Cell, Vol. 63, 729-738,
November
16, 1990, Copyright
0 1990 by Cell Press
Multiple Cell Type-Specific Proteins Differentially Regulate Target Sequence Recognition by the a Retinoic Acid Receptor Christopher K. Glass,” Orly V. Devary,t* and Michael G. Rosenfeldt* l Division of Cellular and Molecular Medicine Department of Medicine t Eukaryotic Regulatory Biology Program *Howard Hughes Medical Institute University of California, San Diego School of Medicine La Jolla, California 92093-0648
Summary Retinoic acid receptors appear to exert profound effects on vertebrate development by regulating the transcription of distinct sets of target genes within different cell types. Several lines of evidence are presented for the existence of multiple, cell type-specific nuclear proteins that function to differentially increase the binding affinity of the a retinoic acid receptor for a variety of response elements. These proteins, which we refer to as retinoic acid receptor coregulators, interact with the retinoic acid receptor via a common dimerization interface that overlaps with its ligand binding domain. These observations raise the intriguing possibility that coregulator proteins serve to restrict and/or direct the effects of retinoic acid receptors on patterns of gene expression during development. Introduction The vitamin A derivative retinoic acid has been implicated in playing a fundamental role in regulating the differentiation of many endodermally, ectodermally, and mesodermally derived tissues (Lotan, 1980; Sporn and Roberts, 1984; Brockes, 1989; and references therein). The direct effects of retinoic acid on patterns of gene expression have been proposed to be mediated by nuclear receptor proteins that are members of the thyroid and steroid hormone superfamily of transcriptional regulators (Giguere et al., 1987; Petkovich et al., 1987). All members of this superfamily are defined by a highly conserved DNA binding domain predicted to form zinc finger structures, and by a less well-conserved domain that is required for ligand binding and regulation of receptor activity (reviewed by Evans, 1988; Beato, 1989). Three distinct retinoic acid receptor genes have been identified, termed a (Giguere et al., 1987; Petkovich et al., 1987), 8 (Brand et al., 1988; Benbrook et al., 1988), and y (Krust et al., 1989; lshikawa et al., 1990). Initial studies of the transcriptional properties of retinoic acid receptors were limited by the lack of characterized retinoic acid-inducible genes and response elements. However, the a retinoic acid receptor was found to activate efficiently transcription from promoters containing certain T3 response elements (TREs), particularly a palindromic variant of the major TRE of the rat growth hormone gene
(Umesono et al., 1988). Although the retinoic acid receptor alone exhibited a relatively low affinity for this element in vitro, high affinity binding was observed upon addition of an F9 teratocarcinoma whnle-cell extract. Further analysis of this effect led to the demonstration that the thyroid hormone receptor could mimic the effects of cell extracts to enhance retinoic acid receptor binding to TREs, increasing its binding affinity by >lO-fold (Glass et al., 1989). DNA binding and cross-linking experiments indicated that the basis for this interaction was the formation of a T3 receptor-retinoic acid receptor heterodimer. Subsequent analysis of retinoic acid-inducible genes, including the laminin Bl (Vasios et al., 1989) and 8 retinoic acid receptor genes (de The et al., 1990; Sucov et al., 1990) has allowed the characterization of naturally occurring retinoic acid response elements. These elements contain two or more degenerate copies of the motif GGTCA, which is also present in estrogen and thyroid hormone response elements, but the orientation, spatial relationship, and precise sequence composition of this motif vary in each element (Figure 1A). DNA binding studies using mutant 8 retinoic acid receptor response elements have demonstrated that both of the tandem GTTCA motifs present in this response element are essential for retinoic acid receptor binding (Sucov et al., 1990). In this article, we present evidence for the existence of multiple proteins, distinct in different cell types, that interact with the a retinoic acid receptor to modulate its binding affinities for several types of response element sequences. The interaction between the retinoic acid receptor and these nuclear proteins is mediated by an extensive C-terminal domain that is required for efficient trans-activation in vivo. These results raise the possibility that a potentially large number of nuclear proteins, which we term retinoic acid receptor coregulators, serve to determine the sets of target genes acted upon by retinoic acid receptors, constituting a combinatorial code influencing the effects of retinoic acid on cell-specific patterns of gene expression. Results Cell Type-Specific Nuclear Factors Differentially Modulate the DNA Binding Properties of the a Retlnoic Acid Receptor Protein-DNA complexes containing the retinoic acid receptor were quantitated by precipitation of 35S-labeled receptor using biotinylated DNA probes (Glass et al., 1987, 1988). Four biotinylated response element sequences were utilized for the majority of these studies: a naturally occurring TRE derived from the rat a myosin heavy chain gene (TRE-mhc; lzumo and Mahdavi, 1988); a palindromic variant of the rat growth hormone TRE (TRE-pal; Glass et al., 1988); a retinoic acid response element from the laminin Bl gene (RARE-lam; Vasios et al., 1989); and a retinoic acid response element from the B retinoic acid receptor gene (RARE-B; de The et al., 1990). The TRE-pal, RARE-lam, and RARE-8 sequences have been demon-
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Figure 1. Binding of CI Retinoic Acid Receptor to TJ and Retinoic Acid Response Elements in the Absence and Presence of HeLa Nuclear Extract (A) Sequences of response elements used in these studies. TRE-pal is a palindromic variant of the rat growth hormone gene TRE (Glass et al., 1988). TRE-mhc is a TRE residing from -152 to-125 bp from the transcriptional start site of the rat a-myosin heavy chain gene (lzumo and Mahdavi, 1999). RARE-D is a retinoic acid response element residing from -59 to -33 bp upstream from the transcriptional start site of the p retinoic acid receptor gene (de Th6 et al., 1990). RARE-lam is a retinoic acid response element residing from -447 to -422 bp upstream of the transcriptional start site of the laminin Bl gene (Vasios et al., 1999). Each of the sequences shown was flanked by 10 bp at each end containing biotin-11-dUTP residues as described in Experimental Procedures. (6) Binding of 35S-labeled a retinoic acid receptor to the biotinylated TRE-pal probe as a function of increasing TRE-pal concentration in the presence (filled circles) and absence (open circles) of HeLa nuclear extract. The negative reciprocal of the slope derived from plots of bound/free versus bound DNA (right) indicates a binding constant of the retinoic acid receptor of 4 m M for this site in the presence of HeLa nuclear extract. (C) Same experiment as in (B), except that the RARE-B was used as a probe. The binding constants of the a retinoic acid receptor for this element in the presence (filled circles) and absence (open circles) of HeLa nuclear extract were 0.9 m M and 11 mM, respectively.
