Molecular Cloning of xSRC-3, a Novel Transcription Coactivator from ...

Molecular Cloning of xSRC-3, a Novel Transcription Coactivator from Xenopus, That Is Related to AIB1, p/CIP, and TIF2

Han-Jong Kim*, Soo-Kyung Lee*, Soon-Young Na, Hueng-Sik Choi, and Jae Woon Lee College of Pharmacy (H.-J.K., S.-K.L., J.W.L.) Department of Biology (S.-Y.N.) Hormone Research Center (H.-S.C., J.W.L.) Chonnam National University Kwangju, South Korea 500–757

Nuclear receptors regulate transcription by binding to specific DNA response elements of target genes. Herein, we report the molecular cloning and characterization of a novel Xenopus cDNA encoding a transcription coactivator xSRC-3 by using retinoid X receptor (RXR) as a bait in the yeast two-hybrid screening. It belongs to a growing coactivator family that includes a steroid receptor coactivator amplified in breast cancer (AIB1), p300/ CREB-binding protein (CBP)-interacting protein (p/ CIP), and transcriptional intermediate factor 2 (TIF2). It also interacts with a series of nuclear receptors including retinoic acid receptor (RAR), thyroid hormone receptor (TR), and orphan nuclear receptors [hepatocyte nuclear receptor 4 (HNF4) and constitutive androstane receptor (CAR)]. However, it does not interact with small heterodimer partner (SHP), an orphan nuclear receptor known to antagonize ligand-dependent transactivation of other nuclear receptors. In CV-1 cells, cotransfection of xSRC-3 differentially stimulates ligand-induced transactivation of RXR, TR, and RAR in a dose-dependent manner. Interestingly, xSRC-3 is highly expressed in adult liver and early stages of oocyte development, suggesting that studies of xSRC-3 may lead to better understanding of the roles nuclear receptors play in oocyte development as well as liver-specific gene expression. (Molecular Endocrinology 12: 1038–1047, 1998)

INTRODUCTION The nuclear receptor superfamily is a group of transcriptional regulatory proteins linked by conserved structure and function (1). The superfamily includes receptors for a variety of small hydrophobic ligands 0888-8809/98/$3.00/0 Molecular Endocrinology Copyright © 1998 by The Endocrine Society

such as steroids, T3, and retinoids, as well as a large number of related proteins that do not have known ligands, referred to as orphan nuclear receptors (2). The receptor proteins are direct regulators of transcription that function by binding to specific DNA sequences named hormone response elements (HREs) in promoters of target genes. While some nuclear receptors apparently bind HREs only as homodimers, retinoic acid receptors (RARs), thyroid hormone receptors (TRs), vitamin D receptor, peroxisomal proliferator-activated receptors (PPARs), and several orphan nuclear receptors bind their specific response elements with high affinity as heterodimers with retinoid X receptors (RXRs) (3–8). Based on this high-affinity binding, such heterodimers have been considered to be the functionally active forms of these receptors in vivo. These heterodimers display distinct HRE specificities to mediate the hormonal responsiveness of target gene transcription, in that distinct HREs are comprised of direct repeats (DRs) of a common halfsite with variable spacing between repeats playing a critical role in mediating specificity (2, 9). Accordingly, RARs activate preferentially through DRs spaced by two or five nucleotides, whereas vitamin D receptor and TR activate through DRs spaced by three and four nucleotides, respectively. RXR-PPAR heterodimers as well as RXR homodimers activate preferentially through DRs spaced by one nucleotide (referred to as DR1). In addition to DRs, response elements composed of palindromes as well as inverted palindromes, referred to as everted repeats, have been shown to mediate transcriptional activation by RXR-RAR and RXR-TR heterodimeric complexes (9). Such DNAbinding flexibility stands in contrast to the steroid hormone receptors, which bind exclusively as homodimers to inverted repeats spaced by three nucleotides (10). Transcriptional activation of nuclear receptors involves at least two separate processes: derepression and activation (2). Repression is mediated in part by 1038

