Octamer Activation of the Calcitonin/Calcitonin Gene-Related Peptide Enhancer

Transcription

1 MOLECULAR AND CELLULAR BIOLOGY, Oct. 1993, p /93/1679-1$2./ Copyright 1993, American Society for Microbiology Vol. 13, No. 1 Retinoic Acid Repression of Cell-Specific Helix-Loop-Helix- Octamer Activation of the Calcitonin/Calcitonin Gene-Related Peptide Enhancer THOMAS M. LANIGAN, LOIS A. TVERBERG, AND ANDREW F. RUSSO* Department ofphysiology and Biophysics, University of Iowa, Iowa City, Iowa Received 6 May 1993/Returned for modification 23 June 1993/Accepted 22 July 1993 We have investigated the mechanism underlying repression of calcitonin/calcitonin gene-related peptide (CT/CGRP) gene expression by retinoic acid. Retinoic acid treatment of the CA77 thyroid C-cell line decreased CT/CGRP promoter activity two- to threefold, which correlates well with the decrease in calcitonin and CGRP mrna levels. Repression is mediated through the nuclear retinoic acid receptors (RAR) on the basis of the retinoid specificity, the sensitivity of repression (half-maximal repression at.2 nm), and the additional repression caused by cotransfection ofan K-RAR expression vector. The sequences required for retinoic acid repression were localized to an 18-bp element containing cell-specific enhancer activity. The enhancer binds helix-loop-helix (HLH) and octamer transcription factors that act synergistically to activate transcription. Retinoic acid repression requires both these factors since mutations in either motif resulted in the loss ofrepression. Furthermore, repression was observed only in cell lines containing enhancer activity. We have used electrophoretic mobility shift assays to show that repression does not involve direct DNA binding of RAR or RAR-retinoid X receptor heterodimers. Instead, repression appears to involve interactions with the stimulatory enhancer factors. Following retinoic acid treatment, there was a specific decrease in an enhancer complex containing both HILH and octamer proteins. Formation of the HLH-octamer complex was also specifically blocked by the addition of exogenous RAR-retinoid X receptor protein. These results demonstrate that RAR can repress CT/CGRP gene transcription by interfering with combinatorial activation by cell-specific HLH and octamer proteins. Retinoic acid and other vitamin A derivatives are known to have profound manifestations on cellular proliferation, differentiation, and pattern formation (5). During development one region that is especially sensitive to the actions of excess retinoids is the vertebrate neural crest. The neural crest is a transitory structure that gives rise to cells that migrate and differentiate into a wide variety of cell types, including thyroid C cells (2). Retinoic acid has an inhibitory effect on neural crest cell migration and can influence the differentiation of crest-derived cells (13, 19, 41). Many of the actions of retinoic acid are mediated through a family of nuclear receptors homologous to steroid and tpyroid hormone receptors (2, 8). In addition to the retinoic acid receptors (RAR) (1, 32), retinoic acid is also bound with lower affinity by cytoplasmic binding proteins and the nuclear retinoid X receptors (RXR) (27). The RXR proteins can also form heterodimers with RAR and other members of the steroid superfamily (16, 21, 51, 52). The neural crest-derived tissues have been shown to contain both classes of nuclear RAR and cytoplasmic binding proteins (6, 26, 34, 35). However, the molecular events involved in RAR regulation of gene expression, and in particular, the repression of gene transcription, are to a large part still unknown. As a tool for studying the mechanisms by which retinoic acid regulates neural crest differentiation, we are studying retinoic acid effects on the rat CA77 thyroid C-cell line. The CA77 cells have a neuronal phenotype characterized by extensive neurites and expression of neurofilaments (36). Retinoic acid treatment of the CA77 cells represses some of these neuronal properties by causing neurite retraction, most likely due to decreased cell adhesion, and causing a two- to * Corresponding author. 679 threefold decrease in calcitonin/calcitonin gene-related peptide (CT/CGRP) mrna levels (36). The CT/CGRP gene provides a useful phenotypic marker, since it is transcriptionally active in a subset of peripheral and central neurons and neuroendocrine cells. Alternative splicing of the primary transcript yields predominantly CT mrna in thyroid C cells and CGRP mrna in neurons (33). Basal transcription of the rat and human CT/CGRP genes is regulated by a cell-specific enhancer about 1 kbp upstream of the initiation site (1, 31, 42-44). Transcription and steady-state mrna levels are further regulated by hormonal stimuli, including cyclic AMP and phorbol ester (4), nerve growth factor (23), glucocorticoids in a cell-specific manner (43), vitamin D (28), and retinoic acid (36). In this study, we addressed the mechanism by which retinoic acid represses CT/CGRP mrna levels in the CA77 thyroid C-cell line. We demonstrate that retinoic acid reduces CIT/CGRP promoter activity and that the repression is mediated through the 18-bp cell-specific enhancer element. To test the significance of colocalization with the cell-specific enhancer, we show that retinoic acid repression occurred only in cell lines containing CT/CGRP enhancer activity. Finally, we use mobility shift assays to demonstrate that retinoic acid repression is not mediated by direct DNA binding but rather involves inhibitory protein interactions. This repression of cell-specific factor activity may represent a common mechanism for retinoic acid repression of gene expression and differentiation in the neural crest and other systems. MATERIALS AND METHODS Cell transfections and luciferase assays. CA77 cells were maintained in Ham's F12-Dulbecco's modified Eagle's medium (DMEM) (low glucose) (1:1)-1% fetal bovine serum

