INTRODUCTION

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The transcription factor nuclear factor B (NFB)¹ is important for the inducible expression of a wide variety of cellular and viral genes (reviewed in Refs. 1 and 2). NFB is composed of homo- and heterodimeric complexes of members of the Rel (NFB) family of polypeptides. 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 also is target of the IB inhibitors, which include IB, IB, IB, Bcl-3, p105, and p100 (3). 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. IB is degraded in response to all of the known inducers of NFB, whereas IB is degraded only when cells are stimulated with inducers such as lipopolysaccharide (LPS) and interleukin-1, which cause persistent activation of NFB (4). Following degradation of the initial pool of IB in response to LPS or interleukin-1, newly synthesized IB accumulates in the nucleus as an unphosphorylated protein that forms a stable complex with NFB and prevents it from binding to newly synthesized IB (5, 6). Bcl-3 is an unusual IB protein in that it can not only inhibit nuclear NFB complexes but can bind to p50 and p52 dimers on DNA and provide the complexes with transactivating activity (7, 8).

Transcription coactivators bridge transcription factors and the components of the basal transcriptional apparatus (reviewed in Ref. 9). Functionally conserved proteins CBP and p300 have been shown to be essential for the activation of transcription by a large number of regulated transcription factors, including nuclear receptors (10-13), CREB (14-16), AP-1 (17, 18), bHLH factors (19), STATs (20, 21), and, most recently, the NFB component p65 (22, 23). In particular, the nuclear receptor superfamily is a group of ligand-dependent transcriptional regulatory proteins that function by binding to specific DNA sequences named hormone response elements in promoters of target genes (reviewed in Ref. 24). Transcriptional regulation by nuclear receptors depends primarily upon a ligand-dependent activation function, AF-2, located in the C terminus and predicted to undergo an allosteric change upon ligand binding (24). Consistent with this, CBP and p300 have been found to interact directly with nuclear receptors in a ligand- and AF-2-dependent manner (10-13). In addition, a series of factors that exhibit ligand- and AF-2-dependent binding to nuclear receptors have been identified both biochemically and by expression cloning. Among these, a group of highly related proteins have been shown to form a complex with CBP and p300 and enhance transcriptional activation by several nuclear receptors, i.e. steroid receptor coactivator-1 (SRC-1) (12, 25), AIB1 (26), TIF2 (27), RAC3 (28), ACTR (29), TRAM-1 (30), p/CIP (31), and XICO (32). Interestingly, SRC-1 (33) and its homologue ACTR (29), along with CBP and p300 (34, 35), were recently shown to contain potent histone acetyltransferase activities themselves and associate with yet another histone acetyltransferase protein p/CAF (36). In contrast, it was shown that SMRT (37) and N-CoR (38), nuclear receptor corepressors, form complexes with Sin3 and histone deacetylase proteins (39, 40). From these results, it was suggested that chromatin remodeling by cofactors may contribute through histone acetylation-deacetylation to transcription factor-mediated transcriptional regulation.

In light of the fact that SRC-1 is capable of forming a complex with CBP and p300 that in turn coactivate the NFB component p65 (22, 23), we tested whether SRC-1 itself participates in the NFB-mediated transactivations as well. Herein, we show that 1) SRC-1 specifically binds to the NFB component p50 but not to p65, 2) SRC-1 coactivates the NFB-mediated transactivations, and 3) p300 synergized with SRC-1 in this coactivation. These results suggest that at least two distinct transcription coactivator molecules with histone acetyltransferase activities (i.e. SRC-1 and CBP-p300) may regulate the NFB-mediated transactivations in vivo, and SRC-1, originally identified as a coactivator for the nuclear receptors, may regulate many different transcription factors.

	EXPERIMENTAL PROCEDURES

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Plasmids-- LexA, B42, T7, or GST vectors to express p50 and p65 were as described previously (41). Polymerase chain reaction-amplified fragments of SRC-1 (A-E as depicted in Fig. 1) were subcloned into EcoRI-SalI restriction sites of the LexA fusion vector pEG202PL (42), EcoRI-XhoI restriction sites of the B42 fusion vector pJG4-5 (42), or EcoRI-XhoI restriction sites of the T7 vector pBS-SK (Stratagene, San Diego, CA). The GST fusion vectors encoding CBP-N (amino acids 1-450) and CBP-C (amino acids 1891-2441) (kind gifts from Dr. Chris Glass, University of California at San Diego) were as described (12). The expression vectors for p300 (kind gift from Dr. David M. Livingston, Dana Farber Cancer Institute, Boston, MA) and SRC-1 (kind gift from Dr. Ming Tsai, Baylor College of Medicine, Houston, TX), along with the transfection indicator construct pRSV--gal and the NFB-responsive reporter construct (B)₄-IL-2-Luc, were as described previously (15, 33, 43).

Yeast Two-hybrid Test-- For the yeast two-hybrid tests, plasmids encoding LexA fusions and B42 fusions were cotransformed into Saccharomyces cerevisiae EGY48 strain containing the LacZ reporter plasmid, SH/18-34 (42). Plate and liquid assays of -gal expression were carried out as described (42, 44-46). Similar results were obtained in more than two similar experiments.

GST Pull Down Assays-- The GST fusions or GST alone was expressed in Escherichia coli, bound to glutathione-Sepharose-4B beads (Amersham Pharmacia Biotech), and incubated with labeled proteins expressed by in vitro translation by using the TNT-coupled transcription-translation system, with conditions as described by the manufacturer (Promega, 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-polyacrylamide gel electrophoresis and autoradiography as described (47).