strated to confer retinoic acid responsiveness to otherwise unregulated promoters by gene transfer experiments. In contrast, the retinoic acid receptor binds with high affinity to the TRE-mhc sequence as a heterodimer with the T3 receptor, but acts to inhibit TBdependent transcription from promoters containing this element in Green monkey kidney (CVl) cells (Glass et al., 1989). The binding of %-labeled a retinoic acid receptor to the TRE-pal and RARE-P sequences as a function of DNA concentration is illustrated in Figures 18 and 1C. In the absence of additional nuclear proteins, the affinity of the retinoic acid receptor for the RARE-P was approximately 11 nM. Its affinity for the TRE-pal was too low to be determined accurately over the same concentration range of DNA. In the presence of HeLa nuclear proteins, the binding of 35S-labeled a retinoic acid receptor to both classes of response elements was significantly increased, particularly at low DNA concentrations. Under these conditions, the binding affinities of in vitro translated a retinoic acid receptor for the RARE-P and TRE-pal sequences were 0.8 mM and 4 nM, respectively. Activities that enhanced retinoic acid receptor binding to T3 and retinoic acid response elements were observed in all cell types of vertebrate origin examined. Figure 2A illustrates the effects of nuclear extracts, derived from a variety of cell types, on retinoic acid receptor binding to the TRE-pal when preincubated with the receptor at equivalent levels of nuclear protein. The effects of these factors were specific for T3 and retinoic acid response elements; enhancement of binding to unrelated &active elements, such as one derived from adenovirus 5 (Ad5; Figure 2B), or other unrelated c&active elements was not observed (data not shown). Intriguingly, differences were observed among the various types of nuclear extracts with respect to their relative effects on retinoic acid receptor binding to T3 and retinoic acid response elements. For example, the binding affinity of the retinoic acid receptor for the TRE-pal was higher than for the RARE-P sequence in the presence of HL60 cell nuclear extracts (0.8 mM and 2.0 nM, respectively), which is the opposite of the hierarchy observed in the presence of HeLa nuclear proteins. The activities present in HeLa and HL60 cells were also distinct from the effects of the p T3 receptor on retinoic acid binding. The p T3 receptor preferentially stimulated retinoic acid receptor binding to the TRE-mhc sequence, as previously reported (Glass et al., 1989), but had no effect on the binding of the retinoic acid receptor to the RARE-lam (Figure 28) or the RARE-P (data not shown). These observations suggested that a variety of cellspecific proteins might interact with the retinoic acid receptor. To obtain more direct evidence for the existence of cell-specific factors, cross-linking experiments were performed with the retinoic acid receptor bound to T3 and retinoic acid response elements in the presence or absence of nuclear extracts prepared from several different cell types. After precipitation of protein-DNA complexes, the cross-linking agent disuccinimidyl suberate (DSS) was added and the products were analyzed by SDS-polyacrylamide gel electrophoresis. As illustrated in Figure 3,
Retinoic Acid Receptor Coregulators 731
-
Figure 2. Factors That Enhance Retinoic Acid Receptor Binding to Specific DNA Sequences Are Ubiquitous (A) Binding of 35S-labelsd a retinoic acid receptor to 100 fmol of the biotinylated TRE-pal sequence was assayed in the absence of nuclear proteins (buffer) or in the presence of 100 ug of nuclear extracts derived from the indicated cell types. (B) Comparison of the effects of HsLa cell nuclear factors and the 3 T3 receptor on the binding of the a retinoic acid receptor to T3 and rstinoic acid response elements. Binding of 35S-labelsd a rstinoic acid receptor to the TRE-pal, RARE-lam, TRE-mhc, and an unrelated cisactive element from adenovirus 5, Ad5, is shown in the absence of additional factors (RAR alone), in the presence of HeLa nuclear extract (RAR + HeLa), and in the presence of an unlabeled in vitro translation product of the b T3 receptor (RAR + TSR beta).
cross-linking products containing the retinoic acid receptor were observed only in samples in which the receptor was incubated with nuclear extract prior to DNA binding. The cross-linked products migrated as multiple bands in the molecular weight range from 95-116 kd. The various nuclear extracts produced cross-linked products that exhibited distinctly different patterns of molecular weights. For example, HeLa cell nuclear extracts produced major cross-linked species of 106 and 116 kd. Subtracting the molecular weight of the retinoic acid receptor (51 -kd) provided molecular weight estimates of the cross-linked proteins in HeLa cells to be 55 kd and 65 kd, hereafter referred to as Hp55 and Hpss. In contrast, cross-linking performed in the presence of HL60 nuclear extract resulted in a major product containing a protein with an apparent molecular weight of approximately 45 kd.
Figure 3. Cross-Linking Experiments Identify Cell Typs-Specific Nuclear Proteins That Interact with the Astinoic Acid Receptor on T3 and Retinoic Acid Response Elements (A) %-labeled rstinoic acid receptor was preincubated with binding buffer alone or approximately 100 ug of nuclear protein extracted from HeLa cells, pituitary tumor cells (GC and 235) Green monkey kidney cells (CVl), mouse lymphoma cells (UO A2), F9 tsratocarcinoma cells, and promyslocytic leukemia cells (HL60). DNA binding reactions were then performed using the RARE-3 probe as described in Figure 1 and Experimental Procedures. Following precipitation of protein-DNA complexes with streptavidin-agaross and extensive washing, the protein cross-linking agent DSS was added at a final concentration of 1 mM. After a 10 min reaction, the samples were analyzed by SDS-polyacrylamide gel electrophoresis. (9) Identical experiment to that in (A), except that the TRE-pal sequence was used in place of the RARE-8 sequence. Arrows denote the following molecular weight standards: myosin heavy chain (200 kd), phosphorylase A (98 kd), bovine serum albumin (66 kd), and ovalbumin (44 kd).
