Department of Biology (M.A.G., Y.L., R.R., M.B.R.) and Institute for Biomolecular Science (M.B.R.), University of South Florida, Tampa, Florida 33620; Departments of Genetics, Cell Biology, and Biochemistry (K.H.K., M.-N.V.), Washington State University, Pullman, Washington 99164; and Department of Biochemistry and Molecular Biology (M.B.R.), UMDNJ-New Jersey Medical School, Newark, New Jersey 07103
Address all correspondence and requests for reprints to: Dr. Melissa Rogers, Biochemistry & Molecular Biology (MSB E627), UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103-2714. E-mail: rogersmb{at}umdnj.edu.
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ABSTRACT |
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INTRODUCTION |
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RA-regulated gene expression is mediated by nuclear receptors that act as retinoid-dependent transcription factors. These receptors are encoded by six different genes, RA receptor (RAR) , ß, and
and retinoid X receptor (RXR)
, ß, and
, and act as homodimers and heterodimers (4). The sequences bound by these receptors are highly pleiotropic. Although a loose consensus sequence of two directly repeated PuG(G/T)TCA motifs (half-sites) separated by five nucleotides (DR5) occurs in many genes, the spacing, relative position, and number of these repeats are highly variable (4). Indeed, RARs have been shown to bind and activate transcription via half-sites separated by up to 150 nucleotides (5). Variation in the consensus half-site sequence is also common. The diversity of receptors and binding elements makes it difficult to identify RA response elements (RAREs) from sequence alone.
Like many other enhancers, RAREs may be located within distant control regions. Although the natural tendency to study regulatory elements near promoters creates a strong bias toward discovering upstream RAREs, RAREs have been identified in introns [e.g. the major histocompatability complex (MHC) H2Kb and CD38 genes (6, 7)] or kilobases downstream of their genes [e.g. erythropoietin and Hoxa1, b1, a4, b4, and d4, (8, 9, 10, 11)]. The pattern of RAREs regulating the Hoxb1 gene strongly supports the need for an RARE screen unbiased by location. The Hoxb1 gene is controlled by three, highly conserved RAREs that independently mediate part of the endogenous expression pattern and response to retinoid exposure in vivo (Ref.11 and references therein). One RARE is upstream and two are 3 kb and 6.5 kb downstream of the transcribed region. The presence and requirement for all three elements were only uncovered after nearly a decade of transgenic mouse labor by several laboratories. Similarly, in vivo Hoxd4 expression requires both upstream and downstream RAREs.
Retinoid-responsive genes have been discovered by serendipity and methods such as differential hybridization (12); subtractive hybridization (13); enhancer/gene traps (14, 15); and PCR-assisted binding site selection from chromatin (16). Because retinoid receptor isoforms and accessory transcription factors differ in various cell types, a gene that is not RA responsive in the chosen cells will be missed. For example, because embryonal carcinoma or embryonic stem cells were used in the published examples, genes induced in adult cells may be underrepresented. Approaches such as differential hybridization rely on differences in mRNA abundance between untreated and RA-treated cells. Because retinoids can induce other transcription factors or signaling molecules, the induction of a particular gene in an RA-treated cell may be only indirectly due to RA. Also, due to an experimental bias toward strongly induced genes, weakly induced genes may be missed. Another difficulty with screening for RA-regulated genes in mammalian cells is that many mammalian cells stop growing or undergo apoptosis in the presence of RA (3). Thus, cell lines must be replicated to a master plate and a test plate containing RA. Although trivial for microbes, replica plating mammalian tissue culture cells is laborious.
Directly regulated genes should be associated with RAREs. Thus, we devised a method to trap RAREs based on their functionality in yeast and have begun to analyze the expression of nearby genes. This approach avoids many of the biases described above.
Yeast lack retinoid receptors and exhibit little retinoid-metabolizing activity. However, if transformed with receptor expression vectors, yeast can synthesize functional receptors. These receptors drive the RA-dependent expression of yeast reporter genes under the control of mammalian RAREs (17, 18, 19). We previously showed that an RARE near the key developmental gene bone morphogenetic protein (Bmp)2 can function in yeast expressing mammalian retinoid receptors (20). A major advantage of using yeast is the absence of retinoid-induced gene-regulatory proteins that might activate mammalian genes. Thus, only elements directly activated by retinoid-bound receptors can function in yeast. We used this approach to screen the mouse genome for RAREs responsive to RARß and RXR homo- or heterodimers. We demonstrated that RAREs trapped in yeast can drive mammalian reporter genes in response to RA and can bind RARß/RXR
directly in vitro. Furthermore, RA induces the transcription of genes associated with these RAREs in mammalian cells.