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interaction of unliganded receptors with corepressors such as N-CoR (nuclear corepressor receptor) (11) and SMRT (12). However, ligand binding triggers dissociation of these corepressors and concomitant recruitment of coactivators. These putative receptor-interacting coactivators include RIP-140 and RIP160 (13, 14), estrogen receptor (ER)-associated proteins ERAP-140 and ERAP-160 (15), TIF1 (transcription intermediary factor 1) (16), TRIP1 (17), ARA70 (18), CBP (CREB-binding protein)/p300 (19–21), SRC-1 (steroid receptor coactivator 1) (19, 22), AIB1 (23), TIF2 (transcriptional intermediate factor 2) (24), RAC3 (25), ACTR (26), TRAM-1 (27), and p/CIP (p300/CBP-interacting protein) (28). In particular, the last seven proteins are highly related to each other and can enhance transcritpional activation by several nuclear receptors (19, 22–28). Functional analysis of nuclear receptors has shown that there are two major activation domains. The N-terminal domain (AF-1) contains a ligand-independent activation function, whereas the extreme C-terminal region of the ligand-binding domain (AF-2) exhibits ligand-dependent transactivation (1). The AF-2 region is conserved among nuclear receptors, and deletion or point mutations in this region impair transcriptional activation without changing ligand and DNA binding affinities (29–31). Recent x-ray crystallographic studies of the ligand-binding domain of nuclear receptors revealed that the ligand binding induces a major conformational change in the AF-2 region (32–34), suggesting that this region may play a critical role in mediating transactivation by a liganddependent interaction with coactivators. These coactivators are postulated to function to transmit the signal of ligand-induced conformational change to the basal transcription machinery. As expected, many coactivators fail to interact with AF-2 mutants of nuclear receptors (13, 16, 24). Targeted chromatin structure change has been postulated to associate with the regulation of gene expression by nuclear receptors (35–37). In particular, recent biochemical and genetic studies support the notion that hyperacetylation of core histones is characteristic to gene activation, while histone deacetylation is involved with transcriptional repression (38). SRC-1 (39) and its homolog ACTR (26), along with CBP/p300 (40, 41), were recently shown to contain potent histone acetyltransferase activities themselves and associate with histone acetyltransferase protein P/CAF (42). CBP/p300 also forms a complex with SRC-1 (43). These results suggest that nuclear receptors target at least three different and self-interacting histone acetyltransferase activities (SRC-1 or related proteins, CBP/p300, and P/CAF) to promoters (26). In contrast, it was shown that SMRT and N-CoR, nuclear receptor corepressors, form complexes with Sin3 and histone deacetylase proteins (44, 45). From these results, it was suggested that chromatin remodeling by cofactors contributes, through histone acetylation-deacetylation, to receptor-mediated transcriptional regulation.

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To understand the processes of ligand-dependent transactivation by nuclear receptors, we screened a Xenopus oocyte cDNA library for RXR-interacting proteins by using the yeast two-hybrid system (46). We describe here the isolation and characterization of a novel Xenopus transcription coactivator xSRC-3. It is a novel member of a growing coactivator family that includes SRC-1 (19, 22), AIB1 (23), p/CIP (28), and TIF2 (24). Studies of xSRC-3 will provide new insights into the molecular mechanisms of transcriptional regulation by nuclear receptors, particularly in oocyte development and liver-specific gene expression, two target tissues where xSRC-3 is highly expressed.

RESULTS Isolation and Expression of xSRC-3 To isolate cDNAs encoding proteins that specifically interact with RXR, we exploited the Gal4-based yeast two-hybrid system that has been previously described (46). We screened a Xenopus oocyte cDNA library (Clontech, Palo Alto, CA) by using a bait containing the ligand binding domain (LBD) of human RXRa. Two independent isolates encoded a novel coactivator molecule that we named xSRC-3. A full-length xSRC-3 cDNA, isolated from rescreening of the identical cDNA library, contains an open reading frame of 1391 amino acids (Fig. 1A). As indicated in Fig. 1B, xSRC-3 is highly related to AIB1 (23), p/CIP (28), and TIF2 (24) (overall, 77%, 71%, and 53% identity, respectively). These proteins share a basic helix-loophelix (bHLH)/PAS domain in the N-terminal, a nuclear receptor-binding domain in the central, and a glutamine (Q) rich sequence in the C-terminal region. In particular, xSRC-3 and its family members have three specific motifs sharing a consensus sequence of LXXLL (where L is leucine and X is any amino acid) within the central receptor-binding domains. These motifs and their neighboring residues are highly charged and well conserved among xSRC-3 and its family members (22–28) (Fig. 1C), and they were recently shown to mediate protein-protein interactions between liganded nuclear receptors and RIP-140, SRC-1, p/CIP, and CBP (28, 47). Northern blot analysis of poly(A)1 RNAs from Xenopus oocytes and adult tissues indicated that the expression of xSRC-3 is highly regulated. As shown in Fig. 2A, xSRC-3 was expressed only in liver among adult tissues examined (;5.8 kb), indicating that xSRC-3 may play specific roles in nuclear receptormediated gene expression in liver. As a control, 28S RNA was probed using the identical blot and found to be expressed in comparable amounts throughout all the tissues (data not shown). In addition, xSRC-3 was highly expressed in only early stages of oocyte development (reviewed in Ref. 48) (Fig. 2B; at least five distinct sizes from approximately 5.8–11.0 kb). The expression was most prominent in stage I, dramati-

