ments indicated that two of these four clones contain related. cDNA inserts ..... DNA binding by the v-erbA oncogene protein of avian erythro- blastosis virus.
MOLECULAR AND CELLULAR BIOLOGY, JUlY 1991, p. 3814-3820 0270-7306/91/073814-07$02.00/0 Copyright C 1991, American Society for Microbiology
Vol. 11, No. 7
NOTES A Retinoic Acid-Responsive Element in the Apolipoprotein Al Gene Distinguishes between Two Different Retinoic Acid Response Pathways JEFFREY N. ROTTMAN,1t RUSSELL L. WIDOM,1 BERNARDO NADAL-GINARD, AND SOTIRIOS K. KARATHANASIS1*
2
VIJAK
MAHDAVI11
Laboratory of Molecular and Cellular Cardiology, Department of Cardiology, Children's Hospital, and Department of Pediatrics, Harvard Medical School,' and Howard Hughes Medical Institute,2 Boston, Massachusetts 02115 Received 26 December 1990/Accepted 10 April 1991
The gene coding for apolipoprotein Al, a plasma protein involved in the transport of cholesterol and other lipids in the plasma, is expressed predominantly in liver and intestine. Previous work in our laboratory has shown that hepatocyte-specific expression is determined by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located in the region -222 to -110 nucleotides upstream of the apolipoprotein AI gene transcription start site (+ 1). In this study, it was found that site A is a highly selective retinoic acid-responsive element (RARE) that responds preferentially to the recently identified retinoic acid receptor RXRa over the previously characterized retinoic acid receptors RARa and RARI. Control experiments indicated that a RARE in the regulatory region of the laminin Bl gene responds preferentially to RARaL and RAR, over RXRa, while a previously described palindromic thyroid hormone-responsive element responds similarly to ail three of these receptors. Gel retardation experiments showed that the activity of these RAREs is concordant with receptor binding. These results indicate that different RAREs may play a fundamental role in defining distinctive retinoic acid cellular response pathways and suggest that retinoic acid response pathways mediated by RXRa play an important role in cholesterol and retinoid transport and metabolism.
Apolipoprotein Al (apoAI) is a major protein constituent of plasma high-density lipoproteins and intestinally derived lipoproteins known as chylomicrons. High-density lipoproteins are involved in a large number of diverse intravascular metabolic processes including the process of reverse cholesterol transport, in which cholesterol from extrahepatic tissues is transported to the liver for conversion to bile acids and eventual excretion (for a recent review, see reference 16). This process is thought to play an important role in protection against premature coronary heart disease (16). Chylomicrons, on the other hand, transport dietary lipids including retinol in the form of retinyl esters to the liver for storage and/or secretion as lipoprotein complexes (reviewed in references 2 and 11). Although several recent studies suggest that dietary, hormonal, and other environmental factors regulate apoAl gene expression, the molecular basis of the mechanisms involved remains poorly understood (16). Based on transient transfection assays, it was recently concluded that in cultured human hepatoma (HepG2) cells, nearly all the transcriptional activity of the apoAl gene is determined by a powerful liver-specific enhancer located in the region -222 to -110 nucleotides upstream of the apoAI gene transcription start site (+1) (36). It was also observed that maximal transcriptional activity of this enhancer was dependent on synergistic interactions between transcription factors bound to three
distinct sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within this enhancer (36). Cloning and characterization of one of the proteins that binds to site A revealed that this protein, which was named apolipoprotein regulatory protein 1 (Arp-1), is a novel member of the steroid-thyroid hormone receptor superfamily of liganddependent transcription factors (20). This observation raised the possibility that signal transduction mechanisms similar to those used for steroid hormone action play an important role in regulation of apoAl gene expression. However, cotransfection assays indicated that overexpression of Arp-1 represses expression of the apoAI gene in HepG2 cells (20). Because Arp-1 was cloned by screening a cDNA library from placenta (20), where the apoAl gene is not expressed, the inhibitory effect of Arp-1 on apoAl gene transcription raised the possibility that Arp-1 is displacing other transcription factors present in liver cells that, similar to Arp-1, bind to site A but, in contrast to Arp-1, activate the apoAl gene enhancer. To search for such transcription factors, we used a cDNA fragment corresponding to the Arp-1 DNA binding domain (coordinates 574 to 851 in reference 20) as a probe for screening of two XgtlO cDNA libraries, one prepared with adult human liver mRNA and another with HepG2 cell mRNA. The hybridization conditions for library screening (hybridization buffer, 5x SET [lx SET is 0.15 NaCl, 30 ,M Tris-HCl (pH 8.0), 2 ,uM EDTA], 0.1 M Na-phosphate [pH 7.5], 0.1% Na-PPi, lOx Denhardt solution, 50% formamide, 1% sodium dodecyl sulfate, 10% dextran sulfate, 150 ,ug of denatured salmon sperm DNA per ml; incubation, 37°C, 16 to 20 h) were adjusted so that the probe, although it
* Corresponding author. t Present address: Cardiology Division, Jewish Hospital at Washington University Medical Center, St. Louis, MO 63130.