Cell Culture and Transfections-- L929 cells were grown in 24-well plates with medium supplemented with 10% fetal calf serum for 24 h and transfected with 100 ng of LacZ expression vector pRSV--gal and 100 ng of a reporter gene (B)₄-IL-2-Luc (43), along with increasing amounts of expression vectors for SRC-1 or p300. Total amounts of expression vectors were kept constant by adding decreasing amounts of pcDNA3 to transfections containing increasing amounts of the SRC-1 or p300 vector. After 12 h, cells were washed and refed with Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Cells were harvested 24 h later, luciferase activity was assayed as described (48), and the results were normalized to the LacZ expression. Similar results were obtained in more than two similar experiments.

	RESULTS AND DISCUSSION

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Interactions of SRC-1 and p50-- We have recently found that XICO (32), a Xenopus homologue of the nuclear receptor coactivator SRC-1 (12, 25), interacts with p50 but not with p65.² The smallest clone among the original isolates of XICO that readily interacted with p50 consisted of only the central nuclear receptor-binding and CBP-p300-binding domains, suggesting that the interaction interface should be included within these two domains.² These interactions were further characterized in the yeast two-hybrid tests (42), in which formations of p50-p50 homodimer and p50-p65 heterodimer were readily detected as previously reported (41) (Table I). A series of SRC-1 fragments were subcloned into B42 and LexA vectors as depicted in Fig. 1. Among the LexA fusions, only SRC-D (SRC-1 amino acids 759-1141) encompassing the previously described CBP-p300-binding domain (12, 25-32) was found to confer autonomous transactivation function to a LacZ reporter construct controlled by upstream LexA-binding sites (42) (Table I). This is consistent with the previous findings in which the CBP-p300-binding domain was shown to be essential for autonomous transactivation functions (12, 25-32). Consistent with an idea that p50 interacts with SRC-D, coexpression of a B42 fusion to the full-length p50 further stimulated the LexA/SRC-D-mediated LacZ expression, whereas coexpression of a B42 fusion to the full-length p65 was without any effects (Table I). In contrast, the LacZ expressions mediated by LexA fusions to SRC-A, -B, -C, or -E were not stimulated by coexpression of B42/p50 or B42/p65. Similar results were also obtained with B42 fusions to SRC-1 fragments and LexA fusions to p50 and p65, in which coexpression of the B42/SRC-D and LexA/p50 pair efficiently stimulated the LacZ reporter expression (data not shown).

View this table: [in this window] [in a new window]   Table I Interactions between SRC-1 and p50 in yeast The indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate LacZ reporter gene. At least six separate transformants from each transformation were transferred to indicator plates containing 5-bromo-4-chloro-3-indolyl -D-galactopyranoside, and reproducible results were obtained using colonies from a separate transformation. +++, strongly blue colonies after 2 days of incubation and strong interaction; ++, light blue colonies after 2 days of incubation and intermediate interaction; +, light blue colonies after more than 2 days of incubation and strong interaction; , white colonies and no interaction.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the SRC-1 constructs. The full-length human SRC-1 (12, 25) and a series of five SRC-1 fragments are as depicted. The nuclear receptor-interacting, CBP-p300-interacting, bHLH/PAS, serine/threonine-rich, and glutamine-rich domains, along with the recently identified histone acetyltransferase domain (33), are as indicated. The amino acid numbers for each construct are shown.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Interactions of p50 with SRC-1. Full-length p50 and p65 (A) as well as various SRC-1 constructs (B) were labeled with [35S]methionine by in vitro translation and incubated with glutathione beads containing GST alone, GST/p50, GST/p65, GST/CBP-N, and GST/CBP-C, as indicated. Beads were washed, and specifically bound material was eluted with reduced glutathione and resolved by SDS-polyacrylamide gel electrophoresis.

Cotransfection of SRC-1 Stimulates the NFB-mediated Transactivations-- To assess the functional consequences of these interactions, SRC-1 was cotransfected into L929 cells along with the reporter construct (B)₄-IL-2-Luc. This reporter construct, previously characterized to efficiently mediate the NFB-dependent transactivations in various cell types, consists of a minimal promoter from the interleukin-2 gene and four upstream B sites from the IL-6 gene (43). Increasing amounts of cotransfected SRC-1 enhanced the reporter gene expressions in an SRC-1 dose-dependent manner, with cotransfection of 800 ng of SRC-1 increasing the fold activation approximately 3-fold (Fig. 3). Consistent with the reports that CBP and p300 are transcription coactivators of the NFB component p65 (22, 23), increasing amounts of cotransfected p300 had stimulatory effects on the reporter gene expressions, with cotransfection of 100 ng of p300 increasing the fold activation approximately 2-fold. Consistent with an idea that SRC-1 and p300 synergize to coactivate the NFB-mediated transactivations, coexpression of p300 and SRC-1 further increased the reporter gene expressions above the levels observed with SRC-1 or p300 alone (Fig. 3). In various cells, SRC-1 also coactivated the LPS- or tumor necrosis factor -induced level of transactivations (data not shown). In contrast, cotransfection of SRC-1 did not affect the LacZ reporter expression of the transfection indicator construct pRSV--gal either in the presence or the absence of LPS or tumor necrosis factor  (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Effects of SRC-1 and p300 cotransfection on the NFB-mediated transactivations. L929 cells were transfected with LacZ expression vector and increasing amounts of SRC-1 or p300 expression vectors along with a reporter gene (B)4-IL-2-Luc (43) as indicated. Normalized luciferase expressions from triplicate samples are presented relative to the LacZ expressions, and the standard deviations are less than 5%.