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cally decreased in stage II, and then gradually disappeared in later stages. In contrast, 28S RNA was expressed in comparable amounts throughout all the stages (data not shown). Consistent with the multiple mRNAs of xSRC-3, we also isolated a number of potential alternative splicing variants of xSRC-3 from the Xenopus oocyte cDNA library (data not shown). Each of these splicing isoforms may play distinct roles in the development of oocytes. Characterization of xSRC-3-Receptor Interactions To examine receptor interaction properties of xSRC-3, we constructed a chimeric LexA protein fused to the central receptor-binding domain of xSRC-3 (amino acids 421-1263) (LexA/xSRC-3-R). As shown in Fig. 3, LexA/xSRC-3-R was transcriptionally inert with a lacZ reporter construct controlled by upstream LexA sites (operators) in yeast cells. However, coexpression of B42 fusions to RAR, RXR-LBD, CAR-LBD (49), and hepatocyte nuclear factor 4 (HNF4)-LBD (50) enhanced the expression of the lacZ reporter, indicating that these receptors interact with xSRC-3 (Fig. 3, A, B, D, and E). Addition of 9-cis-RA had no significant effects on the enhanced expression by B42/RAR and B42/RXR-LBD. Interestingly, coexpression of B42/TRLBD resulted in minimal expression of the lacZ reporter in the absence of T3. However, addition of 1 mM T3 led to significant enhancement of the lacZ reporter expression, indicating that the interaction of xSRC-3 and TR is ligand-dependent (Fig. 3C). In contrast, coexpression of B42 fusion to SHP (51, 52), an orphan nuclear receptor known to antagonize ligand-dependent transactivations of other nuclear receptors (B42/ SHP), was without any effects, indicating that xSRC-3 does not bind to SHP (Fig. 3F). The LXXLL motifs were recently shown to mediate protein-protein interactions between liganded nuclear receptors and p/CIP, RIP-140, SRC-1, and CBP (28, 47). Thus, we examined the effects of specific mutations introduced into each of the three LXXLL motifs in the central receptor interaction domain of xSRC-3. As indicated in Fig. 1C, three mutants, in which the second leucine in the LXXLL motifs was mutated to alanine, were constructed in the context of LexA/xSRC3-R by using PCR (LexA/xSRC-3-LR1, LR2, and LR3,

Fig. 1. Sequence of xSRC-3 A, Amino acid sequences of xSRC-3. Boxed sequences indicate bHLH, and underlined sequences indicate two PAS domains. Shaded sequences are the LXXLL motifs (28, 47), while italicized sequences denote glutamine-rich domain. Two original isolates are indicated as solid and broken arrows, respectively. B, Schematic representation of xSRC-3 and its family members. Regions in AIB1, p/CIP, and TIF2-

with significant homology are aligned with xSRC-3, and percentage of amino acids identical to xSRC-3 in the area of bHLH/PAS and nuclear receptor (NR)-CBP-binding domains, along with the overall similarity percentages, are as indicated. Functional domains including bHLH/PAS, five LXXLL motifs included in the NR- and CBP-binding domains, as well as a stretch of glutamines (poly Q) are as indicated. C, Sequence alignment of the three LXXLL motifs within the receptorinteracting domain. Sequences of AIB, p/CIP, TIF2, and SRC-1 are aligned with xSRC-3. Three point-mutants in these motifs (LXXLL to LXXAL) that are described in Fig. 3 are as indicated (xSRC-3-LR1, LR2, and LR3, respectively). Mutated amino acids are boldface.