These results provided independent evidence for the existence of a potentially large number of distinct and cell type-specific nuclear factors that interact with the a retinoic acid receptor. Comparison of the cross-linked products obtained on the TRE-pal to those obtained on the RARE-b element generally revealed a similar pattern for each particular nuclear extract. However, some extracts exhibited a preference for cross-linking on one of the two elements. For example, cross-linked products were preferentially obtained on the TRE-pal sequence in the presence of HL60 nuclear extract, but preferentially on the RARE-6 sequence in the presence of HeLa nuclear extracts (compare Figure 3A with Figure 38). These differential cross-linking patterns also correlated with the relative affinities of the retinoic acid receptor for the two response elements in the presence of HeLa and HL60 extracts, as described above. Cell fractionation studies indicated that these factors were preferentially localized to the nucleus and required 0.3-0.5 M KCI for efficient extraction. These activities were trypsin sensitive and in all cases could be abolished by
heating at W C for 10 min. These factors were also present in limiting quantities because a 2-fold dilution of each extract generally resulted in an approximate 2-fold decrease in stimulated retinoic acid receptor binding. The ability of nuclear proteins to be cross-linked to the retinoic acid receptor was not reproducibly affected by the addition of retinoic acid (data not shown). However, because reticulocyte lysates used for translation in vitro may contain sufficient retinoic acid to occupy the majority of receptors, dependence of the interaction between the receptor and these proteins on retinoic acid cannot as yet be formally excluded. Fractionation of HeLa nuclear proteins by gel filtration on Superose 6 under high salt conditions was performed to confirm the molecular weight range of proteins interacting with the retinoic acid receptor. The activity profile eluted as an asymmetric peak in the molecular weight range between 55 and 70 kd (Figure 4A), in agreement with the molecular weight range of 55-66 kd predicted on the basis of cross-linking experiments. This activity could be further purified by sequence-specific DNA affinity chromatography on a column containing concatamerized TRE-pal oligonucleotide elements. As shown in Figure 46, passage of HeLa nuclear extract over this column resulted in nearly complete removal of the activity from the flowthrough fraction, while the majority of other nuclear proteins (>95%) passed through the column. After extensive washing, the majority of the initial activity could be recovered by elution of the column with 0.4 M KCI, resulting in an increase in specific activity by more than a factor of 40. The binding of HeLa nuclear factors to this column was sequence specific and of high affinity, because it was performed in the presence of an approximately lo-fold molar excess of salmon sperm DNA and poly(dl-dC). Similar results were obtained using nuclear extracts derived from F9 teratocarcinoma cells, HL60 cells, and WI cells (data not shown). These experiments suggest that, like the TS receptor, other nuclear factors that interact with the retinoic acid receptor also have intrinsic DNA binding properties. Retinoic Acid Receptor Sequences Required for Protein-Protein Interaction Overlap with the Ligand Binding Domain To identify sequences of the retinoic acid receptor necessary for its interactions with cell type-specific nuclear factors on T3 and retinoic acid response elements, the properties of mutant receptors were examined (Figure 5). Deletion of the C-terminus at codon 404 had little or no effect on enhanced DNA binding in the presence of HeLa nuclear extracts (Figure 5A). However, C-terminal deletions ending at codons 351,260, and 204 exhibited no enhanced binding in the presence of HeLa nuclear extracts and demonstrated a slight reduction in basal DNA binding. As expected, the retinoic acid receptor C-terminus (corresponding to codons 167 to 461) was unable to bind to DNA in the presence or absence of HeLa nuclear extract, nor was a point mutant @AR-De*) in which the first conserved cysteine of the DNA binding domain was changed to aspartic acid. Cross-linking experiments per-
Figure 4. Partial Purification of Factors That Interact with the Retinoic Acid Receptor from HeLa Nuclear Extracts (A) Partial purification of factors by gel filtration on a Superose 6 column under high salt conditions (0.4 M KU). Fractions were diluted 4-fold to reduce the KCI concentration to 0.1 M, and tested for their ability to stimulate retinoic acid receptor binding to the TRE-pal (hatched line). The solid line indicates total protein as measured by OC&,. Arrows denote the elution position of the following molecular weight standards: T, thyroglobulin (670 kd); G, y-globulin (158 kd); 0, ovalbumin (44 kd); and M, myoglobulin (17 kd). (B) Partial purification of factors by sequence-specific DNA affinity chromatography. One milliliter of HeLa nuclear extract in binding buffer (20 m M HEPES [pH 7.81, 20% glycerol, 50 m M KCI, 1 m M j3-mercaptoethanol, and 0.1 Nonidet P-40) was combined with 50 ug of salmon sperm DNA and 100 ug of poly(dl-dC) and passed three times through a column containing 1 ml of a DNA affinity matrix consisting of approximately 20 ug of concatamerized TRE-pal covalently linked to Sepharose CL4B. The column was then washed with ten column volumes of binding buffer and eluted with a step gradient of binding buffer adjusted to 0.4 M KCI. Fractions of approximately 0.3 ml were collected and diluted to a final KCI concentration of 100 mM. Crude nuclear extract, the flowthrough fractions, and the fractions eluted with 0.4 M KCI (fractions l-4) were then tested for their ability to activate retinoic acid receptor binding to the TRE-pal. Details of both chromatographic steps are described under Experimental Procedures.
formed with C-terminal deletion mutants demonstrated that enhancement of DNA binding in the presence of HeLa nuclear extracts was correlated precisely with their ability to be cross-linked to Hp55 and Hp65 (Figure 58). These experiments indicate that Hp55 and Hpm mediate the effects of HeLa nuclear extracts on the DNA binding properties of the a retinoic acid receptor and define the
Retinoic Acid Receptor Coregulators 733
!
7
Figure 6. The Protein-Protein Interaction Domain of the Retinoic Acid Receptor Overlaps with the Ligand Binding Domain
Figure 5. Nuclear Factors That Enhance the Binding of the Retinoic Acid Receptor 10 T3 and Retinoic Acid Response Elements Interact with the Retinoic Acid Receptor C-Terminus (A) Influence of HeLa nuclear factors on the binding of wild-type and mutant a retinoic acid receptors to the palindromic TRE. RAR-404, RAR-351, and RAR-204 correspond to C-terminal deletions ending at amino acids 404, 351, and 204, respectively. RAR-N166 represents an amino-terminal deletion in which amino acids 2165 have been removed, including the DNA binding domain. RAR-D* represents a mutant receptor in which the first conserved cysteine of the DNA binding domain has been changed to aspartic acid. In each binding reaction, 100,000 cpm of TCA-precipitable [%]receptor was used for the wild-type and mutant receptors. Binding reactions were performed in the absence or presence of 100 pg of crude HeLa nuclear extract. (B) Cross-linking of HeLa nuclear factors to wild-type and C-terminal deletion mutants of the retinoic acid receptor bound to the palindromic TRE. DNA binding and cross-linking reactions were performed as described in Figure 3 and Experimental Procedures.
