Transcription coactivators either bridge transcription factors and the components ... ducer and activator of transcription (STAT)s (3), acti- vating protein-1 (AP-1) (4), ... tivator molecule of nuclear receptors (designated ASC-2). (18). In particular ...
Activating Protein-1, Nuclear Factor-B, and Serum Response Factor as Novel Target Molecules of the Cancer-Amplified Transcription Coactivator ASC-2
Soo-Kyung Lee, Soon-Young Na, Sung-Yun Jung, Ji-Eun Choi, Byung Hak Jhun, JaeHun Cheong, Paul S. Meltzer, Young Chul Lee, and Jae Woon Lee Center for Ligand and Transcription (S.-K.L., S.-Y.N., S.-Y.J., J.H.C., Y.C.L., J.W.L.) Department of Biology (S.-K.L., S.-Y.N.) and Hormone Research Center (J.H.C., Y.C.L., J.W.L.) Chonnam National University Kwangju 500–757, Korea College of Pharmacy (J.-E.C., B.H.J.) Pusan National University Pusan 609–735, Korea Cancer Genetics Branch (P.S.M.) National Human Genome Research Institute National Institutes of Health Bethesda, Maryland 20892-4470
by SRF, AP-1, and NFB, which may contribute to the putative, ASC-2-mediated tumorigenesis. (Molecular Endocrinology 14: 915–925, 2000)
ASC-2 was recently discovered as a cancer-amplified transcription coactivator molecule of nuclear receptors, which interacts with multifunctional transcription integrators steroid receptor coactivator-1 (SRC-1) and CREB-binding protein (CBP)/ p300. Herein, we report the identification of three mitogenic transcription factors as novel target molecules of ASC-2. First, the C-terminal transactivation domain of serum response factor (SRF) was identified among a series of ASC-2-interacting proteins from the yeast two-hybrid screening. Second, ASC-2 specifically interacted with the activating protein-1 (AP-1) components c-Jun and c-Fos as well as the nuclear factor-B (NFB) components p50 and p65, as demonstrated by the glutathione S-transferase pull-down assays as well as the yeast two-hybrid tests. In cotransfection of mammalian cells, ASC-2 potentiated transactivations by SRF, AP-1, and NFB in a dose-dependent manner, either alone or in conjunction with SRC-1 and p300. In addition, ASC-2 efficiently relieved the previously described transrepression between nuclear receptors and either AP-1 or NFB. Overall, these results suggest that the nuclear receptor coactivator ASC-2 also mediates transactivations
INTRODUCTION Transcription coactivators either bridge transcription factors and the components of the basal transcriptional apparatus and/or remodel the chromatin structures (reviewed in Ref. 1). In particular, CREB binding protein (CBP) and its functional homolog p300 were shown to be essential for the activation of transcription by a large number of regulated transcription factors (reviewed in Ref. 2). Similarly, steroid receptor coactivator-1 (SRC-1) and its family members were recently found to stimulate transactivation by many different transcription factors, including CREB and signal transducer and activator of transcription (STAT)s (3), activating protein-1 (AP-1) (4), nuclear factorB (NFB) (5, 6), p53 (7), and serum response factor (SRF) (8). SRC-1 (9) and its homolog ACTR (10), along with CBP and p300 (11, 12), were recently shown to contain histone acetyltransferase activities and associate with yet another histone acetyltransferase protein p/CAF (13). In contrast, silencing mediator of retinoid and thyroid hormone receptor (SMRT) (14) and nuclear receptor corepressor (N-CoR) (15), nuclear receptor corepres-
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sors, form complexes with Sin3 and histone deacetylase proteins (16, 17). These results are consistent with the notion that acetylation of histones destabilizes nucleosomes and relieves transcriptional repression by allowing transcription factors to access to recognition elements, whereas deacetylation of the histones stabilizes the repressed state (1, 2). We have recently isolated a novel transcription coactivator molecule of nuclear receptors (designated ASC-2) (18). In particular, microinjection of anti-ASC-2 antibody abrogated the ligand-dependent transactivation of retinoic acid receptor, and this repression was fully relieved by coinjection of ASC-2-expression vector, consistent with an idea that ASC-2 is essential for the nuclear receptor function in