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Fig. 2. Northern Blot Analysis of xSRC-3 Northern analyses were performed with Xenopus tissues (A) and oocytes from different developmental stages (B). Tissues and oocyte developmental stages (48) examined are indicated as well as the approximate sizes for each mRNA. RNA loadings are as shown.

Fig. 3. Interactions of xSRC-3 with a Subset of Nuclear Receptors Host cells, in which B-galactosidase expression is dependent on the presence of a transcriptional activator with a LexA DNA-binding domain, were transformed with plasmids expressing the indicated LexA and B42 chimeras. Wild type denotes LexA/xSRC-3-R, a LexA fusion to the central receptor-binding domain of xSRC-3 (amino acids 421-1263). LR1, LR2, and LR3 are identical to LexA/xSRC-3-R except point-mutations within the three LXXLL motifs previously shown to bind nuclear receptors (28, 47), as indicated in Fig. 1C. Open bars indicate coexpression of B42 alone. Hatched bars (no ligand) or black bars (addition of 1 mM ligand) indicate coexpression of indicated B42-receptor fusions. These cells were grown in liquid culture containing galactose, since expression of the B42 chimeras is under the control of the galactose-inducible GAL1 promoter (53). b-Galactosidase readings were determined and corrected for cell density and for time of development (A415 nm/A600 nm) 3 1000/min. The result is the average of at least three different experiments, and the SDs are less than 5%.

respectively). In binding to B42/RAR, LexA/xSRC-3LR1 showed similar results with the wild type. LexA/ xSRC-3-LR2 and LR3 showed significantly impaired interactions in the absence of ligand, while they

showed approximately 25–50% of the wild-type interactions in the presence of 1 mM 9-cis-RA (Fig. 3A). In binding to B42/RXR-LBD, LexA/xSRC-3-LR1 showed significant decrease (;30% of the wild-type interac-

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tions) in the absence of ligand. However, the ligandinduced interaction was comparable to that of the wild type. The results with LexA/xSRC-3-LR2 and LR3 were similar to those of B42/RAR (Fig. 3B). All the mutants showed a significant decrease in binding to B42/TR-LBD (;50–70% of the wild type) and B42/ HNF4-LBD (;30% of the wild type), indicating that all the motifs should be intact for full interactions with TR or HNF4 (Fig. 3, C and D). Surprisingly, interactions with B42/CAR-LBD was not affected by any of the mutations, indicating that CAR interacts with distinct domains of xSRC-3 other than these LXXLL motifs (Fig. 3E). Interactions of xSRC-3 with the AF-2 Domain of TR Recent x-ray crystallographic studies of the ligand binding domain of nuclear receptors revealed that the ligand binding induces a major conformational change in the AF-2 region (32–34), suggesting that this region may play a critical role in mediating transactivation by a ligand-dependent interaction with coactivators. Interestingly, xSRC-3 interacts with TR in a ligand-dependent manner, while it constitutively interacts with RAR and RXR (Fig. 3). However, pointmutations in the LXXLL receptor-interacting motifs revealed cryptic ligand-dependent interactions with RAR and RXR (Fig. 3, A and B). These results suggest that the xSRC-3-receptor interaction interface may contain, at least for a subset of receptors, the AF-2 domain. To test this idea, we constructed a chimeric B42 protein fused to the central receptorbinding domain of xSRC-3 (amino acids 421-1263) (B42/xSRC-3-R). In yeast cells, B42/xSRC-3-R was coexpressed with either LexA/TR-LBD or LexA/TRLBD-F459P, a previously described point-mutant in which the AF-2 domain is specifically disrupted (53). As expected, LexA/TR-LBD-F459P did not show any interactions with B42/xSRC-3-R, while LexA/ TR-LBD efficiently interacted with B42/xSRC-3-R (Fig. 4). These results confirm that xSRC-3 indeed interacts with the AF-2 domain of TR. Autonomous Transactivation of xSRC-3 and Interaction with p300 To examine whether xSRC-3 can directly stimulate transcription when recruited to a specific promoter by linking with a heterologous DNA-binding domain, we constructed a chimeric LexA protein fused to the central receptor binding domain of xSRC-3 to the C terminus (amino acids 421-1391) (LexA/xSRC-3-C). LexA alone did not activate the LacZ reporter controlled by upstream LexA-binding sites, while LexA/ xSRC-3-C efficiently stimulated the reporter gene expression (Fig. 5). In contrast, a similar LexA fusion protein LexA/xSRC-3-R, which lacks the C-terminal 128 amino acids, was transcriptionally inert. These