2 68 LANIGAN ET AL. (Hyclone, Logan, Utah); 44-2C cells were maintained in DMEM (high glucose)-1% equine serum-.1% L-glutamine; and HeLa cells were maintained in Ham's F12-1% fetal bovine serum. In addition, 1 U of penicillin per ml and 1,ug of streptomycin (GIBCO, Grand Island, N.Y.) per ml were added to each medium. All cell types were maintained at 37 C in 5% CO2. Fragments of the rat CT/CGRP 5' flanking sequences used for this study have been previously described (43, 44). The Oct mutant (Oct mut 2) contains TG in place of AT (5'- GGCAGCTGTGCAAMCCT-3') and the HLH mutant contains AC in place of CA (5'-GGACGCTGTGCAAATCCT- 3'). The RARE-TK-luc, RSV-a-RAR, and P-actin-x-hRXR plasmids were generously provided by C. Glass (11, 12). The cells were removed from the dishes by brief trypsin-edta treatment, washed, and resuspended in Dulbecco's phosphate-buffered saline (minus calcium and magnesium) immediately prior to transfection. An apparently critical parameter for retinoic acid inhibition was that the CA77 cells could not be aggregated into clumps. To be consistent, all cell types were subcultured by trypsin treatment 1 day prior to the experiment, except for the 44-2C cells, which often required 2 days to firmly attach. About 4 x 16 to 5 x 16 cells in.8 ml were transfected with supercoiled DNA by electroporation with a Bio-Rad gene pulser apparatus at 2 V for the CA77 cells, 22 V for the HeLa cells, and 26 V for the 44-2C cells, all with a capacitance of 96,iF. The same amount of DNA (2,ug) was used per cuvette unless otherwise indicated. For the a-rxr cotransfection assays, 1,ug of 18-bp CT/CGRP-TK-luc plasmid, 18,ug of RSV-a-RAR plasmid, or 2 jig of P-actin-RXR plasmid (brought to 3,ug of total DNA with CMV-p-gal plasmid) was transfected into the cells. The transfected cells from a single cuvette were then divided between two 6-mm dishes and treated with 1,uM retinoic acid (Sigma) or the vehicle (.1% ethanol). About 5 to 8% cell survival in the electroporation procedure was observed, except for the 44-2C cells, of which approximately 25% were recovered. The cells were harvested 18 to 25 h after transfection and then assayed as previously described (43). Activities are reported per 5,ug of protein unless otherwise noted. Protein measurements and,3-galactosidase assays were performed as described previously (43). The 3-galactosidase activities confirmed that DNA transfection efficiencies were essentially the same (less than 1% standard deviation) within each experiment, although the absolute luciferase activity varied severalfold between experiments because of variations in the transfection efficiency. To compare results from different experiments, the activities were normalized to an internal standard, as indicated in the figure legends. Electrophoretic mobility shift assay. The complementary CT/CGRP HLH-octamer (H/O) enhancer oligonucleotides (5'-GATCCGGCAGCTGTGCAAATCCTG-3') (44) were annealed, and then 1 pmol of DNA was labeled with [al-32p]datp (25,uCi, 3 Ci/mmol) by using Klenow DNA polymerase. The complementary RARE oligonucleotides (5'-GGGTAGGGTICACCGAAAGTTCACTCG-3') (11) (1 pmol) were labeled by phosphorylation with T4 kinase with [y-32p]atp (2,uCi, 3 Ci/mmol), annealed, and purified through a Sephadex G-5 column. The binding reaction mixture (2 RlI) contained.2 pmol of labeled oligonucleotide (generally 5, to 1, cpm), 3 to 6,ug of nuclear extract from CA77 cells or 3,ug from HeLa cells, and binding buffer (1 mm Tris [ph 7.5], 5% glycerol, 5 mm NaCl, 1 mm EDTA, 1 mm dithiothreitol). Poly(dI-dC) (.1 p.g) (Boehringer Mannheim) and.1 pmol of an unrelated poly- MOL. CELL. BIOL. linker oligonucleotide (5'-GATCCTCTAGACATATGGGA TC-3' or 5'-GATCCACTATGTCTAGAGGATC-3') were also included as nonspecific competitors. Nuclear extracts were prepared as previously described (44). For some experiments, extracts were prepared from CA77 cells treated with 1,uM retinoic acid for 24 h. For competition assays, the competitor oligonucleotides were preincubated with the extracts for 15 min prior to the addition of the probe. The competitor sequences have been described previously (44). The reactions were incubated on ice for 15 min and then resolved on a 6% nondenaturing polyacrylamide gel (1:29, bisacrylamide-acrylamide) in.25 x TBE (Tris-borate- EDTA [ph 8.5]) as described previously (44). The dried gels were exposed to film overnight with an intensifying