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RESULTS |
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Selection of RA-Induced Clones
The strategy to screen the library for RARE-containing plasmids is illustrated in Fig. 1. The host strain, BJ5409, is auxotrophic for uracil, tryptophan, and histidine. This allows transformation with two different receptor expression vectors and a reporter vector. The ß-gal reporter plasmid, p
ss, confers the ability to grow on plates lacking uracil (21). The retinoid receptor expression vectors p2HG-RARß and pG1-RXR
confer the ability to grow on plates lacking histidine and tryptophan, respectively (18).
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The activity of the trapped RAREs was quantified using liquid ß-gal assays. Because the yeast clones may have had more than one reporter plasmid, rescued plasmids were transformed back into yeast to confirm that one plasmid was responsible for the observed induction. Thirty-two reporter plasmids exhibited at least 2-fold inducibility upon preliminary analysis.
Receptor Heterodimers Activate the Trapped RAREs Most Efficiently
The isolated RAREs drove RA-responsive ß-gal gene transcription in yeast transformed with both RARß and RXR. Because the in vivo receptor complex can be either heterodimers or homodimers of RARß and RXR
, we examined which receptors optimally activate the isolated RAREs. A subset of trapped clones, selected on the basis of their strength or close proximity to transcribed sequences (A24 and D6), was transformed into yeast expressing no receptor, RARß alone, RXR
alone, or both receptors. These cultures were treated with the RAR/RXR panagonist, 9-cis RA, to activate both the homo- and heterodimeric forms of the receptors. Two control reporter genes also were tested. The YRpßRE plasmid contains a natural RARE from the RARß gene (17). The YRpCRBPII plasmid contains a natural RARE from the cellular retinol binding protein (CRBP) II gene that is induced exclusively by 9-cis RA-activated RXRs in yeast (17). The ß-gal activity of untreated cultures or cultures treated with 1 µM 9-cis RA was compared (Table 1
). The most robust induction occurred in yeast expressing both RARß and RXR
, suggesting that these elements are preferentially activated by RA-bound heterodimers in yeast. However, RARß alone (A18, B12, C13) or RXR
alone (B9, B10, B12) also activated a subset of clones to levels similar to those achieved by these homodimers in ßRE or CRBPII control plasmid-containing yeast, respectively.
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RA Induction of the Nonclassical MHC Class I Gene, T20d
Many genes are induced by retinoids (23). Isolating a known gene would validate our yeast assay. The MHC class I genes encode highly polymorphic membrane antigens involved in the cellular immune response. RA has long been known to induce classical MHC class Ia genes (24) and RAREs have been identified in or near these genes (6, 25). Our genomic clone D7 overlapped an MHC class Ib gene.
Part of clone D7 is 98% identical to the available sequence for exon 5 and flanking intronic sequences of the nonclassical MHC gene T20d formerly known as T15c [Genbank accession no. X16220 (26, 27)]. Six mismatches out of 328 nucleotides may be accounted for by strain differences because our clone was isolated from a strain 129 library and the GenBank sequence was from BALB/c mice.
Although RA has been shown to induce several classical MHC genes in F9 embryonal carcinoma cells, the RA inducibility of the nonclassical MHC gene T20d has not been tested. Because MHC genes readily cross-hybridize, we used the specific RT-PCR assay devised by Eghtesady et al. (28) to measure the abundance of the T20d transcript in RNA isolated from F9 cells treated for 24 h with 250 µM dibutyryl-cAMP, and 500 µM theophylline (CT) alone or with 1.0 µM all-trans-RA (RACT). As shown in Fig. 2A, a 334-bp fragment was specifically amplified from RNA from RACT-treated cells, but not from cells treated with CT. Cells treated for several days with RACT differentiate into parietal endoderm, whereas CT alone has no effect. Differentiation is a stepwise process during which a few directly regulated genes are induced early, within 24 h, before morphological differentiation. In contrast, many genes are not induced until later when cells express the terminally differentiated phenotype. The induction of T20d within 24 h is consistent with direct regulation by RA.
