be introduced 
into cells employing well-known recombinant DNA methods by introducing 
expression plasmids encoding these proteins into the test cells. 
As used herein, the phrase "compounds that increase the amount of reporter 
protein expression relative to expression levels in the absence of said 
test compound" refers to compounds whose presence causes a higher level of 
reporter protein expression driven by an invention RIP-associated or 
RAP-associated expression construct, relative to protein expression in the 
absence of the test compound. 
In accordance with yet a further embodiment of the invention, there is 
provided a method for testing the activity of a test compound as an 
antagonist of ligand for a retinoid receptor, said method comprising: 
(a) culturing host cells containing an invention expression vector in the 
presence of an intracellular retinoid receptor, an intracellular protein 
selected from RIP or RAP, said ligand, and further: 
(i) in the presence of the test compound, or 
(ii) in the absence of the test compound; and thereafter 
(b) selecting test compounds that decrease the amount of reporter protein 
expression relative to expression levels in the absence of said test 
compound. 
The nomenclature used hereafter and the laboratory procedures in 
recombinant DNA technology described below are those well known and 
commonly employed in the art. Standard techniques are used for cloning, 
DNA and RNA isolation, amplification and purification. Enzymatic reactions 
involving DNA ligase, DNA polymerase, restriction endonucleases and the 
like are performed according to the manufacturer's specifications. These 
techniques and various other techniques are generally performed according 
to Sambrook et al., Molecular Cloning--A Laboratory Manual, Cold Spring 
Harbor Laboratory, Cold Spring Harbor, N.Y., (1989). Other general 
references are provided throughout this document. The procedures therein 
are well known in the art and are described herein for the convenience of 
the reader. All the information contained therein is incorporated herein 
by reference. 
The invention will now be described in greater detail by reference to the 
following non-limiting examples. 
METHODS 
DNA fragments containing the TK-Luciferase gene were ligated into the SalI 
site of the vector pBluescript SK+ (Stratagene) using Sal1 linkers to 
produce the plasmid pBS.TK.Luc. The TK promoter of pBS.TK.Luc was replaced 
by various genomic DNA fragments derived from the 5' region of the human 
HOXB1 gene (Acampora et al., 1989, NAR, 17(24):10385-10402). The SpeI-NcoI 
fragment of the 5' region was used as a basal HOXB1 promoter. 
Oligonucleotides used for plasmid construction and gel retardation assay 
were as follows: DR-2A (corresponding to nucleotides 23-51 of SEQ ID 
NO:1); LYRE (corresponding to nucleotides 80-115 of SEQ ID NO:1), and LYRE 
mutant (5'-CAGGCAGACACACTAGTAGGTTACAAATGAGCGTGG-3'; SEQ ID NO:3). 
Cell Cultures and Transfections 
Embryonal carcinoma cell line P19 and NT2/D1 (Andrews et al., 1984, Lab. 
Invest. 50:147-162) were grown in Dulbecco's modified Eagle's medium 
(DMEM) supplemented with 10% fetal bovine serum (Irvine Scientific). 
Twenty-four hours (1 hour for P19 cells) before transfection, cells were 
split in fresh medium described above. Transfections were performed via 
the calcium-phosphate precipitation method as described in Kliewer et al., 
(1992) Nature 355:446-449. 5 .mu.g of the respective reporter luciferase 
plasmid and 7 .mu.g of pCMX-.beta.GAL (as internal control) were 
transfected into appropriate cells. After 12 hours, DNA precipitates were 
washed and cells were cultured with fresh medium containing 1 .mu.M of 
retinoic acid for another 24 hours. Cells were harvested and the 
luciferase assay was carried out according to methods described in De Wet 
et al., (1987) Mol. Cell. Biol. 7:725-737. Transfection efficiency was 
normalized using .beta.-galactosidase activity derived from 
pCMX-.beta.GAL. 