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Fig. 4. Interactions of xSRC-3 with the AF-2 Domain of TR Wild-type and F459P denote LexA/TR-LBD, a LexA fusion to the LBD of rat TRb 1, and a similar LexA fusion protein with a point mutation in the AF-2 domain of TR, respectively, as previously described (53). Open bars indicate coexpression of B42 alone. Hatched bars (no ligand) or black bars (addition of 1 mM ligand) indicate coexpression of B42/xSRC-3-R, a B42 fusion to the central receptor-binding domain of xSRC-3 (amino acids 421-1263). b-Galactosidase readings were determined and corrected for cell density and for time of development (A415 nm/A600 nm) 3 1000/min. The result is the average of at least three different experiments, and the SDs are less than 5%.

Fig. 5. Autonomous Transactivation of xSRC-3 (2) denotes LexA alone. While xSRC-3-C denotes LexA/ xSRC-3-C, a LexA fusion to the central receptor-binding domain to the C terminus of xSRC-3 (amino acids 421-1391), xSRC-3-R denotes LexA/XSRC-3-R, a LexA fusion to the central receptor-binding domain of xSRC-3 (amino acids 421-1263). Open bars indicate coexpression of B42 alone. Black bars indicate coexpression of B42/p300-C, a B42 fusion to the SRC-1-binding domain of p300 (amino acids 2041–2157). b-Galactosidase readings were determined and corrected for cell density and for time of development (A415 nm/A600 nm) 3 1000/min. The result is the average of at least three different experiments, and the SDs are less than 5%.

results indicate that xSRC-3 contains an autonomous transactivation domain, functional in yeast cells, at the C-terminal region including the C-terminal 128 amino acids. Next, we examined whether


the autonomous transactivation function of xSRC-3 is related to interactions with CBP/p300. A chimeric B42 protein fused to the SRC-1-binding domain of p300 (20) (amino acids 2041–2157) was constructed (B42/p300-C). Coexpression of B42/p300-C further enhanced the transcriptional activities of LexA/ xSRC-3-C, while it did not enhance the transcriptional activities of LexA alone or LexA/xSRC-3-R, which lacks the autonomous transactivation function (Fig. 5). These results suggest that xSRC-3 interacts with p300, and the autonomous transactivation may require efficient p300/CBP bindings. In addition, the C-terminal 128 amino acids of xSRC-3

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should be important in binding to CBP/p300, as opposed to the previous results (24–28). Coactivation of Nuclear Receptors by xSRC-3 in CV-1 Cells To examine the coactivator activity of xSRC-3, cotransfection studies were performed in CV-1 cells. As shown in Fig. 6, cotransfection with expression vectors for xSRC-3 significantly increased TR-mediated induction of the TREpal-TK-Luc (49) reporter gene activity by T3. Similarly, xSRC-3 enhanced RXR-mediated induction of the TREpal-TK-Luc reporter gene activity by 9-cis-RA. However, the enhancement of

Fig. 6. Coactivation of Nuclear Receptors by xSRC-3 CV-1 cells were transfected with b-galactosidase expression vector and increasing amount of xSRC-3-expression vectors along with a reporter gene TREpal-TK-LUC (A, B, and D) or b-RARE-TK-LUC (C), as indicated. Cells were unstimulated (open bars) or stimulated (black bars) with 0.1 mM ligand. The cotransfection experiments were also performed with an additional 10 ng of hRXRa or 60 ng of RXRDAF2 (50) expression vectors (D). Normalized luciferase expressions from triplicate samples are presented relative to the b-galactosidase expressions. The result is the average of at least two different experiments, and the SDs are less than 5%.