C-terminal border of the protein-protein interaction domain to be at or near codon 404. Similar results were observed using nuclear extracts derived from CVl, GC, and HL60 cells (data not shown). No enhancement of DNA binding was observed for the retinoic acid receptor truncated at amino acid 351 in the presence of these extracts. Furthermore, no cross-linked products were obtained in the presence of DSS. These results indicate that all of the proteins observed
to interact
with the retinoic acid receptor to enhance its binding to
(A) Cross-linking experiments define an extensive interaction domain. Rabbit reticulocyte lysate containing 100,000 cpm of [%]reUnoic acid receptor or an equivalent amount of the indicated deletion mutants was incubated with 100 ug of HeLa nuclear protein for 30 min. The samples were desalted over a G-25 column equilibrated in 50 m M HEPES (pH 7.6) to remove free methionine and to dilute nonspecific proteins. One microliter of 50 m M DSS was then added to 50 ~1 of the void fraction from each column and reacted for 15 min at room temperature. The reaction was quenched by addition of 5 pl of 1 M ethanolanine. Samples were analyzed by SDS-PAGE on 8% (left panel) or 12.5% (right panel) polyacrylamide gels. Wild-type RAR, lanes l-5; RAR-N167, lane 6; RAR 167-404, lane 7; RAR 246-404, lane 8. Migration positions of the monomeric receptors are shown on the right of each panel. (6) Summary of sequences required for basal DNA binding, enhanced DNA binding, and protein-protein interaction with Hp55 and Hpss,
T3 and retinoic acid response elements do so via a common interface. To define the N-terminal border of this interaction domain, cross-linking experiments were performed in solution with the wild-type
retinoic acid receptor
and a series
of N-terminal deletion mutants (Figure 6A). The wild-type receptor could be efficiently cross-linked to both Hp55 and Hpss in solution, indicating that the receptor can associate with each of these proteins prior to DNA binding. Although unable to bind to DNA, the retinoic acid receptor C-terminus from codon 187 to 461 retained the ability to be cross-linked to both Hps5 and HP~~, indicating that the DNA binding domain was not essential for its interaction with these two factors. Concomitant with the C-terminal
Cdl
734
deletion data, a double deletion mutant of the retinoic acid receptor consisting of amino acids 167 to 404 also retained the ability to be cross-linked to Hp55 and Hps. However, further truncation of the N-terminus to codon 246 severely compromised the interaction with both proteins. The results of these experiments, which are summarized in Figure 66, indicate that a large region of the retinoic acid receptor C-terminus, extending from approximately codons 167 to 404, is involved in protein-protein interactions with Hp55 and Hp65. The Retinoic Acid Receptor Protein-Protein Interaction and Ligand Binding Domains Are Essential for Efficient Ransactivation In Vivo The DNA binding studies described above lead to the prediction that retinoic acid receptors lacking the protein-protein interactionlligand binding domain should exhibit compromised transcriptional activity in vivo because of reduced affinities for their cognate response element sequences. This behavior would distinguish the retinoic acid receptor from the glucocorticoid receptor, which has been demonstrated to activate constitutively transcription upon removal of its ligand binding domain (Hollenberg et al., 1967; Godowski et al., 1966a). We therefore performed a series of experiments to examine the transcriptional activities of retinoic acid receptor mutants lacking the interaction domain. In one type of experiment, increasing amounts of plasmids directing the expression of the wildtype a retinoic acid receptor or a mutant receptor lacking the entire interaction and ligand binding domain were cotransfected into CVl cells. The transcriptional activities of these receptors were assayed using a variant mouse mammary tumor virus (MTV) promoter plasmid containing the TRE-pal in place of sequences containing glucocorticoid response elements (Umesono et al., 1966). This MTV promoter variant was linked to the chloramphenicol acyltransferase gene to facilitate analysis of promoter expression. As shown in Figure 7A, the wild-type a retinoic acid receptor conferred an efficient transcriptional response that was near maximal at 1 m of transfected expression plasmid. In contrast, the truncated retinoic acid receptor was transcriptionally inactive at this concentration of expression plasmid, and exhibited only a weak, constitutive transcriptional effect when 10 vg of expression plasmid was used. The failure of the truncated retinoic acid receptor to efficiently activate transcription could reflect decreased DNA binding affinity, as suggested by in vitro binding experiments, but could also be due to the removal of C-terminal sequences that function as a Pans-activation domain. To examine this question further, we tested the ability of the retinoic acid receptor’s DNA binding domain to mediate transcriptional activation by a potent, heterologous transactivation domain. To do this, we transferred the major trans-activation (tau) domain of the glucocorticoid receptor to the N-terminus of the truncated retinoic acid receptor (RAR-Glau-204). In contrast with previous studies demonstrating that the glucocorticoid receptor tau domain can transfer additional transcriptional activity to the
RA
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RSV-RARLJ 204
I
Figure 7. The C-Terminal Interaction and Ligand Binding Domain of the Retinoic Acid Receptor Is Required for Efficient 7-ans-Activation of Responsive Promoters In Vivo (A) Activity of the MTWRE pal promoter as a function of increasing amounts of cotransfected plasmids directing the expression of the wild-type a retinoic acid receptor (RBV-RAR) or the a retlnoic acid receptor truncated at amino acid 204 (RW-RAR A204). (6) Comparison of the transcriptional adivities of the wild-type and truncated retinoic acid receptors with the activities observed when linked in frame at the N-terminus to the major trans-activation domain of the glucocorticoid receptor. One microgram of each expression vector was used per plate. Retinoic acid receptor-dependent promoter expression was measured using the HBWK promoter linked to the firefly luciferase cDNA in which two copies of the TRE-pal sequence were placed 100 bp upstream of the transcriptional start site. Results are the average of duplicate points (differing
November
16, 1990, Copyright
0 1990 by Cell Press
Multiple Cell Type-Specific Proteins Differentially Regulate Target Sequence Recognition by the a Retinoic Acid Receptor Christopher K. Glass,” Orly V. Devary,t* and Michael G. Rosenfeldt* l Division of Cellular and Molecular Medicine Department of Medicine t Eukaryotic Regulatory Biology Program *Howard Hughes Medical Institute University of California, San Diego School of Medicine La Jolla, California 92093-0648