Gel Retardation Assays 
Gel retardation assays were carried out according to Kliewer et al. (1992, 
supra). The plasmids pCMX-hRAR.alpha. and pCMX-hRXR.alpha. (Kliewer et 
al., 1992, supra) were linearlized and capped mRNA was synthesized in 
vitro using T7 RNA polymerase (Stratagene) according to manufacturer's 
instructions. Aliquots of mRNA were incubated with rabbit reticulocyte 
lysate (Promega) for in vitro translation. For gel retardation assay, 5 
.mu.l of in vitro translated proteins were preincubated in binding butter 
(10 mM TrispH8.0!, 40 mM KCl, 0.05% Nonidet P-40, 6% glycerol, 1 mM DTT, 
5 .mu.g/ml polydI.dC!) on ice for 15 minutes. For competition assays, 20 
fold molar excess of competitor oligonucleotides was mixed at this step. 
Oligonucleotides containing DR-5 (RAR.beta.2RARE), HOXB DR-2A (nucleotides 
30-43 of SEQ ID NO:1) and HOXB DR-2B (nucleotides 41-54 of SEQ ID NO:2) 
were used as competitors and probes. Subsequently, .sup.32 P-labelled 
oligonucleotide probes were added to the reaction mixtures and incubated 
on ice for 15 minutes. The same oligonucleotides used for the construction 
of luciferase plasmids were used. Reaction mixtures were resolved by 5% 
polyacrylamide gel electrophoresis in 0.5.times.TBE. The dried gels were 
autoradiographed at -70.degree. C. 
Nuclear extracts from P19 and NT2/D1 cells were prepared according to 
Digman et al., (1983) NAR 11:1475-1489, and cultured for 2 days in the 
presence or absence of 1 .mu.M of retinoic acid, and subsequently stored 
at -80.degree. C. Approximately 3-5 .mu.g of protein was used for each 
reaction. A 20-fold excess of unlabelled oligonucleotides were used for 
competition experiments. 
EXAMPLE 1 
Isolation of Retinoic Acid Responsive Site in the Promoter Region of the 
HOXB1 Gene 
To analyze the regulatory elements of HOXB1, an overlapping set of large 
chromosomal fragments was isolated and ligated to a luciferase reporter 
gene. As shown in FIG. 1a, a 7 kb fragment spanning the HOXB1 promoter was 
transfected into two embryonal carcinoma cell lines; P19 cells (shaded 
bars) and NT2/D1 cells (solid bars). In P19 cells but not in NT2/D1 cells, 
this plasmid was strongly induced by 1 .mu.M of retinoic acid. Reporter 
constructs containing either -2.0 kb or -1.6 kb of upstream sequence 
retained full retinoic acid responsiveness, whereas deletions containing 
only -1.2 kb of upstream sequence or less produced only marginal 
induction. This data indicates the presence of a specific enhancer (i.e., 
RIP-associated response element) in an approximately 400 bp region between 
PstI and EcoRI restriction endonuclease sites (FIG. 1b). 
This 400 bp region was examined in more detail. Retinoic acid activation 
was localized to a 160 nucleotide SacI-HinfI subfragment (FIG. 1b). A 
single copy of this 160 nucleotide region in front of the HOXB1 promoter 
confers a 13-fold induction to retinoic acid. The nucleotide sequence of 
the enhancer region is shown in FIG. 1c (SEQ ID NO:1). Inspection of the 
sequence reveals the presence of a palindrome referred to as the upstream 
response element (also referred to herein as the RIP-binding-site; 
nucleotides 91-99 of SEQ ID NO:1), as well as a direct repeat of the 
sequence AGGTCA, which forms the core binding site for the retinoic acid 
and retinoid X receptor and is observed in the antisense position at 
nucleotides 30-43 of SEQ ID NO:1 (designated DR-2A). While the 
RIP-binding-site contains no homology to known retinoic acid response 
elements, it has been found to substantially enhance the retinoic acid 
response of the HOX reporter. The 160 bp SacI-HinfI fragment confers 
robust retinoic acid induction on HOXB1 promoter, whereas activation is 
severely repressed (to 25%) following mutation of the RIP-binding-site 
(URE), as shown in FIG. 1c. 
Other RIP-binding-site mutants were tested for the ability to bind RIP. The 
results are shown in Table 1. 
