(Received for publication, August 24, 1994; and in revised form, December 5, 1994)
From the
Human TR3 orphan receptor is a member of the steroid/thyroid hormone receptor superfamily and is the human homologue of the proteins encoded by the rat NGFI-B and mouse nur77 genes. These genes are induced rapidly by androgens/growth factors and may have functions related to cell proliferation, differentiation, and apoptosis. To investigate the TR3 orphan receptor gene transcriptional regulation, a 2.3-kilobase genomic DNA fragment containing the TR3 orphan receptor gene promoter region was isolated, sequenced, and characterized. Sequence homology search within this promoter region revealed some potential cis-acting elements such as cAMP response element, interleukin-6 response element, estrogen response element, and GC box. Deletion analysis and chloramphenicol acetyltransferase assay also showed a novel cis-acting element of TR3 orphan receptor gene (NCAE-TR3), 200-181 base pairs upstream of the transcriptional start site. Gel retardation assay further demonstrated that some nuclear factors can bind to this NCAE-TR3. Together, our data suggest that NCAE-TR3 could be a new enhancer element associated with the transcription of an early response gene for mitogenesis and apoptosis.
TR3 orphan receptor is a member of the steroid/thyroid hormone
receptor
superfamily(1, 2, 3, 4, 5) .
The cDNA of human TR3 orphan receptor was isolated during the screening
of human testis and prostate cDNA libraries for androgen receptor (AR) ()cDNAs using an oligonucleotide probe homologous to the
highly conserved part of the DNA binding domain of glucocorticoid
receptor(6, 7, 8) . This receptor is also a
human homologue of the protein encoded by mouse nur77(9, 10) , N10 gene(11) , and rat NGFI-B gene(12) , which are
thought to be the early response genes immediately induced by androgens
and several growth factors. It has been reported that NGFI-B mRNA can
be rapidly induced by nerve growth factor in PC12 cells(12) .
Our previous reports also showed that the expression of human TR3
orphan receptor mRNA in human prostate LNCaP cell can be rapidly and
biphasically induced by the addition of androgens or growth
factors(13) .
Using domain switch strategy, a chimera receptor (TR3/AR/TR3), in which the DNA binding domain of TR3 orphan receptor was replaced by that of AR, exhibited constitutive activity in the absence of exogenously added ligands in COS-1 monkey kidney and human prostate cancer PC-3 cells. The activation was dependent only on the amount of TR3/AR/TR3 vector transfected and appeared to be independent of the concentration of serum supplement(5) . In contrast, TR2 orphan receptor, another orphan receptor isolated from our lab(1, 8) , was not constitutively active under the same system. However, a chimera receptor (PR/PR/TR2), in which the N-terminal domain and DNA binding domain of TR2 orphan receptor were replaced by that of progesterone receptor, could be activated through a signal transduction pathway initiated at the cell membrane by the neurotransmitter, dopamine(14) . With the similar domain switch strategy, we were able to use another chimeric receptor (AR/TR3/AR), in which the DNA binding domain of AR was replaced by that of TR3 orphan receptor to identify a potential TR3 response element in the promoter region of mouse mammary tumor virus long terminal repeat (15) . Other studies also revealed that 21-hydroxylase gene, a target gene for the NGFI-B (TR3), may contain a potential NGFI-B (TR3) response element(16) . These hormone response elements may provide a much needed tool for the study of the potential functions of TR3 orphan receptor.
The TR3 orphan receptor gene, 8.5-9.0 kb in length, is located on human chromosome 12, band q13.1, and is split into six introns and seven exons. With some notable genomic structure that is different from most of the other known steroid receptor genes, we speculate that the human TR3 orphan receptor may be an evolutionary ancestor in the steroid receptor superfamily(13) .
Sequence analysis of the 5`-flanking region of N10 and NGFI-B genes suggested these genes may contain some potential cis-acting elements(11, 17) . Lau and colleagues (9) also reported that some potential cis-acting elements may play a role in growth factor stimulation. By cloning and sequencing of the 2.3-kb 5`-flanking region of the human TR3 orphan receptor gene, we now report the identification of several potential cis-acting elements. Deletion analysis and CAT assay further reveal, for the first time, that a novel cis-acting element of TR3 orphan receptor gene (NCAE-TR3) may be essential for the transcription of the TR3 orphan receptor gene in HeLa cells.