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A Gene Involved in Spermatogenesis (D6)
A portion of clone D6 matched the sperizin gene (29). Exclusively expressed in round spermatids, sperizin encodes a RING zinc-finger protein. Proteins with these motifs often exhibit ubiquitin E3 ligase activity (30, 31), suggesting sperizin may be involved in the dramatic cellular remodeling occurring during spermiogenesis. The D6 clone includes 390 bp of this intronless gene and 1007 bp of downstream sequence (Fig. 3B). Vitamin A (retinol) is vital in maintaining mammalian spermatogenesis (32). Dietary treatment of vitamin A-deficient animals with retinoids can reinitiate spermatogenesis. Several genes regulated by RA in testes have been identified by various means including gene traps (14, 33, 34, 35, 36). To test the hypothesis that vitamin A can induce sperizin transcription in vivo, we compared the levels of sperizin RNA in the testes of vitamin A-sufficient and vitamin A-deficient rats (Fig. 3A
). Because only A-type spermatogonia and preleptotene spermatocytes remain in vitamin A-deficient rats, it was not surprising that the round spermatid-specific sperizin transcript was undetectable (36). We also measured sperizin RNA in testes in which spermatogenesis was reinitiated by retinol injection. The sperizin transcript was induced within 4 h of injection and levels continued to rise 8 h post injection (Fig. 3A
, lanes 24).
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A Highly Conserved Trapped Gene (A24)
Analysis of the genomic clone A24 revealed a sequence resembling a DR5 RARE: AGGTCAgctggGGGGCA. To test whether or not this element can mediate RA-regulated expression in mammalian cells, several subclones of A24 were inserted into mammalian reporter vectors. The luciferase activity of these sequences in the presence or absence of retinoid was tested. A 145-bp subclone containing the intact DR5 element induced luciferase activity fold in response to all-trans-RA in the context of two different promoters, TK (3.17 ± 0.23, n = 4 in pLucTK2, Fig. 4A) and SV40 (3.87 ± 0.02, n = 2, in pGL3pro, not shown). 9-cis-RA also induced the TK subclone to similar levels (2.87 ± 0.33, n = 2, not shown). In contrast, the fold induction mediated by clones containing only one half-site did not differ significantly from that of the empty vector, pLucTK2 (Fig. 4A
).
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We used the A24 sequence to search GenBank for nearby genes and identified several expressed sequence tags (ESTs) derived from various tissues. IMAGE EST clone 734988 was used to screen a cDNA library prepared from RA-treated P19 cells (37). A 1909-bp clone (S8) overlapping the genomic clone A24 was isolated and sequenced. The DR5 sequence is positioned 85 bp downstream of the 3'-end of this cDNA. Similarly, other RAREs (e.g. Refs.8, 9, 10, 11), are located downstream of genes.
This cDNA clone was used to probe Northern blots of total RNA isolated from tissue culture cells, embryos, and adult organs. As shown in Fig. 4D, RA more than doubled the abundance of a transcript in D3 embryonic stem cells. Thus, the A24 RARE is immediately downstream of a transcript regulated by RA. The transcript was also present in RNA isolated from all tested adult organs and throughout midgestation (Fig. 4E
). After normalization to a constitutively expressed ribosomal protein gene, 36B4 (38), and actin (Fig. 4E
) and comparison to the rRNA intensities (not shown), the transcript abundance was found to be elevated in liver, brain, and cortex.
The A24 cDNA clone contains an open reading frame that may encode a 276-amino-acid peptide. The entire peptide is highly conserved across vertebrate species (Fig. 5). Substantially conserved regions can also be identified in nonvertebrate genomes, specifically the sea squirt, Halocynthia roretzi, the fly, Drosophila melanogaster, and the nematode, Caenorhabditus elegans. Indeed, whereas the overall amino acid identity between the Drosophila and mouse sequences is 40%, several motifs are entirely conserved as shown in Fig. 5
. Interestingly, the A24 open reading frame is 88% identical to that encoded by a human gene located on chromosome 6. A partial sequence of this gene was previously isolated in a screen for brain cDNA clones containing CAG trinucleotide repeats [TNRC5, GenBank accession no. U80744, (39)], indicating expression in human brain as well as mouse.