TABLE 1 
______________________________________ 
RIP 
Binding 
______________________________________ 
##STR1## 
______________________________________ 
URE-wt corresponds to SEQ ID NO:6. The results indicate that either the 
first or third triplet of the 9 base pair RIP-binding site palindrome 
(nucleotides 9-17 of SEQ ID NO:6) can be modified while still retaining 
RIP binding function. Modification of the middle triplet corresponding to 
nucleotides 94-96 of SEQ ID NO:1 abolishes binding to RIP. In addition, 
modification of the 3 nucleotides immediately downstream of the palindrome 
(nucleotides 100-102 of SEQ ID NO:1) reduces the level of RIP binding. 
Interestingly, it has been found that the RIP-binding-site and the RARE, 
when tested independently as single copies, show relatively little 
transcription transactivation activity. Even two tandem copies of the 
RIP-binding-site show only a low-level basal activity and are essentially 
unresponsive to retinoic acid treatment (FIG. 1d). Similarly, two copies 
of the DR-2A site show only modest retinoic acid responsiveness when 
tested in the absence of the RIP-binding-site. However, when the 
RIP-binding-site and DR-2A are combined, full retinoid inducibility is 
synergistically regained. This demonstrates that high levels of retinoic 
acid activation require the co-stimulatory activity of the 
RIP-binding-site (URE), the RARE and their associated binding proteins. 
Gel retardation analysis was used to identify and isolate the 
RIP-binding-site binding activity in nuclear extracts from P19 cells 
(i.e., RIP protein). In the absence of retinoic acid, only background 
levels of activity are observed for protein binding to the 
RIP-binding-site containing fragment. However, following retinoic acid 
treatment of P19 cells, nearly 100-fold induction of RIP binding activity 
is observed. RIP binding is specific for the RIP-binding-site (URE), as 
revealed by competition assays, and is not competed for by the RIP- 
binding-site mutant sequence shown in FIG.1c (SEQ ID NO:3). In contrast, 
no RIP-binding-site binding is observed in nuclear extracts from NT2/D1 
cells (whether or not such cells have been treated with retinoic acid). 
These results demonstrate that in NT2/D1 cells, the isolated HOX reporter 
is totally inactive either before or after retinoic acid treatment (FIG. 
1a, solid bars). 
Interestingly, the NT2/D1 cells are retinoic acid responsive as indicated 
by the control TK reporter containing the ERE (discussed below; FIG. 2b 
lane 1). Furthermore, the NT2/D1 cells are known to undergo 
differentiation in response to retinoic acid (Andrews et al., 1984, Lab. 
Invest. 50:147-162). These results demonstrate that the deficiency of RIP 
precludes the promoter from responding to retinoic acid. This is 
consistent with the results shown in FIG. 1d indicating that the DR-2A 
site alone is incapable of producing a sustained retinoic acid response, 
and further support the view that retinoic acid responsiveness of the 
HOXB1 promoter is based on a cooperative interaction between the DR-2A, 
the RIP-binding-site (URE), and its associated binding protein (RIP). 
Thus, RIP functions as a type of retinoic acid-dependent cofactor. 
These results identify a novel RIP-associated response element composed of 
a retinoic acid receptor response element (DR-2A) in association with a 
RIP-binding-site, which together confer retinoid responsiveness to the 
isolated HOXB1 promoter. Because of the dependence of the RIP-associated 
retinoic acid response on the presence of RIP protein, retinoic acid 
inducibility is observed in P19 cells (which endogenously express RIP) but 
not in NT2/D1 cells (which do not endogenously express RIP). 
EXAMPLE 2 
Isolation of a RA-Responsive Site Downstream of the HOXB1 Gene 
It has been found that while the transfected HOXB1 promoter fails to 
respond to retinoic acid in NT2/D1 cells, the endogenous HOXB1 gene can 
still be activated in NT2/D1 cells (Simeone et al., 1990, Nature 
346:763-767). The retention of inducibility of the intact gene indicated 
the potential existence of a second pathway for retinoic acid 
responsiveness that is not mediated by the promoter. The 3' portion of the 
HOXB1 gene between the promoter and a repetitive DNA cluster was searched 
for such an alternate pathway. Analysis of the 3' portion indicated that 
virtually the entire HOX B cluster, which is more than 100 kb long, is 
essentially free of repetitive DNA. This unique sequence of DNA contains a 
7 kb region downstream of the 3' end of the HOXB1 gene, after which 
numerous repetitive sequence elements are found. 