3814
VOL.
11, 1991
cross-hybridizes to cDNA clones containing the DNA binding domains of various members of the Arp-1 subfamily such as ear-3/COUP-TF and ear-2 (see reference 20), does not cross-hybridize to cDNA clones corresponding to other members of the steroid-thyroid hormone receptor superfamily such as the human estrogen receptor (13), thyroid hormone receptor a-i (32), or the retinoic acid receptors a (RARa [9, 29]) and p (RARP [4]). Several positive clones were obtained, and based on subsequent cross-hybridization and DNA sequencing experiments, it was concluded that most of them corresponded to ear-2, one corresponded to ear-3/COUP-TF and four did not correspond to Arp-1, ear-2, or ear-3/COUP-TF (37). Further cross-hybridization experiments indicated that two of these four clones contain related cDNA inserts, while the other two contain inserts with unrelated sequences. The cDNA sequence of one of the clones with the related inserts was determined and compared with other known members of the steroid-thyroid hormone receptor superfamily. This comparison revealed that this cDNA sequence is identical to the cDNA sequence of a recently identified retinoic acid-responsive receptor referred to as RXRa (26). Specifically, the sequence determined in the current study corresponds to the sequence between residues 43 to 1627 in the published RXRa cDNA sequence (26) and includes the complete RXRa coding region (37). The rationale that led to the cloning of RXRa in the current study together with the observation that, similar to apoAI, RXRa is expressed at high levels in liver (26; data not shown) provided a compelling reason for determining whether RXRa is involved in transcription activation of the apoAI gene in liver cells. To address this possibility, it was decided first to determine whether site A functions as a retinoic acid-responsive element (RARE). Preliminary experiments in HepG2 cells had clearly indicated that site A does not respond to retinoic acid via either RARa, which is constitutively expressed in these cells, or RARP, which is induced severalfold when these cells are treated with retinoic acid (7, 18, 37). Since HepG2 cells do not contain significant amounts of RXRa mRNA (37), it remained possible that site A responds selectively to RXRa. To further evaluate this possibility, we cloned four copies of a previously described (36) double-stranded oligonucleotide corresponding to the apoAl gene site A in tandem and in the same transcriptional orientation with the thymidine kinase (TK) gene promoter in the TK-chloramphenicol acetyltransferase reporter (TK-CAT [25]). The resulting construct (A4-TK-CAT) and an RXRa eukaryotic expression vector (pMT2-RXRa) constructed by cloning the RXR cDNA identified in this study into the previously described vector pMT2 (17) were cotransfected into CV-1 cells. These cells were chosen because the levels of their endogenous RXRa and RARa are below the threshold needed for liganddependent activation of the standard palindromic retinoic acid and thyroid hormone-responsive element TREpal (12, 26, 31, 33). Exogenous expression of RXRa in the presence of 10-6 M retinoic acid activated A4-TK-CAT over 300-fold above baseline levels, which are defined as the levels of expression in the absence of cotransfected receptor and in the absence of retinoic acid (Fig. la and Table 1). This upregulation was dependent on the presence of both RXRa and retinoic acid (Fig. la and Table 1). As a control, the TK-CAT reporter showed no change in expression when cotransfected with pMT2-RXRa in the presence or absence of retinoic acid (data not shown). Similar cotransfection experiments showed that in the presence of retinoic acid, vectors that express RARa (pMT2-RARa) and RARP