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To further delineate the contribution of each half-site to RARß-RXR heterodimer binding, we prepared oligonucleotides containing mutations in each of the half-sites (Fig. 7A
) and performed EMSA (Fig. 7B
). Mutating the first or third half-site did not affect the ability of the RARß-RXR
heterodimer to bind to the oligonucleotide. In contrast, mutating the second half-site abrogated binding by the heterodimer. This indicates that an intact DR1 (mutant 1) or DR5 (mutant 3), but not a DR12, is sufficient for heterodimer binding. RARß or RXR
homodimers did not bind to any of the oligonucleotides.
We next analyzed binding specificity of the half-sites by competition analysis, using wild-type and mutant oligonucleotides (Fig. 7, C and D). As expected, adding cold wild-type oligonucleotide completely inhibited binding of the heterodimer to the wild-type probe. Similarly, mutant 3, which contains an intact DR5, completely inhibited binding to the wild-type probe. Mutant 2 failed to compete with the wild-type probe, consistent with its inability to bind receptor (Fig. 7C
). Mutant 1, containing an intact DR1, inhibited binding only at the highest concentration, indicating that the DR5 site has a higher affinity for the receptors.
EMSAs determined the contribution of each half-site in our composite RARE to RARß-RXR heterodimer binding. To determine the role of each motif in a functional assay, we generated the same mutations in the context of the 155-bp luciferase reporter construct. These constructs were transfected into F9 cells that were then treated with all-trans-RA (Fig. 6
, last four bars). Mutating either the first (MT1) or second (MT2) half-sites reduced RA inducibility to levels approaching background, indicating the importance of these motifs. In contrast, mutation of the third half-site (MT3) had little effect. Together, the EMSA and luciferase data indicate that, although RARß-RXR
heterodimers can bind the DR1, only the DR5 drives RA inducibility in cells. These data are in keeping with previous observations indicating that DR5 elements mediate stronger transcriptional responses than DR1 elements (Ref.5 and references therein).
To identify a gene that the C13 complex RARE might regulate, we queried GenBank with the C13 genomic clone sequence using BlastN. The terminal 740 bp of the mouse genomic clone UUGC1M0544E01 in the GSS database were 98.4% identical to the end of C13. This match ended 166 bp from the DR5/1 RARE. We fully sequenced this clone (6.88 kb) and queried GenBank; however, no additional matches were observed. BlastP analysis indicated that open reading frames within this sequence differed from those of any known proteins. This suggests that the potent C13 RARE regulates either a completely novel gene or one without sequence deposited in GenBank.
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DISCUSSION |
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The number of RAREs activated by specific receptor combinations is unknown. By transforming yeast expressing various receptor combinations with a representative sample of the mouse genomic DNA library, one can estimate the total number of RAREs in the genome by extrapolation. The number of trapped reporter genes in each receptor background provides an estimate of the number of RAREs activated by that receptor combination. For example, our isolation of 32 confirmed RAREs from 9% of the murine genome suggests that RARß and RXR could activate approximately 350 RAREs in the entire genome. This estimate is plausible, considering that RA directly regulates only a subset of the many hundreds of RA-regulated genes. We now have the tools to estimate the number of RAREs activated by specific receptor combinations.
Because the receptor-expressing yeast lack other mammalian transcription factors, this estimate specifically reflects the influence of receptor binding. Thus, this assay is stringent because only those RAREs that can be independently activated by receptors will be isolated. Some RAREs, however, require binding by nonreceptor transcription factors. For example, the tissue plasminogen activator RARE also requires binding by Sp1 (42). We may not isolate these complex RAREs. Alternatively, they may be trapped, but induce weakly in yeast or cells lacking the contributing transcription factor. The RARE associated with sperizin (D6) may fall into this class. In the unique cells normally expressing sperizin, specifically the male germ cells, other transcription factors may stabilize the interaction between retinoid receptors and this RARE. Similarly, the absence of specific transcriptional repressors in yeast would permit the detection of RAREs, the activity of which is masked by repressors in some mammalian cells.