Summary Retinoic acid receptors appear to exert profound effects on vertebrate development by regulating the transcription of distinct sets of target genes within different cell types. Several lines of evidence are presented for the existence of multiple, cell type-specific nuclear proteins that function to differentially increase the binding affinity of the a retinoic acid receptor for a variety of response elements. These proteins, which we refer to as retinoic acid receptor coregulators, interact with the retinoic acid receptor via a common dimerization interface that overlaps with its ligand binding domain. These observations raise the intriguing possibility that coregulator proteins serve to restrict and/or direct the effects of retinoic acid receptors on patterns of gene expression during development. Introduction The vitamin A derivative retinoic acid has been implicated in playing a fundamental role in regulating the differentiation of many endodermally, ectodermally, and mesodermally derived tissues (Lotan, 1980; Sporn and Roberts, 1984; Brockes, 1989; and references therein). The direct effects of retinoic acid on patterns of gene expression have been proposed to be mediated by nuclear receptor proteins that are members of the thyroid and steroid hormone superfamily of transcriptional regulators (Giguere et al., 1987; Petkovich et al., 1987). All members of this superfamily are defined by a highly conserved DNA binding domain predicted to form zinc finger structures, and by a less well-conserved domain that is required for ligand binding and regulation of receptor activity (reviewed by Evans, 1988; Beato, 1989). Three distinct retinoic acid receptor genes have been identified, termed a (Giguere et al., 1987; Petkovich et al., 1987), 8 (Brand et al., 1988; Benbrook et al., 1988), and y (Krust et al., 1989; lshikawa et al., 1990). Initial studies of the transcriptional properties of retinoic acid receptors were limited by the lack of characterized retinoic acid-inducible genes and response elements. However, the a retinoic acid receptor was found to activate efficiently transcription from promoters containing certain T3 response elements (TREs), particularly a palindromic variant of the major TRE of the rat growth hormone gene
(Umesono et al., 1988). Although the retinoic acid receptor alone exhibited a relatively low affinity for this element in vitro, high affinity binding was observed upon addition of an F9 teratocarcinoma whnle-cell extract. Further analysis of this effect led to the demonstration that the thyroid hormone receptor could mimic the effects of cell extracts to enhance retinoic acid receptor binding to TREs, increasing its binding affinity by >lO-fold (Glass et al., 1989). DNA binding and cross-linking experiments indicated that the basis for this interaction was the formation of a T3 receptor-retinoic acid receptor heterodimer. Subsequent analysis of retinoic acid-inducible genes, including the laminin Bl (Vasios et al., 1989) and 8 retinoic acid receptor genes (de The et al., 1990; Sucov et al., 1990) has allowed the characterization of naturally occurring retinoic acid response elements. These elements contain two or more degenerate copies of the motif GGTCA, which is also present in estrogen and thyroid hormone response elements, but the orientation, spatial relationship, and precise sequence composition of this motif vary in each element (Figure 1A). DNA binding studies using mutant 8 retinoic acid receptor response elements have demonstrated that both of the tandem GTTCA motifs present in this response element are essential for retinoic acid receptor binding (Sucov et al., 1990). In this article, we present evidence for the existence of multiple proteins, distinct in different cell types, that interact with the a retinoic acid receptor to modulate its binding affinities for several types of response element sequences. The interaction between the retinoic acid receptor and these nuclear proteins is mediated by an extensive C-terminal domain that is required for efficient trans-activation in vivo. These results raise the possibility that a potentially large number of nuclear proteins, which we term retinoic acid receptor coregulators, serve to determine the sets of target genes acted upon by retinoic acid receptors, constituting a combinatorial code influencing the effects of retinoic acid on cell-specific patterns of gene expression. Results Cell Type-Specific Nuclear Factors Differentially Modulate the DNA Binding Properties of the a Retlnoic Acid Receptor Protein-DNA complexes containing the retinoic acid receptor were quantitated by precipitation of 35S-labeled receptor using biotinylated DNA probes (Glass et al., 1987, 1988). Four biotinylated response element sequences were utilized for the majority of these studies: a naturally occurring TRE derived from the rat a myosin heavy chain gene (TRE-mhc; lzumo and Mahdavi, 1988); a palindromic variant of the rat growth hormone TRE (TRE-pal; Glass et al., 1988); a retinoic acid response element from the laminin Bl gene (RARE-lam; Vasios et al., 1989); and a retinoic acid response element from the B retinoic acid receptor gene (RARE-B; de The et al., 1990). The TRE-pal, RARE-lam, and RARE-8 sequences have been demon-
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IO
15
M x I O9
20
0
40
[RAR bounol
80
120
M x IO”
Figure 1. Binding of CI Retinoic Acid Receptor to TJ and Retinoic Acid Response Elements in the Absence and Presence of HeLa Nuclear Extract (A) Sequences of response elements used in these studies. TRE-pal is a palindromic variant of the rat growth hormone gene TRE (Glass et al., 1988). TRE-mhc is a TRE residing from -152 to-125 bp from the transcriptional start site of the rat a-myosin heavy chain gene (lzumo and Mahdavi, 1999). RARE-D is a retinoic acid response element residing from -59 to -33 bp upstream from the transcriptional start site of the p retinoic acid receptor gene (de Th6 et al., 1990). RARE-lam is a retinoic acid response element residing from -447 to -422 bp upstream of the transcriptional start site of the laminin Bl gene (Vasios et al., 1999). Each of the sequences shown was flanked by 10 bp at each end containing biotin-11-dUTP residues as described in Experimental Procedures. (6) Binding of 35S-labeled a retinoic acid receptor to the biotinylated TRE-pal probe as a function of increasing TRE-pal concentration in the presence (filled circles) and absence (open circles) of HeLa nuclear extract. The negative reciprocal of the slope derived from plots of bound/free versus bound DNA (right) indicates a binding constant of the retinoic acid receptor of 4 m M for this site in the presence of HeLa nuclear extract. (C) Same experiment as in (B), except that the RARE-B was used as a probe. The binding constants of the a retinoic acid receptor for this element in the presence (filled circles) and absence (open circles) of HeLa nuclear extract were 0.9 m M and 11 mM, respectively.