NOTES
3815
(pMT2-RAR,) activated A4-TK-CAT only 20- and 34-fold, respectively, above baseline levels (Fig. la and Table 1). In contrast, under the same cotransfection conditions, RXRa, RARa, and RARP in the presence of retinoic acid all activated a construct containing two copies of the TREpal in TK-CAT (construct M2-TK-CAT [34]) roughly to the same extent, 3.5- to 4.9-fold, above baseline levels (Fig. lc and Table 1). Thus, the preferential response of A4-TK-CAT to RXRa over RARa and RARi was not due to differences in the efficiency of receptor expression. Also note that, according to the data in Table 1, the qualitative aspects of this preferential response remain unchanged irrespective of whether the data are expressed as fold activation above baseline levels or fold stimulation of expression in the presence of receptor and retinoic acid compared with the expression in the presence of the receptor but in the absence of retinoic acid. To ensure that the preferential response of A4-TK-CAT to RXRa was due to site A rather than to a RARE created fortuitously by tetramerization of site A, we cloned a single copy of site A in both orientations in TK-CAT, and the resulting constructs (A1-TK-CAT) were used for cotransfection experiments as described above. The results showed that RXRa in the presence of retinoic acid activates Al-TKCAT 7-fold above baseline, while RARa and RARP activate it only 1.7- and 2.4-fold, respectively (Fig. lb and Table 1). Thus, it appears that site A is a selective RARE that responds preferentially to RXRa over RARa and RAR,. In more recent experiments, we have determined that site A is a truly selective RARE because it does not respond to several other members of the steroid-thyroid hormone receptor superfamily such as thyroid hormone receptor a-1, human estrogen receptor, peroxisome proliferator-activated receptor (15), and the androgen receptor (24) in the presence or absence of their corresponding ligands (37). It is also important to note that the response of site A to RXRa required levels of retinoic acid exceeding 10 -6 M for maximal effect, while the response to RARa was nearly saturated at 10-7 M (Fig. le). This corresponds to the retinoic acid dose-response relationship previously described for these receptors with the TREpal (26). Thus, at the levels of retinoic acid needed to produce a consistent effect of RXRa on the TREpal, a clearly preferential response of site A to RXRa was always observed. As previously suggested, the very high concentrations of retinoic acid required for this transcriptional effect may indicate that the biological ligand for RXRa is not retinoic acid per se but some other retinoid metabolite (26). These observations raised the possibility that different RAREs play an important role in defining distinctive retinoic acid cellular response pathways. This possibility was further evaluated by cotransfection experiments similar to those described above but in which a previously described construct containing a RARE from the laminin B1 gene in the TK-CAT vector (PLAM-TK-CAT [34]) was used as the reporter. The results show that in the presence of retinoic acid, RARa and RAR, activated this reporter by 24- and 17-fold, respectively, above baseline levels, while RXRa activated it only 3.2-fold (Fig. ld and Table 1). Thus, the RARE from the laminin Bi gene displays a pattern of responsiveness opposite to that of site A: it responds preferentially to RARa and RAR,B over RXRa. However, both RARa and RAR, but not RXRa increased expression of this reporter even in the absence of retinoic acid (Fig. ld and Table 1; see also reference 34). This minimizes the observed differential response to these receptors when the data are
3816
MOL. CELL. BIOL.