In mammalian cells, coactivators and corepressors modulate receptor activity by remodeling chromatin (43, 44). Likewise, gene expression in yeast is regulated via highly conserved chromatin remodeling complexes. The general transcription apparatus and many coactivators with histone acetylase activity are highly conserved and are required for the activity of all nuclear receptors. Other coactivators are highly receptor and ligand specific. These factors contribute to the highly cell-specific effects of hormones. Homologs to the steroid receptor coactivator (SRC)/p160 family of coactivators have not been identified in yeast and insects (Refs.45, 46, 47 and our recent Medline searches). However, transfected murine GRIP1 (glucocorticoid receptor-interacting protein 1)/SRC2/transcriptional intermediary factor 2 has been shown to strongly stimulate the activity of transfected RARs on the ßRE element in yeast (46). Interestingly, RARß, but not RAR or RAR
, homodimers did exhibit some GRIP-independent activity and binding in yeast (47). Our initial trapping protocol using RARß would have selectively trapped RAREs that lack a strong SRC/p160 coactivator requirement. It will be interesting to test whether or not GRIP1 can stimulate the activity of these RAREs in yeast.
Unliganded nuclear hormone receptors can repress basal transcription by recruiting corepressors and histone deacetylases (48, 49). Ligand-mediated relief of this repression contributes to the ability of hormones to greatly induce transcription. Hormone-mediated repression has been observed on response elements in various promoter contexts in mammalian cells, but not in yeast. This may be due to the absence of silencing mediator of retinoid and thyroid hormone receptor or nuclear receptor corepressor homologs capable of interacting with mammalian receptors (Ref.47 and our recent Medline searches). We also found that unliganded retinoid receptors failed to repress ß-gal transcription driven by 13 different RAREs in yeast (ßRE, CRBPII, A18, A20, A24, B9, B10, B12, C9, C13, D4, D7, E2; data not shown). This supports the hypothesis that the yeast repressor apparatus cannot interact with mammalian receptors.
We have scanned the 32 trapped sequences for canonical RAREs consisting of two directly repeated PuG(G/T)TCA motifs. Despite being able to activate reporter gene activity only in yeast expressing retinoid receptors, only C13 contained perfect direct repeats in a DR5 pattern (Fig. 7). We demonstrated that several of the sequences lacking canonical RAREs also induced mammalian reporter genes (D7, D6, and A24, Figs. 24
; C9 and E2, not shown). The relative flexibility of the half-site is supported by in vitro measurements of receptor binding. Hauksdóttir and Privalsky (50) compared the in vitro binding of RAR or RXR homodimers or heterodimers to oligonucleotides mutated systematically at each position of each half-site relative to AGGTCA. The A24, D6, and D7 sequences each contain DR5-like elements that vary from the consensus at a single nucleotide. These variations were shown to permit in vitro binding (Fig. 4
and Ref.50). The flexible nature of the RARE highlights the need for a genetic analysis of RAREs based on functionality.
Three of the trapped RAREs are located near RA-regulated genes (Figs. 24). D6 and D7 are proximal to known genes, the round spermatid-specific gene sperizin and the nonclassical MHC gene T20d, respectively. The A24 RARE is 85 bp downstream of a novel and highly conserved gene regulated by RA in embryonic stem cells. In all mammalian sequences, the transcribed region contains an open reading frame that encodes a string of leucines and other nonpolar amino acids typical of a signal peptide (Fig. 5
). In addition, the A24 open reading frame encodes six conserved cysteine residues. An even number of cysteines suggests that the protein can form intramolecular disulfide bonds like many secreted proteins. A consensus N-linked glycosylation site at amino acid 153 is another feature common in secreted proteins. Thus, the trapped A24 RARE may influence the expression of a highly conserved secreted protein in mammalian cells.
In conclusion, we developed a functional assay to efficiently isolate RAREs from whole genomes. We identified 32 sequences that functioned as RAREs in RA-treated yeast expressing RARß and RXR. Analysis of four selected sequences showed that these RAREs also drove RA-dependent transcription in mammalian cells. This approach relies only on the ability of an enhancer to mediate RA-induced reporter gene expression in yeast. Genes proximal to these RAREs are highly likely to be regulated directly by retinoids. Indeed, three of these RAREs (A24, D6, and D7) are located near transcripts induced by RA. Because directly regulated genes may launch signaling processes controlling key cellular decisions, this approach will provide insight into the action of retinoids. Specifically, it should be possible to identify genes whose normal expression requires natural retinoid signals as well as genes induced by teratogenic levels of retinoids. Finally, this method may also be used to identify the target genes for any cellular signal whose receptor can function in yeast.