screen, unless otherwise stated. Purified bacterial glutathione S-transferase (GST) fusion proteins containing nearly full-length human ao-rar and human a-rxr were kindly provided by C. Glass. The control GST fusion protein containing a fragment of a non-dna-binding protein was kindly provided by S. Waters. The integrity and concentrations of the fusion proteins were confirmed by Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels. The GST-RAR and GST-RXR fusion proteins were mixed in a 1:1 ratio at a concentration of.5 to.5,g/pl (diluted as needed in binding buffer with 1,ug of bovine serum albumin per ml) and incubated on ice for 3 min to allow heterodimerization. For DNA-binding assays, the receptor protein (.5 to.5 p,g for CA77 extracts and.5,ug for HeLa extracts) was added directly to a binding reaction mixture containing the radiolabeled DNA probe as described above. For protein interaction studies, the receptor was preincubated in the binding reaction mixture containing CA77 or HeLa nuclear extract for 15 min prior to the addition of the probe. The reaction mixture was then incubated for another 1 to 15 min and analyzed as described above. RESULTS Retinoic acid repression of CT/CGRP promoter activity colocalizes with the cell-specific enhancer. We have recently reported that retinoic acid treatment of CA77 cells causes about a two- to threefold decrease in calcitonin and CGRP mrna levels (36). To determine whether the reduction of mrna levels could be due to a decrease in transcription, the effect of retinoic acid on promoter activity was determined. Luciferase reporter plasmids containing different promoters, including up to 1.9 kb of the 5' flanking region of the rat CT/CGRP gene, were transiently transfected into the CA77 cells. To insure equivalent DNA transfection efficiency between control and retinoic acid-treated cells, the cells were electroporated in a single cuvette and then divided between two dishes for treatments. In addition, in some experiments a CMV-1-gal reporter plasmid was cotransfected along with the luciferase fusion genes as a control for transfection efficiency between different plasmids and retinoic acid treatments. Retinoic acid treatment caused a two- to threefold decrease in the promoter activity of luciferase fusion genes containing 1,125 bp or more of 5' flanking sequences (Fig. 1). In contrast, promoter fragments containing 92 bp or less were not repressed by retinoic acid. This result suggested that the retinoic acid responsive element (RARE) lies between 92 and 1,125 bp 5' of the transcription initiation site. This region has also been localized as the cell-specific enhancer of the rat CT/CGRP gene (42-44). In agreement

3 VOL. 13, 1993 RETINOIC ACID REPRESSION OF HLH-OCTAMER SYNERGY A1 I m nm wmi.n CAL1362 I t l'o'kf''j,",,q. KROME Ig-,-; W-5.7 S.-, :-- -l CAL CAL92 J + CAL163 F - TK g TK RARETK CMV- RA o uc Luciferase Activity (light units) FIG. 1. Retinoic acid repression of CT/CGRP promoter and enhancer activity. CA77 cells were transfected with luciferase fusion genes and treated with 1,uM retinoic acid (RA) (+) or the ethanol vehicle (-) for 24 h and then assayed for luciferase activity. Fusion genes containing the 5' flanking sequences of rat CT/CGRP (CAL), thymidine kinase promoter (TK), CT/CGRP cell-specific enhancer linked to the TK promoter ( TK), tandem elements of the 1-RAR gene RARE linked to the TK promoter (RARE TK), and cytomegalovirus promoter (CMV) are shown schematically. The means from three to six independent experiments normalized to the average activity of TK-luc are shown; error bars indicate standard deviations. The activities per 5 p.g of extract are reported, except CMV-luc activity, which is reported per 1 p.g of extract. D CM 2- with this, we observed that the basal activity of fusion genes containing the 1,125- to 92-bp region was about 1- to 2-fold greater than that of constructs lacking this region (Fig. 1). It should be noted that despite the lower activity, the plasmids lacking enhancer activity still express sufficient luciferase activity (usually 5, light units above the background of about 1 light units) such that repression, if it occurred, could have been detected. As a control to confirm that CA77 cells contain functional RAR that could stimulate transcription and to confirm the specificity of repression, the cells were transfected with a luciferase reporter gene containing two tandem repeats of the RARE from the,3-rar gene linked to the thymidine kinase promoter (RARE-TK-luc) (11). We observed a consistent fivefold increase in RARE-TK-luc activity upon retinoic acid treatment (Fig. 1). As additional controls for the specificity of the retinoic acid effects, we transfected the cells with luciferase fusion genes containing the cytomegalovirus promoter (CMV-luc) or the thymidine kinase promoter (TK-luc), which were not significantly affected by retinoic acid (Fig. 1). The next objective was to document that the sequences required for repression were localized within the region defined by the deletion studies. The 1,125- to 92-bp fragment was linked to the TK promoter-luciferase gene ( TK-luc) and transfected into