vivo (18). Interestingly, ASC-2 was found to be highly amplified and overexpressed in colon, breast, and lung cancers (18), although it was not clear whether the altered expression of ASC-2 directly contributed to the development of cancers. Based on the interactions with multifunctional transcription integrators SRC-1 and CBP/p300 (1, 2), ASC-2 was suspected to mediate transactivations by transcription factors other than nuclear receptors. In this regard, it was interesting to note that high levels of the ASC-2 expression in various human breast cancer cell lines were not strictly correlated with the estrogen receptor-␣ (ER␣) positivity (18), an important criterion in breast and ovarian cancers. Thus, if the altered expression of ASC-2 plays any role in tumorigenesis, it is likely to involve transcription factors other than ER␣. SRF, AP-1, and NFB are known to control a surprisingly diverse set of genes. However, it is interesting to note that these factors share at least one common property, i.e. stimulation of cellular proliferation processes. SRF, along with ternary complex factor (TCF), binds to and activates the serum response element (SRE), present in the upstream regulatory sequences of myogenic genes as well as a number of immediate early genes, including c-fos, which in turn activate genes critical for cell proliferation (reviewed in Ref. 19). SRF belongs to the MADS box family of proteins and recognizes a CArG box in the SRE, whereas TCF does not bind autonomously to the element, but requires the assistance of SRF to efficiently contact the DNA. The AP-1 complex, immediate early response genes, consists of a heterodimer of a Fos family member and a Jun family member (reviewed in Ref. 20). This complex binds the consensus DNA sequence (TGAGTCA) (termed AP1 sites) found in a variety of promoters. The Fos family contains four proteins (c-Fos, Fos-B, Fra-1, and Fra-2), whereas the Jun family is composed of three (c-Jun, Jun-B, and Jun-D). Fos and Jun are members of the basic-leucine zipper (bZIP) family of sequence-specific dimeric DNA-binding proteins. The C-terminal half of the bZIP domain is amphipathic, containing a heptad repeat of leucines that is critical for the dimerization of bZIP proteins. The N-terminal half of the long bipartite-helix is the basic region that is critical for sequence-specific DNA binding. Finally,
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NFB is composed of homo- and heterodimeric complexes of members of the Rel (NFB) family of polypeptides (reviewed in Ref. 21). In vertebrates, this family comprises p50, p65 (RelA), c-rel, p52, and RelB. These proteins share a 300-amino acid region, known as the Rel homology domain, which binds to DNA and mediates homo- and heterodimerization. This domain is also a target of the IB inhibitors, which include IB␣, IB, IB␥, Bcl-3, p105, and p100. In the majority of cells, NFB exists in an inactive form in the cytoplasm, bound to the inhibitory IB proteins. Treatment of cells with various inducers results in the degradation of IB proteins. The bound NFB is released and translocates to the nucleus, where it activates appropriate target genes. Interestingly, several lines of evidence suggested that constitutive activation of NFB contribute to the malignant phenotype of tumor cells. A naturally occurring splice variant of RelA was shown to transform Rat-1 cells (22), whereas antisense oligonucleotides to RelA were shown to inhibit proliferation and tumorigenicity of several tumor cell lines, including the human breast cancer cell lines MCF7 and T47D (23). In addition, activation of NFB through the disruption of IB␣ regulation was shown to result in malignant transformation (24). Herein, we report the identification of SRF, AP-1, and NFB as new target molecules of ASC-2, in which ASC-2 directly interacts with these factors. In cotransfections, ASC-2 potentiated their transactivations in a dose-dependent manner and appeared to be involved with the previously characterized transrepression between nuclear receptors and either AP-1 (25) or NFB (26, 27). These results indicate that ASC-2 may directly regulate the cellular proliferation or tumorigenesis processes in vivo, by acting as a novel coactivator molecule of these mitogenic transcription factors.
