of gene expression, and in particular, the repression of gene ... threefold decrease in calcitonin/calcitonin gene-related pep- ...... Karin, and R L. Wilder. 1990.
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CELLULAR BIOLOGY, Oct. 1993, p. 6079-6088
Vol. 13, No. 10
0270-7306/93/106079-10$02.00/0 Copyright © 1993, American Society for Microbiology
Retinoic Acid Repression of Cell-Specific Helix-Loop-HelixOctamer 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 52242 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 0.2 nM), and the additional repression caused by cotransfection of an 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 of repression. 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 (20). Retinoic acid has an inhibitory effect on neural crest cell migration and can influence the
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.
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) (10, 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 *
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)-10% fetal bovine serum
Corresponding author. 6079
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(Hyclone, Logan, Utah); 44-2C cells were maintained in DMEM (high glucose)-1% equine serum-0.1% L-glutamine; and HeLa cells were maintained in Ham's F12-10% fetal bovine serum. In addition, 100 U of penicillin per ml and 100 ,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'-GGACGCTGTGCAAATCCT3'). 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 106 to 5 x 106 cells in 0.8 ml were transfected with supercoiled DNA by electroporation with a Bio-Rad gene pulser apparatus at 200 V for the CA77 cells, 220 V for the HeLa cells, and 260 V for the 44-2C cells, all with a capacitance of 960 ,iF. The same amount of DNA (20 ,ug) was used per cuvette unless otherwise indicated. For the a-RXR cotransfection assays, 10 ,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 30 ,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 60-mm dishes and treated with 1 ,uM retinoic acid (Sigma) or the vehicle (0.01% ethanol). About 50 to 80% 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 50 ,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 10% 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 10 pmol of DNA was labeled with [al-32P]dATP (25 ,uCi, 3000 Ci/mmol) by using Klenow DNA polymerase. The complementary RARE oligonucleotides (5'-GGGTAGGGTICACCGAAAGTTCACTCG-3') (11) (10 pmol) were labeled by phosphorylation with T4 kinase with [y-32P]ATP (20 ,uCi, 3000 Ci/mmol), annealed, and purified through a Sephadex G-50 column. The binding reaction mixture (20 RlI) contained 0.02 pmol of labeled oligonucleotide (generally 50,000 to 100,000 cpm), 3 to 6 ,ug of nuclear extract from CA77 cells or 3 ,ug from HeLa cells, and binding buffer (10 mM Tris [pH 7.5], 5% glycerol, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol). Poly(dI-dC) (0.1 p.g) (Boehringer Mannheim) and 0.1 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 0.25 x TBE (Tris-borateEDTA [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 0.5 to 0.05 ,g/pl (diluted as needed in binding buffer with 100 ,ug of bovine serum albumin per ml) and incubated on ice for 30 min to allow heterodimerization. For DNA-binding assays, the receptor protein (0.5 to 0.05 p,g for CA77 extracts and 0.05 ,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 10 to 15 min and analyzed as described above. RESULTS acid Retinoic 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 920 bp or less were not repressed by retinoic acid. This result suggested that the retinoic acid responsive element (RARE) lies between 920 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
VOL. 13, 1993
RETINOIC ACID REPRESSION OF HLH-OCTAMER SYNERGY
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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 (1125-920 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 50 p.g of extract are reported, except CMV-luc activity, which is reported per 10 p.g of extract.
with this, we observed that the basal activity of fusion genes containing the 1,125- to 920-bp region was about 10- to 20-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 5,000 light units above the background of about 100 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 920-bp fragment was linked to the TK promoter-luciferase gene (1125920-TK-luc) and transfected into the CA77 cells. As expected, the basal activity of TK-luc was enhanced about 30-fold by the 1,125- to 920-bp region, confirming the presence of enhancer activity. Treatment of the cells with retinoic acid caused a three- to fourfold repression in 1125-
920-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 100 nM retinoic acid. The amount of retinoic acid required for half-maximal repression was about 0.2 nM. This correlates well with previous measurements of the nuclear RAR-binding affinities (Kd, 0.2 nM) (48), suggesting that retinoic acid is working through RAR, rather than a-RXR, which requires at least 10-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 (10, 32). Retinal repressed the CT/CGRP promoter activity only at concentrations of 100 nM or greater, and half-maximal repression occurred at 0.3 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 50 nM) after several days of treatment. In contrast, much greater levels of retinal than retinoic acid are
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LANIGAN ET AL.
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