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RAR-mediated induction of the b-RARE-TK-Luc (49) reporter gene activity by 9-cis-RA was only modest (Fig. 6, A, B and C). In all the cases, the basal level of reporter expression was not significantly affected by cotransfected xSRC-3. We have also performed the cotransfection experiments in the presence of additional expression vectors for rTRb1, hRARa, and hRXRa (Fig. 6D and data not shown). In particular, when 10 ng hRXRa were cotransfected, xSRC-3 dramatically enhanced the 9-cis-RA-induced transactivation of the TREpal-TK-Luc reporter gene activity, with cotransfection of 100 ng xSRC-3 increasing the foldactivation more than 8-fold (Fig. 6D). Furthermore, cotransfection of p300 showed additive effects with xSRC-3 (Fig. 6D), consistent with the interaction of xSRC-3 and p300 (Fig. 5). With cotransfection of 60 ng RXRDAF2, a previously described deletion mutant with a specific defect for the AF2 function (50), xSRC-3 was not able to show any coactivation, consistent with the importance of the AF2 domain to interact with xSRC-3 (Fig. 4). We also cotransfected different cell lines with similar results (data not shown). From these results, we concluded that xSRC-3 is indeed a transcriptional coactivator for nuclear receptors.

DISCUSSION Transcriptional activation of nuclear receptors involves at least two classes of cofactors: corepressors and coactivators (1). Corepressors that associate with unliganded nuclear receptors mediate repression, while coactivators are recruited upon ligand binding and concomitant dissociation of the corepressors. In this report, we added a novel member to a growing family of distinct coactivators that include SRC-1 (19, 22), AIB1 (23), TIF2 (24), and p/CIP (28). We named this factor xSRC-3, which was isolated by using RXR as a bait in the yeast two-hybrid screening of Xenopus oocyte cDNA library. These proteins share the N-terminal bHLH/PAS domain (.60% identity within 350 amino acids), which does not appear to be required for xSRC-3 and its family members to enhance receptormediated transactivation. Even though its function is currently unknown, it may contribute to some functional aspects. Interestingly, xSRC-3 and its family members were shown to have rather conservative, a few alternative splicing isoforms (Refs. 22–28 and data not shown). The conservation of these splicing events suggests that distinct roles may be played by each of these isoforms. It is an interesting hypothesis that xSRC-3 and its family members might be involved with multiple transactivator-mediated signalings, as was the case with CBP/p300 (19–21). Currently, at least two mechanistic details have been proposed to describe the function of these coactivators. First, they are postulated to function to transmit the signal of ligand-induced conformational change to the basal transcription machinery. Second,

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they have been associated with targeted chromatin structure change by nuclear receptors (35–37). Recent biochemical and genetic studies support the notion that hyperacetylation of core histones is characteristic to gene activation, and histone deacetylation is involved with transcriptional repression (38). For instance, it was shown that SMRT (12) and N-CoR (11), nuclear receptor corepressors, form complexes with Sin3 and histone deacetylase proteins (44, 45). In contrast, SRC-1 (39) and its homolog ACTR (26), along with CBP/p300 (40, 41), were recently shown to contain potent histone acetyltransferase activities themselves and associate with histone acetyltransferase protein P/CAF (42), while CBP/p300 can form a complex with SRC-1 (43). These results suggest that nuclear receptors target at least three different and selfinteracting histone acetyltransferase activities (SRC-1 or related proteins, CBP/p300, and P/CAF) to promoters (26). In light of these results, it will be interesting to test whether xSRC-3 itself contains histone acetyltransferase activities. The nuclear receptor and CBP-interaction domains within xSRC-3 and its family members contain a number of a short sequence motif LXXLL (where L is leucine and X is any amino acid) (28, 47). The third helical motif in the receptor interaction domain of NCoA-1 was recently shown to be absolutely required for the RAR function, but not for the ER function (28). Consistent with this, the third helical motif of xSRC-3 seems to be most important for interactions with RAR and RXR (Fig. 3, A and B). However, mutation of the second helical motif of xSRC-3 resulted in reasonably strong interactions with RAR and RXR (Fig. 3), while similar mutation in NcoA-1 completely abolished interactions with RAR and ER (28). These results suggest that these motifs could provide the molecular basis of specificity in nuclear receptor-mediated transcriptional responses by xSRC-3 and its family members. Interactions of xSRC-3 with RAR and RXR were relatively strong and, at least for a partial xSRC-3 (LexA/xSRC-3-R), ligand-independent, while mutations in the LXXLL motifs revealed cryptic ligand-dependent interactions (Fig. 3, A and B). Accordingly, it will be important to test whether a full-length xSRC-3 shows more prominent ligand dependency in interactions with RAR and RXR. The LXXLL motifs within the receptor-binding domains of xSRC-3 and its family members seem to be functionally redundant; i.e. a single mutation within the three motifs only weakens the overall interactions without complete abolishment (Ref. 28 and Fig. 3). Strikingly, none of the interaction mutants affected interactions with CAR (49), indicating that CAR should recognize domains other than these motifs (Fig. 3E). In light of these CAR results, it is noteworthy that TRAM-1 (27) was recently shown to bind subdomains of nuclear receptors including a helix 3 rather than the AF-2 domain, in contrast to SRC-1/ TIF2 (19, 22, 24). Accordingly, it will be interesting to test whether xSRC-3 binds similar subdomains of CAR (49). Most exciting was the finding that xSRC-3