strated to confer retinoic acid responsiveness to otherwise unregulated promoters by gene transfer experiments. In contrast, the retinoic acid receptor binds with high affinity to the TRE-mhc sequence as a heterodimer with the T3 receptor, but acts to inhibit TBdependent transcription from promoters containing this element in Green monkey kidney (CVl) cells (Glass et al., 1989). The binding of %-labeled a retinoic acid receptor to the TRE-pal and RARE-P sequences as a function of DNA concentration is illustrated in Figures 18 and 1C. In the absence of additional nuclear proteins, the affinity of the retinoic acid receptor for the RARE-P was approximately 11 nM. Its affinity for the TRE-pal was too low to be determined accurately over the same concentration range of DNA. In the presence of HeLa nuclear proteins, the binding of 35S-labeled a retinoic acid receptor to both classes of response elements was significantly increased, particularly at low DNA concentrations. Under these conditions, the binding affinities of in vitro translated a retinoic acid receptor for the RARE-P and TRE-pal sequences were 0.8 mM and 4 nM, respectively. Activities that enhanced retinoic acid receptor binding to T3 and retinoic acid response elements were observed in all cell types of vertebrate origin examined. Figure 2A illustrates the effects of nuclear extracts, derived from a variety of cell types, on retinoic acid receptor binding to the TRE-pal when preincubated with the receptor at equivalent levels of nuclear protein. The effects of these factors were specific for T3 and retinoic acid response elements; enhancement of binding to unrelated &active elements, such as one derived from adenovirus 5 (Ad5; Figure 2B), or other unrelated c&active elements was not observed (data not shown). Intriguingly, differences were observed among the various types of nuclear extracts with respect to their relative effects on retinoic acid receptor binding to T3 and retinoic acid response elements. For example, the binding affinity of the retinoic acid receptor for the TRE-pal was higher than for the RARE-P sequence in the presence of HL60 cell nuclear extracts (0.8 mM and 2.0 nM, respectively), which is the opposite of the hierarchy observed in the presence of HeLa nuclear proteins. The activities present in HeLa and HL60 cells were also distinct from the effects of the p T3 receptor on retinoic acid binding. The p T3 receptor preferentially stimulated retinoic acid receptor binding to the TRE-mhc sequence, as previously reported (Glass et al., 1989), but had no effect on the binding of the retinoic acid receptor to the RARE-lam (Figure 28) or the RARE-P (data not shown). These observations suggested that a variety of cellspecific proteins might interact with the retinoic acid receptor. To obtain more direct evidence for the existence of cell-specific factors, cross-linking experiments were performed with the retinoic acid receptor bound to T3 and retinoic acid response elements in the presence or absence of nuclear extracts prepared from several different cell types. After precipitation of protein-DNA complexes, the cross-linking agent disuccinimidyl suberate (DSS) was added and the products were analyzed by SDS-polyacrylamide gel electrophoresis. As illustrated in Figure 3,
Retinoic Acid Receptor Coregulators 731
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Figure 2. Factors That Enhance Retinoic Acid Receptor Binding to Specific DNA Sequences Are Ubiquitous (A) Binding of 35S-labelsd a retinoic acid receptor to 100 fmol of the biotinylated TRE-pal sequence was assayed in the absence of nuclear proteins (buffer) or in the presence of 100 ug of nuclear extracts derived from the indicated cell types. (B) Comparison of the effects of HsLa cell nuclear factors and the 3 T3 receptor on the binding of the a retinoic acid receptor to T3 and rstinoic acid response elements. Binding of 35S-labelsd a rstinoic acid receptor to the TRE-pal, RARE-lam, TRE-mhc, and an unrelated cisactive element from adenovirus 5, Ad5, is shown in the absence of additional factors (RAR alone), in the presence of HeLa nuclear extract (RAR + HeLa), and in the presence of an unlabeled in vitro translation product of the b T3 receptor (RAR + TSR beta).
cross-linking products containing the retinoic acid receptor were observed only in samples in which the receptor was incubated with nuclear extract prior to DNA binding. The cross-linked products migrated as multiple bands in the molecular weight range from 95-116 kd. The various nuclear extracts produced cross-linked products that exhibited distinctly different patterns of molecular weights. For example, HeLa cell nuclear extracts produced major cross-linked species of 106 and 116 kd. Subtracting the molecular weight of the retinoic acid receptor (51 -kd) provided molecular weight estimates of the cross-linked proteins in HeLa cells to be 55 kd and 65 kd, hereafter referred to as Hp55 and Hpss. In contrast, cross-linking performed in the presence of HL60 nuclear extract resulted in a major product containing a protein with an apparent molecular weight of approximately 45 kd.
Figure 3. Cross-Linking Experiments Identify Cell Typs-Specific Nuclear Proteins That Interact with the Astinoic Acid Receptor on T3 and Retinoic Acid Response Elements (A) %-labeled rstinoic acid receptor was preincubated with binding buffer alone or approximately 100 ug of nuclear protein extracted from HeLa cells, pituitary tumor cells (GC and 235) Green monkey kidney cells (CVl), mouse lymphoma cells (UO A2), F9 tsratocarcinoma cells, and promyslocytic leukemia cells (HL60). DNA binding reactions were then performed using the RARE-3 probe as described in Figure 1 and Experimental Procedures. Following precipitation of protein-DNA complexes with streptavidin-agaross and extensive washing, the protein cross-linking agent DSS was added at a final concentration of 1 mM. After a 10 min reaction, the samples were analyzed by SDS-polyacrylamide gel electrophoresis. (9) Identical experiment to that in (A), except that the TRE-pal sequence was used in place of the RARE-8 sequence. Arrows denote the following molecular weight standards: myosin heavy chain (200 kd), phosphorylase A (98 kd), bovine serum albumin (66 kd), and ovalbumin (44 kd).