the CA77 cells. As expected, the basal activity of TK-luc was enhanced about 3-fold by the 1,125- to 92-bp region, confirming the presence of enhancer activity. Treatment of the cells with retinoic acid caused a three- to fourfold repression in TK-luc activity (Fig. 1). Repression was seen with the fragment in both orientations (data not shown). These results demonstrate that the RARE is contained entirely within the cell-specific enhancer region and, furthermore, that retinoic acid repression is transferable to a heterologous promoter. Repression involves the nuclear RAR. We determined the retinoic acid concentrations required for repression of the CT/CGRP promoter (Fig. 2). The dose dependence was measured by transfection of the CAL1125-luc reporter gene into the CA77 cells. The maximal amount of repression was a threefold decrease of control activity and was observed with 1 nm retinoic acid. The amount of retinoic acid required for half-maximal repression was about.2 nm. This correlates well with previous measurements of the nuclear RAR-binding affinities (Kd,.2 nm) (48), suggesting that retinoic acid is working through RAR, rather than a-rxr, which requires at least 1-fold-greater levels of retinoic acid for activation (27). To further characterize the receptor involvement we tested the effect of retinal, a related retinoid that has lower affinity for the RAR (1, 32). Retinal repressed the CT/CGRP promoter activity only at concentrations of 1 nm or greater, and half-maximal repression occurred at.3 pum retinal (Fig. 2). The comparison between retinoic acid and retinal also demonstrated that decreased CT/CGRP promoter activity was not secondary to the decreased cell adhesion caused by retinoic acid treatment (36). Both retinoic acid and retinal decreased cell adhesion at comparable concentrations (greater than 5 nm) after several days of treatment. In contrast, much greater levels of retinal than retinoic acid are

4 682 LANIGAN ET AL. 1.= 8 4) o,6 2 R RAL Retinoic Acid (log M) FIG. 2. Dose dependence and retinoic acid specificity. The CAL1125-luc fusion gene was transfected into CA77 cells and treated with various concentrations of either retinoic acid (RA), retinal (RAL), or the ethanol vehicle as the control. The luciferase activities from retinoic acid treatments (mean and standard deviation of two experiments) and from retinal treatments (mean and standard deviation of duplicate plates from one experiment) were normalized relative to that of control cells. The luciferase activities of all control plates were at least 125, light units per 5 pg of protein extract. A c 6 1.i 4.-.o *wa <r 2 C - -, 1 I C a-rar a-rar - + CAL1125 CMV RARE-TK FIG. 3. Retinoic acid repression upon cotransfection with a-rar. (A) The CAL1125-luc fusion gene (5,ug) was transfected with (+) or without (-) the a-rar expression vector (25,ug) into the CA77 cells and treated with either 1 p.m retinoic acid or the ethanol vehicle control. The mean fold decrease and standard deviation relative to that of control activities following retinoic acid treatment from three experiments are shown. The luciferase activity of CAL1125-luc without retinoic acid and a-rar was at least 1, light units. As a control, CMV-luc activity was essentially unaffected by the cotransfected ax-rar vector, as shown from a single experiment. (B) The RARE-TK-luc fusion gene was transfected as described for panel A. The fold increase in RARE-TK-luc activity following retinoic acid treatment in the presence or absence of cotransfected a-rar expression vector from a single experiment is shown. B =.5 MOL. CELL. BIOL. required for repression of CT/CGRP promoter activity, and no repression is seen at concentrations of retinal that have profound effects on cell adhesion. Furthermore, there was little or no detectable change in cell adhesion following the 1-day treatment periods used for the transient transfection studies. Since retinoic acid appears to work through an RAR to repress CT/CGRP promoter activity, we asked whether cotransfection of an a-rar expression vector in CA77 cells would yield even greater repression. Cotransfection of RAR expression vectors has previously been shown to increase retinoic acid effects on some target genes, for example, repression of the collagenase promoter in HeLa cells (4, 5). Using CAL1125-luc, we observed a fivefold retinoic acid-dependent repression in activity in cultures cotransfected with a-rar, compared with a twofold repression in parallel cultures without cotransfected receptor (Fig. 3). Cotransfection of the a-rar expression vector caused a 15-fold retinoic acid-dependent stimulation of RARE-TK-luc activity, which is threefold greater than the stimulation observed in the absence of cotransfected receptor (Fig. 3). As a control, receptor cotransfection did not significantly affect CMV-luc activity. Furthermore, cotransfection of a-rxr alone had no effect relative to the control, and cotransfection of both a-rxr and a-rar does not increase repression over that seen with a-rar alone (data not shown). These results support the involvement of the receptors and, furthermore, suggest that RAR levels may be a limiting