RESULTS Identification of SRF as an ASC-2-Interactor We have recently described the molecular cloning of ASC-2, a cancer-amplified transcription coactivator molecule of nuclear receptors (18). Based on the functional interactions with multifunctional transcription integrators SRC-1 and CBP/p300 (1, 2), ASC-2 was suspected to function with other transcription factors. In an attempt to identify such factors, we have screened a mouse liver cDNA library by using the yeast two-hybrid screening, in which the previously described ASC-2 fragment (i.e. ASC2–4) (Fig. 1) was used as bait. Consistent with the notion that ASC-2 is a multifunctional transcription integrator molecule, we have isolated a series of different transcription factors and cofactors, identical or homologous to the previously characterized factors (our unpublished results). Among these, two independent isolates encoded the C-terminal region of SRF (i.e. the SRF residues 203– 504, designated SRF⌬N), as shown in Fig. 2A. This interaction was confirmed in the in vitro glutathione
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Fig. 1. Schematic Representation of ASC-2 The full-length human ASC-2 and a series of 10 ASC-2 fragments are as depicted. The glutamic acid/aspartic acid-rich (D/E), glutamine-rich (Q-rich), glutamine/proline-rich (Q/P-rich), serine/threonine-rich (S/T-rich), and basic amino acids (⫹/⫹) domains, as well as two LxxLL motifs (3, 46) that are known to be important for the ligand-dependent interactions between coactivators and nuclear receptors, are as indicated. The previously defined interaction domain for nuclear receptors (RID) (18) and the interaction domains for c-Jun and c-Fos (c-Jun/c-Fos-ID), p65 (p65-ID), SRF (SRF-ID), and p50 (p50-ID), along with the amino acid numbers for each construct, are also shown.
S-transferase (GST) pull-down assays. GST alone and GST-fusions to ASC2–4, ASC2–4.5, and SRF were expressed, purified, and tested for interaction with in vitro translated luciferase, SRF, and ASC-2. As shown in Fig. 2B, the radiolabeled SRF readily interacted with GST-ASC2–4 (the ASC-2 residues 1172–1729) and GST-ASC2–4.5 (the ASC-2 residues 1429–2063), but not with GST alone. Similarly, the radiolabeled ASC-2 interacted with GST-SRF, but not with GST alone. In contrast, the radiolabeled luciferase bound neither of the GST proteins, as expected (results not shown). We have also examined whether the endogenous ASC-2 can bind to SRF in vitro. As shown in Fig. 2C, GSTSRF but not GST alone retained the endogenous ASC-2 from HeLa nuclear extracts, as revealed in Western analysis with the previously described specific monoclonal antibody against ASC-2 (18). As a positive control, GST fusion to thyroid hormone receptor (TR) was shown to retain the endogenous ASC-2 from HeLa nuclear extracts in a T3-dependent manner. From these results, we concluded that ASC-2 specifically binds to SRF. Localization of the SRF-Interacting Interface of ASC-2 To localize the SRF-interaction interface of ASC-2, we generated a series of ASC-2 fragments (Fig. 1). In
the yeast two-hybrid tests, coexpression of a transactivation domain B42 (28) fusion to the full-length SRF (i.e. B42-SRF) further stimulated the LexA-ASC2–4 (the ASC-2 residues 1172–1729), LexA-ASC2–4.5 (the ASC-2 residues 1429–2063), and LexA-ASC2–5 (the ASC-2 residues 1559–2063)-mediated LacZ expressions. However, coexpression of B42-SRF did not affect the LexA-ASC2–1 (the ASC-2 residues 1–557), LexA-ASC2–2 (the ASC-2 residues 391-1057), and LexA-ASC2–3 (the ASC-2 residues 586-1310), and LexA-ASC2–3.5 (the ASC-2 residues 929-1511)-mediated LacZ expressions (Fig. 3A). LexA fusions to ASC2–1 and ASC2–2 showed autonomous transactivation function, as previously noted (18). In addition, transactivation mediated by LexA fusions to ASC2–4a (the ASC-2 residues 1429–1729) and ASC2–4b (the ASC-2 residues 1559–1729) but not ASC2–4c (the ASC-2 residues 1172–1511) was enhanced by coexpression of B42 fusions to the full-length SRF (Fig. 3A). Similar results were also obtained with a B42 fusion to the SRF residues 203–504 (i.e. SRF⌬N). Consistent with these results, B42 fusions to ASC2–4.5, ASC2– 4a, and ASC2–4b but not ASC2–4c stimulated the LexA-SRF and LexA-SRF⌬N-mediated transactivation (Fig. 3B). From these results, we concluded that the SRF transactivation domain interacts with a region containing the ASC-2 residues 1559–1729 (as summarized in Fig. 1).
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Fig. 2. Interactions of SRF with ASC-2 A, The full-length SRF as well as SRF⌬N (i.e. the SRF residues 203–504), which was isolated from the yeast two-hybrid screening as an ASC-2-interactor, are as depicted. The DNA-binding/dimerization (DBD/DD) and transactivation domains as well as the amino acid numbers for each construct are as indicated. B, SRF and ASC-2 were labeled with [35S]methionine by in vitro translation and incubated with glutathione beads containing GST alone, GST-ASC2–4, GST-AS2–4.5, or GST-SRF, as indicated. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-PAGE. Approximately 20% of the total reaction mixture was loaded as input. C, HeLa nuclear extracts were incubated with bacterially expressed GST fusions to TR and SRF or GST alone, as indicated. ⫺ and ⫹ indicate the absence and presence of 0.1 M of T3, respectively. Specifically bound proteins were released by glutathione, resolved by SDS-PAGE, and probed with a monoclonal antibody against ASC-2 (18) in Western analysis. Approximately 20% of the nuclear extracts used in the binding reactions were loaded as input.