constitutively binds HNF4 (51) and CAR, but does not bind SHP (52, 53). These results were consistent with the fact that HNF4 and CAR are constitutive transactivators (49, 51), while SHP, an orphan nuclear receptor known to antagonize ligand-dependent transactivations of other nuclear receptors, contains an active repressor domain (53). Similarly, Rac3 (25) did not interact with COUP-TF (chicken ovalbumin upstream promoter-transcription factor) (55), an orphan nuclear receptor that also antagonizes other receptors. Our results indicate that the C-terminal 128 amino acids of xSRC-3 are essential in binding to p300 and the autonomous transactivation may require efficient p300/CBP bindings (Fig. 6). These results contradict previous results (24–26, 28), in which this C-terminal region seemed unnecessary for the CBP/p300 interactions. Additional, more thorough domain mapping and mutagenesis experiments will be required to provide further insights into the receptor-xSRC-3-p300/ CBP interactions. We clearly demonstrated that xSRC-3 enhances transcriptional activation by RXR and TR in mammalian cells. However, it’s not clear why we observed only a modest enhancement by RAR, while a much higher level of enhancement was observed by its close relative ACTR (26), for instance. This might be due to the fact that, in our experiments with CV-1 cells, a more than 1,200-fold induction was initially obtained with RAR by hormone treatment in the absence of cotransfected xSRC-3. In the experiments with ACTR, only a 10-fold induction was observed with A549 lung carcinoma cells (26). Thus, a saturation effect might have limited our ability to detect a higher level of enhancement. In addition, cotransfection of an additional 10 ng RXR resulted in more than 8-fold activation (compare the results between Fig. 6B and Fig. 6D), suggesting that the ratio between receptor and xSRC-3 can be important. Thus, it is possible that target receptors for xSRC-3 could be present in adequate amounts relative to xSRC-3 in vivo. In conclusion, we identified a novel coactivator xSRC-3 that is related to a growing coactivator family that includes SRC-1 and TIF2. Accordingly, xSRC-3 displays various properties of transcriptional coactivator, including the capacity for ligand-dependent interactions with the receptors, autonomous transactivation, and transcription coactivation. However, xSRC-3 shows very distinct expression patterns from other members. As such, xSRC-3 may lead us to better understand tissue-specific control of nuclear receptormediated gene expressions.

MATERIALS AND METHODS Hormones, Yeast Cells, and Plasmids T3 and 9-cis-RA were obtained from Sigma Chemical Co. (St. Louis, MO). Y190 cells and a parental vector pGBT are as described (Clontech, Palo Alto, CA). A chimeric protein con-