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MATERIALS AND METHODS |
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Plasmids
The yeast reporter vector, pss, contains the ß-gal gene driven by the yeast cytochrome c1 (cyc1) minimal promoter (21). The expression vectors p2HG-RARß and pG1-RXR
encode RARß and RXR
, respectively (18). pLucTK2 was generated by inserting a SmaI-KpnI polylinker fragment from pBluescript II SK(+) (Stratagene, La Jolla, CA) into the SmaI and KpnI site of pLucTK (51).
D7.
The D7 trapped genomic clone (1301-bp Sau3AI fragment) was partially digested with Sau3AI. A 759-bp fragment was filled in with deoxy (d)ATP and dGTP and inserted into the XhoI site of pss, which was partially filled in with deoxythymidine triphosphate and dCTP. This same 759-bp Sau3AI fragment was also inserted into the BamHI site of pBluescriptSK. This plasmid was then digested with XbaI, producing a 417-bp fragment and a 306-bp fragment. These fragments were filled in with Klenow enzyme and deoxynucleotide triphosphates (dNTPs) and inserted into the SmaI site of pLucTK2.
D6.
The D6 trapped genomic clone (1397-bp Sau3AI fragment) was digested with XhoI, releasing an 1179-bp fragment (positions 209-1388) containing two putative RAREs. This fragment was ligated into XhoI-digested pLucTK2.
A24.
A 145-bp SspI-StyI fragment containing the A24 RARE was isolated from the A24 trapped genomic clone. After filling in the StyI end with Klenow enzyme, the fragment was inserted into the SmaI site of pLucTK2 in both orientations. Two additional constructs were prepared by digesting plasmids containing the insert in both orientations with PvuII and XhoI (vector polylinker site). The XhoI site was then filled in and the plasmids were religated, yielding subclones containing inserts of 115 or 30 bp.
C13.
To facilitate subcloning of the C13 genomic clone, positions 15940 were PCR amplified and inserted into the pCRII vector (Invitrogen, San Diego, CA). This 925-bp insert was released by digestion with EcoRI (vector polylinker sites), filled in with dNTPs and Klenow enzyme, and inserted into SmaI-digested pGL3-Promoter (Promega Corp., Madison, WI) in both orientations. The 925-bp fragment in pCRII was also digested with EcoRV (vector polylinker site) and XcmI and treated with mung bean nuclease. The resulting 770-bp fragment was inserted into SmaI-digested pGL3-Promoter in the forward orientation relative to the luciferase gene (see Fig. 6). The 925-bp fragment in pCRII was also digested with EcoRI (vector polylinker site) and XcmI and treated with mung bean nuclease. The resulting 155-bp fragment was inserted into SmaI-digested pGL3-Promoter in the reverse orientation relative to the luciferase gene. This construct was the starting material for the subsequent half-site mutations described below. To reverse the orientation of the 155-bp fragment, the insert was released by digestion with XhoI and NheI (pGL3-Promoter polylinker sites) and ligated into pBluescript digested with XhoI and XbaI. The resulting pBS construct was digested with NotI (pBS polylinker site), filled in with dNTPs and Klenow enzyme, and then digested with KpnI (also in the pBS polylinker). This fragment was then inserted into KpnI-SmaI-digested pGL3-Promoter, producing the plasmid containing the 155-bp fragment in the forward orientation. The C13 half-site mutants were generated by overlap extension PCR as described in Ref.22 . Sequences of the mutated oligonucleotides are shown in Fig. 7A
. To prepare each half-site mutant, two separate PCRs were performed, each using one mutant oligonucleotide and one vector polylinker oligonucleotide to amplify overlapping portions of the 155-bp fragment. These two overlapping products were then mixed and amplified using the two vector polylinker oligonucleotides. The full-length product was then digested with XhoI and SstI (vector polylinker sites) and inserted into XhoI-SstI-digested pGL3-Promoter. Each mutant was verified by sequencing to ensure that only the desired mutations were introduced.
Construction of a Murine Genomic DNA Library in a Yeast Reporter Plasmid
D3 embryonic stem cell DNA was purified as described in Ref.22 . After partial digestion with Sau3AI, fragments of approximately 2 kb were partially filled in with dATP and dGTP and inserted into the XhoI site of pss, which was partially filled in with deoxythymidine triphosphate and dCTP. The ligated DNA was used to transform DH5
competent cells (22). The transformed colonies were harvested and amplified in liquid culture for 2 h before plasmid isolation.