Stimulation of the SRF Transactivation by ASC-2 To assess the functional consequences of these interactions, ASC-2 was cotransfected into HeLa cells along with a reporter construct SRE-c-fos-LUC. This reporter construct, previously characterized to efficiently mediate the SRE-mediated transactivations in various cell types (29), consists of a minimal promoter from the c-fos gene and a single upstream consensus SRE. Serum shock with 20% FCS resulted in approximately 10-fold increase in transactivation of this reporter construct, relative to the level with nonshocked cells (Fig. 4A). Increasing amounts of cotransfected ASC-2 enhanced the reporter gene expressions in an ASC-2 dose-dependent manner, with cotransfection of 100 ng of ASC-2 increasing the fold activation approximately 3-fold (Fig. 4A). As previously reported (8, 30), cotransfected p300 or SRC-1 also had stimulatory effects on the reporter gene expressions. Consistent with an idea that CBP/p300 and SRC-1 functionally cooperate with ASC-2, coexpression of p300 or SRC-1 further increased the reporter gene expressions above the levels observed with ASC-2 alone
(Fig. 4A). In contrast, cotransfection of ASC-2 did not affect the LacZ reporter expression of the transfection indicator construct pRSV--gal either in the presence or absence of serum shock (results not shown). Similar results were also obtained with a reporter construct driven by the previously described SRE-containing, natural c-fos promoter (31) (Fig. 4B). From these results, we concluded that ASC-2 is a bona fide transcription coactivator molecule of SRF. Interaction of ASC-2 with AP-1 and NFB The fact that ASC-2 is overexpressed in human cancers and mediates transactivation by SRF, the wellcharacterized mitogenic transcription factor (19), prompted us to examine whether ASC-2 is also involved with other pro-proliferative transcription factors. Thus, GST alone and GST fusions to the AP-1 components c-Jun and c-Fos as well as the NFB components p50 and p65 were expressed, purified, and tested for interaction with in vitro translated ASC-2. As shown in Fig. 5A, the radiolabeled ASC-2 interacted with all of these GST fusion proteins except
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Fig. 3. Localization of the SRF-Interacting Domain of ASC-2 The indicated B42- and LexA-plasmids were transformed into a yeast strain containing an appropriate LacZ reporter gene, as described (28). Open boxes indicate LexA (A) or B42 (B) alone. Gray and closed boxes indicate the presence of B42 (A) or LexA (B) fusions to SRF and SRF⌬N, respectively, whereas hatched box indicate the presence of LexA fusion to B42, as a positive control. The results with a B42 fusion to SRF⌬N were not determined with regard to LexA fusions to ASC2–1, ASC2–2, and ASC2–3. The data are representative of at least two similar experiments, and the SDs were less than 5%.