factor in retinoic acid repression in the CA77 cells. Localization of the retinoic acid-responsive sequences to an 18-bp HLH-octamer enhancer element. The sequences required for repression were further mapped within the 1,125- to 92-bp region (Fig. 4). The distal and proximal halves of this region, as defined by a convenient PvuII site, were inserted into the TK-luc reporter vector ( TK-luc and TK-luc, respectively). Neither region con- ferred retinoic acid-dependent repression and had little or no enhancer activity (twofold or less). Similar results were seen with the fragments in the opposite orientation (data not shown). Assuming that the PvuII site might have split interdependent enhancer elements, two fragments with lengths of 75 and 18 bp spanning the PvuII site were inserted into the TK-luc vector ( TK-luc and TK-luc, respectively) (Fig. 4). The 75-bp fragment had relatively little enhancer activity (only three- to fourfold), while the 18-bp fragment was more active, with 8- to 12-fold enhancement over TK-luc. For comparison, in parallel experiments the full 25-bp element yielded about 2-fold enhancement. The basis for these apparent differences in fold enhancement was not further investigated but the results suggest that multiple elements may contribute to the enhancer activity in CA77 cells. In contrast to the CA77 cells, the 18-bp element contains all the enhancer activity in the 44-2C C-cell line (43). The important point for this study is that reporter genes containing the 75- and 18-bp fragments exhibited retinoic acid-dependent repression comparable to that of the full 25-bp region (Fig. 4). Consequently, both retinoic acid repression and most enhancer activity were localized to an 18-bp element. These results define a relatively small region that is required for retinoic acid repression. We have recently shown that this 18-bp element binds helix-loop-helix (HLH) proteins and a cell-specific octamer protein that activate transcription in a synergistic manner (44). To address the functional significance of these motifs in repression, site-directed mutations were made in both the octamer motif and HLH motif. Neither of these constructs conferred retinoic acid repression or had very much enhancer activity (twofold or less) (Fig. 4). The greatly reduced enhancement seen upon mutation of either motif is consistent with our results with the 44-2C cell line (44). The loss of retinoic acid-dependent repression with the point mutations

5 VOL. 13, 1993 A RETINOIC ACID REPRESSION OF HLH-OCIAMER SYNERGY 683 nen T K w'* TK MM T T IHLHIO:ctl Oct Mut2 IHLHIOctl TK HLH Mut IHLHjOcCtI TK ~ 8 o 8 g Luciferase Activity (light units) B HLH Octamer GGCAGCTGTGCAAATCCT CCGTCGACACGTTTAGGA FIG. 4. Identification of an 18-bp region containing retinoic acid repression and enhancer activity. (A) Fusion genes containing the TK promoter with the indicated fragments of the CT/CGRP enhancer region were transfected into the CA77 cells. In addition, constructs containing point mutations in the HLH and octamer (Oct) motifs of the 18-bp enhancer element are indicated. The luciferase activities normalized to the average activity of TK-luc from two or three experiments are shown, except for TK activity, which is from one experiment. Error bars indicate standard deviations. (B) Sequence of the 18-bp element containing the cell-specific enhancer and retinoic acid repression activities. The HLH and octamer motifs and point mutations are indicated by closed bars and asterisks, respectively. indicates that both the HLH and octamer motifs are required for repression. Correlation of retinoic acid repression activity and CT/ CGRP gene enhancer activity. To investigate the functional significance of colocalization of the cell-specific enhancer and the retinoic acid repression sequences, we transfected the CT/CGRP enhancer fusion genes into two additional cell lines either containing or lacking enhancer activity. The rat 44-2C thyroid C cells have been shown to express the CT/CGRP gene and have enhancer activity (43). The 1,125- to 92-bp region enhanced the activity of the TK promoter about 5-fold. Retinoic acid caused a twofold repression of TK-luc activity (Fig. 5). Cotransfection with the a-rar expression vector did not appear to increase repression in the 44-2C cells. As controls, RARE-TK-luc increased activity 35-fold, showing that retinoic acid could stimulate transcription in the 44-2C cells, and TK-luc activity did not change, indicating the specificity of the retinoic acid effects. To test whether retinoic acid would repress transcription in cells lacking CT/CGRP enhancer activity, we transfected the luciferase fusion genes into HeLa cells. The HeLa cervical carcinoma cell line has been shown to lack cellspecific CT/CGRP enhancer activity (42, 43) and to have a different set of CT/CGRP enhancer-binding proteins (44). In particular, HeLa cells have little or no OB2 octamer protein. As expected, the 1,125- to 92-bp enhancer increased TK promoter activity five- to sixfold in the HeLa cells. This degree of enhancement was previously seen in HeLa cells and other heterologous cell lines (44) and is most likely due to the close juxtaposition of general transcription factor sites (e.g., Spl) near the TK promoter. This prediction is supported by the observation that the minimal 18-bp element caused minimal enhancement of the TK promoter in HeLa cells (44). We found that retinoic acid had no effect on TK-luc activity in the HeLa cells (Fig. 5). Repression was not observed even with cotransfection of a-rar, a-rxr, or both. It is noteworthy that although the general transcription factor sites within the 1,125- to 92-bp region could enhance expression in HeLa cells, this enhancement was not repressed by retinoic acid treatment. These results