Constructs containing a variety of fragments from this 7 kb 3' downstream 
region were produced, and are shown in FIG. 2a. After addition of a SalI 
linker, the TK.Luciferase gene was inserted into the SalI site of 
pBluescript SK+vector (Stratagene) to produce the plasmid pBS-TK.Luc. The 
human HOXB1 3' region was subcloned into pBS-TK.Luc. The genomic DNA 
fragments derived from the 3' region of HOXB1 were ligated into a TK.luc 
reporter in a fashion that preserves the natural downstream genomic 
configuration. The promoter of the herpes simplex virus thymidine kinase 
gene (TK promoter) was used in the constructs to establish the 
independence of this regulation from the HOX promoter. As shown in FIG. 
2a, construct #1 containing the SacI fragment of +1.0 to +4.5 kb retained 
the same basal luciferase activity as parental Tk.luc, but was activated 
4-fold in response to 1 .mu.M of retinoic acid. The adjacent HindIII 
fragment of +4.0 to +5.5 kb (#2) did not confer responsiveness to retinoic 
acid, while construct #3 (approximately +5.5 to +9.5 kb) displayed weak 
responsiveness. Plasmid tk.beta.RE.luc which has a DR-5 type RARE from 
RAR.beta.2 promoter (Sucov et al., 1990, PNAS, USA 87:5392-5396) was 
induced 11-fold. Parental plasmid Tk.luc was not affected by retinoic 
acid. It can thus be concluded that the +1.0 to +4.5 kb SacI fragment 
contains a retinoic acid response element. 
To localize this putative regulatory sequence, the SacI fragment (+1.0 to 
+4.5) was subdivided into a series of nested constructs. Results using 
these constructs indicate that retinoic acid activates constructs #4 and 
#5, which share 0.2 kb BamHI-SmaI region (shown in FIG. 2A as a thick 
line) while marginal or no inducibility was observed for the fragments in 
constructs #6 and #7 (FIG. 2a). These results indicated that attention 
should be focussed on the 0.2 kb BamHI-SmaI fragment for the 
identification and isolation of the downstream response element. 
Deletion analysis of the 0.2 kb BamHI-SmaI fragment was used to localize 
retinoic acid responsiveness to a 5' 110 nucleotide subfragment. The 
sequence of this region includes a direct repeat of 5'-AGGT(A/C)A-3' (in 
antisense orientation) spaced by 2 nucleotides (referred to herein as 
DR-2B; nucleotides 41-54 of SEQ ID NO:2). No other other RARE motif was 
found from sequence analysis of 1.6 kb spanning this region. To further 
analyze retinoic acid responsiveness, an oligonucleotide containing this 
DR-2B sequence (corresponding to nucleotides 35-59 of SEQ ID NO:2) was 
ligated into the 3' end of the Tk.luc transcription unit as single and 
triple copies (TK.luc(DR2B).sub.1 or TK.luc(DR2B).sub.3, respectively). 
These constructs were transfected into P19 and NT2/D1 cells and examined 
for hormonal response. Following addition of 1 .mu.M of retinoic acid, the 
construct harboring three DR-2B motifs (FIG. 2b, lane 4) displayed robust 
inducibility (24-fold) in P19 cells, yet only a weak response in NT2/D1 
cells. A single DR-2B showed only weak transcriptional activation activity 
in either cell (FIG. 2b, lane 3). The TK.beta.RE.luc control plasmid 
induced luciferase reporter protein expression 10 and 50 fold in P19 and 
NT2/D1 cells, respectively (lane 1), while the parental TK.luc control 
vector (lane 2) did not respond to retinoic acid. 
To test whether the DR-2B response element motif confers retinoic 
acid-inducibility to the HOXB1 promoter, plasmids HXB.Luc and 
HXB(DR-2B).sub.3, which substitutes the SpeI-NcoI promoter fragment (shown 
in FIG. 1a) of HOXB1 (not including the URE) for the TK promoter in the 
plasmids TK.luc and TK.luc(DR2B).sub., were constructed. Transfection and 
luciferase assays were conducted as described in Example 1. A control 
TK.beta.RE.Luc was used that has one copy of DR-5 type motif found in the 
promoter of RAR.beta. gene at 5' side of TK promoter. 