GST alone. In contrast, the radiolabeled SRC-1 bound to GST fusions to c-Jun, c-Fos, and p50 but not p65, as we have previously reported (4, 5). These interactions were further confirmed in yeast. Coexpression of B42 fusions to c-Jun and c-Fos stimulated transactivation by LexA-ASC2–2 but not LexA fusions to ASC2–3, ASC2–3.5, and ASC2–4.5 (Fig. 5B and results not shown). These results strongly suggest that interactions with c-Jun and c-Fos are likely to involve the ASC-2 residues 391–585, although this prediction has yet to be independently confirmed. Coexpression of B42-p50 stimulated transactivation by LexA fusions to ASC2–2, ASC2–3, and ASC2–3.5 but not ASC2–4.5, suggesting that the p50 interactions involve either the ASC-2 residues 929-1057 or multiple regions throughout the ASC-2 residues 391-1429. Finally, coexpression of B42-p65⌬N (i.e. the p65 residues 353–550), which encompasses the p65 transactivation domain, stimulated transactivation by LexA-ASC2–2 but not LexA-ASC2–3.5 and LexA-ASC2–4.5, suggesting that the p65 interactions involve the ASC-2 residues 391– 929. In contrast, B42-p65⌬C (i.e. the p65 residues 1–323) did not show any interactions (Fig. 5B). Overall, these results indicate that specific subregions of
ASC-2 are differentially recognized by the AP-1/NFB components c-Jun, c-Fos, p50, and p65, as summarized in Fig. 1. ASC-2 Stimulates Transactivations by AP-1 and NFB To assess the functional consequences of these interactions, ASC-2 was cotransfected into CV1 cells along with a reporter construct (AP-1)4-TK-LUC (Fig. 6A) and (B)4-interleukin-2 (IL-2)-LUC (Fig. 6C), which consist of a minimal promoter from the thymidine kinase gene and four upstream consensus AP-1 sites (4) and a minimal promoter from the interleukin-2 gene and four upstream B sites from the IL-6 gene (5), respectively. These reporter constructs were previously characterized to efficiently mediate the AP-1 and NFB-dependent transactivations, respectively, in various cell types. Increasing amounts of cotransfected ASC-2 enhanced the reporter gene expressions in an ASC-2 dose-dependent manner. As previously noted (2, 4, 5), cotransfected SRC-1 or p300 also had stimulatory effects on these reporter gene expressions. Consistent with an idea that CBP/p300 and SRC-1 function-
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of cells that expressed the LacZ reporter (i.e. blue cells) was not significant among cells microinjected with control IgG in the absence of TPA but increased to approximately 45% in the presence of TPA. However, only approximately 10% of cells turned blue even in the presence of TPA, when microinjected with antiASC-2 IgG (Fig. 6B). This ␣ASC-2-mediated repression of the TPA response was fully relieved by coinjected ASC-2-expression vector but not by pcDNA3 (Fig. 6B). Taken together with the transient transfection data (Fig. 6A), we concluded that ASC-2 is a molecule pivotal for the function of AP-1 in vivo. These results, along with the results with SRF (Fig. 4), NFB (Fig. 6B), and nuclear receptors (18), strongly suggest that ASC-2 is a multifunctional transcription integrator molecule, like CBP/p300 and SRC-1. ASC-2 Is Involved with the Transrepression between Nuclear Receptors and AP-1/NFB
Fig. 4. ASC-2 Stimulates the SRE-Mediated Transactivation HeLa cells were transfected with LacZ expression vector and ASC-2, p300, or SRC-1-expression vector along with a reporter gene SRE-c-fos-LUC (29) (A) or the natural c-fos promoter-driven reporter construct c-fos-LUC (31) (B), as indicated. Cells were shocked with 20% FCS before harvest, as described (8). Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions, and the results were expressed as fold-activation (nfold) over the value obtained with unstimulated cells. The experiments were repeated at least three times, and the SDs were as shown. Similar results were also obtained with CV-1 and NIH3T3 cells.
ally cooperate with ASC-2, coexpression of p300 or SRC-1 further increased the reporter gene expressions above the levels observed with ASC-2 alone (Fig. 6, A and C). ASC-2 also coactivated the 12-Otetradecanoylphorbol-13 acetate (TPA)- or tumor necrosis factor-␣ (TNF␣)-induced level of transactivations, whereas cotransfection of ASC-2 did not affect the LacZ reporter expression of the transfection indicator construct pRSV--gal either in the presence or absence of TPA or TNF␣ (results not shown). To investigate the function of ASC-2 in vivo, the microinjection technique (32) was further used, in which the LacZ reporter gene was placed under the control of an SV40 minimal promoter containing TPA-responsive AP-1 sites. Remarkably, microinjection of anti-ASC-2 IgG significantly prevented TPA from activating this TPA-dependent transcription unit (Fig. 6B) but had no effect on a promoter under the control of the cytomegalovirus promoter (results not shown). The percentage
Mutual transcriptional inhibitions have been described between various liganded nuclear receptors and either AP-1 or NFB, which was suggested to be due to an interaction among these factors that results in mutual loss of DNA-binding activity (25–27). More recently, competition for common transcription coactivators such as CBP/p300 and SRC-1 was also proposed to be responsible for this mutual antagonism (4, 33). ASC-2 shows similar effects. Coexpression of ASC-2 enhanced the T3-dependent transactivation by TR in a dose-dependent manner (Fig. 7A). As previously noted (4, 5, 27), however, the T3-dependent transactivation by TR was repressed by coexpression of c-Fos or p65 (Fig. 7B), whereas liganded TR efficiently repressed transactivation of both AP-1 and NFB-responsive reporter constructs (Fig. 7, C and D). These inhibitory effects were largely relieved upon addition of increasing amounts of ASC-2. These results strongly indicate that ASC-2 is directly involved with the transrepressions between nuclear receptors and either AP-1 or NFB.