6 684 LANIGAN ET AL. A TK TK B RARE-TK TK RA 1I T 44-2C I.. I I I.. I.. I o o o o to - $cm NCo Luciferase Activity (light units) Probe: RARE H/ / Enhancer a) 1 LU LUJ (1 Competitor: o o < -- z z EC I Z RAR/RXR: t 4 I... 5 RAR/RXR -_ MOL. CELL. BIOL. O LLU LL < -- z Er TK RARE-TK RA Luciferase Activity (light units) FIG. 5. Cell-specific retinoic acid repression of CT/CGRP enhancer activity. (A) The luciferase fusion genes were transfected into the 44-2C cells and treated with either 1 pm retinoic acid (+) or the ethanol vehicle (-). The activities are reported as the means (normalized to the average TK-luc activity) from five experiments. (B) The luciferase fusion genes were transfected into HeLa cells along with the a-rar expression vector and treated with 1,uM retinoic acid (+) or ethanol vehicle (-). The activities were normalized as described for panel A; results are the means from two experiments. Error bars indicate standard deviations. establish that retinoic acid repression requires the presence of cell-specific proteins that bind the 18-bp HLH-octamer enhancer. Lack of RAR binding to the CT/CGRP enhancer element. Sequence analysis of the CT/CGRP enhancer did not reveal an obvious retinoic acid-responsive sequence on the basis of the sequences of other genes that are stimulated by retinoic acid (5). However, the receptor can act through rather degenerate sequences (5, 14). To establish whether RAR bound to the 18-bp CT/CGRP HLH-octamer enhancer, we used an electrophoretic mobility shift assay. The radiolabeled CT/CGRP enhancer oligonucleotide was incubated with bacterially synthesized fusion proteins containing RAR and RXR linked to GST. The GST-RAR and GST-RXR proteins were preincubated to allow RAR-RXR heterodimer formation. We could not detect any binding of GST-RAR- RXR protein to the CT/CGRP enhancer (Fig. 6). As a positive control, we performed the binding assay with the RARE fragment used in the transfection studies since its binding properties have been well characterized (12). A complex containing the radiolabeled RARE oligonucleotide and GST-RAR-RXR protein could be easily detected (Fig. 6). The addition of excess unlabeled competitor RARE oligonucleotides caused a marked reduction in the receptor FIG. 6. Lack of RAR-RXR binding to the CT/CGRP enhancer. Approximately.5,ug each of GST-RAR and GST-RXR fusion proteins was mixed and then added to a binding reaction mixture containing either RARE probe (lanes 2 to 4) or CT/CGRP H/O enhancer (HIO Enh.) probe (lanes 6 to 8). Control lanes contain binding reaction mixtures without RAR-RXR with either the RARE probe (lane 1) or H/O enhancer (lane 5). A 1-fold excess of unlabeled competitor RARE or H/O enhancer DNA was added to reaction mixtures containing RARE (lanes 3 and 4) or H/O enhancer probe (lanes 7 and 8). The gel was exposed to film for 16 h without an intensifying screen. Longer exposures did not reveal any clear binding to the CT/CGRP H/O probe. complex. As expected from the lack of binding seen with the radiolabeled CT/CGRP probe, the addition of a 1-fold excess of unlabeled CT/CGRP oligonucleotide did not affect binding (Fig. 6). Consequently, the RAR-RXR protein has at least 1-fold-lower affinity for the CT/CGRP enhancer than a known RARE. One possible reason for the lack of binding to the CT/ CGRP enhancer seen with bacterially synthesized RAR and RXR proteins is that RAR binding might require a cellular protein other than RXR. This possibility was investigated by using a competition binding assay similar to that described above, except that CA77 nuclear extract was used instead of bacterially synthesized proteins (data not shown). A RARE DNA-RAR protein complex was identified by competition with either a 1-fold excess of unlabeled RARE DNA or the palindromic thyroid hormone response element DNA (TREpal). TRE-pal binds RAR yet has a sequence very different from those of the direct repeats that constitute the RARE probe (12). A second, apparently nonspecific, complex was not fully inhibited, even with 5-fold excess competitors. In contrast to competition with the RARE and TRE-pal oligonucleotides, the RARE DNA-receptor complex was not inhibited, with even up to 5-fold excess of the CT/CGRP enhancer oligonucleotide. These results agree with the lack of binding seen with bacterially synthesized receptors. It should be noted, however, that these competition studies do not rule out the possibility that RAR may be tightly associated with another protein that can recognize the CT/CGRP enhancer but not the RARE motif.