While the promoter alone is only marginally active (FIG. 2b, lane 5), the 
HXB(DR-2B).sub.3 plasmid containing the DR-2B response element confers 
efficient (21-fold) induction of transcription in P19 cells (FIG. 2b, lane 
6). Similar inducibility was observed in F9 cells (ATCC #CRL 1720), while 
NT2/D1 cells show a positive but less effective response. However, as 
shown below, NT2/D1 cells contain low levels of RAR and RXR receptors, and 
response in these cells is markedly potentiated by co-transfection of RAR 
and RXR expression vectors. Together these results demonstrate that the 
HOXB1 DR-2B response element can function as an RARE in two embryonal 
carcinoma cell lines. 
EXAMPLE 3 
Demonstration of RAR:RXR Heterodimer Formation on DR-2A and DR-2B Motifs 
The ability of RAR and RXR to form either homo- or heterodimers on the 
HOXB1 DR-2A and DR-2B response elements was examined using gel retardation 
assays. The expression plasmids pCMX-hRAR.alpha. and pCMX-hRXR.alpha. were 
linearized with NheI restriction enzyme. Subsequently, capped mRNA was 
synthesized in vitro using T7 RNA polymerase (Stratagene) according to the 
manufacturer's instructions. Aliquots of mRNA were incubated with rabbit 
reticulocyte lysate (Promega) for in vitro translation. Human RAR.alpha. 
and RXR.alpha. proteins were synthesized by in vitro translation and mixed 
with P.sup.32 labelled response elements from the RAR.beta.2 RARE (a DR-5 
response element) and the HOXB1 DR-2A and DR-2B response elements followed 
by gel electrophoresis. 5 .mu.l of in vitro translated proteins were used 
for gel retardation assays according to the methods described in Kliewer 
et al., (1992) Nature 355:446--449. 
Results from the gel-retardation assay confirm that high affinity binding 
of RAR and RXR heterodimers to the DR-5 motif as reported previously. 
Similar RAR:RXR heterodimer binding was observed on the HOXB1 DR-2A probe 
and HOXB1 DR-2B probe. Although the specific activity of the probes was 
the same, RAR:RXR heterodimer binding to the DR-2 response elements was 
less intense than the heterodimer binding on the DR-5 response element. 
This is in agreement with previous results that suggest that RAR-RXR 
heterodimers may form lower affinity complexes on the DR-2 motif. The 
heterodimeric nature of these receptor complexes was confirmed using a 
supershift assay in which rabbit polyclonal antibodies against RAR.alpha. 
and RXR.alpha. were incubated with a receptor-DNA mixture. 
To explore the intracellular roles of the RAR:RXR receptor heterodimer on 
HOX gene regulation via the DR-2B RARE, the Tk.luc (DR-2B).sub.2 reporter 
plasmid was transfected with RAR.alpha. and/or RXR.alpha. expression 
vectors into NT2/D1 cells. Transfections and luciferase assays were 
conducted as described above, except 0.1 .mu.g of CMX-hRAR.alpha. and/or 
CMX-hRXR.alpha. were co-transfected. As shown in FIG. 3, dramatic 
synergism was observed when both RAR.alpha. and RXR.alpha. were 
co-transfected. When TK.luc(DR-2B).sub.2 was transfected alone, without 
co-transfection of the RAR.alpha. and RXR.alpha. expression vectors, 
expression of the luciferase reporter proteins was induced only 7-fold by 
retinoic acid. When either the RAR.alpha. or RXR.alpha. were each 
individually co-transfected with TK.luc(DR-2B).sub.2, transactivation of 
luciferase reporter protein expression was enhanced 31-fold and 11-fold, 
respectively. When both RAR.alpha. and RXR.alpha. were co-transfected with 
TK.luc(DR-2B).sub.2, a remarkable synergistic effect on transactivation of 
luciferase expression was observed (74-fold induction). From these DNA 
binding and transactivation assays, it can be concluded that the DR-2B 
serves as an effective target for the RAR:RXR heterodimer. 