NOTES A4-TK-CAT
(a)
A1-TK-CAT
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rtim iilation of e~x nression unon addition ofTA the ligand. Retinoic acid In summary then, of the three RAREs evaluated with receptor regard to their response to RXRa, RARa, and RAR,, the apoAl site A shows a preferential response to RXRot, the None laminin B1 gene RARE shows a preferential response to RARa RARa and RAR,, and the artificial TREpal shows a roughly equal response to all three receptors at the high levels of RAR, retinoic acid used in these experiments. To evaluate whether the observed functional effects inRXRa volved binding of the receptors to these response elements, we performed electrophoretic mobility shift assays using
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1.0 0.7 0.5 1.7 0.6 2.4 0.6 7
1.0 1.0 8.8 24 6.3 17 1.2 3.2
NOTES
VOL. 11, 1991
(a)
TREpal Probe Extract Competitor
X
a
X
cx X
-
-
N
N P
a P
(b) Probe Apo Al Site A aaX X xa a Extract Competitor - - - N N A A
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3817
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2
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FIG. 2. Binding of RARa and RXRa to the TREpal and apoAl site A. Whole-cell extracts (prepared as described in reference 19) from untransfected Cos-1 cells (U) and Cos-1 cells transiently transfected with the retinoic acid receptor expression vector pMT2-RARa (a) or pMT2-RXRa (X) were used for electrophoretic mobility shift assays with [y-32P]ATP-labeled double-stranded oligonucleotides corresponding to the TREpal (a and c) or apoAl gene site A (b and d) as probes. Specifically, 5 ,ug of cell extract was preincubated for 10 min on ice in the presence of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5% glycerol, 1 mM EDTA, 1 mM dithiothreitol, and 2 jig of poly(dI-dC). Then, 50 fmol of probe and a 50-fold molar excess of competitor oligonucleotides (see below) were added together as indicated, and the mixture (final volume, 20 ,jl) was incubated at room temperature for 15 min. Complexes were subsequently resolved by electrophoresis through 6% nondenaturing polyacrylamide gels and visualized by autoradiography. Competitor oligonucleotides were double-stranded oligonucleotides corresponding to the TREpal (P), apoAl gene site A (A), laminin Bi gene RARE (1), or an unrelated sequence (N). Panel d shows retardation complexes formed with the site A probe and the RXRa-enriched Cos-1 cell extracts (extracts X, see above) in the absence (-RA) or the presence (+RA) of retinoic acid at a final concentration of 10' M which was added to the DNA-protein mixture during the 15-min incubation at room temperature prior to electrophoresis. The fraction of total 32p counts in the region of the gel corresponding to specific retardation complexes was determined, and after averaging over several different experiments, it was found to be 85% 16% (P < 0.01) greater in the presence than in the absence of retinoic acid. ±
whole-cell extracts from transiently transfected Cos cells overexpressing RXRa or RARot. Retardation of a single copy of TREpal was observed with extracts programmed with either RARa or RXRa (Fig. 2a, lanes 2 and 3). This binding was specific, since the retardation complex was eliminated by 50-fold self-competition (lanes 6 and 7) but not by 50-fold competition with an unrelated sequence (lanes 4 and 5). Extracts from Cos cells that were mock transfected did not form retardation complexes (lane 8). Specific binding of both RARa and RXRa from these Cos cells was also observed with a single copy of the apoAl gene site A (Fig. 2b). It is noteworthy that site A effectively cross-competed for binding of either RARa or RXRa to the TREpal (Fig. 2c, lanes 4 and 5). This is not surprising since a single copy of site A is functionally comparable to a duplicated copy of the TREpal as an RARa-responsive element and is substantially more powerful as an RXRa-responsive element (compare Fig. lb and c). In contrast, the laminin Bi gene RARE, which effectively cross-competed for binding of RARa to the
TREpal (Fig. 2c, lane 7), failed to compete for binding of RXRa (lane 6). These binding data are consistent with the functional data, since the laminin B1 gene RARE responds to RARa but not to RXRa (Fig. ld). These binding studies suggest but do not conclusively demonstrate that the receptors per se bind directly to these responsive elements, since the complexes observed could instead involve proteins other than, but induced by, the receptors. To address this possibility, RARa and RXRa were translated in vitro and tested for their ability to bind to these RAREs. Reticulocyte lysates programmed with RARa or RXRo mRNA did not form specific retardation complexes with either the TREpal (Fig. 3a, lanes 2 and 3) or with site A (Fig. 3b, lanes 2 and 6). However, when RXRa- or RARaprogrammed lysates were supplemented with untransfected Cos whole-cell extract, specific retardation complexes were formed (Fig. 3a, lanes 4 and 5; Fig. 3b, lanes 3 and 7). The competition experiments in Fig. 3b, lanes 4, 5, 8, and 9, indicate that these complexes are specific. Control experi-
3818
NOTES
MOL. CELL. BIOL. (a) Probe Extract
(b)
TREpal PV R n,
Competitor bound
RtX R o PO
-C
ProID Extract
ApoAl SiteA -
-C -C
_ _ _ _
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.--
X P X R XPXXR, P f -c -C C'C -A N
-.-
Pr; P;
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2
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5 6
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FIG. 3. Electrophoretic mobility shift assays using in vitro-translated receptor proteins and the TREpal (a) or site A (b). Reticulocyte lysates were programmed with RXRa (RX) or RARa (Ra) or mock programmed (RO) and used either directly or after supplementation with untransfected Cos whole-cell extracts (+C). Competitors used at a 50-fold molar excess are described in the legend to Fig. 2. Reticulocyte lysates were programmed with in vitro transcripts of RARa or RXRa according to the supplier's (Promega) protocol. Electrophoretic mobility shift assays were done as described in the legend to Fig. 2, using 2 p.l of lysate and 10-5 M retinoic acid in the binding reaction. Untransfected Cos cell extracts (5 p.g) were added as indicated.