7 VOL. 13, 1993 RETINOIC ACID REPRESSION OF HLH-OCTAMER SYNERGY 685 Extract: Control Rletinoic Acid Competitor: v) 6 c~ I z I I Z fli c (n c c LU (.) C) o M -j m m 8 r- '7 a CD GST-RAR/RXR z a o _ en #I*4WO 4- a --- H/OB : i i HB1 3 - _ Oct-i H/OB HB1 _ a aa zi. a A OB2 -* _ OB2 -_ FIG. 7. Retinoic acid treatment decreases binding of the HLH- OB2 complex in CA77 cell nuclear extracts. A mobility shift assay was done with equal amounts of nuclear extract (6 1Lg) prepared from CA77 cells treated with the ethanol vehicle (lanes 1 to 4) or retinoic acid (lanes 5 to 8). The extracts were preincubated before the addition of the CT/CGRP H/O enhancer (H/O Enh.) probe either without competitor oligonucleotide (lanes 1 and 5) or with a 5-fold excess of competitor oligonucleotides, including the self-competitor (lanes 2 and 6), consensus HLH (HLH cons.) motif (lanes 3 and 7), or consensus octamer (Oct cons.) motif (lanes 4 and 8). FIG. 8. Addition of RAR/RXR to CA77 nuclear extracts prevents formation of the HLH-OB2 complex. Mobility shift assays contained 4,ug of CA77 extract with the Cr/CGRP H/O enhancer probe. For binding assays, the mixtures were incubated without RAR-RXR (lane 1) or were pre-incubated with approximately.5,.125, or.25 p,g of RAR-RXR hybrid complexes (lanes 2 to 5). As a negative control, a control GST fusion protein (.5 p,g) (GST-Con) was incubated in the binding reaction mixture (lane 6). Effect of retinoic acid treatment and RAR on HLH and octamer factor binding to the CT/CGRP enhancer. We then asked whether retinoic acid treatment of the CA77 cells caused reduced binding of the HLH and octamer proteins to the CT/CGRP enhancer. To address this issue, we used electrophoretic mobility shift assays with nuclear extracts prepared from CA77 cells treated for 24 h with either retinoic acid or the ethanol vehicle. We have previously characterized several complexes that form on the radiolabeled CT/ CGRP enhancer (44). These complexes include a cell-specific octamer-binding protein (OB2), an HLH protein (HB1), a complex that contains both an HLH protein and an octamer protein (most likely OB2) (H/OB), and the ubiquitous octamer protein Oct-1. The identities of the complexes were confirmed by competition with the CT/CGRP enhancer and heterologous oligonucleotides containing known HLH and octamer-binding sites (Fig. 7). The HB1 complex is removed by the HLH competitor. The results with the octamer competitor are somewhat more complex since Oct-1 and OB2 have differential affinities for the consensus motif (44). At the concentrations of octamer competitor used in Fig. 7, the Oct-i protein is completely removed, while the OB2 protein is only partly diminished and the H/OB complex is retained. In fact, the HIOB complex is easier to visualize following removal of Oct-i. The effect of retinoic acid treatment on these complexes was then determined by using nuclear extracts prepared from retinoic acid-treated cells. The only detectable difference was a relative diminishment of the H/OB complex (Fig. 7). Densitometric scans indicated that there was about a twofold decrease in H/OB, but no significant change in OB2, HB1, or Oct-i, in the nuclear extracts from retinoic acid-treated cells. To determine whether the RAR were directly interfering with H/OB complex formation, we added bacterial synthesized GST-RAR-RXR proteins to the CA77 nuclear extractbinding reaction (Fig. 8). The addition of RAR-RXR caused a nearly complete loss of the H/OB complex. In contrast, there was no detectable effect on the level of the Oct-1 or HB1 CT/CGRP enhancer complexes. We did notice that the mobility of the OB2 complex was slightly reduced upon the addition of receptors. Although presently tentative, this finding might indicate an interaction between receptors and OB2. It should be noted that at high concentrations (.25 p,g or more), both the RAR and RXR homodimers also specifically removed the H/OB complex, but at lower concentrations (.5,g), only the RAR homodimer and the RAR-RXR heterodimer were effective (data not shown). These data further suggest that retinoic acid repression is mediated through RAR. However, the relative efficacy of the different retinoid receptors and their isoforms, and the ligand dependence of these effects, remains to be fully determined. As a control for any effect the GST moiety might have on the binding reaction, a GST-fusion protein containing a non- DNA-binding protein did not selectively reduce the H/OB complex, even at 1 times the amount of receptor protein (Fig. 8). These results confirm the observations made with the retinoic acid-treated extracts and suggest that the loss of the HLH-octamer complex is directly caused by the RAR protein. To further support the specificity of RAR interference with the H/OB complex, the GST-RAR-RXR proteins were added to a HeLa nuclear extract-binding reaction. We have previously characterized the complexes that bind to the CT/CGRP enhancer from HeLa extracts (44). As expected, two prominent complexes containing Oct-i and an HLH protein were seen. In addition, a very faint third complex

8 686 LANIGAN ET AL. was seen to comigrate with the OB2 complex of CA77 nuclear extracts. The addition of RAR and RXR homodimers, the RAR-RXR heterodimer, or the GST-control fusion protein did not have any effect on the Oct-1 or HLH complexes, as seen with the CA77 extracts. Interestingly, RAR homodimer and the RAR-RXR heterodimer, but not RXR homodimers, did inhibit the faint complex that comigrated with OB2 (data not shown). Preliminary characterization suggests that this complex belongs to the octamer protein family although it remains to be determined whether it represents a small amount of OB2. The selective interference of the RAR homodimer and RAR-RXR heterodimer supports the data that RAR and RAR-RXR specifically interrupt the H/OB complex obtained with CA77 nuclear extracts. DISCUSSION We have investigated the mechanism by which retinoic acid regulates CT/CGRP gene expression in a neural crestderived thyroid C-cell line. Retinoic acid treatment of CA77 rat thyroid C cells repressed CT/CGRP promoter activity two- to threefold. The sequences required for repression were localized to an 18-bp element within the cell-specific enhancer. The finding that repression could be transferred to a heterologous promoter indicates that the repression mediated by this region is not promoter specific. The repression was observed with