Figure 1: Nucleotide sequence of the human TR3 orphan receptor gene promoter region. The location of potential cis-acting elements and comparison with rat NGFI-B and mouse nur77 genes are shown. Nucleotides are numbered relative to the transcriptional start site of genes that were published by Chang et al.(13) . All sequences with potential cis-acting elements are underlined and labeled as SP-1, AP-1, AP-2, CRE, ERE, NF-1, CArG-like, DSE-like, and IL-6-RE. Triangle indicates the existence of first intron. Dot indicates the transcriptional start site.
Figure 2:
The
promoter activity of various deletion mutants of the human TR3 orphan
receptor gene. Deletion constructs are numbered and named according to their length upstream of the transcriptional start
site. CAT activity, after transiently transfected into HeLa cells, was
measured and normalized with -galactosidase activity. Relative CAT
activity of all deletion plasmids and parent vector was calculated with
p-2145TR3CAT as 100%. Results were mean ± S.D. of four separate
experiments.
To further identify cis-acting elements within -314 to -121, several deletion mutants were constructed using exonuclease III (p-199TR3CAT, p-194TR3CAT, p-184TR3CAT, p-151TR3CAT). As shown in Fig. 3, p-314TR3CAT and p-199TR3CAT had considerably higher levels of CAT activities than p-184TR3CAT, p-151TR3CAT, and p-121TR3CAT. Particularly, the CAT activities of p-184TR3CAT and p-151TR3CAT decreased to 23.4 ± 1.3% and 14.8 ± 2.4% of p-314TR3CAT, respectively. The internal deletion mutant, p-314(-200/-171)TR3CAT, in which the region from -200 to -171 was deleted from p-314TR3CAT, reduced CAT activity to 13.7 ± 3.8% of that of p-314TR3CAT and had about the same CAT activity as compared with p-184TR3CAT. These results indicated that the sequence between -200 and -184 is necessary for the transcription of human TR3 orphan receptor gene in the HeLa cells.
Figure 3:
The promoter activities of various
deletion mutants of the human TR3 orphan receptor gene between
-314 and -121. Deletion constructs are numbered and named according to their length upstream of the
transcriptional start site. CAT activities, after being transiently
transfected into HeLa cells, were measured and normalized with
-galactosidase activities. Relative CAT activities of all deletion
plasmids and parent vector were calculated with p-314TR3 as 100%.
Results were mean ± S.D. of four separate
experiments.
Figure 4:
Induction of CAT activities in the SV40
promoter by the NCAE-TR3. Structure orientation of p-314/-121CATp,
p-121/-314CATp, p-200/ -181CATp, and pCAT-promoter and their CAT
activities. The orientations of the NCAE are illustrated by an arrowhead. CAT activity, after transfection into HeLa cells,
was measured and normalized by -galactosidase activity. Relative
CAT activity was calculated with that of HeLa cells transfected with
pCATp as 1.0. Results were mean ± S.D. of four separate
experiments.
Figure 5:
Gel retardation assay of NCAE-TR3 with
HeLa cell nuclear extracts. Gel retardation assay of NCAE-TR3 was
performed by using 11.5 µg of HeLa cell nuclear extract and varying
amounts of competitors. Nuclear extracts were incubated with P-end-labeled NCAE-TR3 (5
10
cpm) and
electrophoresed on a 5% polyacrylamide gel. Lane2 shows probe with 11.5 µg of HeLa nuclear extract. Lanes3 and 4 are the same as lane2 with a 100- and 250-fold molar excess of unlabeled NCAE-TR3,
respectively, used as a specific cold competitor. Lanes5 and 6 are the same as lane2 with a
100- and 250-fold molar excess of unlabeled double-stranded
oligonucleotide (AP-1 oligomer), respectively, used as nonspecific cold
competitors. Lane7 shows NCAE-TR3 probe, and in lane8, AP-1 oligomer probe with 1 footprinting unit
of AP-1 protein is shown.