DISCUSSION ASC-2 was previously shown to have characteristics that are typical of a bona fide transcription coactivator molecule of nuclear receptors (18), i.e. ASC-2 showed a strong ligand- and AF2-dependency in interactions with nuclear receptors, a potent autonomous transactivation function, and specific interactions with the basal transcription machinery. Most strikingly, microinjection of the ASC-2 antibody completely abrogated the ligand-dependent reporter gene activities in Rat-1 fibroblast cells, which was rescued by coexpressed ASC-2 (18). In this report, our original hypothesis (18) that ASC-2 could function as a multifunctional transcription integrator molecule, like CBP/p300 and SRC-1, has been further strengthened. First, we have
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Fig. 5. Interactions of ASC-2 with AP-1 and NFB A, ASC-2 and SRC-1 were labeled with [35S]methionine by in vitro translation and incubated with glutathione beads containing GST alone or GST fusions to c-Jun, c-Fos, p50, and p65, as indicated. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-PAGE. Approximately 20% of the total reaction mixture were loaded as input. B, The indicated B42- and LexA-plasmids were transformed into a yeast strain containing an appropriate LacZ reporter gene, as described (28). Open boxes indicate LexA alone, whereas hatched, stippled, closed and gray boxes indicate the presence of LexA fusions to ASC2–2, ASC2–3, ASC2–3.5, and ASC2–4.5, respectively. The data are representative of at least two similar experiments and the SDs were less than 5%.
isolated a series of transcription factors and cofactors, including SRF (Fig. 2), as ASC-2-interacting proteins from the yeast two-hybrid screening (our unpublished results). Second, ASC-2 was shown to stimulate transactivations by SRF (Fig. 4), AP-1, and NFB (Fig. 6), likely through specific interactions with these factors (as summarized in Fig. 1). Third, ASC-2 appeared to be involved with the previously defined transrepression between nuclear receptors and either AP-1 or NFB (Fig. 7). This ASC-2-mediated reversal of the transrepression between TR and AP-1/NFB (Fig. 7) suggests that a limiting amount of ASC-2 is competitively recruited by these transcription factors. However, it is also possible that ASC-2 may titrate out a putative, secondary factor, which links TR to AP-1/NFB and is required for the transrepression. Alternatively, the exogenously supplied ASC-2 could be blocking the protein-protein interactions of TR with AP-1/NFB (i.e. AP-1 and NFB may directly compete with nuclear receptors to recruit ASC-2). Consistent with this idea, we noticed that the c-Jun/c-Fos, p65, and p50-interaction domains of ASC-2 are clustered around the previously defined receptor interaction domain (as
summarized in Fig. 1). Finally, it’s interesting to note that the ASC-2 interaction domains of SRF (Fig. 2) and p65 (Fig. 5B) overlap with their previously defined autonomous transactivation domains (i.e. the N-terminal region of SRF and the C-terminal region of p65) (20, 21). Considering the fact that ASC-2 is highly amplified in human cancers (18), it’s particularly interesting to note that these newly identified target proteins of ASC-2 are mitogenic transcription factors. Recently, AIB1, an SRC-1 family member, was identified as a gene amplified and overexpressed in breast and ovarian cancers (34). SRC-1 and its family members were also shown to serve as a novel transcription coactivator molecule of AP-1 (4), NFB (5, 6), and SRF (8). Interestingly, ASC-2 maps to 20q11, substantially centromeric to AIB1 (which maps to 20q12) (35), demonstrating that two distinct coactivator molecules of nuclear receptors can be coamplified in cancer cells. Such a co-selection process may favor genes that impinge on the same cellular processes and result in significant effects on transcriptional regulation within tumor cells. In particular, overexpression of these mul-
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Fig. 6. ASC-2 Stimulates the AP-1 and NFB Transactivations A and C, CV-1 cells were transfected with LacZ expression vector and ASC-2, SRC-1 or p300-expression vector along with a reporter gene (AP-1)4-TK-LUC (A) or (B)4-IL-2-LUC (C), as indicated. Transfections were done essentially as described (4, 5). Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions, and the results were expressed as fold-activation (n-fold) over the value obtained with unstimulated cells. Similar results were also obtained with TPA and TNF␣ as well as other cell lines including HeLa and NIH3T3. B, Rat-1 cells were microinjected with control IgG or anti-ASC-2 IgG, along with a reporter construct (i.e. AP-1-SV40--GAL), pcDNA3, and pcDNA3-ASC-2, as indicated. The number of cells that express LacZ is counted relative to the total number of microinjected cells, and expressed as % blue cells, as indicated. Experiments were done at least three times, with ⬎200 cells injected; error bars are ⫾ 2 ⫻ SEM. Open and closed boxes indicate the absence and presence of 0.1 M of TPA, respectively.