EXAMPLE 4 
Assay the Effect of the HOXB1 DR-2B Response Element in Combination With a 
RAP-Binding-Site 
As observed in Example 2 (FIGS. 2a and 2b), a restriction fragment 
including sequences flanking the DR-2B response element motif served as a 
better response element than the synthetic DR-2B motif alone. To test 
whether an additional site in constructs #4 and #5 (FIG. 2a) might act 
cooperatively to enhance the function of the DR-2 response element motif, 
three new constructs were generated and designated TK.Luc(DR-2B).sub.2, 
TK.Luc(DR-2B).sub.2 /90, and TK/90 (FIG. 4). To generate these constructs, 
two copies of DR-2B oligonucleotide were introduced into the 3' side of 
TK.Luciferase transcription unit to create the plasmid TK.Luc(DR2B).sub.2. 
A 90 bp fragment (shown as a double line in FIG. 4) containing the 
RAP-binding-site was generated by PCR and inserted downstream from the 
DR-2B sequence (shown as blocks) to produce the plasmid 
TK.Luc(DR-2B).sub.2 /90. A control plasmid TK.Luc/90 was produced by 
placing only the 90 base pair RAP-binding-site fragment at the downstream 
3' side of TK.Luciferase. 
These plasmids were transfected into NT2/D1 cells and monitored for 
retinoic acid responsiveness in the presence of co-transfected RAR and RXR 
expression plasmids. Transfections and luciferase assays were conducted as 
described in Example 1. As shown in FIG. 3, synergistic transactivation 
was observed on the reporter plasmid TK.Luc(DR-2B).sub.2 by RAR and RXR. 
Unexpectedly, more profound transactivation was observed on the reporter 
plasmid. TK.Luc(DR-2B).sub.2 /90. No transactivation in response to 
retinoic acid was observed in NT2/D1 cells transfected with the TK.Luc/90 
plasmid (only 90 bp fragment was inserted). These results demonstrate the 
enhancement of transactivation on the DR-2B response element by the 90 bp 
fragment containing the invention RAP-binding-site. A similar enhancing 
effect was observed in P19 cells. 
These results demonstrate that the HOXB1 RARE activity is augmented by an 
adjacent RAP-binding-site potentiating sequence. Interestingly, the 90 bp 
fragment containing the RAP-binding-site sequence failed to augment 
retinoic acid responsiveness in CV-1 cells with RAR and/or RXR expression 
vectors (FIG. 4). A gel retardation assay was conducted using the 90 bp 
region (i.e., RAP-binding-site) as a probe, and a protein referred to as 
RAP (Retinoid Activating Protein) was identified and isolated in NT2/D1 
cell nuclear extracts (but not in CV-1 cell extracts) which specifically 
binds to this region. This supports the previous transfection data and 
demonstrates that, in a fashion similar to the 5' RARE (DR-2A), the unique 
combination of DR-2B and RAP-binding-site regulatory sequences gives rise 
to a cell-type specific retinoic acid response element (referred to herein 
as RAP-associated response element). 
While the invention has been described in detail with reference to certain 
preferred embodiments thereof, it will be understood that modifications 
and variations are within the spirit and scope of that which is described 
and claimed. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 7 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 167 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GAGCTCCGTTGTTTATAGAGATCACTCCCTGAACTCTTGCCCTCCTGGACTTGCCCTAGC60 
TTCGGCCCCAGGCTCCGGCCAGGCAGACACCCTGACAGGTTACAAATGAGCGTGGGTGTT120 
GGATTGCCCCAAGCTCTTGCCCTCAAGTTGTCCGGAGGAGGAGAGTC167 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 146 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
(D) TOPOLOGY: both 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GGCGGGCGGAGGAGGCCGAGGTAACCTGGGATCCCGGGCCTGACCTTTTTACCTCGAAGC60 
GCCTCTGGGCTTTCCAAACAAGCCGACAGCGCGCCCGCGGGGGCAGCTATTGTCTCCGGG120 
CCGGTCCCACTGGCAAACCTTTGGTC146 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CAGGCAGACACCCTGACAGGTTACAAATGAGCGTGG36 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
AGGGCATCAGTTCA14 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
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AGGTAATTAGGTCA14 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 26 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: both 
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(A) DESCRIPTION: /desc = "Oligonucleotide" 
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AGCTACACCCTGACAGGTTACAAATA26 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
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RGBNNMNNRGBNNM14 
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