These results provided independent evidence for the existence of a potentially large number of distinct and cell type-specific nuclear factors that interact with the a retinoic acid receptor. Comparison of the cross-linked products obtained on the TRE-pal to those obtained on the RARE-b element generally revealed a similar pattern for each particular nuclear extract. However, some extracts exhibited a preference for cross-linking on one of the two elements. For example, cross-linked products were preferentially obtained on the TRE-pal sequence in the presence of HL60 nuclear extract, but preferentially on the RARE-6 sequence in the presence of HeLa nuclear extracts (compare Figure 3A with Figure 38). These differential cross-linking patterns also correlated with the relative affinities of the retinoic acid receptor for the two response elements in the presence of HeLa and HL60 extracts, as described above. Cell fractionation studies indicated that these factors were preferentially localized to the nucleus and required 0.3-0.5 M KCI for efficient extraction. These activities were trypsin sensitive and in all cases could be abolished by
heating at W C for 10 min. These factors were also present in limiting quantities because a 2-fold dilution of each extract generally resulted in an approximate 2-fold decrease in stimulated retinoic acid receptor binding. The ability of nuclear proteins to be cross-linked to the retinoic acid receptor was not reproducibly affected by the addition of retinoic acid (data not shown). However, because reticulocyte lysates used for translation in vitro may contain sufficient retinoic acid to occupy the majority of receptors, dependence of the interaction between the receptor and these proteins on retinoic acid cannot as yet be formally excluded. Fractionation of HeLa nuclear proteins by gel filtration on Superose 6 under high salt conditions was performed to confirm the molecular weight range of proteins interacting with the retinoic acid receptor. The activity profile eluted as an asymmetric peak in the molecular weight range between 55 and 70 kd (Figure 4A), in agreement with the molecular weight range of 55-66 kd predicted on the basis of cross-linking experiments. This activity could be further purified by sequence-specific DNA affinity chromatography on a column containing concatamerized TRE-pal oligonucleotide elements. As shown in Figure 46, passage of HeLa nuclear extract over this column resulted in nearly complete removal of the activity from the flowthrough fraction, while the majority of other nuclear proteins (>95%) passed through the column. After extensive washing, the majority of the initial activity could be recovered by elution of the column with 0.4 M KCI, resulting in an increase in specific activity by more than a factor of 40. The binding of HeLa nuclear factors to this column was sequence specific and of high affinity, because it was performed in the presence of an approximately lo-fold molar excess of salmon sperm DNA and poly(dl-dC). Similar results were obtained using nuclear extracts derived from F9 teratocarcinoma cells, HL60 cells, and WI cells (data not shown). These experiments suggest that, like the TS receptor, other nuclear factors that interact with the retinoic acid receptor also have intrinsic DNA binding properties. Retinoic Acid Receptor Sequences Required for Protein-Protein Interaction Overlap with the Ligand Binding Domain To identify sequences of the retinoic acid receptor necessary for its interactions with cell type-specific nuclear factors on T3 and retinoic acid response elements, the properties of mutant receptors were examined (Figure 5). Deletion of the C-terminus at codon 404 had little or no effect on enhanced DNA binding in the presence of HeLa nuclear extracts (Figure 5A). However, C-terminal deletions ending at codons 351,260, and 204 exhibited no enhanced binding in the presence of HeLa nuclear extracts and demonstrated a slight reduction in basal DNA binding. As expected, the retinoic acid receptor C-terminus (corresponding to codons 167 to 461) was unable to bind to DNA in the presence or absence of HeLa nuclear extract, nor was a point mutant @AR-De*) in which the first conserved cysteine of the DNA binding domain was changed to aspartic acid. Cross-linking experiments per-
Figure 4. Partial Purification of Factors That Interact with the Retinoic Acid Receptor from HeLa Nuclear Extracts (A) Partial purification of factors by gel filtration on a Superose 6 column under high salt conditions (0.4 M KU). Fractions were diluted 4-fold to reduce the KCI concentration to 0.1 M, and tested for their ability to stimulate retinoic acid receptor binding to the TRE-pal (hatched line). The solid line indicates total protein as measured by OC&,. Arrows denote the elution position of the following molecular weight standards: T, thyroglobulin (670 kd); G, y-globulin (158 kd); 0, ovalbumin (44 kd); and M, myoglobulin (17 kd). (B) Partial purification of factors by sequence-specific DNA affinity chromatography. One milliliter of HeLa nuclear extract in binding buffer (20 m M HEPES [pH 7.81, 20% glycerol, 50 m M KCI, 1 m M j3-mercaptoethanol, and 0.1 Nonidet P-40) was combined with 50 ug of salmon sperm DNA and 100 ug of poly(dl-dC) and passed three times through a column containing 1 ml of a DNA affinity matrix consisting of approximately 20 ug of concatamerized TRE-pal covalently linked to Sepharose CL4B. The column was then washed with ten column volumes of binding buffer and eluted with a step gradient of binding buffer adjusted to 0.4 M KCI. Fractions of approximately 0.3 ml were collected and diluted to a final KCI concentration of 100 mM. Crude nuclear extract, the flowthrough fractions, and the fractions eluted with 0.4 M KCI (fractions l-4) were then tested for their ability to activate retinoic acid receptor binding to the TRE-pal. Details of both chromatographic steps are described under Experimental Procedures.
formed with C-terminal deletion mutants demonstrated that enhancement of DNA binding in the presence of HeLa nuclear extracts was correlated precisely with their ability to be cross-linked to Hp55 and Hp65 (Figure 58). These experiments indicate that Hp55 and Hpm mediate the effects of HeLa nuclear extracts on the DNA binding properties of the a retinoic acid receptor and define the
Retinoic Acid Receptor Coregulators 733
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Figure 6. The Protein-Protein Interaction Domain of the Retinoic Acid Receptor Overlaps with the Ligand Binding Domain
Figure 5. Nuclear Factors That Enhance the Binding of the Retinoic Acid Receptor 10 T3 and Retinoic Acid Response Elements Interact with the Retinoic Acid Receptor C-Terminus (A) Influence of HeLa nuclear factors on the binding of wild-type and mutant a retinoic acid receptors to the palindromic TRE. RAR-404, RAR-351, and RAR-204 correspond to C-terminal deletions ending at amino acids 404, 351, and 204, respectively. RAR-N166 represents an amino-terminal deletion in which amino acids 2165 have been removed, including the DNA binding domain. RAR-D* represents a mutant receptor in which the first conserved cysteine of the DNA binding domain has been changed to aspartic acid. In each binding reaction, 100,000 cpm of TCA-precipitable [%]receptor was used for the wild-type and mutant receptors. Binding reactions were performed in the absence or presence of 100 pg of crude HeLa nuclear extract. (B) Cross-linking of HeLa nuclear factors to wild-type and C-terminal deletion mutants of the retinoic acid receptor bound to the palindromic TRE. DNA binding and cross-linking reactions were performed as described in Figure 3 and Experimental Procedures.