tifunctional integrator molecules may provide a selective advantage for tumor growth. Thus, it remains to be determined whether these mitogenic factors (i.e SRF, NFB, and AP-1) are indeed targeted by increased levels of these coactivator proteins in vivo to sustain tumor growth. However, it is interesting to note that the inhibitory effects of the AP-1 and NFB transactivations by T3 or retinoids can be significantly attenuated when ASC-2 is overexpressed (Fig. 7 and results not shown). These results raise an interesting possibility that overexpressed ASC-2 may contribute to the induction of retinoid resistance in certain human cancers. In breast cancer cells, both overexpression of c-Jun and decreased expression of retinoid receptors were previously shown to result in such retinoid resistance (36, 37). Our recent unpublished data indicate that ASC-2 forms a distinct steady-state coactivator complex in vivo and functionally communicates with other coac-
tivator complexes such as CBP/p300/SRC-1 (38) and ASC-1 (39). The ASC-2 complex was different from the ASC-1, CBP/p300, and SRC-1-complexes (38, 39) when eluted from a sizing column. Interestingly, this endogenous ASC-2 complex was readily retained by GST fusions to SRF, TR, and p50 (Fig. 2C and results not shown). In particular, the TR interactions were strictly T3-dependent, as expected (18). Similar to the previously shown results with nuclear receptors (18), microinjection of ASC-2 antibody effectively disrupted the AP-1 transactivation by TPA (Fig. 6B), suggesting that this ASC-2 complex is essential for at least two classes of transcription factors in vivo, i.e. AP-1 and nuclear receptors. Currently, one of our major focuses is to elucidate the molecular mechanisms by which the ASC-2 complex functions and its activity is regulated. First, we are biochemically dissecting the components of the ASC-2 complex. Second, we have recently found that expression of ASC-2 mRNA is up-regulated
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Fig. 7. Involvement of ASC-2 in Transrepression between Nuclear Receptors and Either AP-1 or NFB HeLa cells were transfected with LacZ expression vector and increasing amount of ASC-2-expression vector along with a reporter gene T3RE-TK-LUC (A and B), (AP-1)4-TK-LUC (C), or (B)4-IL-2-LUC (D). For a T3 response, 10 ng of TR were cotransfected (A and B), whereas 50 ng of c-Fos (C) and p65 (D) were cotransfected to activate the AP-1 and NFB transactivations. To repress the T3 transactivation, 50 ng of c-Fos or p65 were cotransfected (B). Transfections were done essentially as described (4, 5). Open and closed boxes indicate the absence and presence of 0.1 M of T3, respectively. Normalized luciferase expressions from triplicate samples were calculated relative to the LacZ expressions, and the results were expressed as fold-activation (n-fold) over the value obtained with unstimulated cells. Similar results were also obtained with TPA and TNF␣ as well as other cell lines including CV-1 and NIH3T3.
in certain cells by various cytokines and growth factors, including IL-1 and epidermal growth factor (EGF), activating signals for NFB and AP-1/SRF, respectively (our unpublished results). Similarly, transcripts of the SRC-1 family member RAC3 (40) and the nuclear receptor corepressor molecule SMRT (14) were recently shown to be directly up-regulated by retinoids (41, 42). Expression of PGC-1, a thermogenic tissuespecific coactivator of nuclear receptors, was also shown to be temporally regulated (43). To understand this up-regulation of ASC-2, we have recently isolated its full-promoter from a human genomic library, which contains a series of interesting regulatory sites, including AP-1 sites (results not shown). Finally, ASC-2 contains a numerous number of putative phosphorylation sites (18) and, thus, the possible regulation of its inherent activity through various signal transduction pathways is being examined. Recently, this notion has been confirmed with CBP, in which a calcium flux was shown to function as an activating signal for its stimulatory transcriptional activity (44).