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sisting of Gal4 DNA-binding domain fused to the ligand binding domain of human RXRa was constructed by using PCR (Gal4-DBD/RXR-LBD). EGY48 cells, the lexA-b-galactosidase reporter construct, and the LexA- and B42-parental vectors were as reported (52). LexA or B42 fusions to the LBDs of CAR, HNF-4, RXRa, and rat TRb as well as B42 fusions to full-length SHP and RARa were as previously described (52, 54, 56). LexA/xSRC-3-R, a chimeric LexA protein fused to the central receptor-binding domain of xSRC-3 (amino acids 421-1263) and LexA/xSRC-3-C (amino acids 421-1391), a LexA fusion to the central receptor-binding domain to the C terminus of xSRC-3 were similarly constructed. LexA/xSRC-3-LR1, LexA/xSRC-3-LR2, and LexA/ xSRC-3-LR3 were constructed, in the context of LexA/xSRC3-R, by introducing alanines into the second leucines of the three LXXLL motifs (28, 47) within the receptor-binding domains of xSRC-3, respectively. B42/p300-C (amino acids 2041–2157), a B42 fusion to the SRC-1 binding domain of p300 (20) was also constructed. Yeast expression vectors, YEP-RXRa, YEP-RXRb, YEP-RXRg, YEP-RARa, YEP-RARb, YEP-RARg, and YEP-TRa and YEP-TRb were as described (54). Mammalian expression vectors for rTRb1, hRXRa, RXRDAF2 and hRARa, the reporter constructs TREpal-TKLUC and b-RARE-TK-LUC, and the transfection indicator construct pRSV-b-gal are as described (49, 50). Yeast Two-Hybrid Screening Gal4-DBD/RXR-LBD was used as a bait to screen a Xenopus oocyte cDNA library in pGAD10 vector (Clontech) for RXRinteracting proteins in the absence of ligand, as previously described (46). The library plasmids from positive clones that expressed both HIS3 and LacZ reporters were rescued and retransformed into yeast cells, together with the original bait and other constructs, for testing the specificity of proteinprotein interaction. Two independent isolates encoding xSRC-3 was selected for further analysis in this study. Yeast b-Galactosidase Assay The cotransformation and quantitative liquid b-galactosidase assays in yeast were performed with the following changes as described previously (54). The yeast culture was initially diluted to an A600 nm of 0.05, and plated into 96-well culture dishes with the various concentrations of hormone. The cultures were then incubated in the dark at 30 C for 16 h. The A600 nm was determined, and then cells were lysed and substrate was added and A415 nm was read after 10–30 min. The normalized galactosidase values were determined as follows: (A415 nm/A600 nm) 3 1000/min developed. For each experiment, at least three independently derived colonies expressing chimeric receptors were tested. Northern Blot Analysis Poly(A)1 RNAs were isolated from Xenopus multiple tissues and Xenopus oocytes of different developmental stages (48), electrophoresed on a formaldehyde-agarose gel, transferred to membrane, hybridized with a random primed 32P-labeled DNA probes (encompassing amino acids 421-1263 of xSRC3), and exposed on x-ray film, as described previously (57). Cell Culture and Transfection CV-1 cells were grown in 24-well plates with medium supplemented with 10% charcoal-stripped serum. After 24 h incubation, cells were transfected with 100 ng b-galactosidase expression vector pRSV-b-gal and 100 ng of a reporter gene TREpal-TK-LUC (49) or b-RARE-TK-LUC (49), either in the presence or absence of 10 ng of rTRb1-, hRARa-, or

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hRXRa-expression vectors, as previously described (57). Total amounts of expression vectors were kept constant by adding decreasing amounts of the CDM8 expression vector to transfections containing increasing amounts of the TRb-, RARa,- or RXRa-vector. After 12 h, cells were washed and refed with DMEM containing 10% charcoal-stripped FBS. After 12 h, cells were left unstimulated or stimulated with 0.1 mM ligand. Cells were harvested 24 h later, and luciferase activity was assayed as described (57), and the results were normalized to the b-galactosidase expression. Similar results were obtained in more than two similar experiments.

Acknowledgments We thank Drs. Tae-Sung Kim, Wongi Seol, Yoon Kwang Lee, and David D. Moore for valuable advice, plasmids, and critical readings of this manuscript.

Received December 31, 1997. Re-revision received March 31, 1998. Accepted April 2, 1998. Address requests for reprints to: Jae Woon Lee, Ph.D., College of Pharmacy, Hormone Research Center, Chonnam National University, 300 Yongbong-dong Puk-gu, Kwangju 500–757, Korea. E-mail: [email protected]. This research was supported by the academic research fund of Ministry of Education, Republic of Korea (GE 96–81/ 97–143 to J.W.L) and Korean Science and Engineering Foundation (HRC to H.S.C and J.W.L). *The first two authors contributed equally.

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