C-terminal border of the protein-protein interaction domain to be at or near codon 404. Similar results were observed using nuclear extracts derived from CVl, GC, and HL60 cells (data not shown). No enhancement of DNA binding was observed for the retinoic acid receptor truncated at amino acid 351 in the presence of these extracts. Furthermore, no cross-linked products were obtained in the presence of DSS. These results indicate that all of the proteins observed
to interact
with the retinoic acid receptor to enhance its binding to
(A) Cross-linking experiments define an extensive interaction domain. Rabbit reticulocyte lysate containing 100,000 cpm of [%]reUnoic acid receptor or an equivalent amount of the indicated deletion mutants was incubated with 100 ug of HeLa nuclear protein for 30 min. The samples were desalted over a G-25 column equilibrated in 50 m M HEPES (pH 7.6) to remove free methionine and to dilute nonspecific proteins. One microliter of 50 m M DSS was then added to 50 ~1 of the void fraction from each column and reacted for 15 min at room temperature. The reaction was quenched by addition of 5 pl of 1 M ethanolanine. Samples were analyzed by SDS-PAGE on 8% (left panel) or 12.5% (right panel) polyacrylamide gels. Wild-type RAR, lanes l-5; RAR-N167, lane 6; RAR 167-404, lane 7; RAR 246-404, lane 8. Migration positions of the monomeric receptors are shown on the right of each panel. (6) Summary of sequences required for basal DNA binding, enhanced DNA binding, and protein-protein interaction with Hp55 and Hpss,
T3 and retinoic acid response elements do so via a common interface. To define the N-terminal border of this interaction domain, cross-linking experiments were performed in solution with the wild-type
retinoic acid receptor
and a series
of N-terminal deletion mutants (Figure 6A). The wild-type receptor could be efficiently cross-linked to both Hp55 and Hpss in solution, indicating that the receptor can associate with each of these proteins prior to DNA binding. Although unable to bind to DNA, the retinoic acid receptor C-terminus from codon 187 to 461 retained the ability to be cross-linked to both Hps5 and HP~~, indicating that the DNA binding domain was not essential for its interaction with these two factors. Concomitant with the C-terminal
Cdl
734
deletion data, a double deletion mutant of the retinoic acid receptor consisting of amino acids 167 to 404 also retained the ability to be cross-linked to Hp55 and Hps. However, further truncation of the N-terminus to codon 246 severely compromised the interaction with both proteins. The results of these experiments, which are summarized in Figure 66, indicate that a large region of the retinoic acid receptor C-terminus, extending from approximately codons 167 to 404, is involved in protein-protein interactions with Hp55 and Hp65. The Retinoic Acid Receptor Protein-Protein Interaction and Ligand Binding Domains Are Essential for Efficient Ransactivation In Vivo The DNA binding studies described above lead to the prediction that retinoic acid receptors lacking the protein-protein interactionlligand binding domain should exhibit compromised transcriptional activity in vivo because of reduced affinities for their cognate response element sequences. This behavior would distinguish the retinoic acid receptor from the glucocorticoid receptor, which has been demonstrated to activate constitutively transcription upon removal of its ligand binding domain (Hollenberg et al., 1967; Godowski et al., 1966a). We therefore performed a series of experiments to examine the transcriptional activities of retinoic acid receptor mutants lacking the interaction domain. In one type of experiment, increasing amounts of plasmids directing the expression of the wildtype a retinoic acid receptor or a mutant receptor lacking the entire interaction and ligand binding domain were cotransfected into CVl cells. The transcriptional activities of these receptors were assayed using a variant mouse mammary tumor virus (MTV) promoter plasmid containing the TRE-pal in place of sequences containing glucocorticoid response elements (Umesono et al., 1966). This MTV promoter variant was linked to the chloramphenicol acyltransferase gene to facilitate analysis of promoter expression. As shown in Figure 7A, the wild-type a retinoic acid receptor conferred an efficient transcriptional response that was near maximal at 1 m of transfected expression plasmid. In contrast, the truncated retinoic acid receptor was transcriptionally inactive at this concentration of expression plasmid, and exhibited only a weak, constitutive transcriptional effect when 10 vg of expression plasmid was used. The failure of the truncated retinoic acid receptor to efficiently activate transcription could reflect decreased DNA binding affinity, as suggested by in vitro binding experiments, but could also be due to the removal of C-terminal sequences that function as a Pans-activation domain. To examine this question further, we tested the ability of the retinoic acid receptor’s DNA binding domain to mediate transcriptional activation by a potent, heterologous transactivation domain. To do this, we transferred the major trans-activation (tau) domain of the glucocorticoid receptor to the N-terminus of the truncated retinoic acid receptor (RAR-Glau-204). In contrast with previous studies demonstrating that the glucocorticoid receptor tau domain can transfer additional transcriptional activity to the
RA
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Figure 7. The C-Terminal Interaction and Ligand Binding Domain of the Retinoic Acid Receptor Is Required for Efficient 7-ans-Activation of Responsive Promoters In Vivo (A) Activity of the MTWRE pal promoter as a function of increasing amounts of cotransfected plasmids directing the expression of the wild-type a retinoic acid receptor (RBV-RAR) or the a retlnoic acid receptor truncated at amino acid 204 (RW-RAR A204). (6) Comparison of the transcriptional adivities of the wild-type and truncated retinoic acid receptors with the activities observed when linked in frame at the N-terminus to the major trans-activation domain of the glucocorticoid receptor. One microgram of each expression vector was used per plate. Retinoic acid receptor-dependent promoter expression was measured using the HBWK promoter linked to the firefly luciferase cDNA in which two copies of the TRE-pal sequence were placed 100 bp upstream of the transcriptional start site. Results are the average of duplicate points (differing