ments demonstrated that unprogrammed reticulocyte lysates supplemented with untransfected Cos whole-cell extract did not form retardation complexes (Fig. 3a, lane 6; data not shown for site A). Specific complex formation was abolished by preheating the Cos cell extract to 65°C for 5 min (data not shown). These data suggest that the RARa and RXRa receptors themselves, rather than other secondarily induced factors, are present in these DNA-protein complexes. The requirement of untransfected Cos cell extract for receptor binding indicates that other factors not induced by the receptors are necessary for efficient binding. Such a requirement was also noted for other members of the steroidthyroid hormone receptor superfamily (1, 3, 10, 21, 23, 28). In addition, it was recently reported that multiple cellspecific proteins differentially regulate DNA target sequence recognition by RARa (10). Interestingly, preliminary results indicated that in vitro-produced Arp-1 can substitute for Cos cell extracts in facilitating RXRca binding to site A. This raises the possibility that although Arp-1 itself is not responsive to retinoic acid (37), it nevertheless plays an important role in determining distinctive cellular retinoic acid-responsive pathways by directing RXRa binding to specific RAREs. Finally, addition of retinoic acid (105 M) to the DNA binding reaction enhanced binding of Cos cell-produced RXRa to site A by approximately twofold (Fig. 2d). Determination of the dissociation constant (Kd) of the RXRa-site A interaction by Scatchard analysis was consistent with this observation: the Kd value in the absence of retinoic acid was 8.8 nM, while the Kd value in the presence of retinoic acid was 4.0 nM (37). This result provides corroborative evidence for the presence of the receptor in the protein-DNA com-
plex. Taken together, the functional and DNA-protein binding studies presented here demonstrate a preferential responsiveness of apoAl site A to RXRa and a preferential responsiveness of the laminin Bi gene RARE to RARa and RAR,. These two elements thus distinguish between two different retinoic acid-responsive pathways, one in which signal transduction is mediated by RXRa, and another in which signal transduction is mediated by the classical retinoic acid recep-
tors RARa and RAR,. Other transcriptional factors with intermediate affinities to these responsive elements may serve to further enforce and accentuate the distinction between these pathways and permit them to subserve different cellular functions. We speculate that the classical receptors may play a role in cell differentiation, since laminin Bi mRNA is known to increase dramatically upon retinoic acid-induced differentiation of F9 teratocarcinoma cells (35) and RARa suffices to effect granulocytic differentiation of HL60 cells (5). The RXRa, on the other hand, may be involved in processes other than cell differentiation. Indeed, when RXRa was first identified, it was suggested that it played a role in vitamin A metabolism because of its tissue distribution (26). The presence of an element with a preferential response to RXRa in the regulatory region of the apoAl gene, which is involved in lipid transport and metabolism and in particular the transport of vitamin A, supports this hypothesis and suggests that RXRoa or closely related receptors are central to these processes. It should be also emphasized that although several nuclear receptors for retinoids have been identified (22, 26, 38, and references therein), only a few target genes for these receptors have been characterized (6, 8, 14, 27, 31, 34). Functional distinctions among these receptor isoforms have been primarily based on differences in ligand affinity and specificity (4, 26). The existence of RAREs with preferential specificity to different retinoic acid receptors is beginning to be recognized as a potentially important mechanism for the mediation of the diverse biological effects of retinoic acid. Indeed, it has been recently reported that a RARE from the RAR, gene promoter responds preferentially to RARa and RARP over the retinoic acid receptor RAR-y (14). The finding that the apoAl gene site A is a functional RARE taken together with the previous observation that site A is a positive response element for the recently cloned transcription factor HNF4, which is also a member of the steroid-thyroid hormone receptor superfamily (30, 36), and the observation that site A within the context of the apoAl gene promoter-enhancer functions as a negative response element for Arp-1 and ear-3/COUP-TF (20; unpublished data) strongly suggest that multiple regulatory signals con-
VOL . 1 l, 1991 verge onto this site to modulate apoAI gene expression (see also reference 36). It is therefore reasonable to speculate that the level of apoAl gene expression is determined by the intracellular balance of all these signal transduction pathways. Indeed, in preliminary experiments we have observed that the negative effect of Arp-1 or ear-3/COUP-TF on apoAI gene expression in HepG2 cells is overcome by overexpression of RXRa in the presence of retinoic acid (37). These observations raise the possibility that retinoids, or other similar small hydrophobic molecules, play an important role in regulation of apoAl gene expression and suggest that such ligands are fundamental to the signal transduction mechanisms contributing to cholesterol and vitamin A transport and homeostasis. Moreover, these observations raise the intriguing possibility that such molecules acting via these signal transduction pathways are ultimately influencing processes involved in the initiation, progression, and severity of coronary heart disease.