Yeast Transformations and ß-gal Assays
The yeast strain BJ5409 (leu2-, his3
200, ura352, trp1) was transformed as described in Ref.52 . Double or triple transformants were selected by plating on medium lacking the appropriate nutrients. Plates were incubated at 30 C for 24 d to recover transformants. Plate and liquid ß-gal assays are described in Ref.22 . Yeast were RA treated for 24 h as described (20).
Isolation of Reporter Plasmids from Yeast
Yeast reporter plasmids were rescued by preparing DNA as described in Ref.53 and transforming Escherichia coli strain DH5.
RT-PCR
RT-PCR was performed essentially as described in (28). Briefly, oligo-deoxythymidine-primed (Fig. 2A) or random hexamer-primed cDNA (not shown) was synthesized from total cellular RNA. To avoid amplifying genomic DNA that might contaminate the RNA, all reverse transcription (RT) reactions were pretreated with XbaI, which cleaves within the amplified region. T20d-specific (28) or actin-specific primers were used for PCR amplification. The PCR products were Southern blotted and probed with a 252-bp XbaI-BstNI fragment specific for T20d, or with an actin-specific probe (Ambion, Inc., Austin, TX).
F9 Cell Transfections and Luciferase Reporter Assays
F9 cells were grown and transfected as described in Ref.20 with the following modifications. After removing the CaPO4 precipitate, cells were incubated with fresh media containing ethanol vehicle alone or 1 µM all-trans-RA. After 24 h, the cells were lysed and the luciferase activity was measured using the Luciferase Assay System (Promega Corp.). Transfection efficiency was normalized by cotransfecting the ß-gal expression vector (pßAclacZ), which contains the ß-gal-coding region driven by the constitutive ß-actin promoter (54).
cDNA Cloning
A cDNA library (4 x 105 plaques) made from RA-treated P19 embryonal carcinoma cells (37) was screened using a gel-purified insert from IMAGE EST clone 734988 (GenBank accession no. AA260027), corresponding to positions 84656 of the A24 trapped genomic clone. After plaque purification, plasmids were excised by in vivo excision and purified. Six independent clones were isolated and partially mapped and sequenced. These clones differed only by the length of their 5'- and 3'-ends. The longest clone, S8 (1909 bp), was fully sequenced.
Northern Analysis
Northern analysis procedures are described in Ref.55 .
EMSAs
Expression vectors encoding human RARß and murine RXR (obtained from R. Evans) were linearized with BamHI and in vitro transcribed and translated using the TnT T7-coupled wheat germ extract system (Promega Corp.) according to the manufacturers instructions. Binding reactions contained 20 mM HEPES (pH 7.9), 40 mM KCl, 6 mM MgCl2, 1 mM dithiothreitol, 2.55 µg BSA, 1 µg poly-(deoxyinosine-deoxycytidine), 0.5 µg sonicated salmon sperm DNA, 2% Ficoll, 20,000 cpm (
0.2 ng) 32P-labeled oligonucleotide probe, and 4 µl receptor protein or unprogrammed extract. All components except probe were allowed to prebind on ice for 10 min, and then probe was added and the reactions proceeded at room temperature for 20 min. Reactions were loaded on 5% nondenaturing polyacrylamide gels, electrophoresed, dried, and exposed to x-ray film. When used, competitor oligonucleotides were added as indicated in the figure legends. Probes were prepared by end labeling oligonucleotides (Integrated DNA Technologies, Coralville, IA) with T4 polynucleotide kinase and 32P-
ATP. Complementary oligonucleotides were annealed and then purified by passage over Sephadex G50 spin columns. Quantification was performed using ImageQuant Software (Molecular Dynamics, Inc., Sunnyvale, CA).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 M.A.G. and Y.L. contributed equally to this work.
Abbreviations: ß-gal, ß-Galactosidase; BMP, bone morphogenetic protein; CRBP, cellular retinol binding protein; CT, cAMP and theophylline; d, deoxy; dNTP, deoxynucleotide triphosphate; EST, expressed sequence tag; GRIP, glucocorticoid receptor interacting protein; MHC, major histocompatability complex; RA, retinoic acid; RACT, all-trans-RA + CT; RAR, RA receptor; RXR, retinoid X receptor; RARE, RA-responsive element; RT, reverse transcription; SRC, steroid receptor coactivator; SV40, Simian virus 40; TK, thymidine kinase.
Received for publication May 23, 2002. Accepted for publication September 17, 2002.
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REFERENCES |
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