Sequence comparison among human, rat, and mouse TR3 orphan receptor gene promoter regions indicated there are some well conserved sequences within 220 bp upstream of the transcriptional start site. There are four AP-1 binding site-like sequences, five SP-1 binding site sequences, and one CArG-box sequence in this region. However, we could not locate perfect TATA or CCAAT boxes in this region. The lack of a perfect TATA box in the human TR3 orphan receptor gene promoter region is very similar to what was found in the promoter region of the progesterone (29) and androgen receptor genes(30) . The other similarity among these nuclear receptor gene promoter regions is the existence of highly G + C-rich region. As shown in Table 1, the TR3 orphan receptor gene promoter region contains four potential GC boxes within 130 bp of the transcriptional start site (-17, -40, -76, and -91). These GC boxes, like other GC boxes in many housekeeping genes, may interact with the SP-1-TFIID complex to start transcription(31) . Interestingly and surprisingly, based on our CAT assay, a CAT reporter plasmid, p-2149(-314/-121)TR3CAT, which contains these four GC boxes, induces very little CAT activity (Fig. 2). This result suggests that other promoter regions (e.g. -314 to -121) may play an important role in human TR3 orphan receptor gene transcription.
Another potentially important and conserved sequence within human TR3 orphan receptor gene promoter regions is the Dyad symmetry element (DSE)-like sequences (G(A/C)TG(T/C)CCATAT(T/A)TGG(A/C)CA(T/G)CT), which were also found in other immediate early response genes such as c-fos, krox-20, and krox-24(32, 33, 34) . It has been demonstrated that the DSE sequence in these early response genes could be the binding sites for serum response factor and might be needed for serum induction. However, from our data in Fig. 2, this DSE-like sequence at -349 to -333 may not play a significant role in human TR3 orphan receptor gene transcriptional activity, since we obtained similar CAT activity in HeLa cells transfected with either p-427TR3CAT or p-314TR3CAT. Our transient transfection of these CAT reporter genes into HeLa cells also caused constant CAT induction in normal serum; this DSE-like sequence, therefore, may not have an effect on CAT induction like other early response genes.
Other studies (35, 36) suggested that transient transcriptional
activation of early response genes by growth factors may involve a
region with imperfect dyad symmetry called serum response element
(SRE). The SRE is a 22-bp element containing an inner core sequence,
CC(A/T)GG, known as the CArG box. Both CArG box and SRE are
functional and interchangeable(36) , which are not only
required in the transcriptional activation of early response genes but
also are required for the transcriptional repression of the c-fos gene(37, 38, 39) .
These important sequences, however, are not the same sequences (-200 to -181) we identified as the NCAE-TR3 in this report. Our NCAE-TR3 may, therefore, represent an essential sequence for the transcriptional activity of TR3 orphan receptor, an early response gene.
Recently,
Liu et al.(40) demonstrated that mouse nur77 is necessary for induced apoptosis in T-cell hybridomas and can
also be induced during early mitogenesis. The potential sequences
needed for mitogenesis and apoptosis could be located at -378 to
-332 and -332 to -151 upstream of the transcriptional
start site, which share about 85% homology between mouse and human
sequence. Surprisingly, we found NCAE-TR3 located in this region, and
it was completely conserved among human, rat, and mouse, suggesting
that NCAE-TR3 may be also a candidate of cis-acting element related to
the apoptosis. Using VP-16 and calcium ionophore as apoptosis-induced
reagents, we also found the early induction of TR3 orphan receptor mRNA
in the prostate cancer cells, ()which suggests that TR3
orphan receptor could be an early response gene involved in the
androgen-induced mitogenesis and/or apoptosis in prostate.
Our gel retardation assays prove two DNA-protein complexes at NCAE-TR3, which are different from the AP-1 binding complex. It is unclear whether these two complexes are derived from dimerization of one protein or from two different proteins. As this NCAE-TR3 can also activate transcription in SV40 gene promoter, our identification of NCAE-TR3 may represent an important finding in new enhancers required for transcription of an immediate early response gene. Although we were unable to identify the same NCAE-TR3 in other known early response gene promoters, the existence and significance of enhancers in other early response genes are well documented. For example, Deschamps et al.(41) also identified an enhancer in the c-fos gene promoter region. Within this enhancer (-64 to -404 of the cap site) (41) , SRE and cis-inducible factor response element were further proved to function as regulatory elements that may be needed for the induction in response to the stimulus(42, 43, 44) .
Using NCAE-TR3 sequence and Southwestern techniques, we are in the process of identifying proteins that may bind specifically to this unique enhancer. The identification of new transcriptional factors that bind to this important enhancer may help us to understand more about the gene regulation of TR3 orphan receptor at the transcriptional level and may also help us to know more about the mechanism of how androgens regulate cell growth and death.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17590[GenBank].