dosages that correlate well with the binding affinities and activation profiles of the nuclear RAR and was enhanced by coexpression of the a-rar. The RAR can regulate gene transcription by binding specific DNA sequences that are defined by the directional arrangement and spacing of AGGTCA sites (2, 5). However, the CT/ CGRP enhancer does not contain a consensus RARE and lacked any detectable receptor-binding activity, indicating that the inhibition involves protein-protein contacts. The receptors can interact with additional cellular factors, including AP1 cell-specific factors (11, 4, 48, 5). The best characterized of these factors are the RXR proteins, which act as heterodimeric partners with the RAR (16, 21, 51, 52). These studies highlight the capacity of RAR to interact with different proteins. On the basis of the colocalization of retinoic acid repression with the CT/CGRP cell-specific enhancer, we predicted that the repression would also be cell specific. This prediction was confirmed by demonstrating that repression did not occur in cells lacking CT/CGRP HLH-octamer enhancer activity. The significance of these sites for retinoic acid repression was demonstrated by the lack of repression seen with the HLH and octamer point mutants. These results, in combination with the in vitro competition studies, demonstrate that retinoic acid repression requires the HLH-octamer factors that independently bind the CT/CGRP 18-bp enhancer to synergistically activate transcription. We have recently shown that these factors include a cell-specific octamer protein, referred to as OB2 (44). The octamer proteins are a growing family of cell-specific transcription factors that play important roles in development (37). In the case of the CT/CGRP enhancer, we have proposed that retinoic acid blocks the OB2-HLH synergism required for enhancer activation (discussed below) (Fig. 9). Relatively little is known about the mechanisms by which retinoids repress gene transcription. In some systems, repression has been shown to be due to DNA-binding activity (14, 24). Retinoic acid repression has also been shown to be mediated by repression of stimulatory factors in the absence ~~RAR/RXR MOL. CELL. BIOL. SYNERGISMAR HLH O B 2 FIG. 9. Model of cell-specific CT/CGRP enhancer activation and repression. A schematic representation of the CT/CGRP enhancer shows that synergism between an HLH protein and the OB2 octamer protein is required for cell-specific activation. RAR (shown as an RAR-RXR heterodimer) repress the CT/CGRP gene in a cell-specific manner by inhibiting the combinatorial association of the HLH and OB2 proteins, possibly by binding to the OB2 protein. of receptor-dna binding, similar to our observations of the CT/CGRP gene. This type of repression involving inactivation of enhancers is an increasingly common mechanism seen in a number of systems (2, 22). It is particularly interesting that retinoic acid repression of the interleukin-2 gene is mediated at an octamer motif (9). However, the repression mechanism was not clear since retinoic acid treatment did not decrease octamer binding. Likewise, the gene encoding Oct-3 is repressed by retinoic acid action at a 243-bp stem cell-specific enhancer, yet retinoic acid treatment did not affect the DNA-protein binding pattern (3). RAR have also been shown to interfere with AP1 (Fos/Jun) activation of the stromelysin (29) and collagenase (18) genes. Repression of collagenase gene transcription does not involve binding of the RAR to the AP1 site but does reduce binding of the APi/Jun protein, suggesting the formation of nonproductive receptor-jun heterodimers (4, 5). Our working model is that RAR block the cell-specific combinatorial association of the HLH and OB2 factors (Fig. 9). How might this inhibition be occurring? One possibility is that the receptor binds to one of the enhancer proteins and that this association sterically hinders binding of the adjacent protein. This would account for the selective loss of the HLH-octamer complex but not the individual octamer and HLH complexes in the mobility shift assays. This model lays the foundation for investigating the precise mechanism, such as the relative contributions of RAR and RXR and ligand dependency, and, most interesting, for testing the prediction that the receptors bind OB2. The prediction that OB2 interacts with RAR-RXR is consistent with studies showing that octamer proteins (and other POU family members) can interact with coactivators (25, 45, 46), including glucocorticoid receptors (3, 17, 47). Glucocorticoid receptors can physically associate with the Oct-1 homeo domain (17). Interactions between the receptors and the Oct-1 and Oct-2A proteins can increase transcription when a glucocorticoidresponsive element is nearby (3, 47) and repress transcription in the absence of a receptor-binding site (17, 47). Interestingly, we have recently localized the sequences required for cell-specific glucocorticoid repression of the CT/CGRP gene to the same 18-bp element that confers retinoic acid repression (43). Repression of enhancer activity by members of the steroid-retinoid receptor superfamily appears to be an emerging theme. It seems probable that different members of the family may act by similar mechanisms. This prediction is supported by the ability of both glucocorticoid receptors and RAR to repress gene activation by AP1 (7, 15, 18, 29, 38-4,

9 VOL. 13, 1993 RETINOIC ACID REPRESSION OF HLH-OCTAMER SYNERGY , 5) and octamer factors (9, 17, 47, and this report). In addition, the RAR and thyroid hormone and vitamin D receptors share structural motifs, as witnessed by their heterodimerization with a common partner (RXR) (16, 21, 51, 52). Consequently, it is an intriguing correlation not only that the CT/CGRP HLH-octamer enhancer is repressed by retinoic acid and glucocorticoids but also that CT/CGRP transcription is inhibited by vitamin D in vivo (28). These observations suggest that repression of CT/CGRP transcription by several different agents may converge into a common mechanism involving inhibition of cell-specific octamer- HLH synergism. Since members of both the octamer and HLH families play critical roles in development, we predict that retinoic acid repression of cell-specific synergism may underlie some of the activities of retinoids in development, including neural crest cell migration and differentiation. 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