We thank R. M. Evans, S. Green, L. J. Gudas, J. Kakontis, M. Pfahl, and M.-J. Tsai for providing plasmids containing RARa, the peroxisome proliferator-activated receptor, laminin Bi gene RARE, the androgen receptor, RARI, and COUP-TF, respectively. We also thank E. F. Fritsch and D. Kwiatkowski for providing the human liver and HepG2 cDNA libraries, respectively. In addition, we thank Chris Cheney for preparing the manuscript and Emily Flynn-McIntosh for the artwork. This work was supported by Public Health Service grant HL32032 from the National Institutes of Health. J.R. is a Bugher fellow of the AHA Center for Cellular and Molecular Cardiology. B.N.-G. is an investigator of the Howard Hughes Medical Institute. During the early stages of this work, V.M. and S.K.K. were supported by established investigator awards from the American Heart Association. REFERENCES 1. Beebe, J. S., D. S. Darling, and W. W. Chin. 1991. 3,5,3'Triiodothyronine receptor auxiliary protein (TRAP) enhances receptor binding by interactions within the thyroid hormone response element. Mol. Endocrinol. 5:85-93. 2. Blomhoff, R., M. H. Green, T. Berg, and K. R. Norum. 1990. Transport and storage of vitamin A. Science 250:399-404. 3. Bonde, B. G., and M. L. Privalsky. 1990. Sequence-specific DNA binding by the v-erbA oncogene protein of avian erythroblastosis virus. J. Virol. 64:1314-1320. 4. Brand, N., M. Petkovich, A. Krust, P. Chambon, H. de The, A. Marchio, P. Tiollais, and A. Dejean. 1988. Identification of a second human retinoic acid receptor. Nature (London) 332:850853. 5. Collins, S. J., K. A. Robertson, and L. Mueller. 1990. Retinoic acid-induced granulocytic differentiation of HL-60 myeloid leukemia cells is mediated directly through the retinoic acid receptor RARa. Mol. Cell. Biol. 10:2154-2163. 6. de The, H., M. del Mar Vivanco-Ruiz, P. Tiollais, H. Stunnenberg, and A. Dejean. 1990. Identification of a retinoic acid responsive element in the retinoic acid receptor v gene. Nature (London) 343:177-180. 7. de The, H., A. Marchio, P. Tiollais, and A. Dejean. 1989. Differential expression and ligand regulation of the retinoic acid receptor a and genes. EMBO J. 8:429-433. 8. Duester, G., M. L. Shean, M. S. McBride, and M. J. Stewart. 1991. Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis. Mol. Cell. Biol. 11:1638-1646. 9. Giguere, V., E. S. Ong, P. Seguli, and R. M. Evans. 1987. Identification of a receptor for the morphogen retinoic acid. Nature (London) 330:624-629. 10. Glass, C. K., 0. V. Devary, and M. G. Rosenfeld. 1990. Multiple cell type-specific proteins differentially regulate target sequence recognition by the a retinoic acid receptor. Cell 63:729-738. 11. Goodman, D. S. 1984. Vitamin A and retinoids in health and
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