In conclusion, we identified three mitogenic transcription factors (i.e. SRF, AP-1, and NFB) as novel targets for ASC-2, which may contribute to the putative, ASC-2-mediated tumorigenesis. We have also shown that ASC-2 is a multifunctional transcription integrator molecule, like SRC-1 (1, 3–8) and CBP/p300 (2). Thus, further characterization of ASC-2 may provide important insights into the tumorigenesis processes as well as the molecular mechanisms by which multiple transcription factors are coordinately regulated within the cell.
MATERIALS AND METHODS Plasmids To express LexA- and B42-fusions, PCR fragments encoding ASC2–1, ASC2–2, ASC2–3, ASC2–3.5, ASC2–4, ASC2–4.5, ASC2–5, ASC2–4a, ASC2–4b, ASC2–4c, and p65⌬N (i.e. the p65 residues 353–550) and p65⌬C (i.e. the p65 residues
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1–323) were cloned into EcoRI and XhoI/SalI restriction sites of pEG202PL and pJG4–5 (28), respectively. For GST fusion vectors, PCR fragments encoding ASC2–4 and ASC2–4.5 were cloned into EcoRI and XhoI restriction site of pGEX4T (Pharmacia Biotech, Piscataway, NJ). The AP-1-SV40--GAL reporter construct was a gift from Dr. Dave Rose (University of California, San Diego, CA). GST fusion vectors encoding SRF, TR, c-Jun, c-Fos, p50, and p65 as well as the mammalian expression vectors for SRF, ASC-2, p300, SRC-1, c-Fos, p65, and TR, along with the transfection indicator construct pRSV--gal and reporter constructs SRE-c-fos-LUC, c-fosLUC, T3RE-TK-LUC, (AP-1)4-TK-LUC, and (B)4-IL-2-LUC, were as previously described (4, 5, 7, 8, 18, 27, 29, 31, 39). The Yeast Two-Hybrid Screening and Yeast Galactosidase Assay The LexA-ASC2–4 (Fig. 1) was used as a bait to screen a mouse liver cDNA library in pJG4–5 (28) for ASC-2-interacting proteins, and the screening was executed essentially as previously described (45). The yeast -galactosidase assay was done as described (28). For each experiment, at least three independently derived colonies expressing chimeric proteins were tested. GST Pull-Down Assays The GST-fusions or GST alone was expressed in Escherichia coli, bound to glutathione-Sepharose-4B beads (Pharmacia Biotech), and incubated with labeled proteins expressed by in vitro translation by using the TNT-coupled transcriptiontranslation system, with conditions as described by the manufacturer (Promega Corp., Madison, WI). Specifically bound proteins were eluted from beads with 40 mM reduced glutathione in 50 mM Tris (pH 8.0) and analyzed by SDS-PAGE and autoradiography as described (28). Cell Culture and Transfections HeLa, NIH3T3, and CV-1 cells were grown in 24-well plates with medium supplemented with 10% FCS for 24 h and transfected with 100 ng of LacZ expression vector pRSV-gal and 100 ng of an indicated reporter gene, along with indicated amounts of various mammalian expression vectors. Total amounts of expression vectors were kept constant by adding pcDNA3. Transfections and luciferase assays were done as described (4, 5, 8), and the results were normalized to the LacZ expression. Similar results were obtained in more than two similar experiments. Single-Cell Microinjection Assay Rat-1 fibroblast cells, made quiescent by incubating in serum-free medium for 24 h, were microinjected with either preimmune IgG or the affinity-purified anti-ASC-2 IgG along with AP-1-SV40--GAL reporter construct (25 g/ml). About 1 h after injection, cells were stimulated, where indicated, with 0.1 M TPA. After 4 h incubation, cells were fixed and stained to detect injected IgG by using fluorescein isothiocyanate (FITC)-conjugated antibodies and examined for -galactosidase expression as previously described (32).
Acknowledgments We thank Dr. Dave Rose for the LacZ reporter construct. Received November 16, 1999. Revision received January 24, 2000. Accepted February 24, 2000. Address requests for reprints to: Jae Woon Lee, Ph.D., Center for Ligand and Transcription, Chonnam National Uni-
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versity, Kwangju 500–757, Korea. E-mail jlee@chonnam. chonnam.ac.kr. This work was supported by a grant from the National Creative Research Initiatives Program from the Korean Ministry of Science and Technology, Republic of Korea. B.H.J was supported by a grant from the Korean Ministry of Public Health (HMP-98-B-3–0022).
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